Study On The Protective Effect Of Indoletrimethanol On Lipotoxic Injury Of Cardiomyocytes

Objectives:In this study,palmitic acid（PA）was used to stimulate H9C2 cells to induce myocardial cell lipotoxicity injury,so as to establish a cell model and explore the protective effect of indole-3-carbinol（I3C）in high fat induced cardiomyocyte lipotoxicity injury by regulating AMPK/mTORC1/p70S6K pathway.Methods:（1）Establishment of a model of myocardial cell lipotoxicity induced by high fat:In this experiment,H9C2 cardiomyocytes were used for in vitro culture,and different concentrations of PA(0,0.1,0.2,0.4 mmol·L^-1)were used to stimulate H9C2cardiomyocytes for 24 h,and the concentration dependent effects were analyzed;H9C2cardiomyocytes were stimulated with PA 0.1 mmol·L^-1 and PA 0.2 mmol·L^-1（0,12,24,48h）,and the time dependent effects were analyzed;the activity of H9C2 cardiomyocytes was detected by MTT method,and the effective concentration and time were screened out,thus confirming the establishment of a model of cardiomyocyte lipotoxic injury induced by high fat.（2）To explore the mechanism of high fat induced cardiomyocyte lipotoxicity injury:Control group,PA 0.1 mmol·L^-1 group,PA 0.2 mmol·L^-1 group,stimulate H9C2 cardiomy-ocytes for 24 h.DCFH-DA probe was used to detect the level of reactive oxygen species（ROS）in each group of cardiomyocytes;Annexin V-FITC/PI kit was used to detect the apoptosis;JC-1 probe was used to detect the mitochondrial membrane potential;Western blot method was used to detect the expression levels of AMPK/mTORC1/p70S6K pathway and apoptosis pathway related proteins.（3）To explore the protective effect of I3C in high fat induced cardiomyocyte lipotoxicity:H9C2 cardiomyocytes were treated with different concentrations of I3C for 24 h;H9C2 cardiomyocytes were stimulated with different concentrations of I3C for PA 0.2 mmol·L^-1 for 24 h;the activity of H9C2 cardiomyocytes was detected by MTT method,and the optimal concentration of I3C was selected.In the Control group,PA 0.2 mmol·L^-1 group,PA 0.2 mmol·L^-1+I3C 200μmol·L^-1 group,H9C2cardiomyocytes were stimulated for 24 h.DCFH-DA probe was used to detect the level of ROS in cardiomyocytes in each group;Annexin V-FITC/PI kit was used to detect the apoptosis;JC-1 probe was used to detect the level of mitochondrial membrane potential;Western blot method was used to detect the expression levels of AMPK/mTORC1/p70S6K pathway and apoptosis pathway related proteins.Results:（1）After PA stimulated H9C2 cardiomyocytes at different concentrations and time,the cell survival rate showed a concentration dependent and time dependent decreasing trend.（2）In the PA 0.2 mmol·L^-1,H9C2 cardiomyocytes were stimulated for24 h.Compared with the Control group,the ROS level in the cardiomyocytes increased significantly,the mitochondrial membrane potential level decreased significantly,and the apoptosis phenomenon increased significantly.At the same time,AMPK phosphorylated decreased,the expression of p-mTORC1 and p-p70S6K protein increased significantly,the expression of antiapoptotic protein Bcl-2 decreased,and the expression of proapoptotic proteins Bax and Cleaved caspase 3 increased.（3）After stimulation of H9C2 cardiomy-ocytes with different concentrations of I3C for 24 h,the cell survival rate did not change significantly,indicating that I3C has no toxic effect on H9C2 cardiomyocytes.After pre-treatment with I3C,as the concentration of I3C gradually increased,the survival rate of cardiomyocytes showed a concentration dependent increase compared with the PA 0.2mmol·L^-1 group.When pretreatment 200μmol·L^-11 I3C,the survival rate of cardiomyocytes increased most obviously,close to the level of normal control group.At the same time,compared with the PA 0.2 mmol·L^-1 group,the I3C 200μmol·L^-1 pretreatment group can significantly reduce the ROS level in myocardial cells and inhibit the reduction of mitochondrial membrane potential,thereby reducing apoptosis.While I3C can activate the phosphorylation of AMPK,the expression of p-mTORC1 and p-p70S6K protein significantly reduced,the expression of antiapoptotic protein Bcl-2 increased,and the expression of proapoptotic proteins Bax and Cleaved caspase 3 decreased.Conclusion:（1）PA stimulates H9C2 cardiomyocytes can induce myocardial cell lipotoxic injury.（2）I3C may reverse the high fat induced myocardial cell lipotoxicity injury by regulating the AMPK/mTORC1/p70S6K pathway.