Study On The Screening Of Ginsenosides And The Mechanism Of Rc Against Myocardial Ischemia-Reperfusion Injury Based On The Regulation Of Mitochondrial Function By SIRT1

The important effect of ginseng in heart is to "replenish the vitality,reinforce functional activities of the heart,and reassure the spirit".Preclinical studies have shown that ginsenosides,the active ingredient of ginseng,have myocardial protection and antimyocardial ischemia-reperfusion(I/R)injury effects,but their medicinal substances and mechanism have not yet been elucidated.Myocardial I/R injury is cardiomyocyte injury caused by restoration of blood circulation in the treatment of myocardial infarction and the main reason for the high mortality rate of myocardial infarction,which reduce the clinical efficacy.Mitochondria provide 90% of the energy in the heart,and its function determines the ability of cardiomyocytes to resist injury during I/R.Silent information regulator 1(SIRT1)regulates mitochondrial function and metabolism by deacetylating downstream proteins,thereby regulating the progression of myocardial I/R injury.Therefore,the regulation of mitochondrial function based on SIRT1 can promote the survival of cardiomyocytes and improve cardiac function,which is important against myocardial I/R injury.Objective: The study screened the ginsenoside monomers against myocardial I/R injury in ginsenosides(GS)and studied its mechanism based on the extraction,chemical composition analysis,combined with the pharmacodynamic substance evaluation system such as molecular binding ability,surface plasmon resonance(SPR)analysis,enzymatic reaction kinetic analysis,and SIRT1 deacetylation modification to regulate mitochondrial function.This study provides ideas and reliable experimental basis for explaining the core efficacy of ginseng.Methods: 1 GS were extracted by water extraction and alcohol precipitation,and the content of ginsenoside in GS was determined by UV-Vis spectrophotometry.The ginsenoside monomers in GS were qualitatively and quantitatively analyzed by ion mobility high-resolution liquid chromatography-mass spectrometry and other methods.The molecular binding capacity of ginsenoside monomers and SIRT1 protein was evaluated by Glide module of the Schrodinger software.The specific binding sites of ginsenoside monomers and amino acids of SIRT1 protein were further obtained by Py MOL software.Based on the results of molecular docking,the ginsenoside monomers with good binding ability were screened for detection of SIRT1 enzyme activity and SPR system.2 The metabolites in cells were separated,characterized and quantified using UHPLC combined with triple quadrupole mass spectrometer.Protein expression of SIRT1,glycolysis,enzymes of tricarboxylic acid metabolism and mitochondrial complex I-V were analyzed by western blot;intracellular oxygen consumption(OCR)was analyzed by Seahorse multifunctional energy metabolism system;ATP production was detected by luciferase method.In primary cardiomyocytes and H9c2 cells,the number and size of mitochondria were analyzed by transmission electron microscopy and Mito Tracker probe combined with confocal laser microscopy.3 The I/R models were established by culturing without glucose and fetal bovine serum + 0% O2 in primary cardiomyocytes and H9c2 cells.The m RNA expression of mitochondrial biosynthesis-related gene was assessed by real-time quantitative polymerase chain reaction(q PCR)method.Glucose uptake was analyzed by flow cytometry and 2-NBDG probe,GLUT4 protein was quantified and localized by immunofluorescence,and the small molecule inhibitor UK5099 combined with OCR analysis;these methods were used to evaluated the glucose metabolism-related phenotype.4 The SD rat I/R model was established by ligation of the left anterior descending coronary artery.The heart infarct size was analyzed by TTC staining in rat;the morphology of cardiomyocytes in the heart tissue was analyzed by H&E staining;the function of mitochondrial complex I-V was analyzed by enzymatic reaction kinetic system;the expression of SIRT1 was determined by immunohistochemistry.5 The transcription level,protein expression and interaction of Ac-Lys,SIRT1,PGC1α and their downstream proteins in cardiomyocytes were analyzed by q PCR,western blot,immunofluorescence and co-immunoprecipitation.The reverse verification was carried out by RNA interference technology,small molecule inhibitors of SIRT1(NAM and EX-527)combined with phenotype analysis of mitochondrial function and apoptosis.Results: 1 The ratio of GS extracted from ginseng was 6.9%,and the ginsenoside content was 90.5%.The 44 ginsenoside monomers in the extracted GS were identified,and 12 ginsenoside monomers in GS were quantified.Molecular docking software analysis and calculation results show that ginsenosides Rb1(G-score =-9),Rc(G-score =-8.2),and Rb2(G-score =-7.2)have strong binding ability to SIRT1 protein,and Rc can effectively bind with the amino acid sites of LEU205,THR209,ASN226 and GLN222 of SIRT1 protein.The results of enzyme activity and SPR showed that Rc can physically bind to SIRT1 protein and increase the activity of SIRT1.2 The results of targeted metabolomics showed that GS increased glucose and tricarboxylic acid metabolism in H9c2 cells.The results of protein quantification and enzyme activity showed that GS promoted glucose into tricarboxylic acid metabolism in cardiomyocytes.The results of mitochondrial stress experiments showed that GS increased the basal OCR,maximum of OCR and ATP production in H9c2 cells.The results of live cell mitochondrial labeling and transmission electron microscopy showed that GS significantly increased the number of mitochondria,but did not change the size of mitochondria.Ginsenoside Rc in GS significantly promoted the level of OCR and ATP,and increased the expressions of HK-II,MPC1 and SIRT1 proteins in cardiomyocytes.3 Ginsenoside Rc can enhance mitochondrial biosynthesis,ATP production,OCR,the expression of mitochondrial complex II/III/IV and glucose uptake in primary cardiomyocytes and H9c2 cells.In the cardiomyocyte I/R model,ginsenoside Rc can enhance mitochondrial function and ATP production,restore mitochondrial morphology,enhance the expression of mitochondrial complex I-III,reduce the levels of ROS and apoptosis.In the rat I/R injury model,the area of myocardial ischemia was significantly reduced,and the phenotype of myocardial cell injury were alleviated,and the activity of mitochondrial complex III/IV and the number of SIRT1-positive cardiomyocytes in cardiac tissue was significantly recovered after Rc pretreatment.4 Ginsenoside Rc increased the protein expression and interaction of SIRT1 and PGC1α in cardiomyocytes,and also promoted the expression of Nrf1 and Nrf2.Ginsenoside Rc had no significant effect on the expression of SIRT1 protein and mitochondrial biosynthesis in cardiomyocytes with SIRT1 and PGC1α down-expression.After ginsenoside Rc incubation in the I/R-induced cardiomyocyte injury model,the percentage of apoptosis,Cytochrome C protein expression,and glucose uptake were significantly decreased,ant the Bcl-2/Bax ratio,ATP level,and mitochondrial membrane potential were significantly increased.NAM and EX527 can counteract the effect of Rc above.The results of coimmunoprecipitation showed that ginsenoside Rc increases the protein expression of SIRT1 and the physical binding of SIRT1 to PGC1α,and reduces the acetylation of PGC1α in the I/R-induced cardiomyocyte injury model.Conclusions: 1 The 44 ginsenoside monomers in the extracted GS with higher purity was identified,and 12 ginsenoside monomers were quantified.GS can promote the glycolysis,tricarboxylic acid,mitochondrial biosynthesis and mitochondrial function in cardiomyocytes.2 It was screened that ginsenoside Rc could bind to SIRT1,increase the deacetylation activity of SIRT1,mitochondrial function,and energy metabolism in cardiomyocytes.3 Ginsenoside Rc enhanced mitochondrial biosynthesis and function,aerobic glucose metabolism,reduced intracellular and intramitochondrial ROS levels,apoptosis,myocardial ischemia area,and myocardial injury in in vivo and in vitro myocardial I/R models.4 The mechanism of ginsenoside Rc alleviating I/R-induced cardiomyocyte injury by regulating mitochondrial function is mediated by physical binding with SIRT1 protein,which enhances the expression and deacetylation activity of SIRT1 protein,and then modifies PGC1α by deacetylation.