Baoyuan Decoction Regulates The Mechanism Of P38 MAPK-CRYAB Pathway Inhibiting Cardiomyocyte Apoptosis And Preventing HF After AMI

Aim:Heart failure(HF)post acute myocardial infarction(AMI),because of its high morbidity and mortality,has become one of the most serious cardiovascular diseases in worldwide.However,the therapeutic effect is not yet satisfactory.In this study,we aimed to identify the effects and the underlying mechanisms of Baoyuan Decoction(BYD)on oxidative stress(OS)-induced apoptosis in HF post-AMI both in vitro and in vivo.Methods:1.The HF post-AMI model induced by left anterior descending(LAD)ligation was used to screen the effect of the different extraction processes of BYD on improving heart function.Different extraction processes of BYD included:lyophilized powder of whole water decoction(Water decoction),decoction with 65%ethanol precipitation and removal of sugar(65%ethanol),decoction filtered by macroporous resin water elution site(H2O),decoction filtered by macroporous resin 70%ethanol elution site(70%ethanol),decoction with 65%ethanol precipitation and sugar portion(Sugar).After a week of continuous gavage,cardiac function was evaluated by echocardiography;serum biochemical assay was used to determine the levels of lactate dehydrogenase(LDH),creatine kinase(CK),creatine kinase-MB(CK-MB)in the serum;Haematoxylin-eosin(HE)and Masson staining were used to detect inflammatory infiltration and fibrosis.With the indicators of heart function and blood biochemical indicators,the best effective extraction process of BYD on HF post-AMI was determined.2.Animal model of HF post-AMI was established by LAD ligation and administered continuously for one week;echocardiography was used to evaluate the cardiac function of each group of rats;the levels of CK-MB,LDH,superoxide dismutase(SOD)and malondialdehyde(MDA)in serum were detected;HE and TUNEL staining were used to detect inflammatory infiltration and apoptosis;results of above were collected to evaluate the efficacy of BYD on the treatment of HF after AMI.3.Western blot(WB)was used to detect the expressions of B-cell lymphoma-2(Bcl-2)and Bcl-associated protein X(Bax),caspase 3,caspase 9,and other apoptosis-related proteins around the ischemic marginal zone.At the same time,the CM-induced H9C2 cells injury model was established,the viability of H9C2 cell was detected by CCK-8 method;the apoptosis of H9C2 cardiomyocytes was measured by Hoechst 33258 staining method;and the expressions of Bax,caspase 3,caspase 9 and other proteins were detected by WB.The above results illustrate the mechanism of BYD in inhibiting cardiomyocyte apoptosis.In addition,LPS-induced RAW264.7 macrophages injury model was established,CCK-8 method was used to detect viability of the RAW264.7 macrophages and Griess method was used to detect the levels of NO,and the reactive oxygen species(ROS)kit was used to detect the content of ROS in each group of cells;these results were collected to explain the mechanism of BYD in inhibiting oxidative stress-induced cardiomyocyte apoptosis.4.Combined with in vivo and in vitro experiments,WB method and immunohistochemistry method were used to qualitatively and quantitatively evaluate the expressions of regulated proteins such as P38 mitogen-activated protein kinase(MAPK),mitogen-activated protein kinase kinase 6(MKK6),mitogen-activated protein kinase-activated protein kinase 2(MAPKAPK2)and ?B-crystallin(CRYAB)in rat myocardial infarction marginal area.At the same time,in CM induced H9C2 cell,the positive control was induced by the inhibitor of P38 MAPK SB203580;the apoptosis of H9C2 cell in each group was determined by Hoechst 33258 staining;the expressions of related proteins in P38 MAPK-CRYAB pathway were detected by WB.This part was aimed to further explain the role of BYD in the regulation of oxidative stress-induced apoptosis via P38 MAPK-CRYAB pathway.Results:1.Efficacy of BYD of different extraction processes in the treatment of HF after AMI in SD ratsThe results of echocardiography showed that the ejection fraction(EF)and fractional shortening(FS)of rats with HF decreased significantly(P<0.001),suggesting that the HF post-AMI model was successfully induced.After different extraction processes of BYD treatment,EF and FS values showed different changes in different groups,among them,the EF and FS values were increased most significantly in the 65%ethanol group(P<0.05).In the results of blood biochemical tests,it was also shown that the levels of LDH and CK-MB in serum of 65%ethanol group were decreased most significantly(P<0.001,P<0.01,P<0.05).Besides,HE and Masson staining results showed that,in 65%ethanol group,the infiltration of inflammatory cells and fibrosis were significantly decreased.Therefore,the above experimental results suggested that the most effective extraction process on HF post-AMI of BYD is decoctions with 65%ethanol precipitation and removal of sugar.2.Pharmacodynamic study of BYD on HF after AMI in ratsThe values of EF and FS were significantly increased after BYD treatment with different doses of BYD(P<0.001 as an example),suggesting that different doses of BYD can improve heart function in rats with HF post-AMI.In addition,different doses of BYD treatment can improve the levels of left ventricular internal diameter at end-systole(LVIDs)(P<0.001),suggesting that BYD,to a certain extent,can inhibit ventricular remodeling induced by AMI post HF.Serum biochemical assay results showed that BYD could significantly reduce the release of CK-MB and LDH(P<0.001 as an example).In addition,BYD can also significantly increase levels of SOD and reduce levels of MDA(P<0.001 as an example),suggesting that BYD can inhibit ischemia-induced oxidative stress injury.HE and TUNEL staining results showed that BYD could inhibit the infiltration of inflammatory cells and apoptosis.The above results suggested that BYD had the effects of inhibiting oxidative stress,inhibiting myocardial cell apoptosis and improving cardiac function.At the same time,the positive drug Ginaton Tablets also showed good improvement of heart function,redutce inflammatory cell infiltration,inhibit oxidative stress induction and inhibit cardiomyocyte apoptosis.3.Study on pharmacological effects of BYD on OS induced apoptosisIn the infarct marginal myocardium,WB results showed that BYD could significantly down-regulate the expressions of Bax(P<0.01),cleaved-caspase 3(P<0.01),and cleaved-caspase 9(P<0.05)and up-regulate the expression of Bcl-2(P<0.001).At the same time,in vitro,Hoechst 33258 stainning results showed that BYD could inhibit the apoptosis induced by CM(P<0.01),and WB results show that different concentrations of BYD can down-regulate the expression of Bax,cleaved-caspase 3 and cleaved-caspase 9 in H9C2 and inhibit apoptosis(P<0.001 as an example).In addition,in LPS-induced RAW264.7 cell injury model,BYD can inhibit NO and ROS produced by macrophages and then inhibit oxidative stress-induced cardiomyocyte apoptosis(P<0.001 as an example).4.BYD attenuated OS induced apoptosis to prevents HF After AMI via P38 MAPK-CRYAB pathwayActivation of P38 MAPK-CRYAB pathway is a key pathway to regulate oxidative stress-induced cardiomyocyte apoptosis.In the infarct marginal tissue,WB results showed that BYD could activate the expressions of p-MEKK6(P<0.01),p-P38 MAPK(P<0.001)and p-MAPKAPK2(P<0.05)in the P38 MAPK cascade,and significantly up-regulate the expression of CRYAB(P<0.05),suggesting that BYD may activate P38 MAPK-CRYAB pathway to inhibit cardiomyocyte apoptosis.In the CM-induced H9C2 apoptosis model,SB203580,an inhibitor of P38 MAPK,was introduced.Hoechst 33258 staining results suggest that SB203580 can promote the apoptosis induced by CM(P<0.01)and reverse the inhibitory effect of BYD on apoptosis(P<0.05).In addition,BYD can promote the expression of p-P38 MAPK and p-CRYAB in cardiomyocytes induced by CM(P<0.001),and after intervention with SB203580,it can significantly reverse the up-regulation of p-P38 MAPK and p-CRYAB protein expression by BYD(P<0.001).In summary,BYD inhibition of oxidative stress-induced cardiomyocyte apoptosis is mainly achieved through the P38 MAPK-CRYAB pathway.Conclusions:BYD prepared by 65%ethanol precipitation and removal of sugar can effectively improve the acute decline of left ventricular function caused by HF post-AMI;its mechanism is closely related to inhibition of myocardial cell apoptosis induced by oxidative stress injury.Specifically,BYD can reduce the release of MDA in the circulation,reduce the oxidative stress damage,meanwhile,upregulate the P38 MAPK-CRYAB signaling pathway to inhibit cardiomyocyte apoptosis and protect myocardial contractile function.This study not only revealed the specific mechanism of action of BYD in preventing HF post-AMI,but also provided a scientific basis for the clinical use of patients with HF post-AMI.At the same time,it also provided new targets for HF post-AMI drugs.1.The results showed that BYD prepared by 65%alcohol precipitation and sugar removal process can effectively improve the acute decline of left ventricular function caused by HF post-AMI,inhibit the release of LDH,CK,CK-MB,and reduce inflammatory cell infiltration and myocardial fibrosis,suggesting that BYD prepared by 65%alcohol precipitation and sugar removal process showed the best efficacy on prevention and treatment of HF post-AMI.2.BYD prepared by 65%alcohol precipitation and sugar removal process can effectively improve cardiac functions;reduce the release of MDA in circulation,increase the release of SOD,and reduce the OS injury in tissues;decrease of CK-MB and LDH levels in serum of HF rats after AMI;decrease of apoptosis rate in myocardial tissue,suggesting that BYD can improve heart function,reduce OS and inhibite apoptosis in HF post-AMI rats.3.BYD can significantly inhibit mitochondrial apoptotic pathways by inhibiting the expression of the pro-apoptotic proteins Bax,caspase 3,and caspase 9 and promoting the expression of Bcl-2,which may be related to the activation of P38 MAPK-CRYAB signaling pathway.4.This study sheds light on the mechanism of cardiomyocyte apoptosis mediated by oxidative stress injury on HF post-AMI of BYD,and provides new interventions and treatment ideas for the treatment of HF post-AMI.The functional characteristics and pharmacological mechanisms provide experimental evidence.At the same time,this study provides new interventions and targets for the development of drugs for coronary heart disease,HF and other major diseases based on myocardial ischemia.