Effect Of Zhenwu Decoction On Apoptosis Of Cardiomyocytes In CTnT^R141W Transgenic Dilated Cardiomyopathy Mice Based On Network Pharmacolog

Objective:By using the research method of network pharmacology,the active components,core targets and signal pathways of Zhenwu decoction in the treatment of dilated cardiomyopathy were predicted by constructing the network of "drug-target-disease".And verified by animal experimental studies,further reveal the mechanism of Zhenwu decoction on dilated cardiomyopathy mice,and provide reference for clinical treatment and scientific experimental research in the future.Methods:1.The specific steps of predicting the mechanism of Zhenwu decoction in the treatment of dilated cardiomyopathy by network pharmacology are as follows:The main active components and functional targets of Zhenwu decoction are selected based on databases such as traditional Chinese Medicine system Pharmacology Analysis platform（TCMSP）,and then the key targets of dilated cardiomyopathy are obtained by using databases such as Gene Cards and OMIM,and then their common action targets are screened out.Cytoscape3.8.2 software is used to establish the network diagram of "compound-target",that is,"Zhenwu decoction-active ingredient-DCM-target".Through the analysis of String database,the protein interaction（PPI）network was obtained and the key genes were identified,and the GO functional enrichment and KEGG pathway enrichment analysis of the key targets were done in the DAVID database.Finally,molecular docking is done by RCSBPDB,Pub Chem database and so on.2.To predict the mechanism of Zhenwu decoction in the treatment of dilated cardiomyopathy through animal experiments.The specific experimental steps are as follows:In this study,10 C57BL/6J mice were used as blank control group and 30 cTnT^R141W transgenic dilated cardiomyopathy mice according to their body weight were randomly divided into: model control group,Zhenwu decoction group and captopril group and,with 10 mice in each group.The mice in the blank control group and cTnT^R141W transgenic dilated cardiomyopathy group were given the same amount of normal saline twice a day for 4 weeks, the mice in the treatment group were given intragastric administration of Zhenwu decoction at the dose of 18.2g/（kg.d）twice a day for 4 weeks,and the positive control mice were given captopril at the dose of 10mg/（kg.d）twice a day for 4 weeks.Cardiac function ejection fraction（EF%）and fractional shortening（FS%）were measured by echocardiography.The level of apoptosis was evaluated by TUNEL method.The expression of Bax,Bcl-2,caspase-3and caspase-7 protein was measured by Western Blot.Results:1.Network pharmacology and molecular docking:59 main active components and 36 important intersection targets of Zhenwu decoction were obtained.GO enrichment function analysis showed 1776 items and KEGG enrichment pathway analysis.The main active components include kaempferol（MOL000422kaempferol）,B-sitosterol（MOL000358 beta-sitosterol）,stigmasterol（MOL000449Stigmasterol）and ivy sapogenin（MOL000296 hederagenin）;The key targets include AKT1,TNF,CASP3,PPARG,JUN,PTGS2,etc.and mainly involve TNF pathway,MAPK pathway,PI3K-Akt pathway,apoptosis pathway and so on.2.The results of experimental animals:（1）Echocardiographic resultsCompared with the blank group,the diastolic and end-systolic diameters in the model group increased（P < 0.05）,the ejection fraction and short axis shortening decreased（P<0.05）,and the myocardial contractile function decreased.After treatment with Zhenwu decoction,the end-diastolic and end-systolic diameter were decreased（P<0.05）,and the ejection fraction and shortening rate of short axis were increased（P<0.05）.（2）TUNEL apoptosis resultsThe tunel staining pictures of each group were analyzed.The results showed that in the blank group,the cells were arranged orderly,the cytoplasm was uniform,the cell size was normal,and the nucleus was blue;The myocardial tissue of the model group changed significantly,the arrangement of cardiomyocytes was disordered,the nucleus pyknosis was brown,round,crescent or irregular,some cells showed apoptosis,and the apoptosis rate was significantly higher than that in the blank group（P<0.05）;The degree of apoptosis in captopril group and Zhenwu decoction group was significantly lower than that in model group（P<0.05）,which indicated that Zhenwu decoction group and captopril group could effectively inhibit cardiomyocyte apoptosis.To protect the myocardial tissue.（3）WB results Western blot analysis revealed that the protein expression levels of Bax,cleaved-Caspase3 and cleaved-Caspase7 in the captopril and Zhenwu decoction groups were significantly decreased compared with the control group（P<0.05）,while the protein level of Bcl-2 in captopril group and Zhenwu decoction group were significantly increased compared with the control group.Conclusion:1.Zhenwu decoction may effectively regulate cardiomyocyte apoptosis,autophagy and myocardial fibrosis by TNF pathway,MAPK pathway,PI3K-Akt pathway,apoptosis pathway thus play an obvious role in the treatment of dilated cardiomyopathy.2.Zhenwu Decoction improves cardiac function in cTnT^R141W transgenic dilated cardiomyopathy mice.3.Zhenwu Decoction reduces the rate of cardiomyocyte apoptosis and inhibits cardiomyocyte apoptosis in cTnT^R141W transgenic dilated cardiomyopathy mice.4.Zhenwu Decoction increased the protein expression of Bcl-2 and decreased the protein expression of Bax,cleaved-caspase3 and cleaved-caspase7 in cTnT^R141W transgenic dilated cardiomyopathy mice,thus acting to inhibit apoptosis of cardiomyocytes.