The Effect Of MEHP On Cardiovascular Toxicity And Its Mechanism

In this study,oxidative stress and FOXO3 a were set as the entry point to explore the possible molecular mechanism of cardiomyocyte and isolated heart toxicity induced by MEHP.Part I: The effect of MEHP on myocardial damage and the mechanism about oxidative stressObjective: To investigate the oxidative damage of heart and cardiomyocyte cells induced by MEHP.Methods:1.The isolated heart perfusion was used to determine the toxicity of MEHP on rat isolated heart.2.Cell culture and cell model establishment.3.Cell viability was determinrd by MTT assay,ROS levels,cell mitochondrial membrane potential and cell apoptosis of AC16 cells and isolated cardiomyocytes was measured by flow cytometry,DNA damage of AC16 cells after MEHP treatment was measured by SCGE assay,LDH content in Cell Supernatant and Cardiac Perfusate was measured by Automatic Biochemistry Analyzer,histopathological changes was observed under a microscope.Results:1.MEHP could reduce the left ventricular systolic and diastolic function of isolated rat heart,caused the decrease of heart rate and do damage to myocardial cell membrane.The ROS increased after MEHP treatment,and MEHP induced pathological changes of isolated heart of rats.2.MEHP caused a decrease of cell viability and induced cell membrane cytotoxicity in AC16 cells,increased ROS level,DNA damage,apoptosis and mitochondrial damage in AC16 cells.3.NAC attenuated MEHP-induced cardiac damage.4.NAC attenuated the cytotoxicity of AC16 cells induced by MEHP.Part II: The effect of FOXO3 a on oxidative stress and apoptosis induced by MEHP in AC16 cellsObjective: To investigate the role of FOXO3 a in MEHP-induced oxidative stress and apoptosis.Methods:1.Immunofluorescence of FOXO3 a,P-FOXO3 a was determined to observe the nuclear translocation of these two proteins induced by MEHP.2.Cell lines of FOXO3 a over expression and silence were created by transfection of plasmid FOXO3 a TM and targeted silencing siRNA FOXO3 a in AC16 cells.FOXO3 a expression was detected by RT-PCR and Westernblot.3.Effect of FOXO3 a on oxidative stress of AC16 cells.4.FOXO3 a,Mn-SOD,ARC mRNA and protein expression in MEHP treatment group,MEHP-treated FOXO3 aTM group and MEHP-treated siRNA FOXO3 a group were analyzed by Western blot.Results:1.After the treatment of MEHP,FOXO3 a,Mn-SOD and ARC protein expression levels increased and P-FOXO3 a protein decreased significantly compared with the control group.2.After the treatment of MEHP,the mRNA expression levels of FOXO3 a,Mn-SOD and ARC significantly increased in AC16 cells compared with the control group.3.The FOXO3 a high and low expression cell lines in AC16 cells was identified to be successful.4.The ROS level and apoptosis rate decreased in FOXO3 a high expressed AC16 cells.FOXO3 a,Mn-SOD and ARC mRNA and protein expression were further increased in FOXO3 a high expressed AC16 cells.5.The ROS levels and apoptosis rate increased in FOXO3 a silent AC16 cells.FOXO3 a,Mn-SOD and ARC mRNA and protein expression further decreased in FOXO3 a silent AC16 cells compared with cells without FOXO3 a silent.Conclusions:1.MEHP treatment could induce AC16 cell damage.2.MEHP treatment could induce isolated rat heart damage.3.NAC may reduce MEHP-induced heart damage to varying degrees.4.The mechanism of oxidative stress and apoptosis induced by MEHP might be related with the FOXO3 a signals.