Effects Of Corosolic Acid On Post-myocardial Infarction Cardiac Remodeling In Mice

Objective:The aims of this study were to determine whether administration of corosolic acid(CRA)reduces cardiac fibrosis after a myocardial infarction(MI)and to elucidate the underlying therapeutic mechanisms of CRA.C57 BL/6J mice were subjected to either sham surgery or permanent ligation of the left anterior descending artery,The effects of CRA on cardiac function and myocardial fibrosis in mice after infarction,as well as the changes of oxidative stress and macrophages were evaluated by animal experiments,and the possible mechanism of CRA in vitro cell experiments was further explored,so as to find new therapeutic strategies for improving the fibrosis after infarction.Methods: Male C57 BL/6J mice(23.5-27.5g,age 8 weeks)were allowed to acclimatize to the laboratory environment for at least one week,then randomly assigned to control(phosphate-buffered saline-treated)and CRA-treated groups(10mg/kg,20mg/kg).After 14 days of treatment,the mice were subjected to either sham surgery(Sham)or MI by left anterior descending coronary artery ligation.The four groups were named as Sham group,MI group,MI+CRA 10 group,MI+CRA 20 group,respectively.Four weeks after the surgery,fractional shortening(FS),ejection fraction(EF)and hemodynamic measurement of Stroke Volume(SV)and Cardiac output(CO)were performed to evaluate the changes in Cardiac function of mice in each group.Hematoxylin-eosin(HE)staining was performed on paraffin sections to observe the morphological changes of heart tissue,and picrosirius red(PSR)staining was performed to observe the collagen deposition after heart infarction in mice.Heme oxygenase 1(HO-1)and NADPH oxidase 4(Nox4)were observed by immunohistochemical staining to evaluate the oxidative stress level;differences in the number of macrophages in the border area and infarct area were observed by CD68 staining to evaluate the inflammatory level;and cell apoptosis in myocardial tissue was assessed by TUNEL staining.To examine the relative m RNA expression of ANP,BNP,α-MHC,β-MHC,CTGF,Collagen Ⅰα,Collagen Ⅲα,Fibronectin,Nox4,Nox2,HO-1,total RNA was collected using TRIzol reagent and the c DNA was used as a template for reverse transcription polymerase chain reaction(RT-PCR)amplification and detection of the gene expression level.Transforming growth factor-1β(TGF-1β)and Smads signal pathway were detected by Western Blotting to assess fibrosis,levels of oxidative stress were assessed by detecting HO-1,Nox2 and Nox4.MCP-1 and CCR2 were used to assess the infiltration of macrophage.Different concentrations of CRA(1 μM,5 μM and 10 μM)were added to the H9C2 and the cells were cultured in D-Hanks solution in a modular incubator chamber with 1% O2,5% CO2 and 94% N2 for 24 h(hypoxia 24 h),western Blotting was used to detect the expression levels of HO-1,Nox 2,Nox 4 and apoptosis-related proteins Bcl-2 and C-caspase 3.In 24-well plate,2,7-dichlorodihydrofluorescein diacetate(DCFH-DA)staining was used to observe the level of reactive oxygen species(ROS)and TUNEL fluorescence staining was used to observe the apoptotic status;Fibroblast were isolated from neonatal rats,different concentrations of CRA(1 μM,5 μM and 10 μM)and Angiotensin Ⅱ(Ang Ⅱ,100nmol/L)were added to the medium.RT-PCR examined the relative m RNA expression of α-SMA,Collagen Ⅰα and CTGF after Ang Ⅱ stimulation for 24 hours.Results: Statistics of mortality show that CRA could increase the lifetime of mice after myocardial infarction(Figure 1A).More live myocardial cells and reduced infarct size were found in the border zone by HE(Figure 1 B).Echocardiography data analysis showed that fractional shortening(%)and ejection fractional(%)increased significantly after CRA treatment(Figure 1C)(P<0.05),besides,stroke work and cardiac output also increased in CRA treatment groups(Figure 1D)(P<0.05).To explore whether CRA can regulate ECM after MI,PSR straining was performed and results show that CRA limits the level of fibrosis caused by MI.To elucidate the mechanism of diminished collagenous deposition,the RNA levels of cardiac hypertrophy(ANP,BNP,α-MHC,β-MHC)and cardiac fibrosis(CTGF,Collagen Ⅰα,Collagen Ⅲα,FN)were tested.The LV levels of ANP,BNP,β-MHC,CTGF,Collagen Ⅰα,Collagen Ⅲα,and FN were increased in MI group,and these responses were drastically attenuated while CRA administration(Figure 2B)(P<0.05).To examine the molecular mechanisms of CRA in collagen synthesis,we assessed the effect of CRA on TGF-β1/smads cascade activation.The increased levels of TGF-β1,P-smad2 and P-smad3 were attenuated in CRA-pretreated mice after MI surgery(Figure 2 C,D)(P<0.05).To prove that CRA could reduce fibrosis,we examined the effect of CRA on fibroblasts stimulated by Angiotensin Ⅱ.In the Figure 2E,CRA reduced the m RNA expression of α-SMA,Collagen Ⅰα and CTGF,these results indicated that CRA could improve Ang Ⅱ-induced fibrosis.To explore whether CRA regulate the level of cardiac oxidative stress,western blot and RT-PCR were performed and results showed that MI increased the expressions of Nox2 and Nox4,decreased the HO-1 expression,while the levels of Nox2 and Nox4 markly decreased after CRA treatment,in addition,the expression of HO-1 increased in medication group(Figure 3 A,B)(P<0.05),and the same in transcriptional levels(Figure 3 C)(P<0.05).The results of immunohistochemistry for HO-1 and Nox4 showed that HO-1 increasedin MI group but more significantly increased when CRA pretreated.,and the straining of Nox4 was consistent with the results in western bolt and RT-PCR(Figure 3 D).To determine how CRA attenuated oxidative stress,H9C2 cells were exposed to hypoxia(1% O2)for 24 h with or without CRA added.Western blot showd that Nox2 and Nox4 were inhibited by CRA while hypoxia increased the level of them both(Figure 4 A,B)(P<0.05).In the meantime,CRA increased the HO-1 expression(P<0.05).Besides,DCFH straining showd the more ROS in H9C2 cells while hypoxia and this could be blocked by CRA treatment partly(Figure 4 C).The immune system plays an important role in healing response after MI,especially macrophages.We explored the infiltration of macrophage in myocardium.Western blot showed that Mcp-1 and CCR2 were decreased by CRA treatment which increased by MI(Figure 5 A,B)(P<0.05).Besides,the results of immunohistochemistry for CD68 showed that for CRA-pretreated groups,there were less macrophages in both infarcrus zone and border zone than in MI group(Figure 5 C).TUNEL straining showed that a large amount of the TUNEL-positive cells were distributed in the MI group,but the apoptotic cells number significantly decreased in CRA-pretreated groups(Figure 6 A).In addition,in vitro experiment,CRA decreased the apoptosis level caused by hypoxia(Figure 6 B).The results of western blot showed that compared to the hypoxia group,C-caspase3 protein expression was significantly decreased,Bcl-2 protein was increased in CRA-pretreated groups(Figure 6 C,D)(P<0.05).Conclusion: CRA can effectively inhibit the development of fibrosis after myocardial infarction and improve cardiac function.This study demonstrated that CRA can inhibit Nox2 and Nox4 oxidative stress pathways by activating HO-1,and reduce macrophage infiltration and apoptosis,so as to exert the function of inhibiting post-infarction fibrosis and protecting the heart.