Research ArticleEffects of Four Compounds from Gentianella acuta (Michx)Hulten on Hydrogen Peroxide-Induced Injury in H9c2 Cells

Kai Ren1 He Su23 Li-juan Lv4 Le-tai Yi3 Xue Gong1 Lian-sheng Dang5

Rui-fen Zhang23 and Min-hui Li 123

1Baotou Medical College Baotou 014000 China2Inner Mongolia Autonomous Region Hospital of Traditional Chinese Medicine Hohhot 010020 China3Inner Mongolia Institute of Traditional Chinese Medicine Hohhot 010020 China4Department of Basic Science Tianjin Agricultural University Tianjin 300384 China5Department of Geriatrics e first Affiliated Hospital of Baotou Medical College Baotou 014000 China

Correspondence should be addressed to Min-hui Li prof liminhuiyeahnet

Received 14 September 2018 Revised 5 December 2018 Accepted 6 December 2018 Published 20 January 2019

Academic Editor Christina Pabelick

Copyright copy 2019 Kai Ren et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

In previous studies Gentianella acuta (Michx) Hulten was reported to contain xanthones iridoids terpenoids and sterols and ismainly used to cure hepatitis jaundice fever headache and angina pectoris In this study we used bioassay guided fractionationto identify compounds from G acuta and investigated their activity against hydrogen peroxide (H

2O2)-induced apoptosis of H9c2

cells using the 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazoliumbromide (MTT)methodThe levels of nuclear factor erythroid2-related factor 2 (Nrf2) heme oxygenase-1 (HO-1) and glutamate-cysteine ligase catalytic (GCLC) expression were assessed usingquantitative real-time polymerase chain reaction (qRT-PCR) Protein expression was evaluated using western blot The resultsshowed that all four compounds had protective effects on H9c2 cells The transcription levels of HO-1 and GCLC significantlyincreased inH9c2 cells pretreatedwith norswertianolin (1) swetrianolin (2) demethylbellidifolin (3) and bellidifolin (4) Howevercompared to the model group the transcription levels of Nrf2 were not enhanced by pretreatment with compounds 1 2 and 4 Theprotein expression levels of HO-1 and GCLC in H9c2 cells were greater than that in the H

2O2-treated group and the expression of

Nrf2 was not significantly changed except by swetrianolin treatment inhibitors can reverse the protective effect by ZnPP (15 120583M)BSO (10120583M) and brusatol (10120583M) The results indicated that the four compounds isolated from G acuta inhibited the oxidativeinjury induced by H

2O2by activating the Nrf2ARE pathway in H9c2 cells and provide evidence that G acutamay be a potential

therapeutic agent for the treatment of cardiovascular diseases

1 Introduction

Gentianella acuta belongs to the family Gentianaceae and isalso known by the Mongolian name Agute-Qiqige It is anannual herbaceous plant widely distributed to the north ofChina the Mongolia Plateau Siberia and the Far East areasof Russia [1] In previous studies G acuta was reported tocontain xanthenes iridoids terpenoids and sterols [2] Thewhole grass is used as aMongolian native medicinemainly totreat hepatitis jaundice fever and headache A study showedthat G acuta had a significant effect on angina pectoris andother diseases [3]

Angina pectoris the most common type of coronaryheart disease is a clinical syndrome caused by coronaryinsufficiency and acute and temporary myocardial ischemia[4] Myocardial ischemia and reperfusion produce numerousreactive oxygen species (ROS) that can lead to oxidativeinjury of H9c2 cells which is one of the main causes of H9c2cells death after angina [5 6] Hydrogen peroxide (H

2O2) isan important source of ROS which directly oxidize lipids andproteins on the cell membrane which then react with Fe2+ toproduce sdotOH and other more active free radicals to induceapoptosis or necrosis H

2O2is one of the important causes

HindawiBioMed Research InternationalVolume 2019 Article ID 2692970 9 pageshttpsdoiorg10115520192692970

2 BioMed Research International

of myocardial apoptosis through the induction of oxidativestress [7]

In this study we used a bioassay guided method to isolatefour compounds from G acuta We used chromatography toseparate these active ingredients by analyzing the activity dis-carding the inactive components and repeating this processuntil the active ingredients were identified We described theisolation and identification of these compounds along withthe evaluation of their inhibitory effects on H9c2 cells Tothis end we used a combination of high-performance liquidchromatography (HPLC) the 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) and quantitativereal-time polymerase chain reaction (qRT-PCR) methods toinvestigate the protective effects and preliminary mechanismof action of four compounds isolated fromG acuta Further-more the effects on protein expression of relevant pathwaymolecules were evaluated using western blotting

2 Materials and Methods

21 Materials The rat embryonic ventricular myocardial cellline (H9c2)was purchased from Shanghai Ruchu Biotechnol-ogy Co Ltd High glucose DMEM (Gibco USA) fetal bovineserum penicillin and streptomycin (HyClone Thermo Sci-entific) phosphate-buffered saline (PBS) dimethyl sulfoxide(DMSO) purchased from the Sino-American BiotechnologyCompany of Beijing (Beijing China) ultrapure water madeby Gen Pure (Thermo USA) ethanol ethyl acetate n-butanol petroleum chromatographic grade acetonitrile andall chemicals were purchased from Concord TechnologyCo Ltd (Tianjin China) H2O2 was from Tianjin Fu YuReagent Co Ltd (Tianjin China) Trizol was purchasedfrom Invitrogen (Carlsbad CA USA) PrimeScript RTReagent Kit (Perfect Real Time) was obtained from TaKaRaBiotechnology Co (Dalian China) The primers of HO-1GCLC Nrf2 and GAPDH were synthesized from SangonBiotech (Shanghai China)

22 Plant Materials G acuta was collected from GenheCity Inner Mongolia Autonomous Region in July 2013 andidentified byDr Zhang Chunhong (BaotouMedical College)A voucher specimenwas deposited at the Laboratory of Phar-macognosy and Phytochemistry in Baotou Medical College

23 Extraction and Isolation

231 Preparation of Crude Extracts Air-dried powder of Gacuta (301 g) was extracted successively with 70 ethanol toproduce 99 g of the extractThe extract was dissolved in waterand then extracted successively with petroleum ether ethylacetate and 119899-butanol to produce 594 g 1537 g and 1361 gof dry extracts respectively The aqueous phase contained1552 g of the residue

232 Preparation of Fractions from Crude Extracts The ethylacetate fraction (10 g) was separated using a dry column withsilica gel (200-300 mesh [150 g] and a petroleum ether-ethylacetate mixture73 vv) to produce four main fractions (Frs

A1-A4) Fr A2 (691 g) was then further purified using chro-matography with silica gel (200-300 mesh 150 g petroleumether-ethyl acetate73 vv) to produce 14 fractions (Frs B1-B14) We then divided the products into three fractions (B1[14 g] B2-B10 [447 g] and B11-B14 [086 g]) using thin-layerchromatography (TLC)

The 119899-butanol fraction (10 g) was chromatographed usinga silica gel column (200-300 mesh 200 g) to obtain fourfractions (Frs C1-4 [172 57 153 and 048 g respectively])

233 Preparation of Monomeric Compounds Fractions B2-B10 were further purified repeatedly using a Sephadex LH-20column (with petroleum ether-ethyl acetate 73 vv) to obtaincompounds 1 (22mg) and 2 (18mg)

Purification of Fr C2 from the 119899-butanol fractionusing repeated Sephadex LH-20 column chromatography(Sephadex LH-20 8 cm times 80 cm) eluted with 60 methylalcohol provided compounds 3 (21mg) and 4 (15mg)

24 Identification of Antioxidant Compounds

241 Cell Culture and Treatment The H9c2 cells were cul-tured in high glucose Dulbeccorsquos modified Eaglersquos medium(DMEM) supplemented with 10 fetal bovine serum (FBS)and a mixture of penicillin and streptomycin The cells weremaintained in a humidified incubator with an atmosphere of5 CO

2 at 37∘C

242 Determination of H2O2 Model We determined theappropriate concentration of H

2O2using real-time cellular

analysis (RTCA) H9c2 cells were seeded in a CIM-plate-16 microplate in a volume of 100120583L and incubated for 12 hat 37∘C in an atmosphere of 5 CO

2 The H9c2 cells were

divided into control and model groups Then 10 120583L H2O2

(100 200 400 600 and 800 120583molsdotLminus1) was added to themodel groups and the control groups were treated with10120583L DMEM complete medium set up in three complexwellsThe E-Plate 16 was replaced in the xCELLigence RTCAmonitored every 15min and recorded for 30 h The effect ofH2O2on the H9c2 cells was analyzed using the xCELLigence

RTCA data analysis software The cell CI value was used todetermine the optimal H

2O2concentration and duration of

action

243 Cell Viability Assay H9c2 cells were seeded in 96-wellplates at a density of 2 times 104 cellswell at a final volumeof 100120583L and then they were divided into three groupscontrol model and treatment groups Then the treatmentgroups were treated with various concentrations of the crudeextracts (04 02 01 005 and 0025mg ∙ mLminus1) and thefourmonomeric compounds (50 25 125 625 and 3125120583M)at five concentrations After pretreatment for 12 h 400 120583MH2O2solution was added to each well followed by 10 120583L

MTT solution for 4 h at 37∘C and then the supernatant wasdiscarded and 150 120583L dimethyl sulfoxide (DMSO) was addedto eachwellTheplateswere oscillated at a low speed for 5 minat room temperature until all the crystals that had formed

BioMed Research International 3

Table 1 The chromatographic conditions

Time (min) Flow (mL ∙minminus1) Acetonitrile () 01 phosphoric acid water ()0 10 15 855 10 15 8510 10 20 8020 10 30 7040 10 65 3550 10 65 35501 10 15 8555 10 15 85

were fully dissolved The optical density was determined atan absorbance wavelength of 570nm

H9c2 cells were preincubated with zinc protoporphyrinIX (ZnPP 15 120583M) L-buthionine-sulfoximine (BSO 10120583M)and Brusatol (10120583M) for 30min then four compounds(125120583M) were added for 12 h and the cells were incubatedwith 400 120583M H

2O2 After treatment the survival cells were

determined by MTT assay

25 HPLC Detection The following chromatographic condi-tions were used Waters Symmetry C18 column (46mm times150mm 5120583m) mobile phase acetonitrile-01 phosphoricacid water flow rate 10mL ∙ minminus1 detection wavelength254 nm injection volume 10120583L and gradient elution 55min(Table 1) [8 9]

26 qRT-PCR The gene expression levels of HO-1 GCLCand Nrf2 were assessed using qRT-PCR Briefly total RNAwas prepared from cultured H9c2 cells using TRIzol reagentand reverse-transcribed using the PrimeScript RT Master kitaccording to the manufacturerrsquos instructions [10] Aliquotsof the obtained cDNA samples were then amplified usingPCR with the following schedule 40 PCR cycles at 95∘Cfor 5 s primer annealing at 60∘C for 34 s and extensionat 72∘C for 35 s All primers were tested the fluorescentsignals were recorded the Ct values were calculatedand the relative values were compared to control groupThe following equation was used Ct = Ct (sample)-Ct (endogenous control) Ct = Ct (sample)-Ct(untreated) and fold change = 2-Ct Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internalcontrol The sequences of the forwardreverse PCR primersare as follows HO-1 forward 51015840-GCCTGCTAGCCTGGT-TCAAG-31015840 reverse 51015840-AGCGGTGTCTGGGATGAACTA-31015840 GCLC forward 51015840-GTCCTCAGGTGACATTCCAAG-C-31015840 reverse 51015840-TGTTCTTCAGGGGCTCCAGTC-31015840 Nrf2forward 51015840-TTGGCAG AGACATTCCCATTTG-31015840 reverse51015840-AAACTTGCTCCATGTCCTGCTCTA-31015840 GAPDH for-ward 51015840-AAGCTGGTCATCAACGGGAAAC-31015840 reverse 51015840-GAAGACG CCAGTAGACTCCACG-31015840

27 Western Blotting The cells were washed with phosphate-buffered saline (PBS) and lysed on ice using radioimmuno-precipitation assay (RIPA Beyotime Jiangsu China) The

0002040608101214161820

00 50 100 150 200 250 300N

orm

aliz

ed C

ell I

ndex

Time (Hour)

100M 200M400M 600M800M Control

Figure 1 Establishment of H2O2model

protein concentration was determined using a bicinchoninicacid (BCA) protein assay kit according to the manufac-turerrsquos instructions [11] The samples were boiled at 98∘Cfor 3min separated using 12 sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis (PAGE) and thenthe proteins were transferred onto polyvinylidene fluoride(PVDF) membranes which were blocked with 5 non-fat dried milk for 60 min at room temperature The blotswere then incubated with the following rabbit polyclonalantibodies anti-GAPDH (110000) anti-HO-1 (11000) anti-GCLC (11000) and anti-Nrf2 (11000) The blots werethen washed extensively and incubated with horseradishperoxidase (HRP)-conjugated affinity pure goat anti-rabbitIgG (H+L 15000) Visualization was performed using anenhanced chemiluminescence kit according to the instruc-tions of the manufacturer

28 Statistical Analysis The data were analyzed using thestatistical package for the social sciences (SPSS) 190 andthe results are presented as the means plusmn standard deviation(SD) of three independent experiments P-values lt 005 wereconsidered statistically significant

3 Results

31 Establishment of H2O2 Model As shown in Figure 1 600and 800 120583molsdotLminus1 H

2O2were too high to cause the appro-

priate level of cell death whereas 200 120583molsdotLminus1 had littleeffects on the cells Therefore we determined the appropriate

4 BioMed Research International

Control

H2O2

04mgm

l

mgm

l

mgm

l

m

gml

mgml

Control

H2O2

04mgm

l

mgm

l

mgm

l

m

gml

mgml

lowastlowastlowast

Petroleum ether

Ethyl acetate

N-butanol Aqueous phase

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Control

H2O2

04mgm

l

mgm

l

mgm

l

m

gml

mgml

Control

H2O2

04mgm

l

mgm

l

mgm

l

m

gml

mgml

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Figure 2 The activity detection of four crude extracts protected H9c2 cells from H2O2-induced apoptosis (means plusmn SD n = 3) lowast lowast lowastP lt

0001 compared with control P lt 0001 P lt 001 P lt 005 compared with H2O2

Control

H2O2

04mgm

l

mgm

l

mgm

l

m

gml

mgml

control

H2O2

04mgm

l

mgm

l

mgm

l

m

gml

mgml

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

lowastlowastlowast

lowastlowastlowast

B2-B10 C2

Figure 3 The activity detection of fractions B2-B10 and C2 on H2O2-induced apoptosis on H9c2 cells (means plusmn SD n = 3) lowast lowast lowastP lt 0001

compared with control P lt 0001 P lt 001 P lt 005 compared with H2O2

BioMed Research International 5

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Contro

l

M

M

M

M

M

Contro

l

M

M

M

M

M

Contro

l

M

M

M

M

M

Contro

l

M

M

M

M

M

Cel

l via

bilit

y (

)

lowastlowast lowastlowast

lowastlowast

lowast

lowastlowast

(a)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Contro

lH2O

2

M

M

M

M

M

Contro

lH2O

2

M

M

M

M

M

Contro

lH2O

2

M

M

M

M

M

Contro

lH2O

2

M

M

M

M

M

Cel

l via

bilit

y (

)

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

(b)

Figure 4 The activity detection of compounds 1 2 3 and 4 on H2O2-induced apoptosis on H9c2 cells (a) Toxicity of four compounds on

H9c2 cells (b) The active ingredients on H9c2 cells assessed by MTT (means plusmn SD n = 3) lowast lowast lowastP lt 0001 lowastlowastP lt 001 lowastP lt 005 comparedwith control P lt 0001 P lt 001 P lt 005 compared with H

2O2

concentration of H2O2to be 400 120583Mformimicking oxidative

stress-induced injury to H9c2 cells

32 Crude Extracts Prevented H2O2-Induced Reduction in CellViability We used an MTT assay to analyze the effects offour crude extracts on H9c2 cells As shown in Figure 2the number of apoptotic cells significantly increased in themodel group compared to the treatment group Pretreatmentwith the four crude extracts for 12 h showed that the ethylacetate fractions and 119899-butanol extract significantly increasedthe percentage of viable H9c2 cells suggesting that the twofractions protected H9c2 cells from apoptosis induced byH2O2

Based on these results we further evaluated the activityof seven subfractions (B1 B2-B10 B11-B14 C1 C2 C3 andC4) from the ethyl acetate and n-butanol fractions Theresults show that B2-B10 andC2 significantly inhibitedH

2O2-

induced apoptosis of H9c2 cells The protective effects of B2-B10 and C2 are shown in Figure 3 and the results of inactivefractions are not shown in the manuscript

We further evaluated the activity of compounds 1 2 3and 4 obtained from the fractions B2-B10 and C2 Theseresults indicated that the four compounds showed no signif-icant toxic effect on H9c2 cells Compared with the controlgroup H

2O2model group could significantly reduce the cell

viability indicating that the H2O2model is successful The

results showed that the monomeric compounds 1 2 3 and4 had the protective effects on H

2O2-induced H9c2 cells

(Figure 4)

33 HPLC Detection The structures of these compoundswere identified by comparing spectral data (proton nuclear

O

OH

OH

O OH

O

Bellidifolin

O

OH

OH

O OH

OH

Demethylbellidifolin

O

OH

OH

O O

O

O

HOOH

OH

OH

O

OH

OH

O O

OH

O

HO OHOH

OH

SwetrianolinNorswertianolin

Figure 5The chemical structures of four monomeric compounds

magnetic resonance [1H-NMR] 13C-NMR and electrosprayionization [ESI-MS]) with published data as norswertianolin(1) swetrianolin (2) demethylbellidifolin (3) and bellidifolin(4) The chemical structures of the four compounds areshown in Figure 5 We compared the plant contents of thefour monomeric compounds using the area normalizationmethod These data indicated that there was a significantvariation in the contents of the four monomeric compoundsThe fingerprints of the four compounds and crude fractionsare shown in Figure 6 and the relative contents are shown inTable 2

6 BioMed Research International

00 50 100 150 200 250 300 350 400 450 500 550minus100

00

100

200

300

400

500

600

700

1

2

3

4

min

mAUMixed standard

00 50 100 150 200 250 300 350 400 450 500 550minus20

0

20

40

60

80

100

120

140

160 1

2

3 4

min

mAUEthyl acetate

00 50 100 150 200 250 300 350 400 450 500 550minus100

00

100

200

300

400

500

600

700 1

2 3

4

min

mAUB2-B10

00 50 100 150 200 250 300 350 400 450 500 550minus100

00

100

200

300

400

500

600

1

2

3 4

min

mAUN-butanol

00 50 100 150 200 250 300 350 400 450 500 550minus100

00

100

200

300

400

500

600

1

2

3 4

min

mAUC2

1 Norswertianolin 2 Swetrianolin 3 Demethylbellidifolin 4 Bellidifolin

Figure 6 The fingerprints of the four compounds in different extracts

BioMed Research International 7

Control

H2O2

Nrf

2m

RNA

leve

l

lowast

Control

H2O2

HO

-1 m

RNA

leve

l

lowastlowast

lowastlowastlowastlowastlowastlowast

GCL

C m

RNA

leve

l

Control

H2O2

lowastlowast

lowastlowast lowast

1 Bellidifolin 2 Demethylbellidifolin 3 Swetrianolin 4 Norswertianolin

Figure 7 Analysis of HO-1 GCLC and Nrf2 mRNA on H9c2 cells (means plusmn SD n = 3) lowastlowastlowast119875 lt 0001 lowastlowast119875 lt 001 lowast119875 lt 005 compared withH2O2

Table 2 Relative content of four monomeric compounds in different extracts

fractioncompounds

Relative content1 () 2 () 3 () 4 ()

ethyl acetate crude fraction 2643 1985 1118 722B2-B10 fraction 1161 1004 1243 803119899-butanol crude fraction 681 260 017 003C2 fraction 653 323 031 014

34 Transcription Levels of HO-1 GCLC and Nrf2 Weexamined the transcription levels of HO-1 GCLC and Nrf2in H9c2 cells treated with compounds 1 2 3 and 4 Asshown in Figure 7 the transcription levels of HO-1 andGCLC significantly increased in H9c2 cells pretreated withthe indicated concentrations of compounds 1 2 3 and 4for 12 h Furthermore the transcription levels of Nrf2 wereincreased in H9c2 cells pretreated with compound 3 whereascompounds 1 2 and 4 did not alter the express comparedwith the model group

35 Western Blot Analysis of HO-1 GCLC and Nrf2 Figure 8shows the expression of HO-1 and GCLC protein levelsin H9c2 cells after treatment with 400 120583M H

2O2 and the

fluorescent intensities of HO-1 and GCLC in the treatmentgroups were greater than that in the H

2O2-treated group

(Figures 8(b) and 8(c)) However none of the compoundsexcept for swetrianolin significantly changed the expressionof Nrf2 (Figure 8(d)) Pretreatment of H9c2 cells with ZnPPBSO and brusatol for 30 min and then addition of fourcompounds for 12 h followed by exposure toH

2O2 markedly

decreased the cell viability indicating that inhibitors canreverse the protective effects of four compounds (Figure 8(e))

4 Discussion

In this study we established a bioassay-guided method todetect the antioxidative activity of G acuta and found thatH2O2exposure significantly activated oxidative stress in

H9c2 cells This method can also be used to guide themore rapid and accurate separation of active ingredients

than that achieved using conventional separation methodswhich facilitates elucidation of the biological activity ofmedicinal plants and is more conducive to the discoveryof active or lead compounds Therefore the bioassay guidedmodel combined with cell activity evaluation has importantapplicability in natural product research

Numerous studies have shown that the Nrf2ARE path-way is critical to several antioxidant systems [12 13]The tran-scription factorNrf2 regulates the oxidative stress response incells [14 15] Nrf2 combines with Keap1 stably under normalphysiological conditions However following activation Nrf2affects the expression of downstream antioxidant proteinssuch as GCLC and HO-1 to enhance the antioxidant capacityof the cells Therefore we choose the Nrf2ARE pathway toelucidate the underlying protective mechanism in H9c2 cells

We established a method to separate and analyze com-pounds 1 2 3 and 4 from G acuta and conducted stud-ies to validate the efficiency of the method This specificmodel achieved good separation and the four compoundswere successfully eluted from the G acuta sample Thecrude extract had protective effect on H9c2 cells fromH2O2-induced apoptosis The activity of the monomeric

compounds obtained from the fractions was then testedResults of the cytoprotective study showed that the chemicalcomponents in G acuta markedly increased cell viabilitycompared to H

2O2alone

The transcription levels of HO-1 and GCLC significantlyincreased in H9c2 cells pretreated with compounds 1 2 3and4 However compared to themodel group the expressionof HO-1 and GCLC protein levels in H9c2 cells increasedwhereas that of Nrf2 was not significantly changed exceptwith swetrianolin Finally our data demonstrate thatG acuta

8 BioMed Research International

HO-1

GCLC

Nrf 2

GAPDH

HO

Bellidifolin

Demethylbellidifolin

SwetrianolinNorswertianolin

ZnPP

BSOBrusatol

--------

+-------

++------

+-+-----

+--+----

+---+---

++---++-

+-+--++-

+--+-+++

+---+++-

(a)

ZnPP Negative ZnPP PositiveControl

H2O2

HO

-1G

APD

H

ampampampampamp ampamp

amplowast

1 Bellidifolin 2 Demethylbellidifolin 3 Swetrianolin 4 Norswertianolin

(b)

BSO Nagetive BSO PostiveControl

H2O2

GCL

CG

APD

H

ampamp ampampamp amp

lowast

1 Bellidifolin 2 Demethylbellidifolin 3 Swetrianolin 4 Norswertianolin

(c)

Brusatol Nagetive Brusatol PostiveContro

lH2O2

Nrf

2G

APD

H

lowast

1 Bellidifolin 2 Demethylbellidifolin 3 Swetrianolin 4 Norswertianolin

(d)

Cel

l via

bilit

y (

)

Bellidifolin (125M)Demethylbellidifolin (125M)

Swetrianolin (125M)Norswertianolin (125M)

ZnPP (15M)BSO (10M)

Brusatol (10M)

--------

-+------

--+-----

---+----

+-------

++--+---

++---+--

++----+-

+-+-+---

+-+--+--

+-+---+-

+-+----+

++-----+

+--+--+-

HO (400M)

lowastlowastlowast

$$$$$$$$ $$

(e)

Figure 8 Analysis of HO-1 GCLC and Nrf2 protein levels on H9c2 cells (a)Western blotting analysis HO-1 GCLC Nrf2 protein expressionwith or without ZnPP BSO and brusatol (HO-1group only added ZnPP GCLC group only added BSO Nrf2 group only added brusatol)(b) Western blot analysis HO-1 protein expression with or without ZnPP (c) Western blot analysis GCLC protein expression with or withoutBSO (d) Western blot analysis Nrf2 protein expression with or without brusatol (e) Effect of the different treatments on H9c2 cells Dataare expressed as the means plusmn SD n = 3 lowastlowastlowast119875 lt 0001 lowast119875 lt 005 compared with control group P lt 0001 P lt 001 P lt 005 comparedwith H

2O2group $$$119875 lt 0001 $$119875 lt 001 compared with H

2O2group combined with four compounds ampampampP lt 0001 ampampP lt 001 ampP lt 005

compared with H2O2group combined with inhibitors and four compounds GAPDH was used as an internal control

BioMed Research International 9

protects H9c2 cells from oxidative damage via the Nrf2-AREpathway

In conclusion the results of the present study demon-strated that four compounds are able to protect H9c2 cellsfrom H

2O2-induced damage which may be associated with

the activation of Nrf2ARE pathway in H9c2 cells Theseresults provide evidence that G acuta may be a potentialtherapeutic agent for the treatment of cardiovascular diseases

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This study was supported by the research of the effects ofMongolian Danggong-3 on the activity of myocardial cells inrats after myocardial anoxia-reoxygenation (NO 201702026)Natural Science Foundation of Tianjin (15JCQNJC13400)Investigation and Variety Systematization Study of Mon-golian Medicine ldquoDigedardquo (201702121) Chinese NationalNatural Science Foundation (81160504) and Natural ScienceFoundation of Inner Mongolia Autonomous Region (BYJJ-YF 201726)

References

[1] Z Wang Q Wu Y Yu et al ldquoDetermination and phar-macokinetic study of four xanthones inrat plasma after oraladministration of Gentianella acuta extract by UHPLC-ESI-MSMSrdquo Journal of Ethnopharmacology vol 174 pp 261ndash2692015

[2] L J Lv and M H Li ldquoTerpenoids flavonoids and xanthonesfrom Gentianella acuta (Gentianaceae)rdquo Biochemical Systemat-ics and Ecology vol 37 no 4 pp 497ndash500 2009

[3] Y-J Li T-D Bao Q Li et al ldquoProtection of CompoundGranules of Yinxingye onH9c2myocardial cells injury inducedby H2O2rdquo Chinese Traditional and Herbal Drugs vol 47 no 3pp 459ndash465 2016

[4] Y H Bai X Wang L J Zhang et al ldquoAdvances in the Chineseand western medicine treatment of coronary microvascularangina pectorisrdquo Journal of Anhui Health Vocational and Tech-nical College vol 17 no 2 pp 21ndash23 2018

[5] A B Gustafsson and R A Gottlieb ldquoMechanisms of Apoptosisin the Heartrdquo Journal of Clinical Immunology vol 23 no 6 pp447ndash459 2003

[6] Y N Liu W P Wang Z Q Jia P Chen K T Ma and CY Zhou ldquoProtection of -Lipoic Acid Against H

2O2-induced

Oxidative Stress in H9c2 Cellsrdquo Chinese Journal of Biochemistryand Molecular Biology vol 28 no 3 pp 227ndash233 2012

[7] M N Sharikabad K M Oslashstbye andO Broslashrs ldquoEffect of hydro-gen peroxide on reoxygenation-induced Ca2+ accumulation inrat cardiomyocytesrdquo Free Radical Biology amp Medicine vol 37no 4 pp 531ndash538 2004

[8] G D Wu J Zhang D Yang Z W Wang and M H LildquoDetermination of the Norswertianolin in Gentianella acutardquoChinese Journal of Experimental Traditional Medical Formulaevol 18 no 6 pp 69ndash71 2012

[9] L J Lv Y F Wang T Yan H R Han and M H Li ldquoPrepa-ration of swertianolin from Gentianella acuta and its effects onarrhythmia induced by aconitine in micerdquo Chinese Journal ofNew Drugs and Clinical Remedies vol 34 no 8 pp 630ndash6352015

[10] H-J Xu W-L Chen Y Wang M-Z Zheng Y-Y Chen andY-L Shen ldquoS262A mutation abolishes protective effects ofconnexin 43 against hypothermic preservation-induced injuryin cardiomyocytesrdquoe Journal of Heart and Lung Transplanta-tion vol 31 no 6 pp 663ndash669 2012

[11] X Jia L Zhang and X Mao ldquoS-propranolol protected H9C2cells from ischemiareperfusion-induced apoptosis via down-regultion of RACK1 Generdquo International Journal of Clinical andExperimental Pathology vol 8 no 9 pp 10335ndash10344 2015

[12] T Nguyen P Nioi and C B Pickett ldquoThe Nrf2-antioxidantresponse element signaling pathway and its activation byoxidative stressrdquo e Journal of Biological Chemistry vol 284no 20 pp 13291ndash13295 2009

[13] Y-S Keum and B Y Choi ldquoMolecular and chemical regulationof the keap1-Nrf2 signaling pathwayrdquo Molecules vol 19 no 7pp 10074ndash10089 2014

[14] H Li and L Yang ldquoMolecular regulatory mechanism of Nrf2antioxidantrdquo China Journal of Bioinformatics vol 16 no 1 pp1ndash6 2018

[15] H J Sun L Dong J Y Wang M Pan Y Wu and S LildquoProtective effects of Lycium ruthenicum Murr on hydrogenperoxide-induced cellular oxidative stress injury in heart-derived H9c2 cellsrdquo Journal of Beijing University of TraditionalChinese Medicine vol 41 no 4 pp 301ndash305 2018

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

BioMed Research International 3

Table 1 The chromatographic conditions

Time (min) Flow (mL ∙minminus1) Acetonitrile () 01 phosphoric acid water ()0 10 15 855 10 15 8510 10 20 8020 10 30 7040 10 65 3550 10 65 35501 10 15 8555 10 15 85

were fully dissolved The optical density was determined atan absorbance wavelength of 570nm

H9c2 cells were preincubated with zinc protoporphyrinIX (ZnPP 15 120583M) L-buthionine-sulfoximine (BSO 10120583M)and Brusatol (10120583M) for 30min then four compounds(125120583M) were added for 12 h and the cells were incubatedwith 400 120583M H

2O2 After treatment the survival cells were

determined by MTT assay

25 HPLC Detection The following chromatographic condi-tions were used Waters Symmetry C18 column (46mm times150mm 5120583m) mobile phase acetonitrile-01 phosphoricacid water flow rate 10mL ∙ minminus1 detection wavelength254 nm injection volume 10120583L and gradient elution 55min(Table 1) [8 9]

26 qRT-PCR The gene expression levels of HO-1 GCLCand Nrf2 were assessed using qRT-PCR Briefly total RNAwas prepared from cultured H9c2 cells using TRIzol reagentand reverse-transcribed using the PrimeScript RT Master kitaccording to the manufacturerrsquos instructions [10] Aliquotsof the obtained cDNA samples were then amplified usingPCR with the following schedule 40 PCR cycles at 95∘Cfor 5 s primer annealing at 60∘C for 34 s and extensionat 72∘C for 35 s All primers were tested the fluorescentsignals were recorded the Ct values were calculatedand the relative values were compared to control groupThe following equation was used Ct = Ct (sample)-Ct (endogenous control) Ct = Ct (sample)-Ct(untreated) and fold change = 2-Ct Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internalcontrol The sequences of the forwardreverse PCR primersare as follows HO-1 forward 51015840-GCCTGCTAGCCTGGT-TCAAG-31015840 reverse 51015840-AGCGGTGTCTGGGATGAACTA-31015840 GCLC forward 51015840-GTCCTCAGGTGACATTCCAAG-C-31015840 reverse 51015840-TGTTCTTCAGGGGCTCCAGTC-31015840 Nrf2forward 51015840-TTGGCAG AGACATTCCCATTTG-31015840 reverse51015840-AAACTTGCTCCATGTCCTGCTCTA-31015840 GAPDH for-ward 51015840-AAGCTGGTCATCAACGGGAAAC-31015840 reverse 51015840-GAAGACG CCAGTAGACTCCACG-31015840

27 Western Blotting The cells were washed with phosphate-buffered saline (PBS) and lysed on ice using radioimmuno-precipitation assay (RIPA Beyotime Jiangsu China) The

0002040608101214161820

00 50 100 150 200 250 300N

orm

aliz

ed C

ell I

ndex

Time (Hour)

100M 200M400M 600M800M Control

Figure 1 Establishment of H2O2model

protein concentration was determined using a bicinchoninicacid (BCA) protein assay kit according to the manufac-turerrsquos instructions [11] The samples were boiled at 98∘Cfor 3min separated using 12 sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis (PAGE) and thenthe proteins were transferred onto polyvinylidene fluoride(PVDF) membranes which were blocked with 5 non-fat dried milk for 60 min at room temperature The blotswere then incubated with the following rabbit polyclonalantibodies anti-GAPDH (110000) anti-HO-1 (11000) anti-GCLC (11000) and anti-Nrf2 (11000) The blots werethen washed extensively and incubated with horseradishperoxidase (HRP)-conjugated affinity pure goat anti-rabbitIgG (H+L 15000) Visualization was performed using anenhanced chemiluminescence kit according to the instruc-tions of the manufacturer

28 Statistical Analysis The data were analyzed using thestatistical package for the social sciences (SPSS) 190 andthe results are presented as the means plusmn standard deviation(SD) of three independent experiments P-values lt 005 wereconsidered statistically significant

3 Results

31 Establishment of H2O2 Model As shown in Figure 1 600and 800 120583molsdotLminus1 H

2O2were too high to cause the appro-

priate level of cell death whereas 200 120583molsdotLminus1 had littleeffects on the cells Therefore we determined the appropriate

4 BioMed Research International

Control

H2O2

04mgm

l

mgm

l

mgm

l

m

gml

mgml

Control

H2O2

04mgm

l

mgm

l

mgm

l

m

gml

mgml

lowastlowastlowast

Petroleum ether

Ethyl acetate

N-butanol Aqueous phase

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Control

H2O2

04mgm

l

mgm

l

mgm

l

m

gml

mgml

Control

H2O2

04mgm

l

mgm

l

mgm

l

m

gml

mgml

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Figure 2 The activity detection of four crude extracts protected H9c2 cells from H2O2-induced apoptosis (means plusmn SD n = 3) lowast lowast lowastP lt

0001 compared with control P lt 0001 P lt 001 P lt 005 compared with H2O2

Control

H2O2

04mgm

l

mgm

l

mgm

l

m

gml

mgml

control

H2O2

04mgm

l

mgm

l

mgm

l

m

gml

mgml

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

lowastlowastlowast

lowastlowastlowast

B2-B10 C2

Figure 3 The activity detection of fractions B2-B10 and C2 on H2O2-induced apoptosis on H9c2 cells (means plusmn SD n = 3) lowast lowast lowastP lt 0001

compared with control P lt 0001 P lt 001 P lt 005 compared with H2O2

BioMed Research International 5

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Contro

l

M

M

M

M

M

Contro

l

M

M

M

M

M

Contro

l

M

M

M

M

M

Contro

l

M

M

M

M

M

Cel

l via

bilit

y (

)

lowastlowast lowastlowast

lowastlowast

lowast

lowastlowast

(a)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Contro

lH2O

2

M

M

M

M

M

Contro

lH2O

2

M

M

M

M

M

Contro

lH2O

2

M

M

M

M

M

Contro

lH2O

2

M

M

M

M

M

Cel

l via

bilit

y (

)

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

(b)

Figure 4 The activity detection of compounds 1 2 3 and 4 on H2O2-induced apoptosis on H9c2 cells (a) Toxicity of four compounds on

H9c2 cells (b) The active ingredients on H9c2 cells assessed by MTT (means plusmn SD n = 3) lowast lowast lowastP lt 0001 lowastlowastP lt 001 lowastP lt 005 comparedwith control P lt 0001 P lt 001 P lt 005 compared with H

2O2

concentration of H2O2to be 400 120583Mformimicking oxidative

stress-induced injury to H9c2 cells

32 Crude Extracts Prevented H2O2-Induced Reduction in CellViability We used an MTT assay to analyze the effects offour crude extracts on H9c2 cells As shown in Figure 2the number of apoptotic cells significantly increased in themodel group compared to the treatment group Pretreatmentwith the four crude extracts for 12 h showed that the ethylacetate fractions and 119899-butanol extract significantly increasedthe percentage of viable H9c2 cells suggesting that the twofractions protected H9c2 cells from apoptosis induced byH2O2

Based on these results we further evaluated the activityof seven subfractions (B1 B2-B10 B11-B14 C1 C2 C3 andC4) from the ethyl acetate and n-butanol fractions Theresults show that B2-B10 andC2 significantly inhibitedH

2O2-

induced apoptosis of H9c2 cells The protective effects of B2-B10 and C2 are shown in Figure 3 and the results of inactivefractions are not shown in the manuscript

We further evaluated the activity of compounds 1 2 3and 4 obtained from the fractions B2-B10 and C2 Theseresults indicated that the four compounds showed no signif-icant toxic effect on H9c2 cells Compared with the controlgroup H

2O2model group could significantly reduce the cell

viability indicating that the H2O2model is successful The

results showed that the monomeric compounds 1 2 3 and4 had the protective effects on H

2O2-induced H9c2 cells

(Figure 4)

33 HPLC Detection The structures of these compoundswere identified by comparing spectral data (proton nuclear

O

OH

OH

O OH

O

Bellidifolin

O

OH

OH

O OH

OH

Demethylbellidifolin

O

OH

OH

O O

O

O

HOOH

OH

OH

O

OH

OH

O O

OH

O

HO OHOH

OH

SwetrianolinNorswertianolin

Figure 5The chemical structures of four monomeric compounds

magnetic resonance [1H-NMR] 13C-NMR and electrosprayionization [ESI-MS]) with published data as norswertianolin(1) swetrianolin (2) demethylbellidifolin (3) and bellidifolin(4) The chemical structures of the four compounds areshown in Figure 5 We compared the plant contents of thefour monomeric compounds using the area normalizationmethod These data indicated that there was a significantvariation in the contents of the four monomeric compoundsThe fingerprints of the four compounds and crude fractionsare shown in Figure 6 and the relative contents are shown inTable 2

6 BioMed Research International

00 50 100 150 200 250 300 350 400 450 500 550minus100

00

100

200

300

400

500

600

700

1

2

3

4

min

mAUMixed standard

00 50 100 150 200 250 300 350 400 450 500 550minus20

0

20

40

60

80

100

120

140

160 1

2

3 4

min

mAUEthyl acetate

00 50 100 150 200 250 300 350 400 450 500 550minus100

00

100

200

300

400

500

600

700 1

2 3

4

min

mAUB2-B10

00 50 100 150 200 250 300 350 400 450 500 550minus100

00

100

200

300

400

500

600

1

2

3 4

min

mAUN-butanol

00 50 100 150 200 250 300 350 400 450 500 550minus100

00

100

200

300

400

500

600

1

2

3 4

min

mAUC2

1 Norswertianolin 2 Swetrianolin 3 Demethylbellidifolin 4 Bellidifolin

Figure 6 The fingerprints of the four compounds in different extracts

BioMed Research International 7

Control

H2O2

Nrf

2m

RNA

leve

l

lowast

Control

H2O2

HO

-1 m

RNA

leve

l

lowastlowast

lowastlowastlowastlowastlowastlowast

GCL

C m

RNA

leve

l

Control

H2O2

lowastlowast

lowastlowast lowast

1 Bellidifolin 2 Demethylbellidifolin 3 Swetrianolin 4 Norswertianolin

Figure 7 Analysis of HO-1 GCLC and Nrf2 mRNA on H9c2 cells (means plusmn SD n = 3) lowastlowastlowast119875 lt 0001 lowastlowast119875 lt 001 lowast119875 lt 005 compared withH2O2

Table 2 Relative content of four monomeric compounds in different extracts

fractioncompounds

Relative content1 () 2 () 3 () 4 ()

ethyl acetate crude fraction 2643 1985 1118 722B2-B10 fraction 1161 1004 1243 803119899-butanol crude fraction 681 260 017 003C2 fraction 653 323 031 014

34 Transcription Levels of HO-1 GCLC and Nrf2 Weexamined the transcription levels of HO-1 GCLC and Nrf2in H9c2 cells treated with compounds 1 2 3 and 4 Asshown in Figure 7 the transcription levels of HO-1 andGCLC significantly increased in H9c2 cells pretreated withthe indicated concentrations of compounds 1 2 3 and 4for 12 h Furthermore the transcription levels of Nrf2 wereincreased in H9c2 cells pretreated with compound 3 whereascompounds 1 2 and 4 did not alter the express comparedwith the model group

35 Western Blot Analysis of HO-1 GCLC and Nrf2 Figure 8shows the expression of HO-1 and GCLC protein levelsin H9c2 cells after treatment with 400 120583M H

2O2 and the

fluorescent intensities of HO-1 and GCLC in the treatmentgroups were greater than that in the H

2O2-treated group

(Figures 8(b) and 8(c)) However none of the compoundsexcept for swetrianolin significantly changed the expressionof Nrf2 (Figure 8(d)) Pretreatment of H9c2 cells with ZnPPBSO and brusatol for 30 min and then addition of fourcompounds for 12 h followed by exposure toH

2O2 markedly

decreased the cell viability indicating that inhibitors canreverse the protective effects of four compounds (Figure 8(e))

4 Discussion

In this study we established a bioassay-guided method todetect the antioxidative activity of G acuta and found thatH2O2exposure significantly activated oxidative stress in

H9c2 cells This method can also be used to guide themore rapid and accurate separation of active ingredients

than that achieved using conventional separation methodswhich facilitates elucidation of the biological activity ofmedicinal plants and is more conducive to the discoveryof active or lead compounds Therefore the bioassay guidedmodel combined with cell activity evaluation has importantapplicability in natural product research

Numerous studies have shown that the Nrf2ARE path-way is critical to several antioxidant systems [12 13]The tran-scription factorNrf2 regulates the oxidative stress response incells [14 15] Nrf2 combines with Keap1 stably under normalphysiological conditions However following activation Nrf2affects the expression of downstream antioxidant proteinssuch as GCLC and HO-1 to enhance the antioxidant capacityof the cells Therefore we choose the Nrf2ARE pathway toelucidate the underlying protective mechanism in H9c2 cells

We established a method to separate and analyze com-pounds 1 2 3 and 4 from G acuta and conducted stud-ies to validate the efficiency of the method This specificmodel achieved good separation and the four compoundswere successfully eluted from the G acuta sample Thecrude extract had protective effect on H9c2 cells fromH2O2-induced apoptosis The activity of the monomeric

compounds obtained from the fractions was then testedResults of the cytoprotective study showed that the chemicalcomponents in G acuta markedly increased cell viabilitycompared to H

2O2alone

The transcription levels of HO-1 and GCLC significantlyincreased in H9c2 cells pretreated with compounds 1 2 3and4 However compared to themodel group the expressionof HO-1 and GCLC protein levels in H9c2 cells increasedwhereas that of Nrf2 was not significantly changed exceptwith swetrianolin Finally our data demonstrate thatG acuta

8 BioMed Research International

HO-1

GCLC

Nrf 2

GAPDH

HO

Bellidifolin

Demethylbellidifolin

SwetrianolinNorswertianolin

ZnPP

BSOBrusatol

--------

+-------

++------

+-+-----

+--+----

+---+---

++---++-

+-+--++-

+--+-+++

+---+++-

(a)

ZnPP Negative ZnPP PositiveControl

H2O2

HO

-1G

APD

H

ampampampampamp ampamp

amplowast

1 Bellidifolin 2 Demethylbellidifolin 3 Swetrianolin 4 Norswertianolin

(b)

BSO Nagetive BSO PostiveControl

H2O2

GCL

CG

APD

H

ampamp ampampamp amp

lowast

1 Bellidifolin 2 Demethylbellidifolin 3 Swetrianolin 4 Norswertianolin

(c)

Brusatol Nagetive Brusatol PostiveContro

lH2O2

Nrf

2G

APD

H

lowast

1 Bellidifolin 2 Demethylbellidifolin 3 Swetrianolin 4 Norswertianolin

(d)

Cel

l via

bilit

y (

)

Bellidifolin (125M)Demethylbellidifolin (125M)

Swetrianolin (125M)Norswertianolin (125M)

ZnPP (15M)BSO (10M)

Brusatol (10M)

--------

-+------

--+-----

---+----

+-------

++--+---

++---+--

++----+-

+-+-+---

+-+--+--

+-+---+-

+-+----+

++-----+

+--+--+-

HO (400M)

lowastlowastlowast

$$$$$$$$ $$

(e)

Figure 8 Analysis of HO-1 GCLC and Nrf2 protein levels on H9c2 cells (a)Western blotting analysis HO-1 GCLC Nrf2 protein expressionwith or without ZnPP BSO and brusatol (HO-1group only added ZnPP GCLC group only added BSO Nrf2 group only added brusatol)(b) Western blot analysis HO-1 protein expression with or without ZnPP (c) Western blot analysis GCLC protein expression with or withoutBSO (d) Western blot analysis Nrf2 protein expression with or without brusatol (e) Effect of the different treatments on H9c2 cells Dataare expressed as the means plusmn SD n = 3 lowastlowastlowast119875 lt 0001 lowast119875 lt 005 compared with control group P lt 0001 P lt 001 P lt 005 comparedwith H

2O2group $$$119875 lt 0001 $$119875 lt 001 compared with H

2O2group combined with four compounds ampampampP lt 0001 ampampP lt 001 ampP lt 005

compared with H2O2group combined with inhibitors and four compounds GAPDH was used as an internal control

BioMed Research International 9

protects H9c2 cells from oxidative damage via the Nrf2-AREpathway

In conclusion the results of the present study demon-strated that four compounds are able to protect H9c2 cellsfrom H

2O2-induced damage which may be associated with

the activation of Nrf2ARE pathway in H9c2 cells Theseresults provide evidence that G acuta may be a potentialtherapeutic agent for the treatment of cardiovascular diseases

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This study was supported by the research of the effects ofMongolian Danggong-3 on the activity of myocardial cells inrats after myocardial anoxia-reoxygenation (NO 201702026)Natural Science Foundation of Tianjin (15JCQNJC13400)Investigation and Variety Systematization Study of Mon-golian Medicine ldquoDigedardquo (201702121) Chinese NationalNatural Science Foundation (81160504) and Natural ScienceFoundation of Inner Mongolia Autonomous Region (BYJJ-YF 201726)

References

[1] Z Wang Q Wu Y Yu et al ldquoDetermination and phar-macokinetic study of four xanthones inrat plasma after oraladministration of Gentianella acuta extract by UHPLC-ESI-MSMSrdquo Journal of Ethnopharmacology vol 174 pp 261ndash2692015

[2] L J Lv and M H Li ldquoTerpenoids flavonoids and xanthonesfrom Gentianella acuta (Gentianaceae)rdquo Biochemical Systemat-ics and Ecology vol 37 no 4 pp 497ndash500 2009

[3] Y-J Li T-D Bao Q Li et al ldquoProtection of CompoundGranules of Yinxingye onH9c2myocardial cells injury inducedby H2O2rdquo Chinese Traditional and Herbal Drugs vol 47 no 3pp 459ndash465 2016

[4] Y H Bai X Wang L J Zhang et al ldquoAdvances in the Chineseand western medicine treatment of coronary microvascularangina pectorisrdquo Journal of Anhui Health Vocational and Tech-nical College vol 17 no 2 pp 21ndash23 2018

[5] A B Gustafsson and R A Gottlieb ldquoMechanisms of Apoptosisin the Heartrdquo Journal of Clinical Immunology vol 23 no 6 pp447ndash459 2003

[6] Y N Liu W P Wang Z Q Jia P Chen K T Ma and CY Zhou ldquoProtection of -Lipoic Acid Against H

2O2-induced

Oxidative Stress in H9c2 Cellsrdquo Chinese Journal of Biochemistryand Molecular Biology vol 28 no 3 pp 227ndash233 2012

[7] M N Sharikabad K M Oslashstbye andO Broslashrs ldquoEffect of hydro-gen peroxide on reoxygenation-induced Ca2+ accumulation inrat cardiomyocytesrdquo Free Radical Biology amp Medicine vol 37no 4 pp 531ndash538 2004

[8] G D Wu J Zhang D Yang Z W Wang and M H LildquoDetermination of the Norswertianolin in Gentianella acutardquoChinese Journal of Experimental Traditional Medical Formulaevol 18 no 6 pp 69ndash71 2012

[9] L J Lv Y F Wang T Yan H R Han and M H Li ldquoPrepa-ration of swertianolin from Gentianella acuta and its effects onarrhythmia induced by aconitine in micerdquo Chinese Journal ofNew Drugs and Clinical Remedies vol 34 no 8 pp 630ndash6352015

[10] H-J Xu W-L Chen Y Wang M-Z Zheng Y-Y Chen andY-L Shen ldquoS262A mutation abolishes protective effects ofconnexin 43 against hypothermic preservation-induced injuryin cardiomyocytesrdquoe Journal of Heart and Lung Transplanta-tion vol 31 no 6 pp 663ndash669 2012

[11] X Jia L Zhang and X Mao ldquoS-propranolol protected H9C2cells from ischemiareperfusion-induced apoptosis via down-regultion of RACK1 Generdquo International Journal of Clinical andExperimental Pathology vol 8 no 9 pp 10335ndash10344 2015

[12] T Nguyen P Nioi and C B Pickett ldquoThe Nrf2-antioxidantresponse element signaling pathway and its activation byoxidative stressrdquo e Journal of Biological Chemistry vol 284no 20 pp 13291ndash13295 2009

[13] Y-S Keum and B Y Choi ldquoMolecular and chemical regulationof the keap1-Nrf2 signaling pathwayrdquo Molecules vol 19 no 7pp 10074ndash10089 2014

[14] H Li and L Yang ldquoMolecular regulatory mechanism of Nrf2antioxidantrdquo China Journal of Bioinformatics vol 16 no 1 pp1ndash6 2018

[15] H J Sun L Dong J Y Wang M Pan Y Wu and S LildquoProtective effects of Lycium ruthenicum Murr on hydrogenperoxide-induced cellular oxidative stress injury in heart-derived H9c2 cellsrdquo Journal of Beijing University of TraditionalChinese Medicine vol 41 no 4 pp 301ndash305 2018

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

4 BioMed Research International

Control

H2O2

04mgm

l

mgm

l

mgm

l

m

gml

mgml

Control

H2O2

04mgm

l

mgm

l

mgm

l

m

gml

mgml

lowastlowastlowast

Petroleum ether

Ethyl acetate

N-butanol Aqueous phase

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Control

H2O2

04mgm

l

mgm

l

mgm

l

m

gml

mgml

Control

H2O2

04mgm

l

mgm

l

mgm

l

m

gml

mgml

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Figure 2 The activity detection of four crude extracts protected H9c2 cells from H2O2-induced apoptosis (means plusmn SD n = 3) lowast lowast lowastP lt

0001 compared with control P lt 0001 P lt 001 P lt 005 compared with H2O2

Control

H2O2

04mgm

l

mgm

l

mgm

l

m

gml

mgml

control

H2O2

04mgm

l

mgm

l

mgm

l

m

gml

mgml

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

lowastlowastlowast

lowastlowastlowast

B2-B10 C2

Figure 3 The activity detection of fractions B2-B10 and C2 on H2O2-induced apoptosis on H9c2 cells (means plusmn SD n = 3) lowast lowast lowastP lt 0001

compared with control P lt 0001 P lt 001 P lt 005 compared with H2O2

BioMed Research International 5

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Contro

l

M

M

M

M

M

Contro

l

M

M

M

M

M

Contro

l

M

M

M

M

M

Contro

l

M

M

M

M

M

Cel

l via

bilit

y (

)

lowastlowast lowastlowast

lowastlowast

lowast

lowastlowast

(a)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Contro

lH2O

2

M

M

M

M

M

Contro

lH2O

2

M

M

M

M

M

Contro

lH2O

2

M

M

M

M

M

Contro

lH2O

2

M

M

M

M

M

Cel

l via

bilit

y (

)

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

(b)

Figure 4 The activity detection of compounds 1 2 3 and 4 on H2O2-induced apoptosis on H9c2 cells (a) Toxicity of four compounds on

H9c2 cells (b) The active ingredients on H9c2 cells assessed by MTT (means plusmn SD n = 3) lowast lowast lowastP lt 0001 lowastlowastP lt 001 lowastP lt 005 comparedwith control P lt 0001 P lt 001 P lt 005 compared with H

2O2

concentration of H2O2to be 400 120583Mformimicking oxidative

stress-induced injury to H9c2 cells

32 Crude Extracts Prevented H2O2-Induced Reduction in CellViability We used an MTT assay to analyze the effects offour crude extracts on H9c2 cells As shown in Figure 2the number of apoptotic cells significantly increased in themodel group compared to the treatment group Pretreatmentwith the four crude extracts for 12 h showed that the ethylacetate fractions and 119899-butanol extract significantly increasedthe percentage of viable H9c2 cells suggesting that the twofractions protected H9c2 cells from apoptosis induced byH2O2

Based on these results we further evaluated the activityof seven subfractions (B1 B2-B10 B11-B14 C1 C2 C3 andC4) from the ethyl acetate and n-butanol fractions Theresults show that B2-B10 andC2 significantly inhibitedH

2O2-

induced apoptosis of H9c2 cells The protective effects of B2-B10 and C2 are shown in Figure 3 and the results of inactivefractions are not shown in the manuscript

We further evaluated the activity of compounds 1 2 3and 4 obtained from the fractions B2-B10 and C2 Theseresults indicated that the four compounds showed no signif-icant toxic effect on H9c2 cells Compared with the controlgroup H

2O2model group could significantly reduce the cell

viability indicating that the H2O2model is successful The

results showed that the monomeric compounds 1 2 3 and4 had the protective effects on H

2O2-induced H9c2 cells

(Figure 4)

33 HPLC Detection The structures of these compoundswere identified by comparing spectral data (proton nuclear

O

OH

OH

O OH

O

Bellidifolin

O

OH

OH

O OH

OH

Demethylbellidifolin

O

OH

OH

O O

O

O

HOOH

OH

OH

O

OH

OH

O O

OH

O

HO OHOH

OH

SwetrianolinNorswertianolin

Figure 5The chemical structures of four monomeric compounds

magnetic resonance [1H-NMR] 13C-NMR and electrosprayionization [ESI-MS]) with published data as norswertianolin(1) swetrianolin (2) demethylbellidifolin (3) and bellidifolin(4) The chemical structures of the four compounds areshown in Figure 5 We compared the plant contents of thefour monomeric compounds using the area normalizationmethod These data indicated that there was a significantvariation in the contents of the four monomeric compoundsThe fingerprints of the four compounds and crude fractionsare shown in Figure 6 and the relative contents are shown inTable 2

6 BioMed Research International

00 50 100 150 200 250 300 350 400 450 500 550minus100

00

100

200

300

400

500

600

700

1

2

3

4

min

mAUMixed standard

00 50 100 150 200 250 300 350 400 450 500 550minus20

0

20

40

60

80

100

120

140

160 1

2

3 4

min

mAUEthyl acetate

00 50 100 150 200 250 300 350 400 450 500 550minus100

00

100

200

300

400

500

600

700 1

2 3

4

min

mAUB2-B10

00 50 100 150 200 250 300 350 400 450 500 550minus100

00

100

200

300

400

500

600

1

2

3 4

min

mAUN-butanol

00 50 100 150 200 250 300 350 400 450 500 550minus100

00

100

200

300

400

500

600

1

2

3 4

min

mAUC2

1 Norswertianolin 2 Swetrianolin 3 Demethylbellidifolin 4 Bellidifolin

Figure 6 The fingerprints of the four compounds in different extracts

BioMed Research International 7

Control

H2O2

Nrf

2m

RNA

leve

l

lowast

Control

H2O2

HO

-1 m

RNA

leve

l

lowastlowast

lowastlowastlowastlowastlowastlowast

GCL

C m

RNA

leve

l

Control

H2O2

lowastlowast

lowastlowast lowast

1 Bellidifolin 2 Demethylbellidifolin 3 Swetrianolin 4 Norswertianolin

Figure 7 Analysis of HO-1 GCLC and Nrf2 mRNA on H9c2 cells (means plusmn SD n = 3) lowastlowastlowast119875 lt 0001 lowastlowast119875 lt 001 lowast119875 lt 005 compared withH2O2

Table 2 Relative content of four monomeric compounds in different extracts

fractioncompounds

Relative content1 () 2 () 3 () 4 ()

ethyl acetate crude fraction 2643 1985 1118 722B2-B10 fraction 1161 1004 1243 803119899-butanol crude fraction 681 260 017 003C2 fraction 653 323 031 014

34 Transcription Levels of HO-1 GCLC and Nrf2 Weexamined the transcription levels of HO-1 GCLC and Nrf2in H9c2 cells treated with compounds 1 2 3 and 4 Asshown in Figure 7 the transcription levels of HO-1 andGCLC significantly increased in H9c2 cells pretreated withthe indicated concentrations of compounds 1 2 3 and 4for 12 h Furthermore the transcription levels of Nrf2 wereincreased in H9c2 cells pretreated with compound 3 whereascompounds 1 2 and 4 did not alter the express comparedwith the model group

35 Western Blot Analysis of HO-1 GCLC and Nrf2 Figure 8shows the expression of HO-1 and GCLC protein levelsin H9c2 cells after treatment with 400 120583M H

2O2 and the

fluorescent intensities of HO-1 and GCLC in the treatmentgroups were greater than that in the H

2O2-treated group

(Figures 8(b) and 8(c)) However none of the compoundsexcept for swetrianolin significantly changed the expressionof Nrf2 (Figure 8(d)) Pretreatment of H9c2 cells with ZnPPBSO and brusatol for 30 min and then addition of fourcompounds for 12 h followed by exposure toH

2O2 markedly

decreased the cell viability indicating that inhibitors canreverse the protective effects of four compounds (Figure 8(e))

4 Discussion

In this study we established a bioassay-guided method todetect the antioxidative activity of G acuta and found thatH2O2exposure significantly activated oxidative stress in

H9c2 cells This method can also be used to guide themore rapid and accurate separation of active ingredients

than that achieved using conventional separation methodswhich facilitates elucidation of the biological activity ofmedicinal plants and is more conducive to the discoveryof active or lead compounds Therefore the bioassay guidedmodel combined with cell activity evaluation has importantapplicability in natural product research

Numerous studies have shown that the Nrf2ARE path-way is critical to several antioxidant systems [12 13]The tran-scription factorNrf2 regulates the oxidative stress response incells [14 15] Nrf2 combines with Keap1 stably under normalphysiological conditions However following activation Nrf2affects the expression of downstream antioxidant proteinssuch as GCLC and HO-1 to enhance the antioxidant capacityof the cells Therefore we choose the Nrf2ARE pathway toelucidate the underlying protective mechanism in H9c2 cells

We established a method to separate and analyze com-pounds 1 2 3 and 4 from G acuta and conducted stud-ies to validate the efficiency of the method This specificmodel achieved good separation and the four compoundswere successfully eluted from the G acuta sample Thecrude extract had protective effect on H9c2 cells fromH2O2-induced apoptosis The activity of the monomeric

compounds obtained from the fractions was then testedResults of the cytoprotective study showed that the chemicalcomponents in G acuta markedly increased cell viabilitycompared to H

2O2alone

The transcription levels of HO-1 and GCLC significantlyincreased in H9c2 cells pretreated with compounds 1 2 3and4 However compared to themodel group the expressionof HO-1 and GCLC protein levels in H9c2 cells increasedwhereas that of Nrf2 was not significantly changed exceptwith swetrianolin Finally our data demonstrate thatG acuta

8 BioMed Research International

HO-1

GCLC

Nrf 2

GAPDH

HO

Bellidifolin

Demethylbellidifolin

SwetrianolinNorswertianolin

ZnPP

BSOBrusatol

--------

+-------

++------

+-+-----

+--+----

+---+---

++---++-

+-+--++-

+--+-+++

+---+++-

(a)

ZnPP Negative ZnPP PositiveControl

H2O2

HO

-1G

APD

H

ampampampampamp ampamp

amplowast

1 Bellidifolin 2 Demethylbellidifolin 3 Swetrianolin 4 Norswertianolin

(b)

BSO Nagetive BSO PostiveControl

H2O2

GCL

CG

APD

H

ampamp ampampamp amp

lowast

1 Bellidifolin 2 Demethylbellidifolin 3 Swetrianolin 4 Norswertianolin

(c)

Brusatol Nagetive Brusatol PostiveContro

lH2O2

Nrf

2G

APD

H

lowast

1 Bellidifolin 2 Demethylbellidifolin 3 Swetrianolin 4 Norswertianolin

(d)

Cel

l via

bilit

y (

)

Bellidifolin (125M)Demethylbellidifolin (125M)

Swetrianolin (125M)Norswertianolin (125M)

ZnPP (15M)BSO (10M)

Brusatol (10M)

--------

-+------

--+-----

---+----

+-------

++--+---

++---+--

++----+-

+-+-+---

+-+--+--

+-+---+-

+-+----+

++-----+

+--+--+-

HO (400M)

lowastlowastlowast

$$$$$$$$ $$

(e)

Figure 8 Analysis of HO-1 GCLC and Nrf2 protein levels on H9c2 cells (a)Western blotting analysis HO-1 GCLC Nrf2 protein expressionwith or without ZnPP BSO and brusatol (HO-1group only added ZnPP GCLC group only added BSO Nrf2 group only added brusatol)(b) Western blot analysis HO-1 protein expression with or without ZnPP (c) Western blot analysis GCLC protein expression with or withoutBSO (d) Western blot analysis Nrf2 protein expression with or without brusatol (e) Effect of the different treatments on H9c2 cells Dataare expressed as the means plusmn SD n = 3 lowastlowastlowast119875 lt 0001 lowast119875 lt 005 compared with control group P lt 0001 P lt 001 P lt 005 comparedwith H

2O2group $$$119875 lt 0001 $$119875 lt 001 compared with H

2O2group combined with four compounds ampampampP lt 0001 ampampP lt 001 ampP lt 005

compared with H2O2group combined with inhibitors and four compounds GAPDH was used as an internal control

BioMed Research International 9

protects H9c2 cells from oxidative damage via the Nrf2-AREpathway

In conclusion the results of the present study demon-strated that four compounds are able to protect H9c2 cellsfrom H

2O2-induced damage which may be associated with

the activation of Nrf2ARE pathway in H9c2 cells Theseresults provide evidence that G acuta may be a potentialtherapeutic agent for the treatment of cardiovascular diseases

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This study was supported by the research of the effects ofMongolian Danggong-3 on the activity of myocardial cells inrats after myocardial anoxia-reoxygenation (NO 201702026)Natural Science Foundation of Tianjin (15JCQNJC13400)Investigation and Variety Systematization Study of Mon-golian Medicine ldquoDigedardquo (201702121) Chinese NationalNatural Science Foundation (81160504) and Natural ScienceFoundation of Inner Mongolia Autonomous Region (BYJJ-YF 201726)

References

[1] Z Wang Q Wu Y Yu et al ldquoDetermination and phar-macokinetic study of four xanthones inrat plasma after oraladministration of Gentianella acuta extract by UHPLC-ESI-MSMSrdquo Journal of Ethnopharmacology vol 174 pp 261ndash2692015

[2] L J Lv and M H Li ldquoTerpenoids flavonoids and xanthonesfrom Gentianella acuta (Gentianaceae)rdquo Biochemical Systemat-ics and Ecology vol 37 no 4 pp 497ndash500 2009

[3] Y-J Li T-D Bao Q Li et al ldquoProtection of CompoundGranules of Yinxingye onH9c2myocardial cells injury inducedby H2O2rdquo Chinese Traditional and Herbal Drugs vol 47 no 3pp 459ndash465 2016

[4] Y H Bai X Wang L J Zhang et al ldquoAdvances in the Chineseand western medicine treatment of coronary microvascularangina pectorisrdquo Journal of Anhui Health Vocational and Tech-nical College vol 17 no 2 pp 21ndash23 2018

[5] A B Gustafsson and R A Gottlieb ldquoMechanisms of Apoptosisin the Heartrdquo Journal of Clinical Immunology vol 23 no 6 pp447ndash459 2003

[6] Y N Liu W P Wang Z Q Jia P Chen K T Ma and CY Zhou ldquoProtection of -Lipoic Acid Against H

2O2-induced

Oxidative Stress in H9c2 Cellsrdquo Chinese Journal of Biochemistryand Molecular Biology vol 28 no 3 pp 227ndash233 2012

[7] M N Sharikabad K M Oslashstbye andO Broslashrs ldquoEffect of hydro-gen peroxide on reoxygenation-induced Ca2+ accumulation inrat cardiomyocytesrdquo Free Radical Biology amp Medicine vol 37no 4 pp 531ndash538 2004

[8] G D Wu J Zhang D Yang Z W Wang and M H LildquoDetermination of the Norswertianolin in Gentianella acutardquoChinese Journal of Experimental Traditional Medical Formulaevol 18 no 6 pp 69ndash71 2012

[9] L J Lv Y F Wang T Yan H R Han and M H Li ldquoPrepa-ration of swertianolin from Gentianella acuta and its effects onarrhythmia induced by aconitine in micerdquo Chinese Journal ofNew Drugs and Clinical Remedies vol 34 no 8 pp 630ndash6352015

[10] H-J Xu W-L Chen Y Wang M-Z Zheng Y-Y Chen andY-L Shen ldquoS262A mutation abolishes protective effects ofconnexin 43 against hypothermic preservation-induced injuryin cardiomyocytesrdquoe Journal of Heart and Lung Transplanta-tion vol 31 no 6 pp 663ndash669 2012

[11] X Jia L Zhang and X Mao ldquoS-propranolol protected H9C2cells from ischemiareperfusion-induced apoptosis via down-regultion of RACK1 Generdquo International Journal of Clinical andExperimental Pathology vol 8 no 9 pp 10335ndash10344 2015

[12] T Nguyen P Nioi and C B Pickett ldquoThe Nrf2-antioxidantresponse element signaling pathway and its activation byoxidative stressrdquo e Journal of Biological Chemistry vol 284no 20 pp 13291ndash13295 2009

[13] Y-S Keum and B Y Choi ldquoMolecular and chemical regulationof the keap1-Nrf2 signaling pathwayrdquo Molecules vol 19 no 7pp 10074ndash10089 2014

[14] H Li and L Yang ldquoMolecular regulatory mechanism of Nrf2antioxidantrdquo China Journal of Bioinformatics vol 16 no 1 pp1ndash6 2018

[15] H J Sun L Dong J Y Wang M Pan Y Wu and S LildquoProtective effects of Lycium ruthenicum Murr on hydrogenperoxide-induced cellular oxidative stress injury in heart-derived H9c2 cellsrdquo Journal of Beijing University of TraditionalChinese Medicine vol 41 no 4 pp 301ndash305 2018

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Submit your manuscripts atwwwhindawicom

BioMed Research International 5

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Contro

l

M

M

M

M

M

Contro

l

M

M

M

M

M

Contro

l

M

M

M

M

M

Contro

l

M

M

M

M

M

Cel

l via

bilit

y (

)

lowastlowast lowastlowast

lowastlowast

lowast

lowastlowast

(a)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Contro

lH2O

2

M

M

M

M

M

Contro

lH2O

2

M

M

M

M

M

Contro

lH2O

2

M

M

M

M

M

Contro

lH2O

2

M

M

M

M

M

Cel

l via

bilit

y (

)

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

(b)

Figure 4 The activity detection of compounds 1 2 3 and 4 on H2O2-induced apoptosis on H9c2 cells (a) Toxicity of four compounds on

H9c2 cells (b) The active ingredients on H9c2 cells assessed by MTT (means plusmn SD n = 3) lowast lowast lowastP lt 0001 lowastlowastP lt 001 lowastP lt 005 comparedwith control P lt 0001 P lt 001 P lt 005 compared with H

2O2

concentration of H2O2to be 400 120583Mformimicking oxidative

stress-induced injury to H9c2 cells

32 Crude Extracts Prevented H2O2-Induced Reduction in CellViability We used an MTT assay to analyze the effects offour crude extracts on H9c2 cells As shown in Figure 2the number of apoptotic cells significantly increased in themodel group compared to the treatment group Pretreatmentwith the four crude extracts for 12 h showed that the ethylacetate fractions and 119899-butanol extract significantly increasedthe percentage of viable H9c2 cells suggesting that the twofractions protected H9c2 cells from apoptosis induced byH2O2

Based on these results we further evaluated the activityof seven subfractions (B1 B2-B10 B11-B14 C1 C2 C3 andC4) from the ethyl acetate and n-butanol fractions Theresults show that B2-B10 andC2 significantly inhibitedH

2O2-

induced apoptosis of H9c2 cells The protective effects of B2-B10 and C2 are shown in Figure 3 and the results of inactivefractions are not shown in the manuscript

We further evaluated the activity of compounds 1 2 3and 4 obtained from the fractions B2-B10 and C2 Theseresults indicated that the four compounds showed no signif-icant toxic effect on H9c2 cells Compared with the controlgroup H

2O2model group could significantly reduce the cell

viability indicating that the H2O2model is successful The

results showed that the monomeric compounds 1 2 3 and4 had the protective effects on H

2O2-induced H9c2 cells

(Figure 4)

33 HPLC Detection The structures of these compoundswere identified by comparing spectral data (proton nuclear

O

OH

OH

O OH

O

Bellidifolin

O

OH

OH

O OH

OH

Demethylbellidifolin

O

OH

OH

O O

O

O

HOOH

OH

OH

O

OH

OH

O O

OH

O

HO OHOH

OH

SwetrianolinNorswertianolin

Figure 5The chemical structures of four monomeric compounds

magnetic resonance [1H-NMR] 13C-NMR and electrosprayionization [ESI-MS]) with published data as norswertianolin(1) swetrianolin (2) demethylbellidifolin (3) and bellidifolin(4) The chemical structures of the four compounds areshown in Figure 5 We compared the plant contents of thefour monomeric compounds using the area normalizationmethod These data indicated that there was a significantvariation in the contents of the four monomeric compoundsThe fingerprints of the four compounds and crude fractionsare shown in Figure 6 and the relative contents are shown inTable 2

6 BioMed Research International

00 50 100 150 200 250 300 350 400 450 500 550minus100

00

100

200

300

400

500

600

700

1

2

3

4

min

mAUMixed standard

00 50 100 150 200 250 300 350 400 450 500 550minus20

0

20

40

60

80

100

120

140

160 1

2

3 4

min

mAUEthyl acetate

00 50 100 150 200 250 300 350 400 450 500 550minus100

00

100

200

300

400

500

600

700 1

2 3

4

min

mAUB2-B10

00 50 100 150 200 250 300 350 400 450 500 550minus100

00

100

200

300

400

500

600

1

2

3 4

min

mAUN-butanol

00 50 100 150 200 250 300 350 400 450 500 550minus100

00

100

200

300

400

500

600

1

2

3 4

min

mAUC2

1 Norswertianolin 2 Swetrianolin 3 Demethylbellidifolin 4 Bellidifolin

Figure 6 The fingerprints of the four compounds in different extracts

BioMed Research International 7

Control

H2O2

Nrf

2m

RNA

leve

l

lowast

Control

H2O2

HO

-1 m

RNA

leve

l

lowastlowast

lowastlowastlowastlowastlowastlowast

GCL

C m

RNA

leve

l

Control

H2O2

lowastlowast

lowastlowast lowast

1 Bellidifolin 2 Demethylbellidifolin 3 Swetrianolin 4 Norswertianolin

Figure 7 Analysis of HO-1 GCLC and Nrf2 mRNA on H9c2 cells (means plusmn SD n = 3) lowastlowastlowast119875 lt 0001 lowastlowast119875 lt 001 lowast119875 lt 005 compared withH2O2

Table 2 Relative content of four monomeric compounds in different extracts

fractioncompounds

Relative content1 () 2 () 3 () 4 ()

ethyl acetate crude fraction 2643 1985 1118 722B2-B10 fraction 1161 1004 1243 803119899-butanol crude fraction 681 260 017 003C2 fraction 653 323 031 014

34 Transcription Levels of HO-1 GCLC and Nrf2 Weexamined the transcription levels of HO-1 GCLC and Nrf2in H9c2 cells treated with compounds 1 2 3 and 4 Asshown in Figure 7 the transcription levels of HO-1 andGCLC significantly increased in H9c2 cells pretreated withthe indicated concentrations of compounds 1 2 3 and 4for 12 h Furthermore the transcription levels of Nrf2 wereincreased in H9c2 cells pretreated with compound 3 whereascompounds 1 2 and 4 did not alter the express comparedwith the model group

35 Western Blot Analysis of HO-1 GCLC and Nrf2 Figure 8shows the expression of HO-1 and GCLC protein levelsin H9c2 cells after treatment with 400 120583M H

2O2 and the

fluorescent intensities of HO-1 and GCLC in the treatmentgroups were greater than that in the H

2O2-treated group

(Figures 8(b) and 8(c)) However none of the compoundsexcept for swetrianolin significantly changed the expressionof Nrf2 (Figure 8(d)) Pretreatment of H9c2 cells with ZnPPBSO and brusatol for 30 min and then addition of fourcompounds for 12 h followed by exposure toH

2O2 markedly

decreased the cell viability indicating that inhibitors canreverse the protective effects of four compounds (Figure 8(e))

4 Discussion

In this study we established a bioassay-guided method todetect the antioxidative activity of G acuta and found thatH2O2exposure significantly activated oxidative stress in

H9c2 cells This method can also be used to guide themore rapid and accurate separation of active ingredients

than that achieved using conventional separation methodswhich facilitates elucidation of the biological activity ofmedicinal plants and is more conducive to the discoveryof active or lead compounds Therefore the bioassay guidedmodel combined with cell activity evaluation has importantapplicability in natural product research

Numerous studies have shown that the Nrf2ARE path-way is critical to several antioxidant systems [12 13]The tran-scription factorNrf2 regulates the oxidative stress response incells [14 15] Nrf2 combines with Keap1 stably under normalphysiological conditions However following activation Nrf2affects the expression of downstream antioxidant proteinssuch as GCLC and HO-1 to enhance the antioxidant capacityof the cells Therefore we choose the Nrf2ARE pathway toelucidate the underlying protective mechanism in H9c2 cells

We established a method to separate and analyze com-pounds 1 2 3 and 4 from G acuta and conducted stud-ies to validate the efficiency of the method This specificmodel achieved good separation and the four compoundswere successfully eluted from the G acuta sample Thecrude extract had protective effect on H9c2 cells fromH2O2-induced apoptosis The activity of the monomeric

compounds obtained from the fractions was then testedResults of the cytoprotective study showed that the chemicalcomponents in G acuta markedly increased cell viabilitycompared to H

2O2alone

The transcription levels of HO-1 and GCLC significantlyincreased in H9c2 cells pretreated with compounds 1 2 3and4 However compared to themodel group the expressionof HO-1 and GCLC protein levels in H9c2 cells increasedwhereas that of Nrf2 was not significantly changed exceptwith swetrianolin Finally our data demonstrate thatG acuta

8 BioMed Research International

HO-1

GCLC

Nrf 2

GAPDH

HO

Bellidifolin

Demethylbellidifolin

SwetrianolinNorswertianolin

ZnPP

BSOBrusatol

--------

+-------

++------

+-+-----

+--+----

+---+---

++---++-

+-+--++-

+--+-+++

+---+++-

(a)

ZnPP Negative ZnPP PositiveControl

H2O2

HO

-1G

APD

H

ampampampampamp ampamp

amplowast

1 Bellidifolin 2 Demethylbellidifolin 3 Swetrianolin 4 Norswertianolin

(b)

BSO Nagetive BSO PostiveControl

H2O2

GCL

CG

APD

H

ampamp ampampamp amp

lowast

1 Bellidifolin 2 Demethylbellidifolin 3 Swetrianolin 4 Norswertianolin

(c)

Brusatol Nagetive Brusatol PostiveContro

lH2O2

Nrf

2G

APD

H

lowast

1 Bellidifolin 2 Demethylbellidifolin 3 Swetrianolin 4 Norswertianolin

(d)

Cel

l via

bilit

y (

)

Bellidifolin (125M)Demethylbellidifolin (125M)

Swetrianolin (125M)Norswertianolin (125M)

ZnPP (15M)BSO (10M)

Brusatol (10M)

--------

-+------

--+-----

---+----

+-------

++--+---

++---+--

++----+-

+-+-+---

+-+--+--

+-+---+-

+-+----+

++-----+

+--+--+-

HO (400M)

lowastlowastlowast

$$$$$$$$ $$

(e)

Figure 8 Analysis of HO-1 GCLC and Nrf2 protein levels on H9c2 cells (a)Western blotting analysis HO-1 GCLC Nrf2 protein expressionwith or without ZnPP BSO and brusatol (HO-1group only added ZnPP GCLC group only added BSO Nrf2 group only added brusatol)(b) Western blot analysis HO-1 protein expression with or without ZnPP (c) Western blot analysis GCLC protein expression with or withoutBSO (d) Western blot analysis Nrf2 protein expression with or without brusatol (e) Effect of the different treatments on H9c2 cells Dataare expressed as the means plusmn SD n = 3 lowastlowastlowast119875 lt 0001 lowast119875 lt 005 compared with control group P lt 0001 P lt 001 P lt 005 comparedwith H

2O2group $$$119875 lt 0001 $$119875 lt 001 compared with H

2O2group combined with four compounds ampampampP lt 0001 ampampP lt 001 ampP lt 005

compared with H2O2group combined with inhibitors and four compounds GAPDH was used as an internal control

BioMed Research International 9

protects H9c2 cells from oxidative damage via the Nrf2-AREpathway

In conclusion the results of the present study demon-strated that four compounds are able to protect H9c2 cellsfrom H

2O2-induced damage which may be associated with

the activation of Nrf2ARE pathway in H9c2 cells Theseresults provide evidence that G acuta may be a potentialtherapeutic agent for the treatment of cardiovascular diseases

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This study was supported by the research of the effects ofMongolian Danggong-3 on the activity of myocardial cells inrats after myocardial anoxia-reoxygenation (NO 201702026)Natural Science Foundation of Tianjin (15JCQNJC13400)Investigation and Variety Systematization Study of Mon-golian Medicine ldquoDigedardquo (201702121) Chinese NationalNatural Science Foundation (81160504) and Natural ScienceFoundation of Inner Mongolia Autonomous Region (BYJJ-YF 201726)

References

[1] Z Wang Q Wu Y Yu et al ldquoDetermination and phar-macokinetic study of four xanthones inrat plasma after oraladministration of Gentianella acuta extract by UHPLC-ESI-MSMSrdquo Journal of Ethnopharmacology vol 174 pp 261ndash2692015

[2] L J Lv and M H Li ldquoTerpenoids flavonoids and xanthonesfrom Gentianella acuta (Gentianaceae)rdquo Biochemical Systemat-ics and Ecology vol 37 no 4 pp 497ndash500 2009

[3] Y-J Li T-D Bao Q Li et al ldquoProtection of CompoundGranules of Yinxingye onH9c2myocardial cells injury inducedby H2O2rdquo Chinese Traditional and Herbal Drugs vol 47 no 3pp 459ndash465 2016

[4] Y H Bai X Wang L J Zhang et al ldquoAdvances in the Chineseand western medicine treatment of coronary microvascularangina pectorisrdquo Journal of Anhui Health Vocational and Tech-nical College vol 17 no 2 pp 21ndash23 2018

[5] A B Gustafsson and R A Gottlieb ldquoMechanisms of Apoptosisin the Heartrdquo Journal of Clinical Immunology vol 23 no 6 pp447ndash459 2003

[6] Y N Liu W P Wang Z Q Jia P Chen K T Ma and CY Zhou ldquoProtection of -Lipoic Acid Against H

2O2-induced

Oxidative Stress in H9c2 Cellsrdquo Chinese Journal of Biochemistryand Molecular Biology vol 28 no 3 pp 227ndash233 2012

[7] M N Sharikabad K M Oslashstbye andO Broslashrs ldquoEffect of hydro-gen peroxide on reoxygenation-induced Ca2+ accumulation inrat cardiomyocytesrdquo Free Radical Biology amp Medicine vol 37no 4 pp 531ndash538 2004

[8] G D Wu J Zhang D Yang Z W Wang and M H LildquoDetermination of the Norswertianolin in Gentianella acutardquoChinese Journal of Experimental Traditional Medical Formulaevol 18 no 6 pp 69ndash71 2012

[9] L J Lv Y F Wang T Yan H R Han and M H Li ldquoPrepa-ration of swertianolin from Gentianella acuta and its effects onarrhythmia induced by aconitine in micerdquo Chinese Journal ofNew Drugs and Clinical Remedies vol 34 no 8 pp 630ndash6352015

[10] H-J Xu W-L Chen Y Wang M-Z Zheng Y-Y Chen andY-L Shen ldquoS262A mutation abolishes protective effects ofconnexin 43 against hypothermic preservation-induced injuryin cardiomyocytesrdquoe Journal of Heart and Lung Transplanta-tion vol 31 no 6 pp 663ndash669 2012

[11] X Jia L Zhang and X Mao ldquoS-propranolol protected H9C2cells from ischemiareperfusion-induced apoptosis via down-regultion of RACK1 Generdquo International Journal of Clinical andExperimental Pathology vol 8 no 9 pp 10335ndash10344 2015

[12] T Nguyen P Nioi and C B Pickett ldquoThe Nrf2-antioxidantresponse element signaling pathway and its activation byoxidative stressrdquo e Journal of Biological Chemistry vol 284no 20 pp 13291ndash13295 2009

[13] Y-S Keum and B Y Choi ldquoMolecular and chemical regulationof the keap1-Nrf2 signaling pathwayrdquo Molecules vol 19 no 7pp 10074ndash10089 2014

[14] H Li and L Yang ldquoMolecular regulatory mechanism of Nrf2antioxidantrdquo China Journal of Bioinformatics vol 16 no 1 pp1ndash6 2018

[15] H J Sun L Dong J Y Wang M Pan Y Wu and S LildquoProtective effects of Lycium ruthenicum Murr on hydrogenperoxide-induced cellular oxidative stress injury in heart-derived H9c2 cellsrdquo Journal of Beijing University of TraditionalChinese Medicine vol 41 no 4 pp 301ndash305 2018

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

6 BioMed Research International

00 50 100 150 200 250 300 350 400 450 500 550minus100

00

100

200

300

400

500

600

700

1

2

3

4

min

mAUMixed standard

00 50 100 150 200 250 300 350 400 450 500 550minus20

0

20

40

60

80

100

120

140

160 1

2

3 4

min

mAUEthyl acetate

00 50 100 150 200 250 300 350 400 450 500 550minus100

00

100

200

300

400

500

600

700 1

2 3

4

min

mAUB2-B10

00 50 100 150 200 250 300 350 400 450 500 550minus100

00

100

200

300

400

500

600

1

2

3 4

min

mAUN-butanol

00 50 100 150 200 250 300 350 400 450 500 550minus100

00

100

200

300

400

500

600

1

2

3 4

min

mAUC2

1 Norswertianolin 2 Swetrianolin 3 Demethylbellidifolin 4 Bellidifolin

Figure 6 The fingerprints of the four compounds in different extracts

BioMed Research International 7

Control

H2O2

Nrf

2m

RNA

leve

l

lowast

Control

H2O2

HO

-1 m

RNA

leve

l

lowastlowast

lowastlowastlowastlowastlowastlowast

GCL

C m

RNA

leve

l

Control

H2O2

lowastlowast

lowastlowast lowast

1 Bellidifolin 2 Demethylbellidifolin 3 Swetrianolin 4 Norswertianolin

Figure 7 Analysis of HO-1 GCLC and Nrf2 mRNA on H9c2 cells (means plusmn SD n = 3) lowastlowastlowast119875 lt 0001 lowastlowast119875 lt 001 lowast119875 lt 005 compared withH2O2

Table 2 Relative content of four monomeric compounds in different extracts

fractioncompounds

Relative content1 () 2 () 3 () 4 ()

ethyl acetate crude fraction 2643 1985 1118 722B2-B10 fraction 1161 1004 1243 803119899-butanol crude fraction 681 260 017 003C2 fraction 653 323 031 014

34 Transcription Levels of HO-1 GCLC and Nrf2 Weexamined the transcription levels of HO-1 GCLC and Nrf2in H9c2 cells treated with compounds 1 2 3 and 4 Asshown in Figure 7 the transcription levels of HO-1 andGCLC significantly increased in H9c2 cells pretreated withthe indicated concentrations of compounds 1 2 3 and 4for 12 h Furthermore the transcription levels of Nrf2 wereincreased in H9c2 cells pretreated with compound 3 whereascompounds 1 2 and 4 did not alter the express comparedwith the model group

35 Western Blot Analysis of HO-1 GCLC and Nrf2 Figure 8shows the expression of HO-1 and GCLC protein levelsin H9c2 cells after treatment with 400 120583M H

2O2 and the

fluorescent intensities of HO-1 and GCLC in the treatmentgroups were greater than that in the H

2O2-treated group

(Figures 8(b) and 8(c)) However none of the compoundsexcept for swetrianolin significantly changed the expressionof Nrf2 (Figure 8(d)) Pretreatment of H9c2 cells with ZnPPBSO and brusatol for 30 min and then addition of fourcompounds for 12 h followed by exposure toH

2O2 markedly

decreased the cell viability indicating that inhibitors canreverse the protective effects of four compounds (Figure 8(e))

4 Discussion

In this study we established a bioassay-guided method todetect the antioxidative activity of G acuta and found thatH2O2exposure significantly activated oxidative stress in

H9c2 cells This method can also be used to guide themore rapid and accurate separation of active ingredients

than that achieved using conventional separation methodswhich facilitates elucidation of the biological activity ofmedicinal plants and is more conducive to the discoveryof active or lead compounds Therefore the bioassay guidedmodel combined with cell activity evaluation has importantapplicability in natural product research

Numerous studies have shown that the Nrf2ARE path-way is critical to several antioxidant systems [12 13]The tran-scription factorNrf2 regulates the oxidative stress response incells [14 15] Nrf2 combines with Keap1 stably under normalphysiological conditions However following activation Nrf2affects the expression of downstream antioxidant proteinssuch as GCLC and HO-1 to enhance the antioxidant capacityof the cells Therefore we choose the Nrf2ARE pathway toelucidate the underlying protective mechanism in H9c2 cells

We established a method to separate and analyze com-pounds 1 2 3 and 4 from G acuta and conducted stud-ies to validate the efficiency of the method This specificmodel achieved good separation and the four compoundswere successfully eluted from the G acuta sample Thecrude extract had protective effect on H9c2 cells fromH2O2-induced apoptosis The activity of the monomeric

compounds obtained from the fractions was then testedResults of the cytoprotective study showed that the chemicalcomponents in G acuta markedly increased cell viabilitycompared to H

2O2alone

The transcription levels of HO-1 and GCLC significantlyincreased in H9c2 cells pretreated with compounds 1 2 3and4 However compared to themodel group the expressionof HO-1 and GCLC protein levels in H9c2 cells increasedwhereas that of Nrf2 was not significantly changed exceptwith swetrianolin Finally our data demonstrate thatG acuta

8 BioMed Research International

HO-1

GCLC

Nrf 2

GAPDH

HO

Bellidifolin

Demethylbellidifolin

SwetrianolinNorswertianolin

ZnPP

BSOBrusatol

--------

+-------

++------

+-+-----

+--+----

+---+---

++---++-

+-+--++-

+--+-+++

+---+++-

(a)

ZnPP Negative ZnPP PositiveControl

H2O2

HO

-1G

APD

H

ampampampampamp ampamp

amplowast

1 Bellidifolin 2 Demethylbellidifolin 3 Swetrianolin 4 Norswertianolin

(b)

BSO Nagetive BSO PostiveControl

H2O2

GCL

CG

APD

H

ampamp ampampamp amp

lowast

1 Bellidifolin 2 Demethylbellidifolin 3 Swetrianolin 4 Norswertianolin

(c)

Brusatol Nagetive Brusatol PostiveContro

lH2O2

Nrf

2G

APD

H

lowast

1 Bellidifolin 2 Demethylbellidifolin 3 Swetrianolin 4 Norswertianolin

(d)

Cel

l via

bilit

y (

)

Bellidifolin (125M)Demethylbellidifolin (125M)

Swetrianolin (125M)Norswertianolin (125M)

ZnPP (15M)BSO (10M)

Brusatol (10M)

--------

-+------

--+-----

---+----

+-------

++--+---

++---+--

++----+-

+-+-+---

+-+--+--

+-+---+-

+-+----+

++-----+

+--+--+-

HO (400M)

lowastlowastlowast

$$$$$$$$ $$

(e)

Figure 8 Analysis of HO-1 GCLC and Nrf2 protein levels on H9c2 cells (a)Western blotting analysis HO-1 GCLC Nrf2 protein expressionwith or without ZnPP BSO and brusatol (HO-1group only added ZnPP GCLC group only added BSO Nrf2 group only added brusatol)(b) Western blot analysis HO-1 protein expression with or without ZnPP (c) Western blot analysis GCLC protein expression with or withoutBSO (d) Western blot analysis Nrf2 protein expression with or without brusatol (e) Effect of the different treatments on H9c2 cells Dataare expressed as the means plusmn SD n = 3 lowastlowastlowast119875 lt 0001 lowast119875 lt 005 compared with control group P lt 0001 P lt 001 P lt 005 comparedwith H

2O2group $$$119875 lt 0001 $$119875 lt 001 compared with H

2O2group combined with four compounds ampampampP lt 0001 ampampP lt 001 ampP lt 005

compared with H2O2group combined with inhibitors and four compounds GAPDH was used as an internal control

BioMed Research International 9

protects H9c2 cells from oxidative damage via the Nrf2-AREpathway

In conclusion the results of the present study demon-strated that four compounds are able to protect H9c2 cellsfrom H

2O2-induced damage which may be associated with

the activation of Nrf2ARE pathway in H9c2 cells Theseresults provide evidence that G acuta may be a potentialtherapeutic agent for the treatment of cardiovascular diseases

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This study was supported by the research of the effects ofMongolian Danggong-3 on the activity of myocardial cells inrats after myocardial anoxia-reoxygenation (NO 201702026)Natural Science Foundation of Tianjin (15JCQNJC13400)Investigation and Variety Systematization Study of Mon-golian Medicine ldquoDigedardquo (201702121) Chinese NationalNatural Science Foundation (81160504) and Natural ScienceFoundation of Inner Mongolia Autonomous Region (BYJJ-YF 201726)

References

[1] Z Wang Q Wu Y Yu et al ldquoDetermination and phar-macokinetic study of four xanthones inrat plasma after oraladministration of Gentianella acuta extract by UHPLC-ESI-MSMSrdquo Journal of Ethnopharmacology vol 174 pp 261ndash2692015

[2] L J Lv and M H Li ldquoTerpenoids flavonoids and xanthonesfrom Gentianella acuta (Gentianaceae)rdquo Biochemical Systemat-ics and Ecology vol 37 no 4 pp 497ndash500 2009

[3] Y-J Li T-D Bao Q Li et al ldquoProtection of CompoundGranules of Yinxingye onH9c2myocardial cells injury inducedby H2O2rdquo Chinese Traditional and Herbal Drugs vol 47 no 3pp 459ndash465 2016

[4] Y H Bai X Wang L J Zhang et al ldquoAdvances in the Chineseand western medicine treatment of coronary microvascularangina pectorisrdquo Journal of Anhui Health Vocational and Tech-nical College vol 17 no 2 pp 21ndash23 2018

[5] A B Gustafsson and R A Gottlieb ldquoMechanisms of Apoptosisin the Heartrdquo Journal of Clinical Immunology vol 23 no 6 pp447ndash459 2003

[6] Y N Liu W P Wang Z Q Jia P Chen K T Ma and CY Zhou ldquoProtection of -Lipoic Acid Against H

2O2-induced

Oxidative Stress in H9c2 Cellsrdquo Chinese Journal of Biochemistryand Molecular Biology vol 28 no 3 pp 227ndash233 2012

[7] M N Sharikabad K M Oslashstbye andO Broslashrs ldquoEffect of hydro-gen peroxide on reoxygenation-induced Ca2+ accumulation inrat cardiomyocytesrdquo Free Radical Biology amp Medicine vol 37no 4 pp 531ndash538 2004

[8] G D Wu J Zhang D Yang Z W Wang and M H LildquoDetermination of the Norswertianolin in Gentianella acutardquoChinese Journal of Experimental Traditional Medical Formulaevol 18 no 6 pp 69ndash71 2012

[9] L J Lv Y F Wang T Yan H R Han and M H Li ldquoPrepa-ration of swertianolin from Gentianella acuta and its effects onarrhythmia induced by aconitine in micerdquo Chinese Journal ofNew Drugs and Clinical Remedies vol 34 no 8 pp 630ndash6352015

[10] H-J Xu W-L Chen Y Wang M-Z Zheng Y-Y Chen andY-L Shen ldquoS262A mutation abolishes protective effects ofconnexin 43 against hypothermic preservation-induced injuryin cardiomyocytesrdquoe Journal of Heart and Lung Transplanta-tion vol 31 no 6 pp 663ndash669 2012

[11] X Jia L Zhang and X Mao ldquoS-propranolol protected H9C2cells from ischemiareperfusion-induced apoptosis via down-regultion of RACK1 Generdquo International Journal of Clinical andExperimental Pathology vol 8 no 9 pp 10335ndash10344 2015

[12] T Nguyen P Nioi and C B Pickett ldquoThe Nrf2-antioxidantresponse element signaling pathway and its activation byoxidative stressrdquo e Journal of Biological Chemistry vol 284no 20 pp 13291ndash13295 2009

[13] Y-S Keum and B Y Choi ldquoMolecular and chemical regulationof the keap1-Nrf2 signaling pathwayrdquo Molecules vol 19 no 7pp 10074ndash10089 2014

[14] H Li and L Yang ldquoMolecular regulatory mechanism of Nrf2antioxidantrdquo China Journal of Bioinformatics vol 16 no 1 pp1ndash6 2018

[15] H J Sun L Dong J Y Wang M Pan Y Wu and S LildquoProtective effects of Lycium ruthenicum Murr on hydrogenperoxide-induced cellular oxidative stress injury in heart-derived H9c2 cellsrdquo Journal of Beijing University of TraditionalChinese Medicine vol 41 no 4 pp 301ndash305 2018

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

BioMed Research International 7

Control

H2O2

Nrf

2m

RNA

leve

l

lowast

Control

H2O2

HO

-1 m

RNA

leve

l

lowastlowast

lowastlowastlowastlowastlowastlowast

GCL

C m

RNA

leve

l

Control

H2O2

lowastlowast

lowastlowast lowast

1 Bellidifolin 2 Demethylbellidifolin 3 Swetrianolin 4 Norswertianolin

Figure 7 Analysis of HO-1 GCLC and Nrf2 mRNA on H9c2 cells (means plusmn SD n = 3) lowastlowastlowast119875 lt 0001 lowastlowast119875 lt 001 lowast119875 lt 005 compared withH2O2

Table 2 Relative content of four monomeric compounds in different extracts

fractioncompounds

Relative content1 () 2 () 3 () 4 ()

ethyl acetate crude fraction 2643 1985 1118 722B2-B10 fraction 1161 1004 1243 803119899-butanol crude fraction 681 260 017 003C2 fraction 653 323 031 014

34 Transcription Levels of HO-1 GCLC and Nrf2 Weexamined the transcription levels of HO-1 GCLC and Nrf2in H9c2 cells treated with compounds 1 2 3 and 4 Asshown in Figure 7 the transcription levels of HO-1 andGCLC significantly increased in H9c2 cells pretreated withthe indicated concentrations of compounds 1 2 3 and 4for 12 h Furthermore the transcription levels of Nrf2 wereincreased in H9c2 cells pretreated with compound 3 whereascompounds 1 2 and 4 did not alter the express comparedwith the model group

35 Western Blot Analysis of HO-1 GCLC and Nrf2 Figure 8shows the expression of HO-1 and GCLC protein levelsin H9c2 cells after treatment with 400 120583M H

2O2 and the

fluorescent intensities of HO-1 and GCLC in the treatmentgroups were greater than that in the H

2O2-treated group

(Figures 8(b) and 8(c)) However none of the compoundsexcept for swetrianolin significantly changed the expressionof Nrf2 (Figure 8(d)) Pretreatment of H9c2 cells with ZnPPBSO and brusatol for 30 min and then addition of fourcompounds for 12 h followed by exposure toH

2O2 markedly

decreased the cell viability indicating that inhibitors canreverse the protective effects of four compounds (Figure 8(e))

4 Discussion

In this study we established a bioassay-guided method todetect the antioxidative activity of G acuta and found thatH2O2exposure significantly activated oxidative stress in

H9c2 cells This method can also be used to guide themore rapid and accurate separation of active ingredients

than that achieved using conventional separation methodswhich facilitates elucidation of the biological activity ofmedicinal plants and is more conducive to the discoveryof active or lead compounds Therefore the bioassay guidedmodel combined with cell activity evaluation has importantapplicability in natural product research

Numerous studies have shown that the Nrf2ARE path-way is critical to several antioxidant systems [12 13]The tran-scription factorNrf2 regulates the oxidative stress response incells [14 15] Nrf2 combines with Keap1 stably under normalphysiological conditions However following activation Nrf2affects the expression of downstream antioxidant proteinssuch as GCLC and HO-1 to enhance the antioxidant capacityof the cells Therefore we choose the Nrf2ARE pathway toelucidate the underlying protective mechanism in H9c2 cells

We established a method to separate and analyze com-pounds 1 2 3 and 4 from G acuta and conducted stud-ies to validate the efficiency of the method This specificmodel achieved good separation and the four compoundswere successfully eluted from the G acuta sample Thecrude extract had protective effect on H9c2 cells fromH2O2-induced apoptosis The activity of the monomeric

compounds obtained from the fractions was then testedResults of the cytoprotective study showed that the chemicalcomponents in G acuta markedly increased cell viabilitycompared to H

2O2alone

The transcription levels of HO-1 and GCLC significantlyincreased in H9c2 cells pretreated with compounds 1 2 3and4 However compared to themodel group the expressionof HO-1 and GCLC protein levels in H9c2 cells increasedwhereas that of Nrf2 was not significantly changed exceptwith swetrianolin Finally our data demonstrate thatG acuta

8 BioMed Research International

HO-1

GCLC

Nrf 2

GAPDH

HO

Bellidifolin

Demethylbellidifolin

SwetrianolinNorswertianolin

ZnPP

BSOBrusatol

--------

+-------

++------

+-+-----

+--+----

+---+---

++---++-

+-+--++-

+--+-+++

+---+++-

(a)

ZnPP Negative ZnPP PositiveControl

H2O2

HO

-1G

APD

H

ampampampampamp ampamp

amplowast

1 Bellidifolin 2 Demethylbellidifolin 3 Swetrianolin 4 Norswertianolin

(b)

BSO Nagetive BSO PostiveControl

H2O2

GCL

CG

APD

H

ampamp ampampamp amp

lowast

1 Bellidifolin 2 Demethylbellidifolin 3 Swetrianolin 4 Norswertianolin

(c)

Brusatol Nagetive Brusatol PostiveContro

lH2O2

Nrf

2G

APD

H

lowast

1 Bellidifolin 2 Demethylbellidifolin 3 Swetrianolin 4 Norswertianolin

(d)

Cel

l via

bilit

y (

)

Bellidifolin (125M)Demethylbellidifolin (125M)

Swetrianolin (125M)Norswertianolin (125M)

ZnPP (15M)BSO (10M)

Brusatol (10M)

--------

-+------

--+-----

---+----

+-------

++--+---

++---+--

++----+-

+-+-+---

+-+--+--

+-+---+-

+-+----+

++-----+

+--+--+-

HO (400M)

lowastlowastlowast

$$$$$$$$ $$

(e)

Figure 8 Analysis of HO-1 GCLC and Nrf2 protein levels on H9c2 cells (a)Western blotting analysis HO-1 GCLC Nrf2 protein expressionwith or without ZnPP BSO and brusatol (HO-1group only added ZnPP GCLC group only added BSO Nrf2 group only added brusatol)(b) Western blot analysis HO-1 protein expression with or without ZnPP (c) Western blot analysis GCLC protein expression with or withoutBSO (d) Western blot analysis Nrf2 protein expression with or without brusatol (e) Effect of the different treatments on H9c2 cells Dataare expressed as the means plusmn SD n = 3 lowastlowastlowast119875 lt 0001 lowast119875 lt 005 compared with control group P lt 0001 P lt 001 P lt 005 comparedwith H

2O2group $$$119875 lt 0001 $$119875 lt 001 compared with H

2O2group combined with four compounds ampampampP lt 0001 ampampP lt 001 ampP lt 005

compared with H2O2group combined with inhibitors and four compounds GAPDH was used as an internal control

BioMed Research International 9

protects H9c2 cells from oxidative damage via the Nrf2-AREpathway

In conclusion the results of the present study demon-strated that four compounds are able to protect H9c2 cellsfrom H

2O2-induced damage which may be associated with

the activation of Nrf2ARE pathway in H9c2 cells Theseresults provide evidence that G acuta may be a potentialtherapeutic agent for the treatment of cardiovascular diseases

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This study was supported by the research of the effects ofMongolian Danggong-3 on the activity of myocardial cells inrats after myocardial anoxia-reoxygenation (NO 201702026)Natural Science Foundation of Tianjin (15JCQNJC13400)Investigation and Variety Systematization Study of Mon-golian Medicine ldquoDigedardquo (201702121) Chinese NationalNatural Science Foundation (81160504) and Natural ScienceFoundation of Inner Mongolia Autonomous Region (BYJJ-YF 201726)

References

[1] Z Wang Q Wu Y Yu et al ldquoDetermination and phar-macokinetic study of four xanthones inrat plasma after oraladministration of Gentianella acuta extract by UHPLC-ESI-MSMSrdquo Journal of Ethnopharmacology vol 174 pp 261ndash2692015

[2] L J Lv and M H Li ldquoTerpenoids flavonoids and xanthonesfrom Gentianella acuta (Gentianaceae)rdquo Biochemical Systemat-ics and Ecology vol 37 no 4 pp 497ndash500 2009

[3] Y-J Li T-D Bao Q Li et al ldquoProtection of CompoundGranules of Yinxingye onH9c2myocardial cells injury inducedby H2O2rdquo Chinese Traditional and Herbal Drugs vol 47 no 3pp 459ndash465 2016

[4] Y H Bai X Wang L J Zhang et al ldquoAdvances in the Chineseand western medicine treatment of coronary microvascularangina pectorisrdquo Journal of Anhui Health Vocational and Tech-nical College vol 17 no 2 pp 21ndash23 2018

[5] A B Gustafsson and R A Gottlieb ldquoMechanisms of Apoptosisin the Heartrdquo Journal of Clinical Immunology vol 23 no 6 pp447ndash459 2003

[6] Y N Liu W P Wang Z Q Jia P Chen K T Ma and CY Zhou ldquoProtection of -Lipoic Acid Against H

2O2-induced

Oxidative Stress in H9c2 Cellsrdquo Chinese Journal of Biochemistryand Molecular Biology vol 28 no 3 pp 227ndash233 2012

[7] M N Sharikabad K M Oslashstbye andO Broslashrs ldquoEffect of hydro-gen peroxide on reoxygenation-induced Ca2+ accumulation inrat cardiomyocytesrdquo Free Radical Biology amp Medicine vol 37no 4 pp 531ndash538 2004

[8] G D Wu J Zhang D Yang Z W Wang and M H LildquoDetermination of the Norswertianolin in Gentianella acutardquoChinese Journal of Experimental Traditional Medical Formulaevol 18 no 6 pp 69ndash71 2012

[9] L J Lv Y F Wang T Yan H R Han and M H Li ldquoPrepa-ration of swertianolin from Gentianella acuta and its effects onarrhythmia induced by aconitine in micerdquo Chinese Journal ofNew Drugs and Clinical Remedies vol 34 no 8 pp 630ndash6352015

[10] H-J Xu W-L Chen Y Wang M-Z Zheng Y-Y Chen andY-L Shen ldquoS262A mutation abolishes protective effects ofconnexin 43 against hypothermic preservation-induced injuryin cardiomyocytesrdquoe Journal of Heart and Lung Transplanta-tion vol 31 no 6 pp 663ndash669 2012

[11] X Jia L Zhang and X Mao ldquoS-propranolol protected H9C2cells from ischemiareperfusion-induced apoptosis via down-regultion of RACK1 Generdquo International Journal of Clinical andExperimental Pathology vol 8 no 9 pp 10335ndash10344 2015

[12] T Nguyen P Nioi and C B Pickett ldquoThe Nrf2-antioxidantresponse element signaling pathway and its activation byoxidative stressrdquo e Journal of Biological Chemistry vol 284no 20 pp 13291ndash13295 2009

[13] Y-S Keum and B Y Choi ldquoMolecular and chemical regulationof the keap1-Nrf2 signaling pathwayrdquo Molecules vol 19 no 7pp 10074ndash10089 2014

[14] H Li and L Yang ldquoMolecular regulatory mechanism of Nrf2antioxidantrdquo China Journal of Bioinformatics vol 16 no 1 pp1ndash6 2018

[15] H J Sun L Dong J Y Wang M Pan Y Wu and S LildquoProtective effects of Lycium ruthenicum Murr on hydrogenperoxide-induced cellular oxidative stress injury in heart-derived H9c2 cellsrdquo Journal of Beijing University of TraditionalChinese Medicine vol 41 no 4 pp 301ndash305 2018

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

8 BioMed Research International

HO-1

GCLC

Nrf 2

GAPDH

HO

Bellidifolin

Demethylbellidifolin

SwetrianolinNorswertianolin

ZnPP

BSOBrusatol

--------

+-------

++------

+-+-----

+--+----

+---+---

++---++-

+-+--++-

+--+-+++

+---+++-

(a)

ZnPP Negative ZnPP PositiveControl

H2O2

HO

-1G

APD

H

ampampampampamp ampamp

amplowast

1 Bellidifolin 2 Demethylbellidifolin 3 Swetrianolin 4 Norswertianolin

(b)

BSO Nagetive BSO PostiveControl

H2O2

GCL

CG

APD

H

ampamp ampampamp amp

lowast

1 Bellidifolin 2 Demethylbellidifolin 3 Swetrianolin 4 Norswertianolin

(c)

Brusatol Nagetive Brusatol PostiveContro

lH2O2

Nrf

2G

APD

H

lowast

1 Bellidifolin 2 Demethylbellidifolin 3 Swetrianolin 4 Norswertianolin

(d)

Cel

l via

bilit

y (

)

Bellidifolin (125M)Demethylbellidifolin (125M)

Swetrianolin (125M)Norswertianolin (125M)

ZnPP (15M)BSO (10M)

Brusatol (10M)

--------

-+------

--+-----

---+----

+-------

++--+---

++---+--

++----+-

+-+-+---

+-+--+--

+-+---+-

+-+----+

++-----+

+--+--+-

HO (400M)

lowastlowastlowast

$$$$$$$$ $$

(e)

Figure 8 Analysis of HO-1 GCLC and Nrf2 protein levels on H9c2 cells (a)Western blotting analysis HO-1 GCLC Nrf2 protein expressionwith or without ZnPP BSO and brusatol (HO-1group only added ZnPP GCLC group only added BSO Nrf2 group only added brusatol)(b) Western blot analysis HO-1 protein expression with or without ZnPP (c) Western blot analysis GCLC protein expression with or withoutBSO (d) Western blot analysis Nrf2 protein expression with or without brusatol (e) Effect of the different treatments on H9c2 cells Dataare expressed as the means plusmn SD n = 3 lowastlowastlowast119875 lt 0001 lowast119875 lt 005 compared with control group P lt 0001 P lt 001 P lt 005 comparedwith H

2O2group $$$119875 lt 0001 $$119875 lt 001 compared with H

2O2group combined with four compounds ampampampP lt 0001 ampampP lt 001 ampP lt 005

compared with H2O2group combined with inhibitors and four compounds GAPDH was used as an internal control

BioMed Research International 9

protects H9c2 cells from oxidative damage via the Nrf2-AREpathway

In conclusion the results of the present study demon-strated that four compounds are able to protect H9c2 cellsfrom H

2O2-induced damage which may be associated with

the activation of Nrf2ARE pathway in H9c2 cells Theseresults provide evidence that G acuta may be a potentialtherapeutic agent for the treatment of cardiovascular diseases

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This study was supported by the research of the effects ofMongolian Danggong-3 on the activity of myocardial cells inrats after myocardial anoxia-reoxygenation (NO 201702026)Natural Science Foundation of Tianjin (15JCQNJC13400)Investigation and Variety Systematization Study of Mon-golian Medicine ldquoDigedardquo (201702121) Chinese NationalNatural Science Foundation (81160504) and Natural ScienceFoundation of Inner Mongolia Autonomous Region (BYJJ-YF 201726)

References

[1] Z Wang Q Wu Y Yu et al ldquoDetermination and phar-macokinetic study of four xanthones inrat plasma after oraladministration of Gentianella acuta extract by UHPLC-ESI-MSMSrdquo Journal of Ethnopharmacology vol 174 pp 261ndash2692015

[2] L J Lv and M H Li ldquoTerpenoids flavonoids and xanthonesfrom Gentianella acuta (Gentianaceae)rdquo Biochemical Systemat-ics and Ecology vol 37 no 4 pp 497ndash500 2009

[3] Y-J Li T-D Bao Q Li et al ldquoProtection of CompoundGranules of Yinxingye onH9c2myocardial cells injury inducedby H2O2rdquo Chinese Traditional and Herbal Drugs vol 47 no 3pp 459ndash465 2016

[4] Y H Bai X Wang L J Zhang et al ldquoAdvances in the Chineseand western medicine treatment of coronary microvascularangina pectorisrdquo Journal of Anhui Health Vocational and Tech-nical College vol 17 no 2 pp 21ndash23 2018

[5] A B Gustafsson and R A Gottlieb ldquoMechanisms of Apoptosisin the Heartrdquo Journal of Clinical Immunology vol 23 no 6 pp447ndash459 2003

[6] Y N Liu W P Wang Z Q Jia P Chen K T Ma and CY Zhou ldquoProtection of -Lipoic Acid Against H

2O2-induced

Oxidative Stress in H9c2 Cellsrdquo Chinese Journal of Biochemistryand Molecular Biology vol 28 no 3 pp 227ndash233 2012

[7] M N Sharikabad K M Oslashstbye andO Broslashrs ldquoEffect of hydro-gen peroxide on reoxygenation-induced Ca2+ accumulation inrat cardiomyocytesrdquo Free Radical Biology amp Medicine vol 37no 4 pp 531ndash538 2004

[8] G D Wu J Zhang D Yang Z W Wang and M H LildquoDetermination of the Norswertianolin in Gentianella acutardquoChinese Journal of Experimental Traditional Medical Formulaevol 18 no 6 pp 69ndash71 2012

[9] L J Lv Y F Wang T Yan H R Han and M H Li ldquoPrepa-ration of swertianolin from Gentianella acuta and its effects onarrhythmia induced by aconitine in micerdquo Chinese Journal ofNew Drugs and Clinical Remedies vol 34 no 8 pp 630ndash6352015

[10] H-J Xu W-L Chen Y Wang M-Z Zheng Y-Y Chen andY-L Shen ldquoS262A mutation abolishes protective effects ofconnexin 43 against hypothermic preservation-induced injuryin cardiomyocytesrdquoe Journal of Heart and Lung Transplanta-tion vol 31 no 6 pp 663ndash669 2012

[11] X Jia L Zhang and X Mao ldquoS-propranolol protected H9C2cells from ischemiareperfusion-induced apoptosis via down-regultion of RACK1 Generdquo International Journal of Clinical andExperimental Pathology vol 8 no 9 pp 10335ndash10344 2015

[12] T Nguyen P Nioi and C B Pickett ldquoThe Nrf2-antioxidantresponse element signaling pathway and its activation byoxidative stressrdquo e Journal of Biological Chemistry vol 284no 20 pp 13291ndash13295 2009

[13] Y-S Keum and B Y Choi ldquoMolecular and chemical regulationof the keap1-Nrf2 signaling pathwayrdquo Molecules vol 19 no 7pp 10074ndash10089 2014

[14] H Li and L Yang ldquoMolecular regulatory mechanism of Nrf2antioxidantrdquo China Journal of Bioinformatics vol 16 no 1 pp1ndash6 2018

[15] H J Sun L Dong J Y Wang M Pan Y Wu and S LildquoProtective effects of Lycium ruthenicum Murr on hydrogenperoxide-induced cellular oxidative stress injury in heart-derived H9c2 cellsrdquo Journal of Beijing University of TraditionalChinese Medicine vol 41 no 4 pp 301ndash305 2018

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

BioMed Research International 9

protects H9c2 cells from oxidative damage via the Nrf2-AREpathway

In conclusion the results of the present study demon-strated that four compounds are able to protect H9c2 cellsfrom H

2O2-induced damage which may be associated with

the activation of Nrf2ARE pathway in H9c2 cells Theseresults provide evidence that G acuta may be a potentialtherapeutic agent for the treatment of cardiovascular diseases

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This study was supported by the research of the effects ofMongolian Danggong-3 on the activity of myocardial cells inrats after myocardial anoxia-reoxygenation (NO 201702026)Natural Science Foundation of Tianjin (15JCQNJC13400)Investigation and Variety Systematization Study of Mon-golian Medicine ldquoDigedardquo (201702121) Chinese NationalNatural Science Foundation (81160504) and Natural ScienceFoundation of Inner Mongolia Autonomous Region (BYJJ-YF 201726)

References

[1] Z Wang Q Wu Y Yu et al ldquoDetermination and phar-macokinetic study of four xanthones inrat plasma after oraladministration of Gentianella acuta extract by UHPLC-ESI-MSMSrdquo Journal of Ethnopharmacology vol 174 pp 261ndash2692015

[2] L J Lv and M H Li ldquoTerpenoids flavonoids and xanthonesfrom Gentianella acuta (Gentianaceae)rdquo Biochemical Systemat-ics and Ecology vol 37 no 4 pp 497ndash500 2009

[3] Y-J Li T-D Bao Q Li et al ldquoProtection of CompoundGranules of Yinxingye onH9c2myocardial cells injury inducedby H2O2rdquo Chinese Traditional and Herbal Drugs vol 47 no 3pp 459ndash465 2016

[4] Y H Bai X Wang L J Zhang et al ldquoAdvances in the Chineseand western medicine treatment of coronary microvascularangina pectorisrdquo Journal of Anhui Health Vocational and Tech-nical College vol 17 no 2 pp 21ndash23 2018

[5] A B Gustafsson and R A Gottlieb ldquoMechanisms of Apoptosisin the Heartrdquo Journal of Clinical Immunology vol 23 no 6 pp447ndash459 2003

[6] Y N Liu W P Wang Z Q Jia P Chen K T Ma and CY Zhou ldquoProtection of -Lipoic Acid Against H

2O2-induced

Oxidative Stress in H9c2 Cellsrdquo Chinese Journal of Biochemistryand Molecular Biology vol 28 no 3 pp 227ndash233 2012

[7] M N Sharikabad K M Oslashstbye andO Broslashrs ldquoEffect of hydro-gen peroxide on reoxygenation-induced Ca2+ accumulation inrat cardiomyocytesrdquo Free Radical Biology amp Medicine vol 37no 4 pp 531ndash538 2004

[8] G D Wu J Zhang D Yang Z W Wang and M H LildquoDetermination of the Norswertianolin in Gentianella acutardquoChinese Journal of Experimental Traditional Medical Formulaevol 18 no 6 pp 69ndash71 2012

[9] L J Lv Y F Wang T Yan H R Han and M H Li ldquoPrepa-ration of swertianolin from Gentianella acuta and its effects onarrhythmia induced by aconitine in micerdquo Chinese Journal ofNew Drugs and Clinical Remedies vol 34 no 8 pp 630ndash6352015

[10] H-J Xu W-L Chen Y Wang M-Z Zheng Y-Y Chen andY-L Shen ldquoS262A mutation abolishes protective effects ofconnexin 43 against hypothermic preservation-induced injuryin cardiomyocytesrdquoe Journal of Heart and Lung Transplanta-tion vol 31 no 6 pp 663ndash669 2012

[11] X Jia L Zhang and X Mao ldquoS-propranolol protected H9C2cells from ischemiareperfusion-induced apoptosis via down-regultion of RACK1 Generdquo International Journal of Clinical andExperimental Pathology vol 8 no 9 pp 10335ndash10344 2015

[12] T Nguyen P Nioi and C B Pickett ldquoThe Nrf2-antioxidantresponse element signaling pathway and its activation byoxidative stressrdquo e Journal of Biological Chemistry vol 284no 20 pp 13291ndash13295 2009

[13] Y-S Keum and B Y Choi ldquoMolecular and chemical regulationof the keap1-Nrf2 signaling pathwayrdquo Molecules vol 19 no 7pp 10074ndash10089 2014

[14] H Li and L Yang ldquoMolecular regulatory mechanism of Nrf2antioxidantrdquo China Journal of Bioinformatics vol 16 no 1 pp1ndash6 2018

[15] H J Sun L Dong J Y Wang M Pan Y Wu and S LildquoProtective effects of Lycium ruthenicum Murr on hydrogenperoxide-induced cellular oxidative stress injury in heart-derived H9c2 cellsrdquo Journal of Beijing University of TraditionalChinese Medicine vol 41 no 4 pp 301ndash305 2018

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom