Inhibition of Cardiac Hypertrophy Effects in D-Galactose ...downloads.hindawi.com/journals/ecam/2017/2624384.pdf · Inhibition of Cardiac Hypertrophy Effects in D-Galactose-Induced

Research ArticleInhibition of Cardiac Hypertrophy Effects inD-Galactose-Induced Senescent Hearts by Alpinate OxyphyllaeFructus Treatment

Yung-Ming Chang123 Hen-Hong Chang456 Hung-Jen Lin56 Chin-Chuan Tsai12

Chuan-Te Tsai3 Hsin-Nung Chang7 Shu-Luan Lin8 Vijaya PadmaViswanadha9

Ray-Jade Chen10 and Chih-Yang Huang71112

1 The School of Chinese Medicine for Post-Baccalaureate I-Shou University Kaohsiung 840 Taiwan2 Chinese Medicine Department E-Da Hospital Kaohsiung 824 Taiwan3 1PT Biotechnology Co Ltd Taichung 433 Taiwan4 Research Center for Chinese Medicine amp Acupuncture China Medical University Taichung 404 Taiwan5 Department of Chinese Medicine China Medical University Hospital Taichung 404 Taiwan6 School of Post-Baccalaureate Chinese Medicine College of Chinese Medicine China Medical University Taichung 404 Taiwan7 Graduate Institute of Basic Medical Science China Medical University Taichung 404 Taiwan8 1PT Lukang Chinese Medicine Clinics Changhua 505 Taiwan9 Department of Biotechnology Bharathiar University Coimbatore 641046 India10Department of Surgery School of Medicine College of Medicine Taipei Medical University Taipei 110 Taiwan11School of Chinese Medicine China Medical University Taichung 404 Taiwan12Department of Health and Nutrition Biotechnology Asia University Taichung 413 Taiwan

Correspondence should be addressed to Chih-Yang Huang cyhuangmailcmuedutw

Received 29 April 2016 Revised 14 September 2016 Accepted 16 March 2017 Published 5 April 2017

Academic Editor Jairo Kennup Bastos

Copyright copy 2017 Yung-Ming Chang et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Aging is a complex physiological phenomenon accelerated by ROS accumulation with multisystem decline and increasingvulnerability to degenerative diseases and death Cardiac hypertrophy is a key pathophysiological component that accompaniesthe aging process Alpinate Oxyphyllae Fructus (Alpinia oxyphylla MIQ AOF) is a traditional Chinese medicine which providescardioprotective activity against aging hypertension and cerebrovascular disorders In this study we found the protective effect ofAOF against cardiac hypertrophy in D-galactose-induced aging rat model The results showed that treating rats with D-galactoseresulted in pathological hypertrophy as evident from the morphology change increased left ventricular weightwhole heart weightand expression of hypertrophy-related markers (MYH7 and BNP) Both concentric and eccentric cardiac hypertrophy signalingproteins were upregulated in aging rat model However these pathological changes were significantly improved in AOF treatedgroup (AM and AH) in a dose-dependent manner AOF negatively modulated D-galactose-induced cardiac hypertrophy signalingmechanism to attenuate ventricular hypertrophy These enhanced cardioprotective activities following oral administration of AOFreflect the potential use of AOF for antiaging treatments

1 Introduction

Cardiac hypertrophy is an adaptive cellular response associ-ated with increased biomechanical stress such as hyperten-sion hypertrophic cardiomyopathy myocardial infarction

and vascular heart disease [1] It is well known that cardiachypertrophy is an important compensation mechanism inresponse to pathological stress the heart adapts to pressureand volume overload by undergoing hypertrophic enlarge-ment without cell proliferation Even though the initial

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2017 Article ID 2624384 12 pageshttpsdoiorg10115520172624384

2 Evidence-Based Complementary and Alternative Medicine

compensation response may be beneficial continued hyper-trophy eventually leads to heart failure and ultimately death[2ndash4] Pressure and volume overload usually elicits concen-tric hypertrophy and eccentric hypertrophy respectively [56] Cardiac hypertrophies usually accompany the increscentof fetal genes expression including atrial natriuretic peptide(ANP) and brain natriuretic peptide (BNP) both are elevatedin cardiac hypertrophy and have been characterized as a fea-ture of hypertrophy in allmammalian species [7ndash9] CytokineInterleukin-6 (IL6) a potent hypertrophic effecter triggersglycoprotein 130 (gp130) dimerizing and activating down-stream hypertrophic signaling [10 11] Previous study indi-cated that IL6-related pathways play a crucial role in eccen-tric cardiac hypertrophy including IL6-related MEK5-ERK5signaling and JAK2-STAT3 signaling These hypertrophy-related molecular signals were activated simultaneously withIL6 expression and lead to morphological changes [12 13]Recently reports showed that the hypoxia marker BNIP3(Bcl-2adenovirus E1B nineteen-kDa interacting protein 3)induced concentric cardiac hypertrophy by targeting themitochondria and endoplasmic reticulum (ER) activating thecalcium-dependent signaling calcineurinNFAT3 [13 14]Another hypertrophic mediator p38 MAPK was found tomediate concentric cardiac hypertrophy by phosphorylatedGATA4 to further expressed hypertrophic response genessuch as ANP and BNP [13 15ndash17]

Mitochondria are generally considered the major sourceof reactive oxygen species (ROS) production [18 19] otherpotential sources of ROS include NADPH oxidase xanthineoxidase and uncoupled nitric oxide synthase (NOS) [20ndash22]Accumulating evidence demonstrates that oxidative stressimpaired antioxidant defense due toROSoverproduction andhave been implicated in the development and subsequentprogression of related heart diseases especially cardiac hyper-trophy [23ndash26] ROS production increase has been shown tobe involved in the hypertrophy of isolated cardiomyocytesinduced by angiotensin II (AngII) tumor necrosis factor-a (TNFa) cyclic stretch or a-adrenergic agonists [27ndash29]Additionally NADPH oxidase 4 (Nox4) a major source ofoxidative stress in the failing heart which has been identifiedinduces cardiac hypertrophy through activating AktmTORand NF120581B signaling [30 31] It has also been reported thatNox4 directly mediates mitochondrial dysfunction oxidativestress and myocardial cell death during pressure overload-induced cardiac hypertrophy [31 32]

D-Galactose (D-gal) is a reducing sugar that has beenwidely used in age-related oxidative damage and aging phar-macology research [33ndash35] D-Gal generates ROS during itsmetabolism in vivo by reacting readily with the free amines inproteins lipids and nucleic acids to form advanced glycationend products (AGEs) [36 37] Growing evidence suggeststhat AGEs are interacting receptors for AGE (RAGE) inmanycell types and induce downstreamNF-120581B and other signalingpathways eventually lead to ROS generation and acceleratethe aging process [38ndash41]

Alpinate Oxyphyllae Fructus (Alpinia oxyphylla MIQAOF) is one of the important traditional Chinese medicineswhich has antiaging and sexual-reinforcing activity [42]

According to the Chinese pharmacopoeia AOF has beenwidely used for treating gastralgia diarrhea ulcerationantitumor hypertension accompanying symptoms and cere-brovascular disorders [42ndash46] An increasing number ofevidences indicate that AOF extracts exhibit cardioprotectiveand neuroprotective activity against oxidative stress-inducedapoptosis [44 47] Subsequent studies demonstrated thatAOF extracts protect againstAng II induced cardiac apoptosisin H9c2 cardiomyoblast cell [48] We here further investigatewhether AOF ameliorate the ROS-induced aging heart prob-lem and related signaling paths and mechanisms

2 Materials and Methods

21 AOF Extraction Fragmented Alpinate Oxyphyllae Fruc-tus (Alpinia oxyphylla MIQ AOF) was obtained from Shin-Long Pharmaceutical Company (Taichung Taiwan)

The AOF fragment (150 g) was extracted by boiling in06 L of boiling water for 2 hThe water extract was filtered bygravity-flow procedure at reduced pressure for convenienceand then stored at 4∘C The water extract was spray dried toproduce a powdered extract The product yield percentagewas 74

22 Animals and Experimental Design Thirty-four male 8-week-old Sprague-Dawley rats weighing 220 plusmn 20 g were pro-vided by the National Institutes of Health (NIH) colony andcared for at the Comparative Biology facility at Texas AampMUniversity in accordance with NIH and ULACC (UniversityLaboratory Animal Care Committee) standards Rats weremaintained on standard laboratory conditions of temperature(23 plusmn 2∘C) and a 12 12 h light-dark cycle with water and ratchow available ad libitum for the duration of the study After 2weeks of acclimatization the rats were randomly divided into5 groups Onewas normal control group (NC)The other fourwere induced aging groups injected with D-galactose (150mgkgday for 8 weeks) in induced aging groups the three AOFgroups were orally administered with AOF of 50 (AOF lowAL) 100 (AOF medium AM) and 150 (AOF high AH)mgkgday respectively the normal control groupwere giventhe same volume of control solution After the animals weresacrificed the heart tissue was immediately collected andstored at minus80∘C until further use

23 Cardiac Characteristics The whole hearts of animalswere weighed after being excised and cleaned with PBS Theleft ventricle tissues were isolated and weighed The tibialength was measured by the electronic digital Vernier caliperto adjust the whole heart weightThe ratios of the whole heartweight to the tibia length and the left ventricle weight to tibialength were calculated

24 Echocardiography Transthoracic echocardiographic ima-ges of ratswere performedusingHewlett-Packard Sonos 5500ultrasound machine with a 15MHz linear-array transducerLeft ventricular M-mode measurements at the level of thepapillary muscles include interventricular septal thickness atend-diastole (IVSD) left ventricular internal dimension at

Evidence-Based Complementary and Alternative Medicine 3

end-diastole (LVIDd) left ventricular posterior wall thick-ness at end-diastole (LVPWd) and left ventricular internaldimension at end-systole (LVIDs)

25 Tissue Extraction The left ventricle tissues were isolatedand washed 3 times in PBS buffer and then weighed Approx-imately 01 g tissue was added by 1mL lysis buffer (01SDS 05 Na-deoxycholate 1 NP-40 2mM EDTA 50mMTrisHCL 50mM NaF and 150mM NaCl) into the mixtureThe tissue was homogenized for 20min and centrifugedat 1200 rpm at 4∘C After stirring and centrifugation at12500 rpm a clean upper layer suspensionwas extractedThehomogenization was repeated and a clean upper layer sus-pension was extracted

3 Senescence-Associated120573-Galactosidase Staining

The tissue sections were fixed in 02 glutaraldehyde and2 formaldehyde at room temperature for 15min Sectionswere washed three times in PBS and incubated in freshlysenescence-associated 120573-galactosidase (SA-120573-gal) stainingsolution (1mgmL X-gal 40mM citric acidsodium phos-phate (pH 60) 5mM potassium ferricyanide 5mM potas-sium ferrocyanide 150mM NaCl and 2mMMgCl2) for 14 hat 37∘C without CO2 SA-120573-gal staining was visualized underan Olympus (Tokyo Japan) microscope

31 Hematoxylin-Eosin (HampE) Staining The tissue sectionswere dyed using hematoxylin and eosin (HampE) After thehearts were excised tissue sections were stained using hema-toxylin and eosin (HampE) Sections were dewaxed by immer-sion in xylene and dyed with hematoxylin for 5min thesections were washed three times in double-distilled water(DDW) and soaked in 85 alcohol for 2min The sectionswere dyed with eosin for 5min and dehydrated throughgraded alcohols (100 95 and 75) Finally the sectionswere soaked in 100alcohol for 5min and two times in xylenefor 1min The sections were then sorted and photograph-ically analyzed using a microscope (Olympus Microscope)

32 Electrophoresis and Western Blot Cardiac tissues extractprotein concentration was determined by the Lowry proteinassay Tissue protein samples (40 120583glane) and 5x loading dyewere mixed and placed on 95∘C for 5min and then separatedon sodiumdodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) with a constant voltage of 70VThe upper SDS-PAGE layer was a 5 stacking gel and the bottom layer was8 or 10 or 12 separating gel

Electrophoresed proteins were transferred to a polyvinyl-idene difluoride (PVDF) membrane (045120583m pore sizeMillipore Bedford MA USA) with vertical electrophoresissystem (Bio-Rad Laboratories Inc Berkeley CA USA) ThePVDFmembranes were incubated in 5 fat-freemilk in Tris-buffered saline (TBS) and shaken for 1 h at RT After shakingthe PVDF membranes were incubated with the primaryantibody overnight at 4∘CThe primary antibody was dilutedto 1 1000 in antibody binding buffer The immunoblots werewashed with TBS buffer 3 times for 10min each and then

incubated with the secondary antibody solution containinggoat anti-mouse IgG-HRP goat anti-rabbit IgG-HRP ordonkey anti-goat IgG-HRP (Santa Cruz Biotechnology) for1 hr at room temperatureThe secondary antibodywas dilutedto 1 3000 in TBS buffer The membrane was washed againwith washing buffer Finally the membrane was coloredwith an enhanced chemiluminescence ECLWestern blottingluminal reagent (Santa Cruz Biotechnology) and membranedata was collected using an LAS-4000 mini (GE HealthcareLife Sciences) The data were quantified using Image J

33 Statistical Analysis All experiments were repeated atleast three times One-way ANOVA was used for compar-isons between multiple groups Studentrsquos 119905-test was used tocompare two differences groups Con and IA (induced aging)served as negative control (normal rats) and positive control(aging rats) groups respectively 119901 lt 005 was consideredsignificant

4 Results

41 Body Weight and Cardiac Characteristics Rat heartweight and cardiac functional parameters were analyzedto compare differences between groups The whole heartweight (WHW) left ventricular weight (LVW) WHWtibiaLVWtibia and the echocardiographic parameter LVPWdincreased in the IA group when compared to the controlgroup (119901 lt 005) AOF treatment for 10 weeks significantlydecreased the aging effects of the treated IA rats as comparedwith the IA group (119901 lt 005) as shown in Table 1 Theseresults indicated that the AOF treatment was effective in D-galactose induced aging rat model

42 AOF Treatment Reduced Senescent Changes in InducedAging Rat Hearts We examined the expression of SA-120573-gal awell-defined in vivo senescencemarker [49ndash51]The intensityof SA-120573-gal staining in induced aging (IA) rat hearts was sig-nificant increase in the percentage of SA-120573-gal accumulationcompared to the control group (119901 lt 005 Figure 1(a)) How-ever in the IA + AOF treatment group (AL AM and AH)the percentage of SA-120573-gal accumulation was significantlyreduced (119901 lt 005 Figure 1) Furthermore the senescencebiomarkers SA-120573-gal and p21 were significantly increased inthe IA group compared with those in control group but wereseen to be regulated in the groups treated with AOF (Fig-ure 1(b))

D-Galactose treatment remarkably decreased the proteinlevels of HO1 and SOD1 but AOF treatment amelioratedthe effect in a dose-dependent manner (Figure 1(c)) Thesefindings suggest that AOF treatment can protect cardiac cellagainst senescence

43 Cardiac Histopathological Changes Analyses of CardiacTissue In order to investigate whether AOF treatmentimproves cardiac architecture we performed histopatho-logical tomography analyses of whole heart tissues withhematoxylin-eosin staining (Figure 2) HampE stained sectionsof the IA group showed increased LV wall thickening com-pared to that of the control group but this abnormality


Control

IA + AL IA + AM IA + AH

Induced aging (IA)

(a)

훽-gal

p21

훽-Actin

Control kDaIA + AL IA + AM IA + AH

IA IA + AL IA + AM IA + AHConIA IA + AL IA + AM IA + AHCon

lowastlowastlowastlowastlowast

Induced aging (IA)

0

05

1

15

2

25

훽-G

al훽

-act

in

0

1

2

3

4

5

6

p21훽

-act

in

54

21

43

(b)

HO1 32

SOD1 23

37GADPH

002040608

11214

HO

1G

AD

PH

IA IA + AL IA + AM IA + AHCon IA IA + AL IA + AM IA + AHCon0

02040608

11214

SOD

1G

AD

PH lowastlowast

lowastlowastlowast

Control kDaIA + AL IA + AM IA + AHInduced aging (IA)

(c)

Figure 1 AOF treatment reduced senescent changes in aged rat hearts (a) SA-b-gal staining results for cardiac tissues from study groupsof rats Blue precipitation in the cytoplasm was observed in the senescent cells times200 magnification (b) Representative aging-related proteinproducts extracted from the left ventricles of study rats in each group (c) Representative antioxidants protein products extracted from the leftventricles of study rats in each group control rats IA induced aging rats AL AOF low AM AOF medium AH AOF high were measuredusing Western blotting 120573-Actin and GADPH were used as an internal control lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 compared with the controlgroup 119901 lt 005 119901 lt 001 and 119901 lt 0001 compared with the induced aging 119899 = three independent experiments for each data point


Table 1 Heart weight and echocardiographic indices

Control Induced aging (IA) IA + AL IA + AM IA + AH119899 = 4 119899 = 4 119899 = 4 119899 = 4 119899 = 4

Body weight (g) 456 plusmn 3105 462 plusmn 3232 437 plusmn 6102 444 plusmn 314 464 plusmn 156WHW (g) 139 plusmn 006 155 plusmn 007lowast 139 plusmn 010 137 plusmn 003 140 plusmn 003

LVW (g) 099 plusmn 006 111 plusmn 006lowast 095 plusmn 011 096 plusmn 002 097 plusmn 002

WHWtibia (102) 310 plusmn 009 352 plusmn 02lowastlowast 335 plusmn 023 325 plusmn 008 337 plusmn 010lowast

LVWtibia (102) 220 plusmn 009 254 plusmn 013lowastlowast 228 plusmn 024 230 plusmn 005 233 plusmn 008

IVSD (mm) 137 plusmn 008 131 plusmn 012 143 plusmn 016 131 plusmn 012 138 plusmn 010LVIDd (mm) 915 plusmn 062 967 plusmn 073 889 plusmn 034 920 plusmn 012 902 plusmn 050LVPWd (mm) 132 plusmn 012 150 plusmn 014lowast 135 plusmn 008 110 plusmn 009 123 plusmn 007

LVIDs (mm) 609 plusmn 075 617 plusmn 083 512 plusmn 027 599 plusmn 031 505 plusmn 025lowast

Values are means plusmn SD 119899 = 4 at least in each group AL AOF low AM AOF medium AH AOF high WHW whole heart weight LVW left ventricularweightWHWtibia whole heart weight normalized by tibia length LVWtibia left ventricular weight normalized by tibia length IVSD interventricular septalthickness at end-diastole LVIDd left ventricular internal dimension at end-diastole LVPWd left ventricular posterior wall thickness at end-diastole LVIDsleft ventricular internal dimension at end-systole lowast119901 lt 005 and lowastlowast119901 lt 001 compared to the control group 119901 lt 005 119901 lt 001 and 119901 lt 0001 comparedto the induced aging group respectively

Control

0

RVRV

LV LV

RV RV RV

LV LV LV


Induced aging (IA)

1 cm0 1 cm

0 1 cm 0 1 cm 0 1 cm(a)

IA IA + AL IA + AM IA + AHCon0

05

1

15

2

Fold

chan

ge

lowastlowast

(b)

Figure 2 Hematoxylin and eosin staining (HampE stain) Cardiac tissue sections stained with hematoxylin and eosin (a) The myocardialarchitecture images were magnified times10The scale bar is 1 cm RV right ventricle and LV left ventricle (b) Quantization of cardiac ventriculararea (lowastlowast119901 lt 001 compared with the control group 119901 lt 001 compared with the IA group) 119899 = three independent experiments for eachdata point


was reduced in AOF treatment groups (AL AM and AH)(Figure 2)

44 AOF Treatment Suppressed Cardiac Hypertrophy in D-Galactose-Induced Senescence Effects in SD Rat Hearts Tofurther identify whether pathological hypertrophy signalingpathways were altered in AOF treated aging rats the genesinvolved in pathological hypertrophy were examined byWestern blotting The data indicated that the concentrichypertrophy-related MAPKs such as p-ERK12 p-c-JUN p-JNK and p-p38 were significantly increased in the IA groupcompared with those in control group Moreover the patho-logical hypertrophy associated transcription factors such asNFATc3 and p-GATA4 were increased in the aging ratshearts (Figure 3) However the p-ERK12 p-c-JUN p-JNKp-p38 NFATc3 and p-GATA4 in the AOF treatment groups(AM and AH) were significantly lower than those in the IAgroup (Figure 3) The eccentric pathological related proteinp-MEK5 p-ERK5 and transcription factors STAT3 were alsosignificantly increased in aging rats models However allthese pathological changes were reversed by AOF in a dosedependent manner (Figure 4)

45 Dose-Dependent Effects of AOF on BNP MYH6 andMYH7 Protein Levels After confirming the effects of AOFtreatment on pathological hypertrophy associated signalingpathways we investigated the physiological hypertrophy-related markers BNP MYH6 and MYH7 [52ndash54] proteinlevels by Western blot analysis Our results showed that D-galactose-induced aging effects caused a significant increasein the protein expression levels of both BNP and MYH7by approximately twofold and 40 respectively HoweverMYH6 protein level was upregulated by AOF treatment ina dose-dependent manner to reach approximately 30 of itscontrol value (Figure 5)

5 Discussion

D-Galactose induced oxidative stress in animal models tomimic natural aging has been wildly used in antiaging phar-macological studies [55] Oxidative stress is increased due toabnormal metabolism such as glucose autoxidation and gen-eration of AGE that results in the increase of reactive oxygenspecies (ROS) and eventually contributes to cardiac hypertro-phy [56 57] It is well known that oxidative stress is generatedvia ROS and has been implicated in the development ofcardiac hypertrophy [57 58]The aimof the present studywasto investigate the cardiac protective role and the mechanismof AOF against pathological hypertrophy in D-galactoseinduced aging rats Senescence staining and immunoblotshow that D-galactose induces oxidative stress resulting incardiac aging Additionally D-galactose treatment remark-ably decreased the protein level of SOD1 and HO1 and AOFsupplementation showed significant protection effect by ele-vating these antioxidants Furthermore whole heart weightleft ventricular weight and left ventricular posterior wallthickness at end-diastole significantly increased in the aginggroups However AOF treatments ameliorate age-relatedfunctional deterioration and promoted the hearts of IA group

rats tomaintain a relatively healthy physiological stateThere-fore AOF treatment potentially provides positive effects oncardiac cell morphology and cardiac contraction AOF is atraditional Chinese medicine that exhibits cardioprotectiveactivity against oxidative stress-induced apoptosis [44 47]Accumulating evidence demonstrates that AOF possessesdiverse pharmacologic effects including antioxidant anti-inflammatory antitumor and antiapoptosis [59ndash62] Thuswe expected AOF may provide a recovery effect againstcardiac hypertrophy inD-galactose-induced aged ratWe fur-ther examined the mechanism of aging-induced cardiomyo-cyte hypertrophy which occurred through both concentricand eccentric hypertrophy pathways

Previous studies indicate that ERK12 and JNK12 phos-phorylate various substrates such as transcription factorsGATA4 andNFATs subsequently triggering the transcriptionof hypertrophy-related genes and eventually result in cardiachypertrophy [63 64] Our current results showed that theexpression of cardiac pathological and concentric hypertro-phy proteins ERK12JNK p-P38 and NFATc3 was markedlyincreased in rat hearts with D-galactose treatment Wefurther observed that the eccentric-related MEK5-ERK5 sig-naling and the transcription factor STAT3 protein were alsoupregulated in aging rats heartsNotably AOF treatment con-ferred protection against cardiac hypertrophy via downregu-lation of both concentric- and eccentric-related hypertrophysignaling pathway

In mammals the myosin heavy-chain isoforms MYH6and MYH7 have been identified as cardiac motor proteinsthat are crucial determinants of contractile performance incardiac muscle tissue [65 66] Emerging evidence demon-strated that MYH7 protein expression was significantlyincreased in cardiac hypertrophy and has been identified ashypertrophy-related marker [67] Subsequent studies indi-cate that cardiac hypertrophy usually accompanies upregu-lation of BNP and MYH7 andor downregulation of MYH6expression levels [68] In the present study high BNP andMYH7 expressions were observed following D-galactosetreatment in the IA groups Treatment with AOF in IA ratssignificantly decreased the hypertrophy-relatedmarkers BNPand MYH7 in a dose-dependent manner in cardiomyocytesSystematic survey on literatures shows that over the pastdecade at least 80 chemical constituents were reported tobe identified from A oxyphylla that include sesquiterpenesflavonoids diarylheptanoids steroids volatile oil and theirglycosides [62 69ndash78] Recently Chen et al [79] evaluated theabundance of nine secondarymetabolites inA oxyphylla cap-sular fruits including flavonoids (eg tectochrysin izalpininchrysin apigenin-410158407-dimethyl ether and kaempferide)diarylheptanoids (eg yakuchinone A yakuchinone B andoxyphyllacinol) and sesquiterpenes (eg nootkatone) Thenine secondary metabolites were differentially concentratedin seeds and fruit capsules Furthermore the optimized con-dition for the extraction and the content levels of nine com-pounds of different harvest time has been previously evalu-ated by Li et al [80] and the remarkable differences in chem-ical markers at different harvest time by Li et al [80] Addi-tionally protocatechuic acid (PCA) one of the major activeingredients in the kernels of AOF has been reported as


54

kDa

NFATc3 130

p-ERK12 4244

p-c-JUN 39

38

p-JNK

p-P38

4654

훼-Tub

50p-GATA4

ERK12 4244

JNK 4654

c-JUN 39

Control IA + AL IA + AM IA + AHInduced aging (IA)

Con IA AL AM AHCon IA AL AM AH



002040608

1121416

p-ER

K12

훼-tu

bulin

lowastlowastlowast lowastlowastlowast

0

05

1

15

2

25

3N

FATc

3훼

-tubu

lin

0

05

1

15

2

25

3

p-JN

K훼

-tubu

lin

005

115

225

335

p-c-

JUN

훼-tu

bulin

lowastlowastlowastlowastlowastlowast


0

05

1

15

2

25

p-G

ATA

4훼

-tubu

lin

0

05

1

15

2

25

p-P3

8훼

-tubu

lin

Figure 3 Representative concentric hypertrophy protein products extracted from the left ventricles of study rats in each group control ratsIA induced aging rats AL AOF low AM AOF medium AH AOF high were measured using Western blotting 120572-Tubulin was used as aninternal control lowastlowastlowast119901 lt 0001 compared with the control group 119901 lt 005 119901 lt 001 and 119901 lt 0001 compared with the induced aging119899 = three independent experiments for each data point


8691STAT3

p-MEK5

p-ERK5

훽-Actin 43

54

115

ERK5 115

MEK5 54

kDaControl IA + AL IA + AM IA + AHInduced aging (IA)

0

05

1

15

2

25

3

35

p-M

EK5훽

-act

in

0

05

1

15

2

25

p-ER

K5훽

-act

in

005

115

225

335

4

STAT

3훽

-act

in

IA IA + AL IA + AM IA + AHCon




lowastlowastlowast

Figure 4 Representative eccentric hypertrophy protein products extracted from the left ventricles of study rats in each group control ratsIA induced aging rats AL AOF low AM AOF medium AH AOF high were measured using Western blotting 120573-Actin was used as aninternal control lowastlowastlowast119901 lt 0001 compared with the control group 119901 lt 001 and 119901 lt 0001 compared with the induced aging 119899 = threeindependent experiments for each data point

an efficient cytoprotective agent against oxidative stress neu-rotoxicity and apoptosis [42ndash44 46] Recently we reportedthat PCA effectively promoted RSC96 cell survival and prolif-eration by activating IGF-IR-PI3K-Akt signaling [81] More-over PCA induced the migration and regeneration of RSC96Schwann cells via ERK12 JNK12 and p38 MAPK pathwaysactivation [82] The protective effects associated with thechemical constituents of PCA correlate with the pharmaco-logical activities of AOF as defined in the present study

In conclusion our findings suggest the beneficial effectsof AOF on cardiac hypertrophy The mechanism by whichAOF protects against cardiac hypertrophy may be partly viablockade of both concentric and eccentric signaling path-ways D-Galactose-induced pathological hypertrophy associ-ated proteins as well as expression levels of BNP and MYH7could attenuate by AOF treatment Therefore AOF might be

a potential candidate for the treatment of aging-inducedcardiovascular hypertrophy and heart failure

Abbreviations

BW Body weightWHW Whole heart weightLVW Left ventricle weightIVSD Interventricular septal thickness at

end-diastoleLVIDd Left ventricular internal dimension at

end-diastoleLVPWd Left ventricular posterior wall thickness at

end-diastoleLVIDs Left ventricular internal dimension at

end-systole


224

BNP 36

kDa

223

MYH7

MYH6

훼-Tub 54

BNPMYH7MYH6


05

1

15

2

25Pr

otei

n le

vel (

fold

of c

hang

e)

lowastlowast

lowastlowast

lowastlowastlowast


Figure 5 Dose-dependent effect of AOF on the hypertrophic protein expression levels from the left ventricles of study rats in each groupBNP MYH6 and MYH7 protein levels were determined by Western blot analysis 120572-Tubulin was used as an internal control lowastlowast119901 lt 001and lowastlowastlowast119901 lt 0001 compared with the control group 119901 lt 005 119901 lt 001 and 119901 lt 0001 compared with the induced aging 119899 = threeindependent experiments for each data point

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This study is supported in part by Taiwan Ministry ofHealth and Welfare Clinical Trial and Research Center ofExcellence (MOHW105-TDU-B-212-133019) and also sup-ported by China Medical University under the Aim for TopUniversity Plan of the Ministry of Education Taiwan

References

[1] J A Hill and E N Olson ldquoCardiac plasticityrdquoTheNew EnglandJournal of Medicine vol 358 no 13 pp 1370ndash1380 2008

[2] A M Feldman E O Weinberg P E Ray and B H LorellldquoSelective changes in cardiac gene expression during compen-sated hypertrophy and the transition to cardiac decompensa-tion in rats with chronic aortic bandingrdquo Circulation Researchvol 73 no 1 pp 184ndash192 1993

[3] P W de Leeuw and A A Kroon ldquoHypertension and thedevelopment of heart failurerdquo Journal of Cardiovascular Phar-macology vol 32 supplement 1 pp S9ndashS15 1998

[4] I Kehat and J D Molkentin ldquoMolecular pathways underlyingcardiac remodeling during pathophysiological stimulationrdquoCirculation vol 122 no 25 pp 2727ndash2735 2010

[5] W Grossman and W J Paulus ldquoMyocardial stress and hyper-trophy a complex interface between biophysics and cardiacremodelingrdquo The Journal of Clinical Investigation vol 123 no9 pp 3701ndash3703 2013

[6] J J Hunter and K R Chien ldquoSignaling pathways for cardiachypertrophy and failurerdquoTheNew England Journal of Medicinevol 341 no 17 pp 1276ndash1283 1999

[7] K R Chien H Zhu K U Knowlton et al ldquoTranscriptionalregulation during cardiac growth and developmentrdquo AnnualReview of Physiology vol 55 pp 77ndash95 1993

[8] N Hama H Itoh G Shirakami et al ldquoRapid ventricularinduction of brain natriuretic peptide gene expression inexperimental acute myocardial infarctionrdquo Circulation vol 92no 6 pp 1558ndash1564 1995

[9] J R Dietz ldquoMechanisms of atrial natriuretic peptide secretionfrom the atriumrdquo Cardiovascular Research vol 68 no 1 pp 8ndash17 2005

[10] T Kanda and T Takahashi ldquoInterleukin-6 and cardiovasculardiseasesrdquo Japanese Heart Journal vol 45 no 2 pp 183ndash1932004

[11] J Pan K Fukuda H Kodama et al ldquoInvolvement of gp130-mediated signaling in pressure overload-induced activation of


the JAKSTAT pathway in rodent heartrdquo Heart and Vessels vol13 no 4 pp 199ndash208 1998

[12] L-M Chen W-W Kuo J-J Yang et al ldquoEccentric cardiachypertrophy was induced by long-term intermittent hypoxia inratsrdquo Experimental Physiology vol 92 no 2 pp 409ndash416 2007

[13] Y-J Weng W-W Kuo C-H Kuo et al ldquoBNIP3 induces IL6and calcineurinNFAT3 hypertrophic-related pathways inH9c2cardiomyoblast cellsrdquoMolecular and Cellular Biochemistry vol345 no 1-2 pp 241ndash247 2010

[14] L Zhang L Li H Liu J L Borowitz and G E Isom ldquoBNIP3mediates cell death by different pathways following localizationto endoplasmic reticulum and mitochondrionrdquo The FASEBJournal vol 23 no 10 pp 3405ndash3414 2009

[15] H-C Wu Y-L Yen W-W Kuo et al ldquoP38 mitogen-activatedprotein kinase pathways are involved in the hypertrophy andapoptosis of cardiomyocytes induced by Porphyromonas gingi-valis conditionedmediumrdquoCell Biochemistry and Function vol26 no 2 pp 246ndash255 2008

[16] Q Liang and J D Molkentin ldquoDivergent signaling pathwaysconverge on GATA4 to regulate cardiac hypertrophic geneexpressionrdquo Journal of Molecular and Cellular Cardiology vol34 no 6 pp 611ndash616 2002

[17] J D Molkentin and E N Olson ldquoGATA4 a novel transcrip-tional regulator of cardiac hypertrophyrdquo Circulation vol 96no 11 pp 3833ndash3835 1997

[18] F Di Lisa N Kaludercic A Carpi R Menabo and M Gior-gio ldquoMitochondria and vascular pathologyrdquo PharmacologicalReports vol 61 no 1 pp 123ndash130 2009

[19] M Rigoulet E D Yoboue and A Devin ldquoMitochondrial ROSgeneration and its regulation mechanisms involved in H2O2signalingrdquo Antioxidants and Redox Signaling vol 14 no 3 pp459ndash468 2011

[20] F J Giordano ldquoOxygen oxidative stress hypoxia and heartfailurerdquo Journal of Clinical Investigation vol 115 no 3 pp 500ndash508 2005

[21] J-M Li N P Gall D J Grieve M Chen and A M ShahldquoActivation of NADPH oxidase during progression of cardiachypertrophy to failurerdquo Hypertension vol 40 no 4 pp 477ndash484 2002

[22] C E Murdoch M Zhang A C Cave and A M ShahldquoNADPH oxidase-dependent redox signalling in cardiac hyper-trophy remodelling and failurerdquo Cardiovascular Research vol71 no 2 pp 208ndash215 2006

[23] N S Dhalla R M Temsah and T Netticadan ldquoRole of oxida-tive stress in cardiovascular diseasesrdquo Journal of Hypertensionvol 18 no 6 pp 655ndash673 2000

[24] P H Sugden and A Clerk ldquoCellular mechanisms of cardiachypertrophyrdquo Journal of Molecular Medicine vol 76 no 11 pp725ndash746 1998

[25] C X C Santos N Anilkumar M Zhang A C Brewer and AM Shah ldquoRedox signaling in cardiac myocytesrdquo Free RadicalBiology and Medicine vol 50 no 7 pp 777ndash793 2011

[26] H Tsutsui S Kinugawa and S Matsushima ldquoOxidative stressand heart failurerdquo American Journal of PhysiologymdashHeart andCirculatory Physiology vol 301 no 6 pp H2181ndashH2190 2011

[27] K Nakamura K Fushimi H Kouchi et al ldquoInhibitory effectsof antioxidants on neonatal rat cardiac myocyte hypertrophyinduced by tumor necrosis factor-120572 and angiotensin IIrdquo Circu-lation vol 98 no 8 pp 794ndash799 1998

[28] S Hirotani K Otsu K Nishida et al ldquoInvolvement of nuclearfactor-120581B and apoptosis signal-regulating kinase 1 inG-protein-coupled receptor agonist-induced cardiomyocyte hypertrophyrdquoCirculation vol 105 no 4 pp 509ndash515 2002

[29] D R Pimentel J K Amin L Xiao et al ldquoReactive oxygenspecies mediate amplitude-dependent hypertrophic and apop-totic responses to mechanical stretch in cardiac myocytesrdquoCirculation Research vol 89 no 5 pp 453ndash460 2001

[30] Q D Zhao S Viswanadhapalli P Williams et al ldquoNADPHoxidase 4 induces cardiac fibrosis and hypertrophy throughactivating AktmTOR and NF120581B signaling pathwaysrdquo Circula-tion vol 131 no 7 pp 643ndash655 2015

[31] J Kuroda T Ago S Matsushima P Zhai M D Schneider andJ Sadoshima ldquoNADPH oxidase 4 (Nox4) is a major source ofoxidative stress in the failing heartrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 107 no35 pp 15565ndash15570 2010

[32] EDeMarchi F Baldassari A BononiMRWieckowski andPPinton ldquoOxidative stress in cardiovascular diseases and obesityrole of p66Shc and protein kinase Crdquo Oxidative Medicine andCellular Longevity vol 2013 Article ID 564961 11 pages 2013

[33] Z-F Zhang S-H Fan Y-L Zheng et al ldquoPurple sweet potatocolor attenuates oxidative stress and inflammatory responseinduced by d-galactose in mouse liverrdquo Food and ChemicalToxicology vol 47 no 2 pp 496ndash501 2009

[34] Y-Y Zhou X-F Ji J-P Fu et al ldquoGene transcriptional andmetabolic profile changes in mimetic aging mice induced by D-galactoserdquo PLoS ONE vol 10 no 7 Article ID e0132088 2015

[35] X L Zhang B Jiang Z B Li S Hao and L J An ldquoCatalpolameliorates cognition deficits and attenuates oxidative damagein the brain of senescent mice induced by D-galactoserdquo Phar-macology Biochemistry and Behavior vol 88 no 1 pp 64ndash722007

[36] R Bucala and A Cerami ldquoAdvanced glycosylation chemistrybiology and implications for diabetes and agingrdquo Advances inPharmacology vol 23 pp 1ndash34 1992

[37] H Vlassara R Bucala and L Striker ldquoPathogenic effectsof advanced glycosylation biochemical biologic and clinicalimplications for diabetes and agingrdquo Laboratory Investigationvol 70 no 2 pp 138ndash151 1994

[38] G Munch BWestcott T Menini and A Gugliucci ldquoAdvancedglycation endproducts and their pathogenic roles in neurologi-cal disordersrdquo Amino Acids vol 42 no 4 pp 1221ndash1236 2012

[39] N A Calcutt M E Cooper T S Kern and A M SchmidtldquoTherapies for hyperglycaemia-induced diabetic complicationsfrom animal models to clinical trialsrdquo Nature Reviews DrugDiscovery vol 8 no 5 pp 417ndash429 2009

[40] X Song M Bao D Li and Y M Li ldquoAdvanced glycation in D-galactose induced mouse aging modelrdquo Mechanisms of Ageingand Development vol 108 no 3 pp 239ndash251 1999

[41] X Zhang C Jin Y Li S Guan F Han and S ZhangldquoCatalpol improves cholinergic function and reduces inflamma-tory cytokines in the senescent mice induced by D-galactoserdquoFood and Chemical Toxicology vol 58 pp 50ndash55 2013

[42] B-S Koo W-C Lee Y-C Chang and C-H Kim ldquoProtectiveeffects of alpinae oxyphyllae fructus (Alpinia oxyphylla MIQ)water-extracts on neurons from ischemic damage and neuronalcell toxicityrdquo Phytotherapy Research vol 18 no 2 pp 142ndash1482004

[43] G-F Shi L-J An B Jiang S Guan and Y-M Bao ldquoAlpiniaprotocatechuic acid protects against oxidative damage in vitro


and reduces oxidative stress in vivordquo Neuroscience Letters vol403 no 3 pp 206ndash210 2006

[44] L J An S Guan G F Shi Y M Bao Y L Duan andB Jiang ldquoProtocatechuic acid from Alpinia oxyphylla againstMPP+-induced neurotoxicity in PC12 cellsrdquo Food and ChemicalToxicology vol 44 no 3 pp 436ndash443 2006

[45] G Shui Y M Bao J Bo and L J An ldquoProtective effectof protocatechuic acid from Alpinia oxyphylla on hydrogenperoxide-induced oxidative PC12 cell deathrdquo European Journalof Pharmacology vol 538 no 1ndash3 pp 73ndash79 2006

[46] X Yu L An Y Wang H Zhao and C Gao ldquoNeuroprotec-tive effect of Alpinia oxyphylla Miq fruits against glutamate-induced apoptosis in cortical neuronsrdquo Toxicology Letters vol144 no 2 pp 205ndash212 2003

[47] N Shoji A Umeyama T Takemoto and Y Ohizumi ldquoIsolationof a cardiotonic principle from Alpinia oxyphyllardquo PlantaMedica vol 50 no 2 pp 186ndash187 1984

[48] Y-M Chang C-T Tsai C-C RWang et al ldquoAlpinate oxyphyl-lae fructus (Alpinia oxyphyllaMiq) extracts inhibit angiotensin-II induced cardiac apoptosis in H9c2 cardiomyoblast cellsrdquoBioscience Biotechnology and Biochemistry vol 77 no 2 pp229ndash234 2013

[49] K Itahana J Campisi and G P Dimri ldquoMethods to detectbiomarkers of cellular senescence the senescence-associated 120573-galactosidase assayrdquoMethods in Molecular Biology vol 371 pp21ndash31 2007

[50] E Sikora T Arendt M Bennett and M Narita ldquoImpactof cellular senescence signature on ageing researchrdquo AgeingResearch Reviews vol 10 no 1 pp 146ndash152 2011

[51] I Gorenne M Kavurma S Scott and M Bennett ldquoVascularsmooth muscle cell senescence in atherosclerosisrdquo Cardiovas-cular Research vol 72 no 1 pp 9ndash17 2006

[52] L Monserrat M Hermida-Prieto X Fernandez et alldquoMutation in the alpha-cardiac actin gene associated withapical hypertrophic cardiomyopathy left ventricular non-compaction and septal defectsrdquo European Heart Journal vol28 no 16 pp 1953ndash1961 2007

[53] S P Barry S M Davidson and P A Townsend ldquoMolecularregulation of cardiac hypertrophyrdquo International Journal ofBiochemistry and Cell Biology vol 40 no 10 pp 2023ndash20392008

[54] H M Korashy H A Al-Suwayeh Z H Maayah M A AnsariS F Ahmad and S A Bakheet ldquoMitogen-activated proteinkinases pathwaysmediate the sunitinib-induced hypertrophy inrat cardiomyocyte H9c2 cellsrdquo Cardiovascular Toxicology vol15 no 1 pp 41ndash51 2015

[55] S-CHo J-H Liu andR-YWu ldquoEstablishment of themimeticaging effect in mice caused by D-galactoserdquo Biogerontology vol4 no 1 pp 15ndash18 2003

[56] M J Sheetz and G L King ldquoMolecular understanding ofhyperglycemiarsquos adverse effects for diabetic complicationsrdquoTheJournal of the AmericanMedical Association vol 288 no 20 pp2579ndash2588 2002

[57] M Seddon Y H Looi and A M Shah ldquoOxidative stressand redox signalling in cardiac hypertrophy and heart failurerdquoHeart vol 93 no 8 pp 903ndash907 2007

[58] K Tanaka M Honda and T Takabatake ldquoRedox regulation ofMAPK pathways and cardiac hypertrophy in adult rat cardiacmyocyterdquo Journal of the American College of Cardiology vol 37no 2 pp 676ndash685 2001

[59] C-Z Wang H-H Yuan X-L Bao and M-B Lan ldquoIn vitroantioxidant and cytotoxic properties of ethanol extract ofAlpinia oxyphylla fruitsrdquo Pharmaceutical Biology vol 51 no 11pp 1419ndash1425 2013

[60] K-S Chun K-K Park J Lee M Kang and Y-J Surh ldquoInhi-bition of mouse skin tumor promotion by anti-inflammatorydiarylheptanoids derived from Alpinia oxyphylla Miquel (Zin-giberaceae)rdquo Oncology Research vol 13 no 1 pp 37ndash45 2002

[61] Z-HHeWGeGG-L Yue C B-S LauM-FHe andP P-HBut ldquoAnti-angiogenic effects of the fruit of Alpinia oxyphyllardquoJournal of Ethnopharmacology vol 132 no 2 pp 443ndash449 2010

[62] Q Zhang C Cui C-Q Chen et al ldquoAnti-proliferative and pro-apoptotic activities ofAlpinia oxyphylla onHepG2 cells throughROS-mediated signaling pathwayrdquo Journal of Ethnopharmacol-ogy vol 169 pp 99ndash108 2015

[63] E Bisping S Ikeda S W Kong et al ldquoGata4 is requiredfor maintenance of postnatal cardiac function and protectionfrom pressure overload-induced heart failurerdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 103 no 39 pp 14471ndash14476 2006

[64] J DMolkentin ldquoCalcineurin-NFAT signaling regulates the car-diac hypertrophic response in coordination with the MAPKsrdquoCardiovascular Research vol 63 no 3 pp 467ndash475 2004

[65] J E Stelzer S L Brickson M R Locher and R L Moss ldquoRoleof myosin heavy chain composition in the stretch activationresponse of rat myocardiumrdquo Journal of Physiology vol 579 no1 pp 161ndash173 2007

[66] S Galler E Puchert B Gohlsch D Schmid and D PetteldquoKinetic properties of cardiac myosin heavy chain isoforms inratrdquo Pflugers Archiv vol 445 no 2 pp 218ndash223 2002

[67] S Chugh M Ouzounian Z Lu et al ldquoPilot study identifyingmyosin heavy chain 7 desmin insulin-like growth factor 7 andannexin A2 as circulating biomarkers of human heart failurerdquoProteomics vol 13 no 15 pp 2324ndash2334 2013

[68] W ZhangV ElimbanM SNijjar S KGupta andN SDhallaldquoRole of mitogen-activated protein kinase in cardiac hypertro-phy and heart failurerdquo Experimental and Clinical Cardiologyvol 8 no 4 pp 173ndash183 2003

[69] M Miyazawa Y Nakamura and Y Ishikawa ldquoInsectici-dal sesquiterpene from Alpinia oxyphylla against Drosophilamelanogasterrdquo Journal of Agricultural and Food Chemistry vol48 no 8 pp 3639ndash3641 2000

[70] T Morikawa H Matsuda I Toguchida K Ueda and MYoshikawa ldquoAbsolute stereostructures of three new sesquiter-penes from the fruit of Alpinia oxyphylla with inhibitory effectson nitric oxide production and degranulation in RBL-2H3cellsrdquo Journal of Natural Products vol 65 no 10 pp 1468ndash14742002

[71] N Shoji A Umeyama Y Asakawa T Takemoto K Nomotoand Y Ohizumi ldquoStructural determination of nootkatol a newsesquiterpene isolated from Alpinia oxyphylla miquel possess-ing calcium-antagonistic activityrdquo Journal of PharmaceuticalSciences vol 73 no 6 pp 843ndash844 1984

[72] X-Q Lv J-G Luo X-B Wang J-S Wang J Luo and L-YKong ldquoFour new sesquiterpenoids from the fruits of Alpiniaoxyphyllardquo Chemical and Pharmaceutical Bulletin vol 59 no3 pp 402ndash406 2011

[73] Z J Qing W Yong L Y Hui et al ldquoTwo new natural productsfrom the fruits of Alpinia oxyphylla with inhibitory effectson nitric oxide production in lipopolysaccharide-activatedRAW2647 macrophage cellsrdquo Archives of Pharmacal Researchvol 35 no 12 pp 2143ndash2146 2012


[74] J Xu C Ji Y Zhang J Su Y Li and N Tan ldquoInhibitoryactivity of eudesmane sesquiterpenes fromAlpinia oxyphylla onproduction of nitric oxiderdquo Bioorganic andMedicinal ChemistryLetters vol 22 no 4 pp 1660ndash1663 2012

[75] B Jiang W-J Wang M-P Li et al ldquoNew eudesmanesesquiterpenes from Alpinia oxyphylla and determination oftheir inhibitory effects on microgliardquo Bioorganic and MedicinalChemistry Letters vol 23 no 13 pp 3879ndash3883 2013

[76] O Muraoka M Fujimoto G Tanabe et al ldquoAbsolute stere-ostructures of novel norcadinane- and trinoreudesmane-typesesquiterpenes with nitric oxide production inhibitory activityfrom Alpinia oxyphyllardquo Bioorganic and Medicinal ChemistryLetters vol 11 no 16 pp 2217ndash2220 2001

[77] L Hou G Ding B Guo et al ldquoNew sesquiterpenoids and aditerpenoid from Alpinia oxyphyllardquo Molecules vol 20 no 1pp 1551ndash1559 2015

[78] W Song Y Li J Wang Z Li and J Zhang ldquoCharacterizationof nucleobases and nucleosides in the fruit of Alpinia oxyphyllacollected from different cultivation regionsrdquo Drug Testing andAnalysis vol 6 no 3 pp 239ndash245 2014

[79] F Chen H-L Li Y-F Tan et al ldquoDifferent accumulationprofiles of multiple components between pericarp and seedof Alpinia oxyphylla capsular fruit as determined by UFLC-MSMSrdquoMolecules vol 19 no 4 pp 4510ndash4523 2014

[80] Y-H Li F Chen J-F Wang Y Wang J-Q Zhang and T GuoldquoAnalysis of nine compounds from Alpinia oxyphylla fruit atdifferent harvest time using UFLC-MSMS and an extractionmethod optimized by orthogonal designrdquo Chemistry CentralJournal vol 7 no 1 article 134 2013

[81] D-T Ju H-E Liao M A Shibu et al ldquoNerve regenerationpotential of protocatechuic acid in RSC96 schwann cells byinduction of cellular proliferation and migration through IGF-IR-PI3K-Akt signalingrdquo Chinese Journal of Physiology vol 58no 6 pp 412ndash419 2015

[82] D-T Ju W-W Kuo T-J Ho et al ldquoProtocatechuic acid fromAlpinia oxyphylla induces schwann cell migration via ERK12JNK and p38 activationrdquoAmerican Journal of Chinese Medicinevol 43 no 4 pp 653ndash665 2015

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


compensation response may be beneficial continued hyper-trophy eventually leads to heart failure and ultimately death[2ndash4] Pressure and volume overload usually elicits concen-tric hypertrophy and eccentric hypertrophy respectively [56] Cardiac hypertrophies usually accompany the increscentof fetal genes expression including atrial natriuretic peptide(ANP) and brain natriuretic peptide (BNP) both are elevatedin cardiac hypertrophy and have been characterized as a fea-ture of hypertrophy in allmammalian species [7ndash9] CytokineInterleukin-6 (IL6) a potent hypertrophic effecter triggersglycoprotein 130 (gp130) dimerizing and activating down-stream hypertrophic signaling [10 11] Previous study indi-cated that IL6-related pathways play a crucial role in eccen-tric cardiac hypertrophy including IL6-related MEK5-ERK5signaling and JAK2-STAT3 signaling These hypertrophy-related molecular signals were activated simultaneously withIL6 expression and lead to morphological changes [12 13]Recently reports showed that the hypoxia marker BNIP3(Bcl-2adenovirus E1B nineteen-kDa interacting protein 3)induced concentric cardiac hypertrophy by targeting themitochondria and endoplasmic reticulum (ER) activating thecalcium-dependent signaling calcineurinNFAT3 [13 14]Another hypertrophic mediator p38 MAPK was found tomediate concentric cardiac hypertrophy by phosphorylatedGATA4 to further expressed hypertrophic response genessuch as ANP and BNP [13 15ndash17]

Mitochondria are generally considered the major sourceof reactive oxygen species (ROS) production [18 19] otherpotential sources of ROS include NADPH oxidase xanthineoxidase and uncoupled nitric oxide synthase (NOS) [20ndash22]Accumulating evidence demonstrates that oxidative stressimpaired antioxidant defense due toROSoverproduction andhave been implicated in the development and subsequentprogression of related heart diseases especially cardiac hyper-trophy [23ndash26] ROS production increase has been shown tobe involved in the hypertrophy of isolated cardiomyocytesinduced by angiotensin II (AngII) tumor necrosis factor-a (TNFa) cyclic stretch or a-adrenergic agonists [27ndash29]Additionally NADPH oxidase 4 (Nox4) a major source ofoxidative stress in the failing heart which has been identifiedinduces cardiac hypertrophy through activating AktmTORand NF120581B signaling [30 31] It has also been reported thatNox4 directly mediates mitochondrial dysfunction oxidativestress and myocardial cell death during pressure overload-induced cardiac hypertrophy [31 32]

D-Galactose (D-gal) is a reducing sugar that has beenwidely used in age-related oxidative damage and aging phar-macology research [33ndash35] D-Gal generates ROS during itsmetabolism in vivo by reacting readily with the free amines inproteins lipids and nucleic acids to form advanced glycationend products (AGEs) [36 37] Growing evidence suggeststhat AGEs are interacting receptors for AGE (RAGE) inmanycell types and induce downstreamNF-120581B and other signalingpathways eventually lead to ROS generation and acceleratethe aging process [38ndash41]

Alpinate Oxyphyllae Fructus (Alpinia oxyphylla MIQAOF) is one of the important traditional Chinese medicineswhich has antiaging and sexual-reinforcing activity [42]

According to the Chinese pharmacopoeia AOF has beenwidely used for treating gastralgia diarrhea ulcerationantitumor hypertension accompanying symptoms and cere-brovascular disorders [42ndash46] An increasing number ofevidences indicate that AOF extracts exhibit cardioprotectiveand neuroprotective activity against oxidative stress-inducedapoptosis [44 47] Subsequent studies demonstrated thatAOF extracts protect againstAng II induced cardiac apoptosisin H9c2 cardiomyoblast cell [48] We here further investigatewhether AOF ameliorate the ROS-induced aging heart prob-lem and related signaling paths and mechanisms

2 Materials and Methods

21 AOF Extraction Fragmented Alpinate Oxyphyllae Fruc-tus (Alpinia oxyphylla MIQ AOF) was obtained from Shin-Long Pharmaceutical Company (Taichung Taiwan)

The AOF fragment (150 g) was extracted by boiling in06 L of boiling water for 2 hThe water extract was filtered bygravity-flow procedure at reduced pressure for convenienceand then stored at 4∘C The water extract was spray dried toproduce a powdered extract The product yield percentagewas 74

22 Animals and Experimental Design Thirty-four male 8-week-old Sprague-Dawley rats weighing 220 plusmn 20 g were pro-vided by the National Institutes of Health (NIH) colony andcared for at the Comparative Biology facility at Texas AampMUniversity in accordance with NIH and ULACC (UniversityLaboratory Animal Care Committee) standards Rats weremaintained on standard laboratory conditions of temperature(23 plusmn 2∘C) and a 12 12 h light-dark cycle with water and ratchow available ad libitum for the duration of the study After 2weeks of acclimatization the rats were randomly divided into5 groups Onewas normal control group (NC)The other fourwere induced aging groups injected with D-galactose (150mgkgday for 8 weeks) in induced aging groups the three AOFgroups were orally administered with AOF of 50 (AOF lowAL) 100 (AOF medium AM) and 150 (AOF high AH)mgkgday respectively the normal control groupwere giventhe same volume of control solution After the animals weresacrificed the heart tissue was immediately collected andstored at minus80∘C until further use

23 Cardiac Characteristics The whole hearts of animalswere weighed after being excised and cleaned with PBS Theleft ventricle tissues were isolated and weighed The tibialength was measured by the electronic digital Vernier caliperto adjust the whole heart weightThe ratios of the whole heartweight to the tibia length and the left ventricle weight to tibialength were calculated

24 Echocardiography Transthoracic echocardiographic ima-ges of ratswere performedusingHewlett-Packard Sonos 5500ultrasound machine with a 15MHz linear-array transducerLeft ventricular M-mode measurements at the level of thepapillary muscles include interventricular septal thickness atend-diastole (IVSD) left ventricular internal dimension at











4 Results






Control


Induced aging (IA)

(a)

훽-gal

p21

훽-Actin




Induced aging (IA)

0

05

1

15

2

25

훽-G

al훽

-act

in

0

1

2

3

4

5

6

p21훽

-act

in

54

21

43

(b)

HO1 32

SOD1 23

37GADPH

002040608

11214

HO

1G

AD

PH


02040608

11214

SOD

1G

AD

PH lowastlowast

lowastlowastlowast


(c)












Control

0

RVRV

LV LV

RV RV RV

LV LV LV


Induced aging (IA)

1 cm0 1 cm

0 1 cm 0 1 cm 0 1 cm(a)


05

1

15

2

Fold

chan

ge

lowastlowast

(b)






5 Discussion






54

kDa

NFATc3 130

p-ERK12 4244

p-c-JUN 39

38

p-JNK

p-P38

4654

훼-Tub

50p-GATA4

ERK12 4244

JNK 4654

c-JUN 39





002040608

1121416

p-ER

K12

훼-tu

bulin


0

05

1

15

2

25

3N

FATc

3훼

-tubu

lin

0

05

1

15

2

25

3

p-JN

K훼

-tubu

lin

005

115

225

335

p-c-

JUN

훼-tu

bulin



0

05

1

15

2

25

p-G

ATA

4훼

-tubu

lin

0

05

1

15

2

25

p-P3

8훼

-tubu

lin



8691STAT3

p-MEK5

p-ERK5

훽-Actin 43

54

115

ERK5 115

MEK5 54


0

05

1

15

2

25

3

35

p-M

EK5훽

-act

in

0

05

1

15

2

25

p-ER

K5훽

-act

in

005

115

225

335

4

STAT

3훽

-act

in





lowastlowastlowast





Abbreviations





end-systole


224

BNP 36

kDa

223

MYH7

MYH6

훼-Tub 54

BNPMYH7MYH6


05

1

15

2

25Pr

otei

n le

vel (

fold

of c

hang

e)

lowastlowast

lowastlowast

lowastlowastlowast





Acknowledgments


References





























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























4 Results






Control


Induced aging (IA)

(a)

훽-gal

p21

훽-Actin




Induced aging (IA)

0

05

1

15

2

25

훽-G

al훽

-act

in

0

1

2

3

4

5

6

p21훽

-act

in

54

21

43

(b)

HO1 32

SOD1 23

37GADPH

002040608

11214

HO

1G

AD

PH


02040608

11214

SOD

1G

AD

PH lowastlowast

lowastlowastlowast


(c)












Control

0

RVRV

LV LV

RV RV RV

LV LV LV


Induced aging (IA)

1 cm0 1 cm

0 1 cm 0 1 cm 0 1 cm(a)


05

1

15

2

Fold

chan

ge

lowastlowast

(b)






5 Discussion






54

kDa

NFATc3 130

p-ERK12 4244

p-c-JUN 39

38

p-JNK

p-P38

4654

훼-Tub

50p-GATA4

ERK12 4244

JNK 4654

c-JUN 39





002040608

1121416

p-ER

K12

훼-tu

bulin


0

05

1

15

2

25

3N

FATc

3훼

-tubu

lin

0

05

1

15

2

25

3

p-JN

K훼

-tubu

lin

005

115

225

335

p-c-

JUN

훼-tu

bulin



0

05

1

15

2

25

p-G

ATA

4훼

-tubu

lin

0

05

1

15

2

25

p-P3

8훼

-tubu

lin



8691STAT3

p-MEK5

p-ERK5

훽-Actin 43

54

115

ERK5 115

MEK5 54


0

05

1

15

2

25

3

35

p-M

EK5훽

-act

in

0

05

1

15

2

25

p-ER

K5훽

-act

in

005

115

225

335

4

STAT

3훽

-act

in





lowastlowastlowast





Abbreviations





end-systole


224

BNP 36

kDa

223

MYH7

MYH6

훼-Tub 54

BNPMYH7MYH6


05

1

15

2

25Pr

otei

n le

vel (

fold

of c

hang

e)

lowastlowast

lowastlowast

lowastlowastlowast





Acknowledgments


References





























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control


Induced aging (IA)

(a)

훽-gal

p21

훽-Actin




Induced aging (IA)

0

05

1

15

2

25

훽-G

al훽

-act

in

0

1

2

3

4

5

6

p21훽

-act

in

54

21

43

(b)

HO1 32

SOD1 23

37GADPH

002040608

11214

HO

1G

AD

PH


02040608

11214

SOD

1G

AD

PH lowastlowast

lowastlowastlowast


(c)












Control

0

RVRV

LV LV

RV RV RV

LV LV LV


Induced aging (IA)

1 cm0 1 cm

0 1 cm 0 1 cm 0 1 cm(a)


05

1

15

2

Fold

chan

ge

lowastlowast

(b)






5 Discussion






54

kDa

NFATc3 130

p-ERK12 4244

p-c-JUN 39

38

p-JNK

p-P38

4654

훼-Tub

50p-GATA4

ERK12 4244

JNK 4654

c-JUN 39





002040608

1121416

p-ER

K12

훼-tu

bulin


0

05

1

15

2

25

3N

FATc

3훼

-tubu

lin

0

05

1

15

2

25

3

p-JN

K훼

-tubu

lin

005

115

225

335

p-c-

JUN

훼-tu

bulin



0

05

1

15

2

25

p-G

ATA

4훼

-tubu

lin

0

05

1

15

2

25

p-P3

8훼

-tubu

lin



8691STAT3

p-MEK5

p-ERK5

훽-Actin 43

54

115

ERK5 115

MEK5 54


0

05

1

15

2

25

3

35

p-M

EK5훽

-act

in

0

05

1

15

2

25

p-ER

K5훽

-act

in

005

115

225

335

4

STAT

3훽

-act

in





lowastlowastlowast





Abbreviations





end-systole


224

BNP 36

kDa

223

MYH7

MYH6

훼-Tub 54

BNPMYH7MYH6


05

1

15

2

25Pr

otei

n le

vel (

fold

of c

hang

e)

lowastlowast

lowastlowast

lowastlowastlowast





Acknowledgments


References





























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























Control

0

RVRV

LV LV

RV RV RV

LV LV LV


Induced aging (IA)

1 cm0 1 cm

0 1 cm 0 1 cm 0 1 cm(a)


05

1

15

2

Fold

chan

ge

lowastlowast

(b)






5 Discussion






54

kDa

NFATc3 130

p-ERK12 4244

p-c-JUN 39

38

p-JNK

p-P38

4654

훼-Tub

50p-GATA4

ERK12 4244

JNK 4654

c-JUN 39





002040608

1121416

p-ER

K12

훼-tu

bulin


0

05

1

15

2

25

3N

FATc

3훼

-tubu

lin

0

05

1

15

2

25

3

p-JN

K훼

-tubu

lin

005

115

225

335

p-c-

JUN

훼-tu

bulin



0

05

1

15

2

25

p-G

ATA

4훼

-tubu

lin

0

05

1

15

2

25

p-P3

8훼

-tubu

lin



8691STAT3

p-MEK5

p-ERK5

훽-Actin 43

54

115

ERK5 115

MEK5 54


0

05

1

15

2

25

3

35

p-M

EK5훽

-act

in

0

05

1

15

2

25

p-ER

K5훽

-act

in

005

115

225

335

4

STAT

3훽

-act

in





lowastlowastlowast





Abbreviations





end-systole


224

BNP 36

kDa

223

MYH7

MYH6

훼-Tub 54

BNPMYH7MYH6


05

1

15

2

25Pr

otei

n le

vel (

fold

of c

hang

e)

lowastlowast

lowastlowast

lowastlowastlowast





Acknowledgments


References





























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















5 Discussion






54

kDa

NFATc3 130

p-ERK12 4244

p-c-JUN 39

38

p-JNK

p-P38

4654

훼-Tub

50p-GATA4

ERK12 4244

JNK 4654

c-JUN 39





002040608

1121416

p-ER

K12

훼-tu

bulin


0

05

1

15

2

25

3N

FATc

3훼

-tubu

lin

0

05

1

15

2

25

3

p-JN

K훼

-tubu

lin

005

115

225

335

p-c-

JUN

훼-tu

bulin



0

05

1

15

2

25

p-G

ATA

4훼

-tubu

lin

0

05

1

15

2

25

p-P3

8훼

-tubu

lin



8691STAT3

p-MEK5

p-ERK5

훽-Actin 43

54

115

ERK5 115

MEK5 54


0

05

1

15

2

25

3

35

p-M

EK5훽

-act

in

0

05

1

15

2

25

p-ER

K5훽

-act

in

005

115

225

335

4

STAT

3훽

-act

in





lowastlowastlowast





Abbreviations





end-systole


224

BNP 36

kDa

223

MYH7

MYH6

훼-Tub 54

BNPMYH7MYH6


05

1

15

2

25Pr

otei

n le

vel (

fold

of c

hang

e)

lowastlowast

lowastlowast

lowastlowastlowast





Acknowledgments


References





























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















54

kDa

NFATc3 130

p-ERK12 4244

p-c-JUN 39

38

p-JNK

p-P38

4654

훼-Tub

50p-GATA4

ERK12 4244

JNK 4654

c-JUN 39





002040608

1121416

p-ER

K12

훼-tu

bulin


0

05

1

15

2

25

3N

FATc

3훼

-tubu

lin

0

05

1

15

2

25

3

p-JN

K훼

-tubu

lin

005

115

225

335

p-c-

JUN

훼-tu

bulin



0

05

1

15

2

25

p-G

ATA

4훼

-tubu

lin

0

05

1

15

2

25

p-P3

8훼

-tubu

lin



8691STAT3

p-MEK5

p-ERK5

훽-Actin 43

54

115

ERK5 115

MEK5 54


0

05

1

15

2

25

3

35

p-M

EK5훽

-act

in

0

05

1

15

2

25

p-ER

K5훽

-act

in

005

115

225

335

4

STAT

3훽

-act

in





lowastlowastlowast





Abbreviations





end-systole


224

BNP 36

kDa

223

MYH7

MYH6

훼-Tub 54

BNPMYH7MYH6


05

1

15

2

25Pr

otei

n le

vel (

fold

of c

hang

e)

lowastlowast

lowastlowast

lowastlowastlowast





Acknowledgments


References





























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















8691STAT3

p-MEK5

p-ERK5

훽-Actin 43

54

115

ERK5 115

MEK5 54


0

05

1

15

2

25

3

35

p-M

EK5훽

-act

in

0

05

1

15

2

25

p-ER

K5훽

-act

in

005

115

225

335

4

STAT

3훽

-act

in





lowastlowastlowast





Abbreviations





end-systole


224

BNP 36

kDa

223

MYH7

MYH6

훼-Tub 54

BNPMYH7MYH6


05

1

15

2

25Pr

otei

n le

vel (

fold

of c

hang

e)

lowastlowast

lowastlowast

lowastlowastlowast





Acknowledgments


References





























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















224

BNP 36

kDa

223

MYH7

MYH6

훼-Tub 54

BNPMYH7MYH6


05

1

15

2

25Pr

otei

n le

vel (

fold

of c

hang

e)

lowastlowast

lowastlowast

lowastlowastlowast





Acknowledgments


References





























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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