Study On Mechanism Of Effect Of Baoyuan Decoction On Adriamycin Induced Myocardial Injury And Endoplasmic Reticulum Stress Induced Apoptosis In Rats

Objective: Based on the method of Network pharmacology and Molecular docking,The main active components,Target points and Mechanism of Baoyuan Decoction in the treatment of Chronic heart failure were clarified.The activity,apoptosis,pathological changes of Myocardial tissue,Serum related indexes of the rats with Chronic heart failure were observed The effect of Baoyuan Decoction on Chronic heart failure and its mechanism were discussed.Methods:1.Mechanism of Baoyuan Decoction in the treatment of chronic heart failure based on network pharmacologyThe effective components and targets of Baoyuan Decoction were searched by tcmsp database and related literatures;the CHF disease targets were screened by genecards,OMIM,TTD,drugbank and Pharm GKB databases;the intersection targets of Baoyuan Decoction and CHF were obtained by data processing,and the screened intersection targets were imported into string database to construct PPI network diagram To form a visual network diagram of "Baoyuan Decoction drug activity target",and further screen out the core target of Baoyuan Decoction in the treatment of CHF;to carry out go enrichment analysis and KEGG enrichment analysis on the intersection target of Baoyuan Decoction and CHF;to carry out molecular docking on the key components and core target of Baoyuan decoction.2.Effect of Baoyuan Decoction on adriamycin induced cardiomyocyte injury and endoplasmic reticulum stress induced apoptosis in vitroCardiomyocytes were cultured from 1-3 days old suckling mice.Cardiomyocytes were randomly divided into 5 groups: normal control group(normal cell culture,with equal amount of medium,without any drugs);Adriamycin injury group(add adriamycin 1μg/m L);Adriamycin + Baoyuan Decoction low concentration group(add adriamycin 1μg/m L and Baoyuan Decoction 5μg/m L);Adriamycin + Baoyuan Decoction medium concentration group(add adriamycin 1μg/ m L and Baoyuan Decoction 10μg/m L);Adriamycin + Baoyuan Decoction high concentration group(add adriamycin 1μg/m L and Baoyuan Decoction20μg/m L).After 48 hours,the effects of different concentrations of Baoyuan Decoction on the activity of cardiomyocytes were detected by MTT assay;Apoptosis was detected by TUNEL;Real time PCR was used to detect the expression of endoplasmic reticulum stress and apoptosis related proteins The expression levels of GRP78,CHOP,JNK and Caspase12 were detected by PCR.3.Experimental study of Baoyuan Decoction on adriamycin induced myocardial injury and endoplasmic reticulum stress induced apoptosis in rats.90 healthy SD rats were randomly divided into blank group,model group,Baoyuan Decoction low dose group,Baoyuan Decoction medium dose group,Baoyuan Decoction high dose group and captopril group,with 15 rats in each group.Except for the blank group,the other five groups were given adriamycin solution by intraperitoneal injection at the dose of 1.5mg/kg twice a week for 8 weeks according to the 24mg/kg body weight.After the end of administration,the samples were taken,he staining and Masson staining were used to observe the pathological changes and fibrosis of myocardial tissue;ELISA was used to detect the levels of BNP,SOD and MDA;Western blot was used to detect the protein expression of endoplasmic reticulum stress and apoptosis related proteins GRP78,PERK,e IF2α,CHOP,JNK and Caspase12;Real time PCR was used to detect the expression of endoplasmic reticulum stress and apoptosis related proteins The expression levels of GRP78,PERK,e IF2α,CHOP,JNK and Caspase12 were detected by PCR.Results:1.The effective active components of Baoyuan decoction are 144,with 253 target points.The key components mainly include quercetin,grapefruit,glaxon and other flavonoids;the intersection target of Baoyuan Decoction and CHF is 240,and its core targets include Jun,STAT3,TP53,rela,mapk1,ESR1,etc;the enrichment analysis results of GO and KEGG show that it mainly involves 2475 biological processes,104 cell components,253 molecular functions,174 related signaling pathways,It mainly regulates the biological processes such as oxidative stress,inflammation,calcium channel and apoptosis signal pathway through cell components such as cytoplasm,cell membrane and endoplasmic reticulum,and give full play to DNA binding transcription factor binding,RNA polymerase II specific DNA binding transcription factor binding,serine / threonine kinase activity,ubiquitin like protein ligase junction The molecular functions of the combination play a role in the treatment of CHF through signal pathways such as PI3K-Akt,MAPK,AGE-RAGE,IL-17,Apoptosis,Cancer and so on.2.Effect of Baoyuan Decoction on adriamycin induced cardiomyocyte injury and endoplasmic reticulum stress induced apoptosis.(1)MTT assay results showed that compared with the normal control group,the cell viability of adriamycin injury group and Baoyuan Decoction low concentration group was significantly decreased(P<0.05),and compared with adriamycin injury group,the cell viability of Baoyuan Decoction medium concentration group,high concentration group was significantly increased(P<0.05).(2)TUNEL results showed that apoptosis positive staining cells were rare in the observation field of normal control group;green positive staining cells were distributed among normal cells in adriamycin injury group and Baoyuan Decoction low concentration group,the number of apoptosis positive staining cells was significantly increased,and the nucleus was significantly decreased;the number of green positive staining cells was significantly increased in Baoyuan Decoction medium concentration group,high concentration group The number of nuclei increased significantly.(3)Real time PCR results showed that different concentrations of Baoyuan Decoction could down regulate the m RNA expression of Endoplasmic reticulum stress apoptotic pathway related factors.The m RNA expressions of GRP78,JNK and Caspase12 in Baoyuan Decoction low concentration group were not significantly different from those in adriamycin induced group(P>0.05),but the m RNA expressions of GRP78,JNK and Caspase 12 in Baoyuan Decoction high concentration group were significantly decreased(P<0.01,P<0.05);CHOP in low and medium concentration groups of Baoyuan Decoction There was no significant difference in chop m RNA expression between the two groups(P>0.05);The expression of CHOP m RNA in the high concentration group of Baoyuan Decoction decreased significantly(P< 0.05).3.Experimental study of Baoyuan Decoction on adriamycin induced myocardial injury and endoplasmic reticulum stress induced apoptosis in rats(1)HE staining results of myocardial tissue showed that in the model group,the arrangement of myocardial cells was disordered,the morphology was diverse,a large number of myocardial cells were hypertrophic,inflammatory cells were infiltrated,the muscle gap was widened,the myocardial fiber was broken,the myocardial fiber staining was uneven,the myocardial interstitial space was expanded,and the myocardial interstitial vessels were dilated and congested.After intervention with Baoyuan decoction,the pathological changes of myocardial tissue in the medium and high dose Baoyuan Decoction groups were significantly alleviated compared with those in the model group,with only a few inflammatory infiltration of myocardial cells,basically neat arrangement of myocardial cells,a few hypertrophy of myocardial cells,occasionally congestion of myocardial interstitial vessels and rupture of myocardial fibers.(2)Masson staining results showed that the collagen fibers in the myocardium of the model group were significantly proliferated,and there were more blue stained fibers and increased extracellular matrix around the vessels.After the intervention with Baoyuan decoction,the blue stained collagen fibers between myocardium were reduced,a large area of normal myocardial tissue was visible,and the muscle fiber space was significantly narrowed.(3)The level of BNP was detected by ELISA,Compared with the blank group,the serum BNP levels of each group were significantly increased(P<0.01),indicating that there were significant changes in heart failure state.After intervention with Baoyuan decoction,the serum BNP levels of Baoyuan Decoction medium dose group,high dose group and captopril group were significantly decreased(P<0.01),There was no significant difference in serum BNP level between Baoyuan Decoction high-dose group and captopril group(P >0.05).(4)The levels of SOD and MDA were detected by ELISA.The results showed that compared with the model group,the levels of SOD in low and medium dose Baoyuan Decoction groups were not significantly increased(P>0.05),the levels of SOD in high dose Baoyuan Decoction group and captopril group were significantly increased(P<0.01),and there was no significant difference between high dose Baoyuan Decoction group and captopril group(P>0.05);Compared with the model group,the MDA level of low-dose Baoyuan Decoction group was not significantly decreased(P>0.05),the MDA level of medium dose,high-dose Baoyuan Decoction group and captopril group was significantly decreased(P<0.01,P<0.05),and the MDA level of high-dose Baoyuan Decoction group and captopril group was not significantly different(P>0.05).(5)The results of Western blot and real-time PCR showed that compared with the model group,GRP78,PERK and e IF2α were related to endoplasmic reticulum stress apoptotic pathway,The protein and m RNA expressions of CHOP,JNK and Caspase12 were significantly increased(P<0.05,P<0.01);There was no significant difference in protein and m RNA expression between low-dose Baoyuan Decoction group and model group(P>0.05);The expression of Endoplasmic reticulum stress and Apoptosis related protein and m RNA were significantly down regulated in the high dose Baoyuan Decoction group(P<0.05,P<0.01);There was no significant difference in protein and m RNA expression between high-dose Baoyuan Decoction group and captopril group(P>0.05).Conclusion:1.Through the analysis of the results of network pharmacology,it is preliminarily clear that the active components of Baoyuan Decoction in treating CHF are saponins and flavonoids.Through cell components such as cytoplasm,cell membrane and endoplasmic reticulum,PI3K-Akt,MAPK,AGE-RAGE,TNF,Cancer and other pathways can be regulated to inhibit inflammatory response,oxidation resistance and apoptosis,Thus,it can play a role in the treatment of CHF.2.Baoyuan Decoction can improve the cell activity of myocardial cells after adriamycin induced injury,and improve the activity of myocardial cells,and significantly inhibit the apoptosis of cardiomyocytes induced by adriamycin.The improvement of myocardial cell activity and the inhibition of myocardial cell apoptosis are closely related to the concentration and dose of drug.3.Baoyuan Decoction can improve the myocardial tissue injury induced by adriamycin,improve the degree of myocardial fibrosis,reduce the level of BNP and MDA,improve SOD level,It has significant antioxidant effect and improves the state of oxidative stress.4.Baoyuan Decoction can inhibit GRP78,PERK and e IF2α by inhibiting it The expression of protein and m RNA of isoendoplasmic reticulum stress-related factors,the expression of CHOP,JNK and Caspase-12 m RNA related factors of endoplasmic reticulum stress pathway and then play a role in inhibiting the apoptosis of cardiac cells mediated by endoplasmic reticulum stress.