Study On The Protective Effect Of Honokiol On Lipotoxic Myocardial Injury And Related Mechanisms

Objective:In this study,H9C2 cardiomyocytes were cultured in vitro.The aim to investigate the protective effect of honokiol（HON）on myocardial lipotoxicity injury induced by palmitic acid（PA）,and its association with endoplasmic reticulum stress-mitochondrial signal pathway through detect cell apoptosis,intracellular reactive oxygen species level,mitochondrial membrane potential changes and endoplasmic reticulum stress（ERS）and mitochondrial related protein expression levels.Methods:（1）Establishment of H9C2 myocardial lipotoxicity injury model:H9C2myocardial cells were stimulated with different concentrations of PA(0,0.1,0.2,0.4,0.8,1mmol·L^-1)for 24 h;in different time（0,12,24,48 h）,administer PA 0.2 mmol·L^-1 to stimulate myocardial cells,and evaluate cell proliferation ability by MTT method,so as to select effective concentration and time,and determine the appropriate conditions for establishing a lipotoxicity model of H9C2 myocardial cells.（2）Clarify the mechanism of myocardial lipotoxicity:detect the level of reactive oxygen species（ROS）in myocardial cells by DCFH-DA probe,and detect cell apoptosis by Annexin V-FITC/PI double staining method situation,JC-1 probe staining was used to detect changes in mitochondrial membrane potentials of myocardial cells,and Western blot was used to detect the relate proteins expression of ERS and mitochondrial apoptotic pathway.（3）To explore the protective mechanism of HON on lipotoxic myocardial damage:The modeling conditions for myocardial lipotoxic injury were selected as PA 0.2 mmol·L^-1 to stimulate myocardial cells for 24 h.After pretreatment with HON,the cell proliferation ability was evaluated by MTT method,DCFH-DA probe was used to detect the level of ROS in myocardial cells,and Annexin V-FITC/PI double staining method was used to detect the cell apoptosis.Mortality,JC-1 probe staining was used to detect changes in mitochondrial membrane potential of myocardial cells,and Western blot was used to detect the relate proteins expression of ERS and mitochondrial apoptotic pathway.Results:The proliferation ability of H9C2 cardiomyocytes gradually decreased with the increase of PA concentration and prolonged the stimulation time,showing a concentration and time dependence.When PA 0.2 mmol·L^-1 stimulated cardiomyocytes for 24 h,the rate of cardiomyocyte proliferation decreased significantly（P<0.05）.Therefore,we chose PA concentration of 0.2 mmol·L^-1 and stimulation time of 24 h as the modeling conditions,and continued subsequent experiments.Compared with the control group,the ROS level in myocardial cells increased significantly,the apoptosis rate increased significantly,and the mitochondrial membrane potential level decreased significantly in the PA 0.2 mmol·L^-1group.Western blot analysis showed that the expression of endoplasmic reticulum stress-related proteins p-PERK,p-IRE1,GRP78,and CHOP significantly increased,the expression of mitochondrial apoptosis pathway-related proteins Bax,and Cleaved caspase3 increased,while Bcl-2 expression decline.It can be seen that PA-induced myocardial lipotoxicity is related to ERS and mitochondrial apoptotic pathway.When we pretreated with different concentrations of HON,the value-added rate of cells gradually increased,and showed a concentration-dependent.When HON 20μmol·L^-1 pretreated myocardial cells,the cell proliferation ability increased significantly,so that the cell proliferation ability reached almost normal level,and when pretreated with HON 40μmol·L^-1,compared with PA+HON 20μmol·L^-1 group,The rate of cell appreciation has declined.The ROS level,apoptotic rate,and mitochondrial membrane potential level in myocardial cells were HON concentration-dependent.Compared with the PA 0.2 mmol·L^-1 group,the ROS level and apoptosis rate in myocardial cells was significantly reduced,the mitochondrial membrane potential level was significantly increased,and the cell morphology was close to normal in the PA+HON 20μmol·L^-1 group.Western blot detection of ERS-related proteins p-PERK,p-IRE1,GRP78,CHOP expression significantly decreased,mitochondrial apoptosis pathway-related proteins Bax,Cleaved caspase 3 expression levels decreased,while Bcl-2 expression increase.Conclusion:PA induces apoptosis of H9C2 cardiomyocytes through the ERS-mitochondrial apoptosis pathway.HON can significantly improve myocardial cell injury induced by PA.The specific mechanism of HON reduce lipid-induced myocardial injury and improves cardiac function through the ERS-mitochondrial apoptosis pathway.