Isoliquiritigenin Attenuates Monocrotaline-Induced Pulmonary …downloads.hindawi.com/journals/ecam/2019/4568198.pdf · 2019-07-30 · ResearchArticle Isoliquiritigenin Attenuates

Research ArticleIsoliquiritigenin Attenuates Monocrotaline-InducedPulmonary Hypertension via Inhibition of the InflammatoryResponse and PASMCs Proliferation

Haifeng Jin 12 Yang Jiang1 Fengxia Du3 Linna Guo1 GuanWang1 Sang Chan Kim 4

ChulWon Lee 4 Lei Shen 12 and Rongjie Zhao 25

1Department of Anatomy Qiqihar Medical University Qiqihar Heilongjiang 161006 China2Qiqihar Institute of Medical and Pharmaceutical Sciences Qiqihar Medical University Qiqihar Heilongjiang 161006 China3Department of Etiology Qiqihar Medical University Qiqihar Heilongjiang 161006 China4MRC-GHF College of Korean Medicine Daegu Haany University Gyeongsan 38610 Republic of Korea5Department of Psychopharmacology Qiqihar Medical University Qiqihar Heilongjiang 161006 China

Correspondence should be addressed to Lei Shen shenlei815yahoocom and Rongjie Zhao zhao rongjieyahoocom

Received 21 February 2019 Accepted 16 May 2019 Published 26 May 2019

Academic Editor Olumayokun A Olajide

Copyright copy 2019 Haifeng Jin 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 properly cited

Pulmonary hypertension (PH) is a progressive and serious disease where exacerbated inflammatory response plays a criticalrole Isoliquiritigenin (ISL) an important flavonoid isolated from Glycyrrhizae radix exhibits a wide range of pharmacologicalactions including anti-inflammation Previously we found ISL alleviated hypoxia-induced PH in the present study to extendthis we evaluated the effects of ISL on monocrotaline (MCT)-induced PH and the relevant mechanisms Rats received a singleintraperitoneal injection of MCT followed by intragastric treatments with ISL (10 mgkgd or 30 mgkgd) once a day for 28 daysThe MCT administration increased the right ventricular systolic pressure (RVSP) (p lt 0001) the median width of pulmonaryarteries (plt 001) and the weight ratio of the right ventricular wallleft ventricular wall plus septum (Fulton index) (plt 001) in ratshowever these changes were inhibited by both doses of ISL (p lt 005) In addition treatment with ISL suppressed the upregulatedproduction of serum interleukin-6 (p lt 001) and tumor necrosis factor-120572 (p lt 005) by MCT and reversed the increases in thenumbers of proliferating cell nuclear antigen (PCNA)-positive cells (p lt 001) in the medial wall of pulmonary arteries In in vitroexperiments ISL (10 120583M 30 120583M and 100 120583M) inhibited excessive proliferation of cultured primary pulmonary artery smoothmuscle cells (PASMCs) (p lt 005 p lt 001 and p lt 0001) in a dose-dependentmanner and prevented an increase in the expressionsof PCNA (p lt 001) and phospho-Akt (p lt 005) in PASMCs induced by hypoxia These results suggest that ISL can attenuateMCT-induced PH via its anti-inflammatory and antiproliferative actions

1 Introduction

Pulmonary hypertension (PH) is a progressive and severedisease characterized pathophysiologically by increased pul-monary pressure pulmonary artery structural remodelingand right ventricular hypertrophy and finally leads to death[1 2] Nowadays there are three major classes of drugsprostaglandins phosphodiesterase inhibitors and endothelinreceptor antagonists available to treat PH but they all presentlimited effectiveness and even produce unwanted conse-quences such as dysregulated pulmonary vasoconstriction

response and worsened gas exchange [3 4] Therefore thereis an urgent need to develop new agents that are moreeffective and less adverse in treating PH Fortunately there areseveral types of animal PH models including monocrotaline(MCT)-induced rat PH which pathophysiologically mimichuman PH and help screen new chemical candidates forthe treatment of PH Studies have shown that a singledose of MCT can elicit the typical PH pathophysiologies inrat cardiopulmonary system which share similar processeswith those in human PH For example MCT administra-tion engenders a persistent elevation of pulmonary vascular

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2019 Article ID 4568198 10 pageshttpsdoiorg10115520194568198

2 Evidence-Based Complementary and Alternative Medicine

resistance and a progressive increase in the artery pressurefollowed by pulmonary artery structural remodeling andright ventricular hypertrophy consequently results in collapseof respiratory and circulatory systems [5] Thus impeding orreversing the pathophysiological progress of MCT-inducedPH is a promising approach to treat chemicals-inducedPH

So far the molecular mechanisms of PH are not fullyunderstood but clinical and preclinical evidences showinflammation is a key player in mediating the pathophys-iological process of PH [6] In PH patients there are sig-nificantly increased circulating levels of proinflammatorycytokines such as interleukin-6 (IL-6) and tumor necrosisfactor-120572 (TNF-120572) and the high levels of these factors areassociated with the increased risk of death in the patients[7] Likewise in animal models the degree of perivascularinflammation positively correlates with pulmonary vascularremodeling [8] IL-6 and TNF-120572 can induce the proliferationmigration and differentiation of pulmonary vascular cellsharmfully promoting vascular remodeling [9] In additionproinflammatory factors directly disrupt endothelial mem-brane integrity and enhance hyperplasia in pulmonary arterysmooth muscle cells (PASMCs) and the latter is anotherkey player in the progress of PH [9] It is turned out thatthe excessive PASMCs proliferation is mainly responsiblefor the pulmonary vascular medial hyperplasia elevatedvascular resistance and right ventricular hypertrophy [110] Therefore dampening the inflammatory response andinhibiting the abnormal proliferation of PASMCs can providean effective way to halt the development of PH pathology

Glycyrrhizae radix (G radix licorice) is most frequentlyused in traditional Chinese medicine to treat a variety ofpathological states due to its superb detoxification effects [11]Isoliquiritigenin (ISL) is an important bioactive ingredientin G radix mediates the therapeutic effects of G radix andpossesses a wide range of pharmacological properties such asanti-inflammatory antioxidant antitumor and immunoreg-ulatory activities [12ndash16] ISL inhibits carbon tetrachloride-induced hepatic damage and the proliferation of humanprostate cancer LNCaP cells through its antioxidant and anti-inflammatory actions [17 18] ISL improves atherosclerosisand hypoxia-induced cardiomyocytes contractile dysfunc-tion by decreasing the levels of reactive oxygen species(ROS) and proinflammatory factors [19ndash21] These findingscollectively indicate that ISL may have a therapeutic effect onPH Indeed in a recent study we have found ISL alleviatedhypoxic PH through inhibition of oxidative stress indicatinga potential application of ISL to treat PH As mentionedabove PH presents common pathophysiologies in its finalphase but involves different etiologies that complicate itspharmacotherapies Therefore to support the therapeuticeffect of ISL on hypoxic PH and further expand the ther-apeutic applications of ISL in treating PH in the presentstudy we evaluated the effects of ISL on chemicals-inducedPH in rats For this we first established an MCT-inducedPH rat model where exacerbated inflammation and excessiveproliferation of PASMCs play a key role and then by usingthis model examined the effect of ISL on rat MCT-inducedPH and investigated the relevant mechanisms

2 Materials and Methods

21 Reagents and Antibodies ISL gt 99 purity was providedby Shanghai Yuanye Biotechnology Co Ltd (ShanghaiChina) Monocrotaline (MCT) was purchased from SigmaSigmandashAldrich Inc (St LouisMOUSA) Fetal bovine serum(FBS) Dulbeccorsquos modified Eaglersquos medium (DMEM) Trizolreagent and trypsin solution were obtained from GibcoLaboratories (Grand Island NY USA) Antibodies against120572-smooth muscle actin (120572-SMA) proliferating cell nuclearantigen (PCNA) Akt phospho-Akt (p-Akt) and GAPDHwere purchased from Cell Signaling Technology (BeverlyMA USA)

22 Animals and Experimental Design Eight-week-old maleSpraguendashDawley rats (body weight 220ndash250 g) were obtainedfrom the Animal Center of Qiqihar Medical UniversityChina The rats were caged (3 rats per cage) and suppliedby filtered pathogen-free air unlimited commercial rat chowand water at a temperature between 22 and 24∘C with12 h lightdark cycles and relative humidity of 50 Allanimal experiments were approved by the Animal Careand Use Committee of the Qiqihar Medical University andconducted in accordance with the National Institutes ofHealth guidelines concerning the care and use of labora-tory animals After 7 days of acclimatization period therats were randomly divided into 4 groups saline-treatedgroup (n = 8) MCT-treated control group (n = 8) MCT+ 10 mgkgd ISL (MCT+ISL10) group (n = 8) and MCT+ 30 mgkgd ISL (MCT+ISL30) group (n = 8) For theestablishment of an MCT-induced PH rat model rats weregiven a single intraperitoneal injection of MCT (50 mgkg)which was dissolved in 1N hydrochloric acid diluted insterile saline and adjusted to pH 74 with 1N sodiumhydroxide Thereafter the rats stayed in their home cages for28 days and received ISL treatments ISL was dissolved in5 Tween-80 and intragastrically administered to the ratsat the dose of 10 mgkgd or 30 mgkgd once a day for28 days while an equal volume of 5 Tween-80 was givento the rats in the control groups Thirty minutes after thefinal dose of ISL the rats were subjected to hemodynamicanalysis and then blood samples and lungs of the ratswere collected for further biochemical and morphologicassays

23 Hemodynamic Assay and Assessment of the Right Ven-tricular Hypertrophy The rats were anesthetized with anintraperitoneal injection of 75 mLkg 48 tribromoethanoland a polyethylene catheter was inserted into the rightventricle through the right jugular vein The right ventriclesystolic pressure (RVSP) was measured via a fluid-filledpressure transducer and recorded using PowerLab Software(ADInstruments Castle Hill Australia) For assessment ofright ventricular hypertrophy the left ventricular wall plusseptum and the right ventricular wall were harvested andthe weight ratio of the right ventricular wallleft ventricularwall plus septum (Fulton index) was calculated to quantifythe right ventricular hypertrophy

Evidence-Based Complementary and Alternative Medicine 3

24 Histological Observation A lobe of the right lung fromeach rat was fixed in 4 neutral-buffered formalin subjectedto paraffin embedding sliced into 4-120583m thick sections andthen stained with hematoxylin and eosin (HE) Morphologicchanges in small pulmonary arteries (range 50ndash200120583m)wereobserved For assessment of pulmonary artery structuralremodeling the total vessel area the medial wall area theinside diameter and outside diameter of pulmonary arterieswere measured Pulmonary artery structural remodeling wasevaluated by the percentage medial wall area (WA) =(medial wall area)(total vessel area) times 100 and the percentagemedial wall thickness (WT) = (outside diameterminusinsidediameter)(outside diameter)times100

25 Immunohistochemical Staining Lung sections weredeparaffinized in xylene and rehydrated with ethanol andantigens were retrieved After blocking unspecific proteinbinding with 5 bovine serum albumin for 30 min at roomtemperature the lung sections were incubated overnightat 4∘C with one of the following antibodies anti-PCNAantibody (11500) or anti-120572-SMA antibody (1500) Thensections were further incubated with a biotinylated anti-mouse IgG antibody for 2 h at room temperature Finallythe sections were visualized using diaminobenzidine andcounterstained with hematoxylin Quantitative assessmentsof 120572-SMA and PCNA were carried out as describedpreviously [22]

26 Assay of IL-6 and TNF-120572 The blood samples werecentrifuged at 1500 x g at 4∘C for 20 minutes and thesera were collected and the serum levels of IL-6 and TNF-120572were measured using enzyme-linked immunosorbent assay(ELISA) kits (Shanghai Bluegene Biotech Co Ltd ShanghaiChina) according to the manufacturerrsquos instructions

27 Quantitative Real-Time RT-PCR Analysis Total RNAwas extracted from lung tissues using Trizol agent Quan-titative real-time RT-PCR (qPCR) was performed to assessmRNA expression of the following genes The primers forthe genes were as follows IL-6 (101 bp) forward 51015840-AAG-TCGGAGGCTTAATTACACATGT-31015840 reverse 51015840-AAG-TGCATCATCGTTGTTCATACA-31015840 TNF-120572 (110 bp) for-ward 51015840-AAATGGGCTCCCTCTATCAGTTC-31015840 reverse51015840-TCTGCTTGGTGGTTTGCTACGAC-31015840 housekeepinggene GAPDH (123 bp) forward 51015840-AGGTCGGTGTGA-ACGGATTTG-31015840 reverse 51015840- TGTAGACCATGTAGT-TGAGGTCA -31015840 respectively

28 Primary PASMCs Culture and In Vitro Hypoxia Ratprimary PASMCs were obtained by explants method andcultured as described previously [22] Smooth muscle cellidentity was verified by 120572-SMA immunocytochemical stain-ing and the cells of passages 3ndash7 were used PASMCs weredivided into 6 groups normoxia hypoxia hypoxia + 10 120583MISL hypoxia + 30 120583M ISL and hypoxia + 100 120583M ISL Thehypoxia or normoxia was implemented by exposing PASMCsto either in 3 oxygen or 21 oxygen condition for 24 hrespectively

29 Cell Proliferation Assay PASMCs proliferation was mea-sured by the 3-(45-dimethylthiazol-2-yl)-2 5-diphenyltet-razolium bromide (MTT) assay PASMCs were seeded in 96-well culture plates (2times104 cells per well) and incubated for24 h under the condition of either normoxia or hypoxia plusISL and then 10 120583L MTT (5 mgmL) was added to each wellAfter being incubated with MTT for 4 h the media of eachwell was removed and the produced formazan crystals inthe wells were dissolved by adding 150 120583L dimethyl sulfoxide(DMSO) The optical density value of each well was readat 490 nm wavelength using a spectrophotometer which isdirectly proportional to the number of living cells

210Western Blotting Analysis PASMCs were lysed in a pro-tein extraction buffer A cocktail of protease and phosphataseinhibitors was added to the buffer in advance Then thelysates were centrifuged at 12000 rpm for 15 min at 4∘C andthe supernatants were collected The protein concentrationwas determined by using the Lowry method (Bio-Rad)Twenty-five 120583g of protein was separated by electrophoresison 10 sodium dodecyl sulfate polyacrylamide gels andtransferred to a nitrocellulose membrane The membranewas blocked and incubated with the appropriate primaryantibody to PCNA (11000) p-Akt (11000) and total Akt(11000) Immunoreactive proteins were detected by chemi-luminescence with an ECL detection system

211 Statistical Analyses All values are expressed as meansplusmn SEM Statistical analysis was performed using the com-mercially available software GraphPad Prism 50 (GraphPadSoftware San Diego CA USA) The statistical significanceof differences between groups was evaluated by one-wayanalysis of variance (ANOVA)withNewman-Keulsmultiple-comparison tests Significant difference was accepted at p lt005

3 Results

31 ISL Attenuated MCT-Induced PH and Pulmonary ArteryRemodeling As seen in Figure 1(a) a single MCT caused asubstantial PH as evidenced by significant increases in theaverage RVSP of the MCT-treated control rats [F(3 28) = 859p lt 0001 saline group vs MCT group p lt 0001] Howeverboth doses of ISL (10 mgkgd and 30 mgkgd) preventedthese increases (MCT group vs MCT+ISL10 p lt 005 MCTgroup vs MCT+ISL30 p lt 001) (Figure 1(a)) In agreementwith the RVSP results MCT induced a significant elevationof the Fulton indices [F(3 28) = 459 p lt 001 saline group vsMCT group p lt 001] which was also blocked by both dosesof ISL treatment (MCT group vs MCT+ISL10 p lt 005MCTgroup vs MCT+ISL30 p lt 005) (Figure 1(b))

In the present study MCT-induced pulmonary arteryremodeling was indexed by WT and WA of pulmonaryarteries As shown in Figure 2MCTmarkedly elevatedWTand WA [WT F(3 28) = 641 p lt 001 saline group vsMCT group p lt 001 WA F(3 28) = 451 p lt 005 salinegroup vs MCT group p lt 001] while treatment with ISLimproved these pathological changes (WT MCT group vs


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Figure 1 Effects of ISL on pulmonary hypertension and right ventricular hypertrophy in MCT-induced PH (a) Right ventricular systolicpressure (RVSP) in rats (b) Fulton index in rats Values are means plusmn SEM (n = 8) p lt 001 p lt 0001 compared with saline group $ p lt005 $$ p lt 001 compared with MCT group

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Figure 2 Effects of ISL on pulmonary artery structural remodeling (a) Hematoxylin and eosin staining of pulmonary arteries (scale bar =50 120583m) (b)Medial wall thickness (WT) of pulmonary arteries (c) Medial wall area (WA) of pulmonary arteries Values are means plusmn SEM(n = 8) p lt 001 compared with saline group $ p lt 005 compared with MCT group

MCT+ISL10 p lt 005 MCT group vs MCT+ISL30 p lt 005WA MCT group vs MCT+ISL10 p lt 005 MCT group vsMCT+ISL30 p lt 005) (Figure 2)

32 ISL Blocked an Elevation of 120572-SMA Expressions Thehyperplastic smooth muscularization was examined in lungsections using an antibody against 120572-SMA to evaluate thecellular mechanism for the increased thickness and areaof pulmonary arteries As seen in Figure 3 the integrated

optical density (OD) value of 120572-SMA in the MCT controlgroup was higher than in the saline control group [F(3 16)= 493 p lt 005 saline group vs MCT group p lt 005]reflecting the association of increased thickness and area ofpulmonary arteries with enhanced proliferation of SMCsHowever in accordance with the abovementioned morpho-logical observation both doses of ISL blocked the MCT-induced enhancement of the OD value of 120572-SMA (MCTgroup vs MCT+ISL10 p lt 005MCT group vs MCT+ISL30p lt 005) (Figure 3)



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Figure 3 Analysis of optical density (OD) value of 120572-SMA in pulmonary arteries (a) Immunohistochemical staining of 120572-SMAof pulmonaryarteries (scale bar = 50 120583m) (b) Quantitative analysis of OD value of 120572-SMA immunoreactivity in pulmonary arteries Values are means plusmnSEM (n = 5) p lt 005 compared with saline group $ p lt 005 compared with MCT group

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Figure 4 Effects of ISL on the levels of IL-6 and TNF-120572 in serum (a) IL-6 levels in serum (b) TNF-120572 levels in serum Values are means plusmnSEM (n = 8) p lt 001 p lt 0001 compared with saline group $ p lt 005 $$ p lt 001 compared with MCT group

33 ISL Reduced MCT-Induced Plasma IL-6 and TNF-120572Secretion The ELISA analysis revealed that the serum levelsof both IL-6 and TNF-120572 in the MCT control group weresignificantly higher than those in the saline control group[IL-6 F(3 28) = 866 p lt 0001 saline group vs MCT groupp lt 0001 TNF-120572 F(3 28) = 479 p lt 001 saline groupvs MCT group p lt 001] However these increases in theinflammatory factor levels were reduced by treatment withboth doses of ISL (IL-6MCT group vs MCT+ISL10 p lt 001MCT group vsMCT+ISL30 plt 001 TNF-120572 MCT group vsMCT+ISL10 p lt 005 MCT group vs MCT+ISL30 p lt 005)(Figure 4)

34 ISL Prevented MCT-Induced Upregulation of PulmonaryExpressions of IL-6 and TNF-120572mRNA Being paralleled withthe plasma ELISA results the qPCR analysis showed that asingle MCT increased the mRNA levels of IL-6 and TNF-120572in lung tissues [IL-6 mRNA F(3 12) = 1217 p lt 0001 salinegroup vs MCT group p lt 0001 TNF-120572 mRNA F(3 12) =1005 p lt 001 saline group vs MCT group p lt 0001]However the same qPCR assay also revealed both doses ofISL prevented this upregulation of the mRNA expressions(IL-6 mRNA MCT group vs MCT+ISL10 p lt 001 MCTgroup vs MCT+ISL30 p lt 001 TNF-120572mRNA MCT group

vs MCT+ISL10 p lt 001 MCT group vs MCT+ISL30 p lt001) (Figure 5)

35 ISL Inhibited PASMCs Proliferation PCNA is a markerfor cell proliferation To determine the effect of ISL onPASMCs proliferation the PCNA-positive cells in the medialwall of pulmonary arteries were quantified via the immuno-histochemistry staining There were increased numbers ofPCNA-positive cells in pulmonary arteries from the MCT-challenged rats compared to the saline-treated control rats[F(3 16) = 712 p lt 001 saline group vs MCT group p lt 001]which was inhibited by treatment with ISL (MCT group vsMCT+ISL10 p lt 001 MCT group vs MCT+ISL30 p lt 001)(Figure 6)

In addition in the in vitro experiments the proliferationof the PASMCs under the hypoxia condition was greaterthan that under the normoxia condition [F(4 20) = 1329p lt 0001 normoxia group vs hypoxia group p lt 0001]but treatment with all three doses of ISL inhibited thisincreased proliferation in a dose-dependent way (Hypoxiavs Hypoxia+ISL 10 120583M p lt 005 Hypoxia vs Hypoxia+ISL30 120583M p lt 001 Hypoxia vs Hypoxia+ISL 100 120583M plt 0001 Hypoxia+ISL 10 120583M vs Hypoxia+ISL 100 120583M plt 005 Hypoxia+ISL 30 120583M vs Hypoxia+ISL 100 120583M p


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Figure 5 Effects of ISL on IL-6 and TNF-120572mRNA expressions in rat lungs (a) IL-6 mRNA levels in rat lungs (b) TNF-120572mRNA levels inrat lungs Values are means plusmn SEM (n = 4) p lt 0001 compared with saline group $$ p lt 001 compared with MCT group


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Figure 6 Analysis of the percentage of PCNA-positive cells in the medial wall of pulmonary arteries (a) Immunohistochemical staining ofPCNA of pulmonary arteries (scale bar = 50 120583m) (b)The percentage of the PCNA-positive cells in the total smoothmuscle cells in the medialwall of pulmonary arteries Values are means plusmn SEM (n = 5) p lt 001 compared with saline group $$ p lt 001 compared with MCT group

lt 005) (Figure 7) Moreover in Western blotting analysishypoxia elevated the PCNA and p-Akt protein expressionsin the cultured PASMCs [PCNA F(4 15) = 760 p lt 001Normoxia vs Hypoxia p lt 001 p-Akt F(4 15) = 679 p lt001 normoxia group vs hypoxia group p lt 001] but did notsignificantly affect the total Akt expressions while again ISLtreatment suppressed these increments (PCNA Hypoxia vsHypoxia+ISL 10 120583M p lt 001 Hypoxia vs Hypoxia+ISL 30120583M p lt 001 Hypoxia vs Hypoxia+ISL 100 120583M p lt 001 p-Akt Hypoxia vs Hypoxia+ISL 10 120583M p lt 005 Hypoxia vsHypoxia+ISL 30 120583M p lt 005 Hypoxia vs Hypoxia+ISL 100120583M p lt 005) (Figure 8)

4 Discussion

In this study when tested at 28 days after a single doseof MCT there were significant increases in pulmonaryartery pressure and structural remodeling of pulmonaryarteries along with exacerbated right ventricular hypertro-phy in the MCT-challenged rats marking the successfulestablishment of MCT-induced PH However in this studyoral treatment with both doses of ISL (10 mgkgd and 30mgkgd) prevented these pathophysiological changes Inaddition ISL inhibited MCT-induced increases in serumlevels and pulmonary gene expressions of IL-6 and TNF-120572


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Figure 7 Effects of ISL on hypoxia-induced PASMCs proliferationValues are means plusmn SEM (n = 5) p lt 005 p lt 001 and p lt0001 compared with normoxia group $ p lt 005 $$ p lt 001 and $$$

p lt 0001 compared with hypoxia group p lt 005 compared withHypoxia+ISL 100 120583M group

Moreover in the in vitro experiment ISL dose-dependentlyinhibited hypoxia-induced increases of PASMCs prolifer-ation and suppressed hypoxia-induced PCNA and p-Aktprotein expressionsThese results collectively suggest that ISLproduces therapeutic effects on MCT-induced PH and thatthe effects are mediated through its anti-inflammatory andantiproliferative actions

In the present study we have found both doses ofISL reduced MCT-induced elevation of the RVSP which isconsistent with the previous founding that ISL attenuatedchronic hypoxia-induced pulmonary hypertension [23]Thiseffect appears to be very important because the increasedpulmonary artery pressure is not only a typical resultingpathophysiology of PH but also an important causative factorfor PH development Increased pulmonary artery pressureevokes adjustment mechanisms in the body which gradu-ally lead to pathological changes in the cardiopulmonarysystem Evidence has shown that ISL directly relaxes rataorta smooth muscle and guinea-pig tracheal smooth muscle[24 25] therefore the inhibitory effect of ISL on the RVSPmay be an initial mechanism that underlies the effects ofISL on the structural remodeling of pulmonary arteriesand right ventricular hypertrophy in the MCT-induced PHrats

Right ventricular hypertrophy is universally measuredby the Fulton index In the present study a single dose ofMCT resulted in a significant increase in the Fulton indicesindicating the occurrence of right ventricular hypertrophyhowever this was reversed by treatment with both doses ofISL Moreover in the present study MCT administrationinduced pulmonary artery remodeling manifested by thesignificant increases in the WT and WA of pulmonaryarteries whichwere also inhibited by both doses of ISLTheseeffects conjunction with the effect of ISL on the RVSP suggest

that ISL can attenuate all the three major pathophysiologiesof PH induced by MCT in rats

An excessively increased inflammatory state induced byadverse stimuli such as hypoxia and MCT is critical inthe initiation and development of the pathophysiologicalchanges in PH particularly in MCT-induced PH rats [26]Dulce et al have reported MCT administration significantlyincreased the plasma IL-6 and TNF-120572 levels in rats [27]Tiago et al have found MCT markedly elevated the IL-6mRNA expressions in lung tissues which was associatedwith exacerbated hemodynamic and pathological changes inrat cardiopulmonary system [28] In agreement with thesereports in the present study the ELISA revealed the MCTenhanced serum levels of IL-6 and TNF-120572 and the qPCRanalysis found the MCT promoted pulmonary gene expres-sions of these two proinflammatory factors Modulation ofIL-6 and TNF-120572 can dampen or facilitate the process ofPH For example immunosuppressive steroids decrease IL-6levels and reduce pulmonary artery pressures [29] high levelsof TNF-120572 suppress the mRNA expression of the vasodilatingprostacyclin and increase pulmonary vascular reactivity [3031] and in the aforementioned studies done by Dulce etal and Tiago et al the pulmonary hemodynamics and theright ventricular hypertrophy in PH rats were amelioratedby antagonizing the overactivated IL-6 and TNF-120572 systemsSimilarly in the present study treatment with ISL blockedMCT-induced augmentation of IL-6 and TNF-120572 expressionsin the general circulation and lung tissues It has beenreported that ISL inhibits lipopolysaccharide-induced IL-6and TNF-120572 production in bone marrow-derived dendriticcells [32] and suppressed carbon tetrachloride-induced hep-atic TNF-120572 and cyclooxygenase-2 expressions [17]Thereforeour results suggest that ISL can inhibit the inflammatoryresponse in MCT-induced PH rats by which ISL improvesthe pathophysiologies of the PH

The pulmonary vascular wall consists of the fibrob-last cells (adventitia) smooth muscle cells (media) andendothelial cells (intima) Of these 3 cell types abnor-mal proliferation of medial smooth muscle cells is themain determinant of pulmonary vascular resistance andconsidered as a hallmark of pulmonary artery struc-tural remodeling [33] Systemically and locally producedinflammatory cytokines recruit a variety of intracellularsignaling pathways including protein kinase C mitogen-activated protein kinases phosphatidylinositol-3 kinases(PI3K) and Ca2+calmodulin-dependent protein kinases tocontrol PASMCs contractility differentiation and prolifera-tion [6 9] PCNA an acidic nuclear protein is expressed dur-ing the G1S phase in the cell cycle and used as an operationalmarker for cell proliferation Activation of PI3KAkt inducesproproliferative and antiapoptotic responses in PASMCs[34] In the present study the immunofluorescence stainingshowed that the MCT significantly increased the numbers ofPCNA-positive cells in the medial wall of pulmonary arteriesand theMTT analysis revealed 24-h hypoxia notably elevatedthe proliferation of the primary cultured PASMCs Further-more in the Western blotting analysis the hypoxia enhancedthe PCNA and p-Akt protein expressions However all thesechanges were inhibited by treatment with ISL in the present


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Figure 8 Effects of ISL on hypoxia-induced protein expressions of PCNA and p-Akt in PASMCs (a) PCNA protein expressions (relative tonormoxia group) (b) p-Akt protein expressions (relative to normoxia group) Values are means plusmn SEM (n = 4) p lt 001 compared withnormoxia group $ p lt 005 and $$ p lt 001 compared with hypoxia group

study Evidence has shown that ISL inhibits the proliferationof some cancerous and noncancerous cells including humanarterial smooth muscle cells [11 35] and this effect is medi-ated through the PI3KAkt signaling pathway [36]Thereforethese results indicate that ISL can inhibit hypoxia-inducedproliferation of PASMCs via modulation of the PI3KAktpathway which may be linked to the therapeutic effect of ISLon MCT-induced PH

It is worth noting that the doses of ISL used in the invivo or in the in vitro experiments in the present studyeffectively improved pathophysiological and biochemicalchanges related to PH but an expected dose-dependentfashion was not statistically observed in the checked effectsexcept for the effect on the proliferation of the PASMCsThis failure to observe a dose-dependent response in thepresent study may be mainly ascribed to the inadequatedose escalation Considering the importance of the dose-response relationship in the pharmacodynamics of a drugthis limitation must be improved in the future work

In summary in the present study 10 and 30 mgkgd ISLattenuated the pathophysiological changes in the hemody-namics pulmonary artery structural remodeling and rightventricle hypertrophy induced by MCT administration inrats In addition ISL inhibited MCT-induced inflammatory

response reversed abnormal PASMCs proliferation in in vivoand in vitro and moreover blocked hypoxia-induced Aktphosphorylation in vitro These observations suggest that ISLexerts therapeutic effects on MCT-induced PH via inhibitionof the inflammatory response and PASMCs proliferation andfurther provide an experimental basis for expanding thetherapeutic applications of ISL to treatment of chemicals-induced PH

Data Availability

The data supporting the conclusions of the present studyare properly analyzed and included in Results section andare available from the corresponding author upon reasonablerequest

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Haifeng Jin Lei Shen and Rongjie Zhao designed theresearch Haifeng Jin Yang Jiang Fengxia Du Linna Guo


Guan Wang Sang Chan Kim and Lei Shen conducted theexperiments Haifeng Jin and ChulWonLee did the statisticalanalysis Rongjie Zhao Lei Shen and Haifeng Jin wrote themanuscript

Acknowledgments

This work was supported by the Science Research Founda-tion of Qiqihar Medical University (no QY2016M-02) theHeilongjiang Postdoctoral Financial Assistance (no LBH-Z17213) China and the National Research Foundation ofKorea (no 2018R1A5A2025272) Korea

References

[1] MMHoeperHGhofrani EGrunigHKloseHOlschewskiand S Rosenkranz ldquoPulmonary hypertensionrdquo DeutschesArzteblatt International vol 114 no 5 pp 73ndash84 2017

[2] I S Bazan andWH Fares ldquoPulmonary hypertension diagnos-tic and therapeutic challengesrdquo Therapeutics and Clinical RiskManagement vol 11 pp 1221ndash1233 2015

[3] R T Zamanian K T Kudelko Y K Sung V de Jesus PerezJ Liu and E Spiekerkoetter ldquoCurrent clinical management ofpulmonary arterial hypertensionrdquoCirculation Research vol 115no 1 pp 131ndash147 2014

[4] N Hambly F Alawf and S Mehta ldquoPulmonary hypertensiondiagnostic approach andoptimalmanagementrdquoCanadianMed-ical Association Journal vol 188 no 11 pp 804ndash812 2016

[5] A Hussain M S Suleiman S J George M Loubani and AMorice ldquoHypoxic pulmonary vasoconstriction in humans taleor mythrdquoTheOpen CardiovascularMedicine Journal vol 11 pp1ndash13 2017

[6] S C Pugliese J M Poth M A Fini A Olschewski K CEl Kasmi and K R Stenmark ldquoThe role of inflammation inhypoxic pulmonary hypertension from cellular mechanismsto clinical phenotypesrdquo American Journal of Physiology-LungCellular and Molecular Physiology vol 308 no 3 pp L229ndashL252 2015

[7] E Soon A M Holmes C M Treacy et al ldquoElevated levelsof inflammatory cytokines predict survival in idiopathic andfamilial pulmonary arterial hypertensionrdquo Circulation vol 122no 9 pp 920ndash927 2010

[8] A Groth B Vrugt M Brock R Speich S Ulrich and L CHuber ldquoInflammatory cytokines in pulmonary hypertensionrdquoRespiratory Research vol 15 no 1 article no 47 2014

[9] M Rabinovitch C GuignabertMHumbert andMR NicollsldquoInflammation and immunity in the pathogenesis of pulmonaryarterial hypertensionrdquo Circulation Research vol 115 no 1 pp165ndash175 2014

[10] S Malenfant A-S Neyron R Paulin et al ldquoSignal transduc-tion in the development of pulmonary arterial hypertensionrdquoPulmonary Circulation vol 3 no 2 pp 278ndash293 2013

[11] F Peng Q Du C Peng et al ldquoA review the pharmacology ofisoliquiritigeninrdquo Phytotherapy Research vol 29 no 7 pp 969ndash977 2015

[12] R Gaur S Kumar P Trivedi et al ldquoLiquiritigenin derivativesand their hepatotoprotective activityrdquoNatural Product Commu-nications (NPC) vol 5 no 8 pp 1243ndash1246 2010

[13] S C Kim S J Park J R Lee J C Seo C H Yang andS H Byun ldquoCytoprotective activity of Glycyrrhizae radix

extract against arsenite-induced cytotoxicityrdquo Evidence-BasedComplementary and Alternative Medicine vol 5 no 2 pp 165ndash171 2008

[14] E J Yang J S Min H Y Ku et al ldquoIsoliquiritigenin isolatedfrom Glycyrrhiza uralensis protects neuronal cells againstglutamate-induced mitochondrial dysfunctionrdquo Biochemicaland Biophysical Research Communications vol 421 no 4 pp658ndash664 2012

[15] S H Lee J Y Kim G S Seo Y-C Kim and D HSohn ldquoIsoliquiritigenin fromDalbergia odorifera up-regulatesanti-inflammatory heme oxygenase-1 expression in RAW2647macrophagesrdquo Inflammation Research vol 58 no 5 pp 257ndash262 2009

[16] G T Kwon H J Cho W Chung K Park A Moon and JH Park ldquoIsoliquiritigenin inhibits migration and invasion ofprostate cancer cells possible mediation by decreased JNKAP-1 signalingrdquoThe Journal of Nutritional Biochemistry vol 20 no9 pp 663ndash676 2009

[17] Z Zhao S M Park L Guan et al ldquoIsoliquiritigenin attenuatesoxidative hepatic damage induced by carbon tetrachloridewith or without buthionine sulfoximinerdquo Chemico-BiologicalInteractions vol 225 pp 13ndash20 2015

[18] X Zhang E D Yeung J Wang et al ldquoIsoliquiritigenin anatural anti-oxidant selectively inhibits the proliferation ofprostate cancer cellsrdquo Clinical Experimental Pharmacollogy ampPhysiology vol 37 no 8 pp 841ndash847 2010

[19] X Zhang P Zhu X Zhang et al ldquoNatural antioxidant-isoliquiritigenin ameliorates contractile dysfunction of hypoxiccardiomyocytes via AMPK signaling pathwayrdquo Mediators ofInflammation vol 2013 Article ID 390890 10 pages 2013

[20] F Du Q Gesang J Cao et al ldquoIsoliquiritigenin attenuatesatherogenesis in apolipoprotein E-deficientmicerdquo InternationalJournal of Molecular Sciences vol 17 no 11 p 1932 2016

[21] C Noguchi J Yang K Sakamoto et al ldquoInhibitory effects ofisoliquiritigenin and licorice extract on voltage-dependent K+currents in H9c2 cellsrdquo Journal of Pharmacological Sciences vol108 no 4 pp 439ndash445 2008

[22] H Jin M Liu X Zhang et al ldquoGrape seed procyanidinextract attenuates hypoxic pulmonary hypertension by inhibit-ing oxidative stress and pulmonary arterial smooth muscle cellsproliferationrdquo The Journal of Nutritional Biochemistry vol 36pp 81ndash88 2016

[23] S Zhang X Li L Yao et al ldquoEffects of isoliquiritigeninon pulmonary vascular remodeling in chronic hypoxia ratmodelrdquoActa Anatomica Sinica vol 49 no 4 pp 492ndash496 2018(Chinese)

[24] S Yu and S Kuo ldquoVasorelaxant effect of isoliquiritigenin anovel soluble guanylate cyclase activator in rat aortardquo BritishJournal of Pharmacology vol 114 no 8 pp 1587ndash1594 1995

[25] B Liu J Yang Q Wen and Y Li ldquoIsoliquiritigenin a flavonoidfrom licorice relaxes guinea-pig tracheal smooth muscle invitro and in vivo role of cGMPPKG pathwayrdquo EuropeanJournal of Pharmacology vol 587 no 1-3 pp 257ndash266 2008

[26] R Nogueira-Ferreira R Vitorino R Ferreira and THenriques-Coelho ldquoExploring the monocrotaline animalmodel for the study of pulmonary arterial hypertension anetwork approachrdquoPulmonary Pharmacology andTherapeuticsvol 35 pp 8ndash16 2015

[27] D Fontoura J Oliveira-Pinto M Tavares-Silva et al ldquoMyocar-dial and anti-inflammatory effects of chronic bosentan therapyin monocrotaline-induced pulmonary hypertensionrdquo Revista


Portuguesa de Cardiologia (English Edition) vol 33 no 4 pp213ndash222 2014

[28] T Henriques-Coelho S M Oliveira R S Moura et alldquoThymulin inhibits monocrotaline-induced pulmonary hyper-tension modulating interleukin-6 expression and suppressingp38 pathwayrdquo Endocrinology vol 149 no 9 pp 4367ndash43732008

[29] A Bhargava A Kumar N Yuan M H Gewitz and R MathewldquoMonocrotaline induces interleukin-6 mRNA expression in ratlungsrdquo Heart Disease vol 1 no 3 pp 126ndash132 1999

[30] A Itoh J Nishihira H Makita K Miyamoto E Yamaguchiand M Nishimura ldquoEffects of IL-1120573 TNF-120572 and macrophagemigration inhibitory factor on prostacyclin synthesis in ratpulmonary artery smooth muscle cellsrdquo Respirology vol 8 no4 pp 467ndash472 2003

[31] M Fujita J M Shannon C G Irvin et al ldquoOverexpression oftumor necrosis factor-120572 produces an increase in lung volumesand pulmonary hypertensionrdquo American Journal of Physiology-Lung Cellular andMolecular Physiology vol 280 no 1 pp L39ndashL49 2001

[32] W Li Y N Sun X T Yan et al ldquoFlavonoids from Astragalusmembranaceus and their inhibitory effects on LPS-stimulatedpro-inflammatory cytokine production in bone marrow-derived dendritic cellsrdquo Archives of Pharmacal Research vol 37no 2 pp 186ndash192 2014

[33] L A Shimoda and S S Laurie ldquoVascular remodeling inpulmonary hypertensionrdquo Journal of Molecular Medicine vol91 no 3 pp 297ndash309 2013

[34] C V Garat J T Crossno Jr T M Sullivan J E BReusch and D J Klemm ldquoInhibition of phosphatidylinositol3-kinaseakt signaling attenuates hypoxia-induced pulmonaryartery remodeling and suppresses CREB depletion in arterialsmooth muscle cellsrdquo Journal of Cardiovascular Pharmacologyvol 62 no 6 pp 539ndash548 2013

[35] T Chen S Deng and R Lin ldquoThe inhibitory effect of Isoliquir-itigenin on the proliferation of human arterial smooth musclecellrdquo BMC Pharmacology amp Toxicology vol 18 no 1 p 57 2017

[36] J Chen C Liu Q-Q Yang et al ldquoIsoliquiritigenin suppressesosteosarcoma U2OS Cell proliferation and invasion by regulat-ing the PI3KAkt signalling pathwayrdquo Chemotherapy vol 63no 3 pp 155ndash161 2018

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


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Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


resistance and a progressive increase in the artery pressurefollowed by pulmonary artery structural remodeling andright ventricular hypertrophy consequently results in collapseof respiratory and circulatory systems [5] Thus impeding orreversing the pathophysiological progress of MCT-inducedPH is a promising approach to treat chemicals-inducedPH

So far the molecular mechanisms of PH are not fullyunderstood but clinical and preclinical evidences showinflammation is a key player in mediating the pathophys-iological process of PH [6] In PH patients there are sig-nificantly increased circulating levels of proinflammatorycytokines such as interleukin-6 (IL-6) and tumor necrosisfactor-120572 (TNF-120572) and the high levels of these factors areassociated with the increased risk of death in the patients[7] Likewise in animal models the degree of perivascularinflammation positively correlates with pulmonary vascularremodeling [8] IL-6 and TNF-120572 can induce the proliferationmigration and differentiation of pulmonary vascular cellsharmfully promoting vascular remodeling [9] In additionproinflammatory factors directly disrupt endothelial mem-brane integrity and enhance hyperplasia in pulmonary arterysmooth muscle cells (PASMCs) and the latter is anotherkey player in the progress of PH [9] It is turned out thatthe excessive PASMCs proliferation is mainly responsiblefor the pulmonary vascular medial hyperplasia elevatedvascular resistance and right ventricular hypertrophy [110] Therefore dampening the inflammatory response andinhibiting the abnormal proliferation of PASMCs can providean effective way to halt the development of PH pathology

Glycyrrhizae radix (G radix licorice) is most frequentlyused in traditional Chinese medicine to treat a variety ofpathological states due to its superb detoxification effects [11]Isoliquiritigenin (ISL) is an important bioactive ingredientin G radix mediates the therapeutic effects of G radix andpossesses a wide range of pharmacological properties such asanti-inflammatory antioxidant antitumor and immunoreg-ulatory activities [12ndash16] ISL inhibits carbon tetrachloride-induced hepatic damage and the proliferation of humanprostate cancer LNCaP cells through its antioxidant and anti-inflammatory actions [17 18] ISL improves atherosclerosisand hypoxia-induced cardiomyocytes contractile dysfunc-tion by decreasing the levels of reactive oxygen species(ROS) and proinflammatory factors [19ndash21] These findingscollectively indicate that ISL may have a therapeutic effect onPH Indeed in a recent study we have found ISL alleviatedhypoxic PH through inhibition of oxidative stress indicatinga potential application of ISL to treat PH As mentionedabove PH presents common pathophysiologies in its finalphase but involves different etiologies that complicate itspharmacotherapies Therefore to support the therapeuticeffect of ISL on hypoxic PH and further expand the ther-apeutic applications of ISL in treating PH in the presentstudy we evaluated the effects of ISL on chemicals-inducedPH in rats For this we first established an MCT-inducedPH rat model where exacerbated inflammation and excessiveproliferation of PASMCs play a key role and then by usingthis model examined the effect of ISL on rat MCT-inducedPH and investigated the relevant mechanisms

2 Materials and Methods

21 Reagents and Antibodies ISL gt 99 purity was providedby Shanghai Yuanye Biotechnology Co Ltd (ShanghaiChina) Monocrotaline (MCT) was purchased from SigmaSigmandashAldrich Inc (St LouisMOUSA) Fetal bovine serum(FBS) Dulbeccorsquos modified Eaglersquos medium (DMEM) Trizolreagent and trypsin solution were obtained from GibcoLaboratories (Grand Island NY USA) Antibodies against120572-smooth muscle actin (120572-SMA) proliferating cell nuclearantigen (PCNA) Akt phospho-Akt (p-Akt) and GAPDHwere purchased from Cell Signaling Technology (BeverlyMA USA)

22 Animals and Experimental Design Eight-week-old maleSpraguendashDawley rats (body weight 220ndash250 g) were obtainedfrom the Animal Center of Qiqihar Medical UniversityChina The rats were caged (3 rats per cage) and suppliedby filtered pathogen-free air unlimited commercial rat chowand water at a temperature between 22 and 24∘C with12 h lightdark cycles and relative humidity of 50 Allanimal experiments were approved by the Animal Careand Use Committee of the Qiqihar Medical University andconducted in accordance with the National Institutes ofHealth guidelines concerning the care and use of labora-tory animals After 7 days of acclimatization period therats were randomly divided into 4 groups saline-treatedgroup (n = 8) MCT-treated control group (n = 8) MCT+ 10 mgkgd ISL (MCT+ISL10) group (n = 8) and MCT+ 30 mgkgd ISL (MCT+ISL30) group (n = 8) For theestablishment of an MCT-induced PH rat model rats weregiven a single intraperitoneal injection of MCT (50 mgkg)which was dissolved in 1N hydrochloric acid diluted insterile saline and adjusted to pH 74 with 1N sodiumhydroxide Thereafter the rats stayed in their home cages for28 days and received ISL treatments ISL was dissolved in5 Tween-80 and intragastrically administered to the ratsat the dose of 10 mgkgd or 30 mgkgd once a day for28 days while an equal volume of 5 Tween-80 was givento the rats in the control groups Thirty minutes after thefinal dose of ISL the rats were subjected to hemodynamicanalysis and then blood samples and lungs of the ratswere collected for further biochemical and morphologicassays

23 Hemodynamic Assay and Assessment of the Right Ven-tricular Hypertrophy The rats were anesthetized with anintraperitoneal injection of 75 mLkg 48 tribromoethanoland a polyethylene catheter was inserted into the rightventricle through the right jugular vein The right ventriclesystolic pressure (RVSP) was measured via a fluid-filledpressure transducer and recorded using PowerLab Software(ADInstruments Castle Hill Australia) For assessment ofright ventricular hypertrophy the left ventricular wall plusseptum and the right ventricular wall were harvested andthe weight ratio of the right ventricular wallleft ventricularwall plus septum (Fulton index) was calculated to quantifythe right ventricular hypertrophy










3 Results




0

10

20

30

40

1 2 3 4

1 Saline

2 MCT

3 MCT+ISL10

4 MCT+ISL30

RVSP

(mm

Hg)

$$$

(a)

0

01

02

03

04

1 2 3 4

1 Saline

2 MCT

3 MCT+ISL10

4 MCT+ISL30

Fulto

n in

dex

$ $

(b)



0

10

20

30

40

50

60

1 2 3 4

WT

of p

ulm

onar

y ar

terie

s (

)

$$

(b)

0

20

40

60

80

100

1 2 3 4

WA

of p

ulm

onar

y ar

terie

s (

)

$$

(c)







0

200

400

600

800

1000

1 2 3 4

OD

val

ue (

-SM

A)

$ $

(b)


0100200300400500600

1 2 3 4

Seru

m IL

-6 le

vel (

pgm

L)

$$$$

1 Saline2 MCT


(a)

0

20

40

60

80

1 2 3 4Seru

m T

NF-

le

vel (

pgm

L)

$ $

1 Saline2 MCT


(b)








0100200300400500600

1 2 3 4Lu

ng IL

-6 m

RNA

leve

l (

Sa

line g

roup

)

$$$$

1 Saline

2 MCT

3 MCT+ISL10

4 MCT+ISL30(a)

0

100

200

300

400

500

1 2 3 4

Lung

TN

F-

mRN

A le

vel

(

Salin

e gro

up)

$$ $$

1 Saline

2 MCT

3 MCT+ISL10

4 MCT+ISL30(b)



0

10

20

30

40

50

1 2 3 4PCN

A-po

sitiv

e cel

ls l

ocal

cells

()

$$$$

(b)



4 Discussion



00

02

04

06

08

10

1 2 3 4 5

OD

val

ue

$$

$$$

$












0

50

100

150

200

250

1 2 3 4 50

50

100

150

200

250

1 2 3 4 5

PCNA

p-AKT

total AKT

GAPDH


4 Hypoxia+ISL 30 M

5 Hypoxia+ISL 100 M

PCN

A ex

pres

sion

( o

f Nor

mox

ia g

roup

)

p-A

KT ex

pres

sion

( o

f Nor

mox

ia g

roup

)

(a) (b)

$$$$

$$

$

$ $






Data Availability








Acknowledgments


References












































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




























3 Results




0

10

20

30

40

1 2 3 4

1 Saline

2 MCT

3 MCT+ISL10

4 MCT+ISL30

RVSP

(mm

Hg)

$$$

(a)

0

01

02

03

04

1 2 3 4

1 Saline

2 MCT

3 MCT+ISL10

4 MCT+ISL30

Fulto

n in

dex

$ $

(b)



0

10

20

30

40

50

60

1 2 3 4

WT

of p

ulm

onar

y ar

terie

s (

)

$$

(b)

0

20

40

60

80

100

1 2 3 4

WA

of p

ulm

onar

y ar

terie

s (

)

$$

(c)







0

200

400

600

800

1000

1 2 3 4

OD

val

ue (

-SM

A)

$ $

(b)


0100200300400500600

1 2 3 4

Seru

m IL

-6 le

vel (

pgm

L)

$$$$

1 Saline2 MCT


(a)

0

20

40

60

80

1 2 3 4Seru

m T

NF-

le

vel (

pgm

L)

$ $

1 Saline2 MCT


(b)








0100200300400500600

1 2 3 4Lu

ng IL

-6 m

RNA

leve

l (

Sa

line g

roup

)

$$$$

1 Saline

2 MCT

3 MCT+ISL10

4 MCT+ISL30(a)

0

100

200

300

400

500

1 2 3 4

Lung

TN

F-

mRN

A le

vel

(

Salin

e gro

up)

$$ $$

1 Saline

2 MCT

3 MCT+ISL10

4 MCT+ISL30(b)



0

10

20

30

40

50

1 2 3 4PCN

A-po

sitiv

e cel

ls l

ocal

cells

()

$$$$

(b)



4 Discussion



00

02

04

06

08

10

1 2 3 4 5

OD

val

ue

$$

$$$

$












0

50

100

150

200

250

1 2 3 4 50

50

100

150

200

250

1 2 3 4 5

PCNA

p-AKT

total AKT

GAPDH


4 Hypoxia+ISL 30 M

5 Hypoxia+ISL 100 M

PCN

A ex

pres

sion

( o

f Nor

mox

ia g

roup

)

p-A

KT ex

pres

sion

( o

f Nor

mox

ia g

roup

)

(a) (b)

$$$$

$$

$

$ $






Data Availability








Acknowledgments


References












































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















0

10

20

30

40

1 2 3 4

1 Saline

2 MCT

3 MCT+ISL10

4 MCT+ISL30

RVSP

(mm

Hg)

$$$

(a)

0

01

02

03

04

1 2 3 4

1 Saline

2 MCT

3 MCT+ISL10

4 MCT+ISL30

Fulto

n in

dex

$ $

(b)



0

10

20

30

40

50

60

1 2 3 4

WT

of p

ulm

onar

y ar

terie

s (

)

$$

(b)

0

20

40

60

80

100

1 2 3 4

WA

of p

ulm

onar

y ar

terie

s (

)

$$

(c)







0

200

400

600

800

1000

1 2 3 4

OD

val

ue (

-SM

A)

$ $

(b)


0100200300400500600

1 2 3 4

Seru

m IL

-6 le

vel (

pgm

L)

$$$$

1 Saline2 MCT


(a)

0

20

40

60

80

1 2 3 4Seru

m T

NF-

le

vel (

pgm

L)

$ $

1 Saline2 MCT


(b)








0100200300400500600

1 2 3 4Lu

ng IL

-6 m

RNA

leve

l (

Sa

line g

roup

)

$$$$

1 Saline

2 MCT

3 MCT+ISL10

4 MCT+ISL30(a)

0

100

200

300

400

500

1 2 3 4

Lung

TN

F-

mRN

A le

vel

(

Salin

e gro

up)

$$ $$

1 Saline

2 MCT

3 MCT+ISL10

4 MCT+ISL30(b)



0

10

20

30

40

50

1 2 3 4PCN

A-po

sitiv

e cel

ls l

ocal

cells

()

$$$$

(b)



4 Discussion



00

02

04

06

08

10

1 2 3 4 5

OD

val

ue

$$

$$$

$












0

50

100

150

200

250

1 2 3 4 50

50

100

150

200

250

1 2 3 4 5

PCNA

p-AKT

total AKT

GAPDH


4 Hypoxia+ISL 30 M

5 Hypoxia+ISL 100 M

PCN

A ex

pres

sion

( o

f Nor

mox

ia g

roup

)

p-A

KT ex

pres

sion

( o

f Nor

mox

ia g

roup

)

(a) (b)

$$$$

$$

$

$ $






Data Availability








Acknowledgments


References












































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















0

200

400

600

800

1000

1 2 3 4

OD

val

ue (

-SM

A)

$ $

(b)


0100200300400500600

1 2 3 4

Seru

m IL

-6 le

vel (

pgm

L)

$$$$

1 Saline2 MCT


(a)

0

20

40

60

80

1 2 3 4Seru

m T

NF-

le

vel (

pgm

L)

$ $

1 Saline2 MCT


(b)








0100200300400500600

1 2 3 4Lu

ng IL

-6 m

RNA

leve

l (

Sa

line g

roup

)

$$$$

1 Saline

2 MCT

3 MCT+ISL10

4 MCT+ISL30(a)

0

100

200

300

400

500

1 2 3 4

Lung

TN

F-

mRN

A le

vel

(

Salin

e gro

up)

$$ $$

1 Saline

2 MCT

3 MCT+ISL10

4 MCT+ISL30(b)



0

10

20

30

40

50

1 2 3 4PCN

A-po

sitiv

e cel

ls l

ocal

cells

()

$$$$

(b)



4 Discussion



00

02

04

06

08

10

1 2 3 4 5

OD

val

ue

$$

$$$

$












0

50

100

150

200

250

1 2 3 4 50

50

100

150

200

250

1 2 3 4 5

PCNA

p-AKT

total AKT

GAPDH


4 Hypoxia+ISL 30 M

5 Hypoxia+ISL 100 M

PCN

A ex

pres

sion

( o

f Nor

mox

ia g

roup

)

p-A

KT ex

pres

sion

( o

f Nor

mox

ia g

roup

)

(a) (b)

$$$$

$$

$

$ $






Data Availability








Acknowledgments


References












































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















0100200300400500600

1 2 3 4Lu

ng IL

-6 m

RNA

leve

l (

Sa

line g

roup

)

$$$$

1 Saline

2 MCT

3 MCT+ISL10

4 MCT+ISL30(a)

0

100

200

300

400

500

1 2 3 4

Lung

TN

F-

mRN

A le

vel

(

Salin

e gro

up)

$$ $$

1 Saline

2 MCT

3 MCT+ISL10

4 MCT+ISL30(b)



0

10

20

30

40

50

1 2 3 4PCN

A-po

sitiv

e cel

ls l

ocal

cells

()

$$$$

(b)



4 Discussion



00

02

04

06

08

10

1 2 3 4 5

OD

val

ue

$$

$$$

$












0

50

100

150

200

250

1 2 3 4 50

50

100

150

200

250

1 2 3 4 5

PCNA

p-AKT

total AKT

GAPDH


4 Hypoxia+ISL 30 M

5 Hypoxia+ISL 100 M

PCN

A ex

pres

sion

( o

f Nor

mox

ia g

roup

)

p-A

KT ex

pres

sion

( o

f Nor

mox

ia g

roup

)

(a) (b)

$$$$

$$

$

$ $






Data Availability








Acknowledgments


References












































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















00

02

04

06

08

10

1 2 3 4 5

OD

val

ue

$$

$$$

$












0

50

100

150

200

250

1 2 3 4 50

50

100

150

200

250

1 2 3 4 5

PCNA

p-AKT

total AKT

GAPDH


4 Hypoxia+ISL 30 M

5 Hypoxia+ISL 100 M

PCN

A ex

pres

sion

( o

f Nor

mox

ia g

roup

)

p-A

KT ex

pres

sion

( o

f Nor

mox

ia g

roup

)

(a) (b)

$$$$

$$

$

$ $






Data Availability








Acknowledgments


References












































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















0

50

100

150

200

250

1 2 3 4 50

50

100

150

200

250

1 2 3 4 5

PCNA

p-AKT

total AKT

GAPDH


4 Hypoxia+ISL 30 M

5 Hypoxia+ISL 100 M

PCN

A ex

pres

sion

( o

f Nor

mox

ia g

roup

)

p-A

KT ex

pres

sion

( o

f Nor

mox

ia g

roup

)

(a) (b)

$$$$

$$

$

$ $






Data Availability








Acknowledgments


References












































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















Acknowledgments


References












































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of


































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















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