Study On The Mechanism Of Baoyuantang Regulating AT1-CARP Pathway To Inhibit Ventricular Remodeling And Preventing Heart Failure After Myocardial Infarction

Aim:HF after AMI has become a hot and difficult topic in the field of cardiovascular research because of its serious complications and high mortality.VR after AMI is considered to be the main pathological basis of HF.Therefore,mitigating VR after AMI is an important intervention in the treatment of HF.Although the existing therapeutic drugs have been widely used in clinic,their efficacy and safety are still unsatisfactory.Traditional Chinese medicine(TCM)has the characteristics of improving activity and quality of life of patients with HF,and has high safety of medication.Therefore,it has become an important field in searching for new therapeutic drugs for HF.In this study,we aimed to identify the effects and the underlying mechanisms of BYD on Ang Ⅱ-induced apoptosis and hypertrophy in HF after AMI both in vitro and in vivo.Methods:1.The HF after AMI models were prepared by ligating of LAD of artery in rats.Rats were orally administered for 28 d.Cardiac function of rats in each group was evaluated by echocardiography;serum levels of CK and LDH were measured by blood biochemical assay;the levels of HF markers such as ANP,BNP and NT-proBNP in plasma were detected by ELISA;the content of Ang Ⅱ in plasma was detected by Radioimmunoassay;inflammatory cell infiltration and cardiomyocytes hypertrophy were detected by HE staining;cardiomyocytes apoptosis was detected by TUNEL staining;and the deposition of collagen was detected by Sirius red staining.2.Mechanism of BYD in inhibiting cardiomyocytes apoptosis of HF after AMIIn vivo,the content of Ang Ⅱ in myocardial tissue was detected by IHC method;the expression of RAAS-related proteins,CARP and downstream apoptotic protein were detected by WB;and the mitochondrial structure was observed by transmission electron microscopy.In vitro,apoptotic model of H9C2 cells was induced by Ang Ⅱ stimulation;CCK-8 and Hoechst 33258 methods were combined to detect the viability and the apoptosis of H9C2 cells in each group after the intervention of BYD;WB method was used to detect the expressions of related proteins;and the effect of BYD on mitochondria injury was evaluated by Rhodamine 123 staining.3.Mechanisms of BYD in inhibiting cardiomyocytes hypertrophy of HF after AMIIn vivo,the expressions of AT1,Calpain 1,CARP and its downstream related hypertrophic proteins such as Calcineurin,GATA4 and Erkl/2 in myocardial tissues were detected by WB.In vitro,hypertrophic model of H9C2 cells was induced by Ang II stimulation;Rhodamine phalloidin and DAPI staining were combined to evaluate the hypertrophy of H9C2cells in each group;WB method was used to detect the expressions of AT1,Calpain 1,CARP,p-GATA4 and p-Erk1/2,and immunofluorescence method was used to detect the expression of CARP in cytoplasm.4.To further verify the pathway of cardiomyocytes apoptosis and hypertrophy regulated by CARP and whether BYD could regulate apoptosis and hypertrophy by interfering with CARP,the overexpressed CARP cell model was prepared by transfection of pcDNA3.1-Ankrd1 plasmid in vitro.Hoechst 33258 and Rhodamine phalloidin staining were used to detect cardiomyocytes apoptosis and hypertrophy.The expression of CARP and related apoptotic and hypertrophic proteins in cells were detected by WB.Based on the above results,the mechanism of BYD regulating cardiomyocytes apoptosis and hypertrophy by regulating CARP was explained.Results:1.Pharmacodynamic study of BYD on VR in rats with HF after AMIAfter treatment with BYD,the EF and FS of rats in each group were improved to varying degrees(P<0.05,P<0.01),suggesting that BYD could effectively inhibit the decline of cardiac function caused by AMI injury.In addition,BYD could significantly inhibit the increase of LVIDd and LVIDs(P<0.01),suggesting that BYD could inhibit VR in HF rats to some extent.The results of myocardial zymogram showed that BYD could inhibit the release of CK and LDH from cardiomyocytes(P<0.01).Meanwhile,it could suppress the plasma levels of HF markers and Ang Ⅱ(P<0.05),suggesting that BYD could inhibit myocardial injury caused by AMI.HE staining results showed that BYD could significantly inhibit inflammatory cells infiltration and cardiomyocytes hypertrophy(P<0.01).TUNEL staining showed that BYD could effectively inhibit cardiomyocytes apoptosis in the infarcted border area.The results of Sirius red staining showed that BYD could inhibit the excessive deposition of myocardial interstitial collagen.The above results suggested that BYD could inhibit VR and improve cardiac function in rats with HF after AMI.The positive drug Fosinopril also showed a better effect on improving cardiac function,reducing inflammatory cells infiltration and inhibiting apoptosis and hypertrophy.2.BYD attenuated cardiomyocytes apoptosis to prevent HF after AMI via AT1-CARP pathwayIHC staining results showed that BYD could reduce Ang Ⅱ levels in myocardial tissue(P<0.01).WB results showed that BYD could significantly down-regulate the expressions of AT1(P<0.01),ACE1(P<0.001)and CARP(P<0.01),inhibit the expressions of downstream apoptotic proteins such as P53(P<0.001),Bax(P<0.001)and Cleaved caspase-3(P<0.001),and up-regulate the expression of anti-apoptotic proteins such as Bcl-2(P<0.05)and Mdm2(P<0.01).Besides,electron microscopy results showed that BYD could inhibit mitochondrial structural damage in HF rats.In vitro,MTT and Hoechst 33258 results both showed that BYD could inhibit the apoptosis of H9C2 cells induced by Ang Ⅱ stimulation.The results of WB showed that BYD could down-regulate the expressions of AT1,CARP,P53 and Bax.In addition,Rhodamine 123 staining showed that BYD could inhibit mitochondrial membranepotential damage induced by Ang Ⅱ stimulation.3.BYD attenuated cardiomyocytes hypertrophy to prevent HF after AMI via AT1-CARP pathwayWB results showed that BYD could significantly down-regulate the expression of AT1(P<0.01),Calpain 1(P<0.01)and CARP(P<0.01),inhibit the phosphorylation of GATA4(P<0.001)and Erk1/2(P<0.01),which were the downstream proteins of CARP.However,BYD had no obvious regulatory effect on the expression of Calcineurin.In vitro,Rhodamine phalloidin staining showed that BYD could inhibit the hypertrophy of H9C2 cells induced by Ang Ⅱ stimulation.WB results showed that BYD could significantly down-regulate the expression of AT1,Calpain 1 and CARP,and then ameliorate cardiomyocytes hypertrophy by inhibiting the phosphorylation of GATA4 and Erkl/2.In addition,immunofluorescence staining also showed that BYD could inhibit the expression of CARP in cytoplasm induced by Ang Ⅱ stimulation.4.Mechanism validation of BYD attenuating cardiomyocytes apoptosis and hypertrophy via CARPThe results of Hoechst 33258 and Rhodamine phalloidin staining showed that the pcDNA3.1-Ankrdl plasmid transfection group had obvious apoptosis and increased cell surface area,respectively.However,BYD could significantly inhibit cardiomyocytes apoptosis induced by overexpression of CARP,and could effectively reverse the cardiomyocytes hypertrophy.The results of WB showed that the expression of CARP was up-regulated in the plasmid-transfected group,and it could promote the expression of P53 and inhibit the expression of Mdm2.In addition,the phosphorylation levels of CARP-related hypertrophic proteins GATA4 and Erkl/2 were also significantly increased.BYD not only significantly inhibited the overexpression of CARP,but also down-regulated the expression of its downstream apoptotic protein P53,and also significantly inhibited the phosphoiylation of hypertrophic related proteins GATA4 and Erkl/2.Conclusions:1.Pharmacodynamic studies suggest that BYD can effectively improve the acute decline of cardiac function in rats with HF after AMI,inhibit the increase of left ventricular diameter caused by VR,reduce the level of CK and LDH in serum,and effectively reduce the levels of HF markers and Ang Ⅱ in plasma.In addition,BYD can effectively inhibit inflammatory cells infiltration,cardiomyocytes hypertrophy,apoptosis and excessive deposition of collagen in myocardial tissue in myocardial tissues.The above results suggest that the effect of BYD on improving heart function of rats with HF after AMI is closely related to the inhibition of cardiomyocytes apoptosis and hypertrophy and other pathological processes of VR,which provide a direction for subsequent mechanism research.2.The results both in vivo and in vitro of BYD on attenuating cardiomyocytes apoptosis indicate that the level of Ang Ⅱ is elevated in myocardial tissue because of myocardial ischemia injury,and further activates the expression of AT1,which leads to the high expression of CARP in myocardial tissues.P53-mitochondrial apoptosis pathway is then initiated by CARP,which increases the expressions of P53,Bax and Cleaved caspase-3 and decreases the expression of Bcl-2 and Mdm2.After the intervention of BYD,the level of Ang II and the expression of AT1 and CARP are all inhibited.Meanwhile,BYD can promote the expressions of anti-apoptotic proteins Bcl-2 and Mdm2,and inhibit the expression of pro-apoptotic proteins P53,Bax and Cleaved caspase-3,thus play an anti-apoptotic role in cardiomyocytes.At the same time,WB results show that BYD can effectively inhibit the overexpression of CARP in H9C2 cells prepared by pcDNA3.1-Ankrdl plasmid transfection,and exert inhibitory effect on its downstream regulatory protein P53.In conclusion,BYD can inhibit cardiomyocytes apoptosis in rats with HF after AMI by regulating AT1-CARP pathway.CARP has become a key protein and potential target for BYD to improve cardiac function and inhibit cardiomyocytes apoptosis.3.The results both in vivo and in vitro of BYD on attenuating cardiomyocytes hypertrophy show that while the myocardial ischemia continued,GATA4/Erkl/2 which is a pathway downstream of CARP regulating hypertrophy is activated,which in turn induces an increase in myocardial cell surface area and aggravation of HF.After the intervention of BYD,the phosphorylation of related proteins in GATA4/Erkl/2 hypertrophic pathway is inhibited by regulating AT1-CARP pathway,maintaining the normal size and diameter of cardiomyocytes,preventing the occurrence of VR and delaying the deterioration of HF.Meanwhile,WB results show that BYD can directly inhibit the overexpression of CARP in H9C2 cells transfected with pcDNA3.1-Ankrd1 plasmid,and further inhibit the phosphorylation of the hypertrophic protein GATA4 which indicate that CARP is key protein and potential target for BYD to exert anti-cardiomyocytes hypertrophy.4.This study elucidates the efficacy of BYD on cardiomyocytes apoptosis and hypertrophy in rats with HF after AMI and the mechanism of anti-apoptosis and anti-hypertrophy by regulating AT1-CARP pathway.Especially,the mechanism verification of cardiomyocytes apoptosis and hypertrophy regulated by CARP and the clarification of the direct regulation effect of BYD on CARP provide a new intervention for the treatment of HF by inhibiting VR with CARP as a new key protein and potential target,and provide a powerful experiment basis for the clinical application of BYD in the prevention and treatment of HF,5 as well as the development of new drugs and even the secondary optimization of its formulation.