The Mechanism Research Of Proteceive Effect Of SGLT2 Inhibitors On Cardiomyocytes Injury Induced By Palmitic Acid

Objective:In this study,H9c2 cardiomyocytes were treated with palmitic acid（PA）to establish myocardial lipid toxic injury model,and to explore the protective effect of sodium glucose cotransporter 2（SGLT2）inhibitors empagliflozin（EMPA）and dapagliflozin（DAPA）on PA-induced cardiomyocyte injury and its possible mechanism.Methods:1.CCK-8 method was used to detect the effects of different concentrations of PA on the survival rate of H9c2 cardiomyocytes,and the optimal concentration of PA was selected.The cultured H9c2 cardiomyocytes were randomly divided into control group and PA group(25μmol·L^-1,50μmol·L^-1,100μmol·L^-1,200μmol·L^-1)for 24 h;2.CCK-8 method was used to detect effects of different concentrations of EMPA and DAPA on the survival rate of H9c2 cardiomyocytes treated with PA,and the optimal concentration of EMPA and DAPA was selected.H9c2 cardiomyocytes were randomly divided into control group,PA group,PA+EMPA(0.1μmol·L^-1,1μmol·L^-1,10μmol·L^-1)group,and PA+DAPA(0.1μmol·L^-1,1μmol·L^-1,10μmol·L^-1)group;3.The content of reactive oxygen species（ROS）in H9c2 cardiomyocytes treated with different concentrations of PA(100μmol·L^-1,150μmol·L^-1,200μmol·L^-1)was detected by flow cytometry and fluorescence microscopy,and the optimal concentration of ROS induced by PA was selected;4.Flow cytometry and fluorescence microscopy were used to detect ROS content in H9c2 cardiomyocytes treated with EMPA and DAPA on PA;5.The cultured H9c2 cardiomyocytes were randomly divided into control group,PA group,PA+EMPA(1,10μmol·L^-1)group,PA+DAPA(1,10μmol·L^-1)group,PA+LY294002 group and PA+RAPA group.The protein expressions of TLR4,NF-κB,AKT and m TOR in H9c2 cardiomyocyte were detected by Western blot experiments and data analysis was performed.Results:1.CCK-8 experimental results showed that the cell survival rate was significantly decreased when PA concentration was 100μmol·L^-1and 200μmol·L^-1,and there was a significant difference compared with control group（P<0.01）,PA 100μmol·L^-1was selected as the subsequent experimental concentration;2.CCK-8 results showed that EMPA and DAPA at 1 and 10μmol·L^-1significantly increased the survival rate of H9c2 cardiomyocytes treated with PA（P<0.01）;3.ROS experiments showed that H9c2 cardiomyocytes treated with PA 150μmol·L^-1and 200μmol·L^-1for 12 h could significantly induce ROS production（P<0.01）.PA 200μmol·L^-1was used for follow-up experiments;4.ROS experiments showed that pretreatment of EMPA and DAPA significantly inhibited the production of ROS induced by PA,and the effect of DAPA is more obvious（P<0.01）;5.Western blot experiments showed that pretreatment EMPA and DAPA could down-regulate the protein expression of TLR4 and phosphorylated m TOR induced by PA（P<0.05 or P<0.01）,enhanced the expression of phosphorylated AKT（P<0.01）,but had no effect on phosphorylated NF-κB p65 protein expression.Pretreatment AKT inhibitor LY294002 and m TOR inhibitor RAPA could inhibit the expression of phosphorylated AKT and phosphorylated m TOR proteins（P<0.01）.Conclusion:EMPA and DAPA can inhibit ROS production,DAPA has a more significant effect than EMPA,and by down-regulating the expression of TLR4 and phosphorylated m TOR protein,increasing the expression of phosphorylated AKT protein,thereby protecting the cardiomyocyte injury induced by PA.These experimental results indicated that EMPA and DAPA had certain protective effect on lipotoxic myocardial injury,providing theoretical basis for the treatment of lipotoxic cardiomyopathy.