Study On The Protective Effect Of Ershen Granules On Myocardial Ischemia Model Based On Mitochondrial Function And Related Mechanisms

Objective:In this project,through in vivo and in vitro experiments,using analysis methods such as pathology and molecular biology,the mechanism of action of Ershen particles is mainly confirmed from the aspects of oxidative stress,apoptosis,mitochondrial energy metabolism,and mitochondrial biogenesis.To explore the mechanism of Ershen Granules in the targeted regulation of SIRT1-PGC-1?-PPAR?signaling pathway in myocardial ischemia.It provides experimental basis for the clinical promotion and application of Ershen Granules in the treatment of coronary heart disease myocardial ischemia..Methods:1.Refer to the method for determination of the content of each medicinal material in the Chinese Pharmacopoeia 2020 version and the liquid-phase fingerprint test method of each medicinal material in the"Hong Kong Pharmacopoeia",perform HPLC fingerprint test on Ershen Granules,and use standard reference substances to confirm the active ingredients The characteristic peak.Reuse network pharmacology to explore the effective chemical components of Ershen granules through the TCMSP platform,search for genes related to coronary heart disease myocardial ischemia in the Genen Card database,and construct a network diagram of chemical components-target points and PPI network diagrams;use DAVID for KEGG and GO enrichment Analyze,use Schrodinger Maestro software and online tools for molecular docking verification and predict core targets.2.Use cobalt chloride to establish a hypoxia-ischemia model of cardiomyocytes,use enzyme-linked immunoassay to detect LDH,CK content,SOD,MDA activity,use TUNEL method to detect cardiomyocyte apoptosis expression,use flow cytometry to detect intracellular activity The oxygen content and apoptosis level were analyzed by Western blot to analyze the expression levels of Cleaved PARP,Cleaved Caspase-3,Cleaved Caspase-9and Bcl-2/Bax.3.Enzyme-linked immunosorbent assay was used to detect the activity of mitochondrial respiratory chain enzyme complexes I,II,III,and V,Cytation5 was used to detect ATP content,use flow cytometry to detect myocardial cell mitochondrial membrane potential(MMP),and western blotting was used to analyze cells The expression levels of PGC-1?,PPAR?,and SIRT1.RT-PCR detection method was used to detect the expression levels of SIRT1,PPAR?and PGC-1?genes.4.The rat myocardial ischemia model was formed by intraperitoneal injection of pituitrin.The effect of Ershen Granules on the electrocardiogram of myocardial ischemia rats was detected.The pathological changes of myocardial tissue were observed by HE staining method,the degree of myocardial tissue fibrosis was observed by Masson staining method,and AMPK,p-AMPK,and PAMPK in rat myocardial tissue were analyzed by Western blot.The expression levels of SIRT1,PGC-1?,and PPAR?.RT-PCR detection method was used to detect the expression levels of SIRT1,PGC-1?and PPAR?genes in rat myocardial tissue.Results:1.Using"Medicine Chromatographic Fingerprint Similarity Evaluation System 2012.1 Edition"to calculate the fingerprints of 10 batches of samples,the similarity was above 0.997,with good reproducibility and stable ingredients.According to the results of network pharmacology,a total of 221 compounds and 264 target proteins were screened to participate in network construction.Gene enrichment analysis showed that 960 GO entries were involved,and a total of 726 KEGG signaling pathways were involved.The molecular docking results showed,The core target has strong binding ability with chemical composition.It is concluded that the SIRT1-PGC-1?-PPAR?signaling pathway may be the core potential target of Ershen Granules to improve coronary heart disease myocardial ischemia.2.Ershen Granules can effectively reduce cell LDH,MDA,CK levels(P<0.05),increase SOD levels(P<0.01 or P<0.001),and significantly reduce intracellular ROS content(P<0.01 or P<0.001),Reduce the expression of apoptotic cells(P<0.01 or P<0.001),down-regulate the expression of apoptotic proteins Cleaved Caspase-3,Cleaved Caspase-9and Cleaved PARP,and increase the expression of Bcl-2/Bax(P<0.01 or P<0.001).3.Ershen granule can effectively increase the activity of mitochondrial Complex?,?,?,?(P<0.05),increase the content of ATP(P<0.001),increase the level of mitochondrial membrane potential,and increase the target protein in myocardial cells The expression of PGC-1?and PPAR?(P<0.01 or P<0.001)increased the expression of target genes SIRT1,PGC-1?and PPAR?in cardiomyocytes(P<0.05,P<0.01 or P<0.001).4.Ershen granules can effectively improve the changes of electrocardiogram in rats with myocardial ischemia.HE staining results showed that in the model group,fatty degeneration of myocardial cells,edema of myocardial fibers,lightening of coloration,punctate infiltration of lymphocytes and mast cells,dissolution of multiple myocardial fibers and hyperplastic connective tissue were seen in the model group;compared with the model group,the low-dose group was seen Fatty degeneration of cardiomyocytes,punctate infiltration of lymphocytes and mast cells,dissolution of multiple myocardial fibers and hyperplasia of connective tissue.In the middle-dose group,myocardial fibers were decomposed clearly,stained evenly,with a small amount of edema and lymphocytes.In the high-dose group,the tissues were uniformly stained,the myocardial fiber morphology and structure were clearly demarcated,and the interstitium was not abnormal.Masson staining results showed that in the model group,there were increased collagen fibers in the myocardial tissue,disordered arrangement of myocardial fibers,increased and thicker collagen fibers,and obvious myocardial fibrosis.After the intervention of different doses of Ershen Granules,the muscles of the low-dose Ershen Granules group still showed increased collagen fibers and thickening,and myocardial fibrosis was obvious.Only mild collagen fiber hyperplasia was seen in the myocardial tissue of rats in the middle and high dose groups of Ershen granule,and the degree of fibrosis was significantly reduced.Ershen Granules can significantly increase the expression of target proteins SIRT1,PGC-1?,PPAR?(P<0.01 or P<0.001),and increase the expression of target genes SIRT1,PGC-1?,PPAR?(P<0.05 or P<0.01).Conclusion:1.It is preliminarily clear that Ershen Granules can inhibit the ROS level of cardiomyocytes induced by Co Cl₂,thereby achieving the effect of reducing the apoptosis of cardiomyocytes mediated by oxidative stress.2.By promoting the activation of SIRT1-PGC-1?-PPAR?signaling pathway,Ershen Granules,Ershen Granules can improve the morphological changes of myocardial ischemia rats and the degree of myocardial tissue fibrosis,increase the activity of mitochondrial respiratory chain complex,and promote ATP.Synthesized,so as to achieve the effect of improving heart function.