Protective Effect Of Ginsenoside Rg3 On Heart Failure Based On SUMOylation Of SERCA2a Mediate Intracellular Calcium Homeostasis

Objective:1.To observe the protective effect of ginsenoside Rg3 on the heart of mice with heart failure,and explore the effect of ginsenoside Rg3 on abnormal Ca²⁺cycle homeostasis of cardiomyocytes.2.To clarify the role of SUMOylation of SERCA2a in mediate the cardioprotective effect of ginsenoside Rg3 on heart failure.3.To explore if ginsenoside Rg3 enhances the SUMOylation of SERCA2a to improve the cardiac function after heart failure is mediated by SUMO1.Methods:We established a mouse model of heart failure induced by aortic arch constriction（TAC）and a model of HL-1 cell hypertrophy induced by isoproterenol（ISO）,and treated with ginsenoside Rg3.We detect the cardiac function of mice by using a high-resolution small animal ultrasound instrument;and detect the gene expression levels of ANP and BNP by using real-time quantitative PCR（Q-PCR）;and detect the level of SUMOylation of SERCA2a by using co-immunoprecipitation technology（Co-IP）;and detect the protein expression levels of NCX,PLB,Ry R2,ATF6 and CHOP in heart tissue and HL-1 cells by using Western blotting;Detect the positioning of SERCA2a and SUMO1 on the built-in network by using a laser confocal microscope;detect the hypertrophy state of HL-1 cells by immunofluorescence staining withα-actin;and detect intracellular Ca²⁺overload by using Fluo-3AM dye;and detect the level of reactive oxygen species（ROS）in cells by using DCFH-DA dye;We established a SERCA2a SUMOylation site mutation plasmid,and transfected HL-1 cells with ginsenoside Rg3 treatment,and extracted the endoplasmic reticulum to detect the activity level of SERCA2a;HE staining was used to characterize myocardial tissue in mice with heart failure,and Masson staining was used to evaluate cardiac tissue collagenization.Results:1.The experimental results of the high-resolution small animal ultrasound instrument show that ginsenoside Rg3 significantly improves the cardiac function of TAC-induced wild-type heart failure mice,including enhancing the ejection fraction（EF）and short axis shortening of mice with heart failure.2.HE staining results showed that ginsenoside Rg3 significantly inhibited the arrangement of myocardial fibers in the heart of mice after heart failure,and reduced the degree of inflammatory cell infiltration in the hearts of wild-type heart failure mice.Masson staining results showed that ginsenoside Rg3 significantly reduced the degree of collagen deposition in the hearts of wild-type heart failure mice.3.RT-PCR results showed that the m RNA expression of ANP and BNP increased after heart failure in wild-type mice,and the administration of ginsenoside Rg3 inhibited the m RNA expression level of ANP and BNP.4.In the ISO-induced HL-1 cell injury model,immunofluorescence results showed that compared with the model group,ginsenoside Rg3 significantly reduced the hypertrophy area of ISO-induced HL-1 cells;Fluo-3AM was used to detect intracellular Ca²⁺content,the results showed that the intracellular Ca²⁺content of the ISO group was significantly higher than that of the PBS treatment group,and the intracellular Ca²⁺overload of the HL-1 cells was stimulated by ISO.The results of Tunel staining showed that the apoptotic rate of cells in the ISO group was significantly increased,and administration of ginsenoside Rg3 significantly inhibited the intracellular Ca²⁺overload and apoptosis induced by ISO.5.Western blotting results showed that ginsenoside Rg3 significantly reduced the expression levels of NCX,ATF6 and CHOP proteins in the hearts of wild-type heart failure mice.The results of detection of Co-IP and SERCA2a activity showed that the SUMO level of SERCA2a in the heart of wild-type heart failure mice and in ISO-induced HL-1 cells decreased,accompanied by the decreased activity of SERCA2a,and the administration of ginsenoside Rg3 increased the level of SERCA2a.The level and activity of SUMO of SERCA2a.6.The results of immunofluorescence double staining showed that ginsenoside Rg3enhanced the expression of SUMO1 in HL-1 cells.The results of confocal laser microscope showed that ginsenoside Rg3 promoted the co-localization of SUMO1 and SERCA2a on the endoplasmic reticulum.7.After SUMO1 gene knockout,ginsenoside Rg3 failed to enhance the cardiac function of SUMO1^-/^-heart failure mice.The staining results of tissue sections showed that ginsenoside Rg3 could not significantly inhibit the degree of inflammatory cell infiltration in the heart of SUMO1^-/^-mice with heart failure.The results of Masson staining showed that ginsenoside Rg3 had no significant difference in the degree of cardiac collagen deposition in the SUMO1^-/^-heart failure mice treatment group and the model group.Western blotting results showed that ginsenoside Rg3 failed to reduce the expression levels of NCX,ATF6 and CHOP proteins in the hearts of SUMO1^-/^-heart failure mice.The results of detection of Co-IP and SERCA2a activity showed that compared with the heart of SUMO^-/^-heart failure mice,the administration of ginsenoside Rg3 failed to significantly improve the level and activity of SERCA2a.8.Transfect HL-1 cells with the SUMOylation site mutation plasmid（ATP2A2 KR）of SERCA2a,and perform ISO induction and ginsenoside Rg3 treatment.Western blotting results showed that after transfection of ATP2A2 KR,ginsenoside Rg3 failed to reduce the protein expression levels of ATF6 and CHOP induced by ISO.The results of Co-IP and RT-PCR experiments showed that after transfecting HL-1 cells with the ATP2A2KR plasmid,ginsenoside Rg3 failed to enhance the ISO-induced reduction in the level of SUMO of SERCA2a,and failed to reduce the m RNA expression levels of ANP and BNP in ATP2A2 KR group.Fluorescence results showed that after transfection with ATP2A2 KR,ginsenoside Rg3 failed to inhibit the cell hypertrophy induced by ISO,which was not significantly different from the ISO group.In addition,ATP2A2 KR transfection caused ginsenoside Rg3 to fail to inhibit the intracellular Ca²⁺overload and apoptosis induced by ISO.The DCFH-DA fluorescent probe was used to detect intracellular ROS levels.The results showed that ginsenoside Rg3 significantly reduced the ROS levels in HL-1 cells induced by ISO in the Vector group.After transfection of ATP2A2 KR,ginsenoside Rg3 failed to inhibit the excessive ROS levels in cells induced by ISO.JC-1 fluorescent probe was used to detect the effect of ginsenoside Rg3 on mitochondrial membrane potential.The results showed that ginsenoside Rg3significantly reduced the decrease in mitochondrial membrane potential of HL-1 cells induced by ISO in the Vector group.After transfection of Myc-ATP2A2 KR,ginsenoside Rg3 failed to improve the reduction of mitochondrial membrane potential induced by ISO,which was not significantly different from the ISO group.Conclusions:1.Ginsenoside Rg3 significantly improved cardiac function and reduced myocardial damage in mice with heart failure.And significantly inhibited the cell hypertrophy and apoptosis induced by ISO.2.Ginsenoside Rg3 enhanced the SUMOylation level and activity of SERCA2a in the heart of mice with heart failure and ISO-induced HL-1 cells,and improved the damaged Ca²⁺homeostasis and endoplasmic reticulum stress response in cardiomyocytes.3.Ginsenoside Rg3 enhanced the activity of SERCA2a by enhancing the SUMO of SERCA2a to play a cardioprotective effect after heart failure.SUMO1 mediates the protective effect of ginsenoside Rg3 on cardiomyocyte Ca²⁺cycle homeostasis.