Protective Effect And Molecular Mechanism Of Ginsenosides On Injured H9C2 Cells Induced By Hypoxia

Objective:The protective effects of ginsenosides on the hypoxia injury of H9c2 cells induced by cobalt chloride were studied by a series of biological cell detection methods.The effects of ginsenosides were confirmed from mitochondrial membrane potential,oxidative stress,aerobic respiration,ATP,NAD⁺,SIRT1/PGC1?,glucoses uptake and mitochondrial biogenesis.To explore the target regulation of SIRT1 in ginsenosides hypoxia tolerance,provide experimental basis for finding out the material basis of ginsenosides hypoxia tolerance,expounding the mechanism of ginsenosides heart protection and product development.Methods:Firstly,the content of ginsenoside was analyzed qualitatively and quantitatively.First of all,the content of total saponins was determined by UV-vis spectrophotometry,then the content and kinds of ginsenoside monomer were determined and calculated by HPLC.Finally,the content of ginsenoside was analyzed qualitatively by LC-MS.Secondly,establish the hypoxia model of H9c2 cells induced by cobalt chloride in vitro:JC-1 was used to detect the mitochondrial membrane potential,DCFH-DA was used to detect the active oxygen to screen the action time and concentration of cobalt chloride.Then the therapeutic effect of ginsenoside was tested:the cells were randomly divided into control?Ctrl?group,model?CoCl₂?group and treatment?GS?group.MMP,ROS,glucose uptake and mitochondrial biogenesis were detected by flow cytometry,OCR,ECAR,ATP and NAD⁺/NADH were detected by Cytation 5,SIRT1 and PGC1?protein were detected by WesternBlot.Next,nicotinamide?NAM,SIRT1 inhibitor?was used to verify the hypoxia tolerance mechanism of ginsenosides:cells were randomly divided into control?Ctrl?group,model?CoCl₂?group,treatment?GS?group and inhibitor?NAM?group.MMP,ROS,glucose uptake and mitochondrial biogenesis were still detected by flow cytometry.OCR,ECAR,ATP and NAD⁺/NADH were detected by Cytation 5,SIRT1 and PGC1?were detected by WesternBlot.Finally,in order to find out the material basis of ginsenosides effect:according to the content percentage of ginsenoside to intervene hypoxia,cells were randomly divided into control group?Ctrl?,model group?CoCl₂?and treatment group?GS/saponin monomer?.MMP was detected by flow cytometry.Results:?1?The content of total saponins was 81.76%,the content of Rg1,Re,Rf,Rb1,Rc,Rh1,Rb2,Rb3,F1,Rd,F2,Rk3,Rg3,Rg3?R?,PPT,Rk1,Rg5 and Rh2 were 11.23%,18.32%,4.37%,5.96%,10.34%,1.40%,3.15%,0.57%,0.22%,7.42%,2.83%,6.30%,2.46%,1.00%,0.16%,1.73%,2.31%and 1.68%.?2?Cobalt chloride decreased the mitochondrial membrane potential and increased the active oxygen in H9c2 cells.800?M and 12h cobalt chloride were selected as the suitable conditions for the establishment of the model.?3?Compared with the Ctrl group,CoCl₂ significantly increased ROS and ECAR levels,while GS decreased them;compared with the Ctrl group,CoCl₂ significantly decreased MMP,glucose uptake,mitochondrial biogenic level,OCR,ATP,NAD⁺/NADH,SIRT1 and PGC1?protein,while GS increased them.?4?Compared with the Ctrl group,CoCl₂ significantly increased ROS and ECAR levels,while GS decreased them and NAM increased them;compared with the Ctrl group,CoCl₂ significantly decreased MMP,glucose uptake,mitochondrial biogenesis,OCR,ATP,NAD⁺/NADH,SIRT1 and PGC1?protein,while GS increased them and NAM decreased them.?5?Compared with the Ctrl group,CoCl₂ significantly reduced MMP,while GS,Rg1,Re,Rf,Rb1,Rc and Rb2 increased MMP.Conclusion:Ginsenosides has a well protective effect on the injury of H9c2 cells induced by cobalt chloride.SIRT1/PGC1?signaling pathway plays an important role in this process,among which Rg1,Re,Rf,Rb1,Rc and Rb2 may be the material basis of its hypoxia resistance.