| 1.Purpose:H9C2 cardiomyocytes were cultured in vitro to establish glucose oxygen deprivation /glucose reoxygenation(OGD/R)model.On the basis of clarifying the efficacy of Shengmai Injection(SMI),a representative formula for supplementing qi and nourishing Yin,to improve the OGD/R injury of H9C2 cardiomyocytes,the mechanism of action was predicted with the help of network pharmacology,and key genes and signal pathways were analyzed by high-throughput transcriptome sequencing,At the level of gene and protein,the molecular mechanism of Yiqi Yangyin recipe Shengmai Injection in improving OGD/R injury of H9C2 cardiomyocytes was explained to enrich the connotation of Yiqi Yangyin treatment principle.2.Method:2.1 Efficacy evaluation of Yiqi Yangyin recipe Shengmai Injection in reducing OGD/R injury of H9C2 cardiomyocyteBy drawing the growth curve,the modeling and intervention time of cardiomyocytes were determined;The myocardial cell injury model of H9C2 rats was established by OGD / R.the myocardial cell injury related enzymes CK and CK-MB were detected by LDH release method and ELISA;Taking the gradient concentration of SMI as the intervention drug,the efficacy of SMI in reducing OGD / R injury of cardiomyocytes in H9C2 rats was determined by detecting the contents of muscle cell injury related enzymes CK and CK-MB in the supernatant and the expression levels of AMP and ATP in cells.2.2 Prediction of mechanism of Yiqi Yangyin recipe Shengmai Yin in reducing OGD / R injury of H9C2 cardiomyocytesUPLC-Q/TOF-MS was used to analyze the components of SMI extract.By searching Pub Med and CNKI databases,and according to the chemical composition mass spectrometry cleavage secondary fragment data collected by MSE mode,compared with the literature results,the components of SMI extract were identified.Network pharmacology was used to predict the mechanism of SMI reducing myocardial ischemia-reperfusion(MIRI).Collect the action targets of SMI extract components in tcmsp database;Genecards database was used to screen the action targets related to Miri.The "drug target" PPI network and "disease target" PPI network were constructed by using Cytoscape 3.7.0 software to obtain the network of SMI intervention Miri.The topological characteristics of the network are analyzed and the target points are obtained;The go and KEGG enrichment results of core targets were analyzed by metascape online analysis tool and "weishengxin" website.Identify the key effects and their corresponding regulatory indicators,and predict the mechanism of SMI in reducing cardiomyocyte injury in H9C2 rats.2.3 Verification of mechanism of Yiqi Yangyin recipe Shengmai injection to reduce OGD/R injury of H9C2 cardiomyocytesTranscriptomics predicts the candidate key genes and signal pathways that SMI of Yiqi Yangyin recipe plays a therapeutic role on OGD/R of H9C2 cardiomyocytes at the gene level.The total RNA extraction reverse transcription DNA text database was constructed respectively.The transcriptome sample database of H9C2 rat cardiomyocytes was constructed by high-throughput sequencing on the hi SEQ x-ten platform.Through the process of go function annotation,KEGG enrichment analysis and expression difference analysis,the candidate key genes and signal pathways for the therapeutic effect of SMI on OGD/R of H9C2 rat cardiomyocytes were screened.The changes of mitochondrial membrane potential in H9C2 rat cardiomyocytes were detected by mitochondrial membrane potential;RT-q PCR detection: CREB,Bcl2,RXRα/SHC/TNF-α.The expression level of IL-10 m RNA and the expression levels of p-CREB,CREB and Caspase3 protein were detected by Western Blotting to clarify the mechanism of SMI inhibiting apoptosis and alleviating cardiomyocyte injury in H9C2 rats.RT-q PCR detection: AMPK,PGC-1 α.The expression levels of GLUT4,PDK1 and HK2 m RNA;Western blotting: AMPK,PGC-1 α.Protein expression level to clarify the mechanism of SMI regulating energy metabolism and reducing cardiomyocyte injury in H9C2 rats3.Result:3.1 OGD 3H / R 6h can successfully replicate H9C2 rat cardiomyocyte injury model(1)The cell growth curve showed "s" type on the 7th day,and the growth rate increased significantly on the 3rd day,showing exponential growth,which is most suitable for drug intervention.(2)Model identification: analyze and compare OGD/R at different times,and confirm that OGD3h/R6 h can successfully establish H9C2 rat cardiomyocyte injury model.Compared with control,the contents of LDH,CK and CK-MB in the supernatant of OGD3h/R6 h group were increased,the expression level of AMP was up-regulated and the expression level of ATP was down regulated.3.2 Yiqi Yangyin recipe Shengmai injection 5μL/m L can reduce OGD/R injury of H9c2cardiomyocytesAnalysis and comparison of different drug concentrations confirmed that SMI5 μL/m L can significantly reduce the OGD/R injury of cardiomyocytes in H9C2 rats.Compared with the control group,the cell viability of OGD/ R group decreased significantly,SMI 5/10/15μL/m L The cell viability increased in group.Compared with the control group,the contents of LDH,CK and CK-MB in the supernatant of OGD/R group were increased,SMI5/10/15μL/m LThe contents of LDH/CK and CK-MB in the supernatant of L/ m L group decreased,SMI 5μL/m L is particularly significant.Compared with the control group,the expression level of AMP was up-regulated and the expression level of ATP was down regulated in the OGD/R group and TMZ12 five μ The expression level of AMP was down-regulated and the expression level of ATP was up-regulated in mol/l group,and the efficacy of SMI was equivalent to that of TMZ.3.3 Prediction of mechanism of Yiqi Yangyin recipe Shengmai Yin in reducing OGD/R injury of H9C2 cardiomyocytesTo establish a database of chemical constituents of Shengmai Decoction for supplementing qi and nourishing Yin,and the results of automatic analysis were further checked and corrected by using masslynx 4.1 software.A total of 64 compounds were identified.Secondly,109 action targets of SMI extract were obtained by searching tcmsp database,1384 MIRI related targets were collected by genecards database,and the network of SMI intervention MIRI was constructed by network pharmacology.Through the analysis of network topology characteristics,23 core targets of the network were obtained;The go and KEGG enrichment analysis of the core targets were carried out,and the network core targets and core pathways were analyzed by using the pathway analysis method based on outcome effect.It was found that SMI reduced myocardial ischemia-reperfusion injury from the perspectives of apoptosis and energy metabolism.Based on this,the key targets of SMI regulating apoptosis and energy metabolism were determined,and the mechanism of SMI reducing myocardial ischemia-reperfusion injury was predicted.3.4 Verification of mechanism of Yiqi Yangyin recipe Shengmai injection to reduce OGD/R injury of H9C2 cardiomyocytesThe results of go functional enrichment of transcriptomics showed that the mechanism of SMI of Yiqi Yangyin recipe in reducing H9C2 cardiomyocyte injury induced by OGD/R may be related to the regulation of apoptosis and energy metabolism.The KEGG pathway enrichment analysis of 34 core targets showed that Shengmaiyin inhibited apoptosis and regulated energy metabolism through PI3 K Akt signal pathway,TNF signal pathway and MAPK signal pathway,and alleviated the OGD/R injury of cardiomyocytes in H9C2 rats.Yiqi Yangyin recipe SMI inhibits apoptosis and reduces OGD/R injury of H9C2cardiomyocytes: compared with control group,OGD/R group ΔΨ M decreased significantly;Compared with OGD/R group,SMI group ΔΨM up regulation.Compared with control,IL-10,Bcl2 and RXR in OGD/R group α The m RNA level of OGD/R group was significantly down regulated-α The m RNA expression level of was significantly up-regulated;Compared with OGD/R group,IL-10,Shc and RXRα in SMI group The m RNA level of SMI group was significantly up-regulated-α The m RNA expression level of was significantly down regulated.The expression levels of creep3 and creepb protein were up-regulated compared with those of creepb group;Compared with model group,the expression levels of p-CREB/CREB protein and p-CREB,CREB and casebase3 protein in SMI group were down regulated.Yiqi Yangyin recipe SMI regulates energy metabolism and reduces OGD/R injury of H9C2 cardiomyocytes: compared with control,PGC-1 in OGD/R group α The m RNA levels of and GLUT4 were significantly down-regulated,and the m RNA expression level of HK2 was significantly up-regulated in OGD/R group;Compared with OGD/R group,SMI group PGC-1α The m RNA levels of and GLUT4 were significantly up-regulated,and the m RNA expression level of HK2 in SMI group was significantly down regulated.Compared with the control group,the expression level of AMPK protein in the model group was down regulated and PGC-1α was decreased The protein expression level was down regulated;Compared with model group,the expression level of AMPK protein in SMI group was up-regulated and PGC-1α was increased The protein expression level was up-regulated.4.Conclusion 4.1 OGD 3h/R6 h can successfully replicate H9C2 rat cardiomyocyte injury model.SMI can improve cell viability,reduce the contents of LDH,CK and CK-MB related to cardiomyocyte injury,down regulate the expression level of AMP and up regulate the expression level of ATP,so as to reduce cardiomyocyte injury in H9C2 rats,and the SMI and TMZ of Yiqi Yangyin recipe have the same effect.4.2 Based on the network pharmacology prediction,transcriptomics and later validation,it is found that SMI of Yiqi Yangyin recipe can inhibit apoptosis,regulate energy metabolism and reduce the OGD/R injury of H9C2 cardiomyocytes by regulating AMPK/CREB,which enriches the connotation of Yiqi Yangyin recipe. |
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