The Effect Of LACTB On Cardiomyocytes Damage Induced By High Free Fatty Acids

Objective: 1.To investigate the role of LACTB in cardiomyocytes damage induced by high free fatty acids.2.To determine whether oxidative stress is an intrinsic mechanism of LACTB’s protection.3.To clarify whether LACTB and its effects on oxidative stress mediates acipimox’s protection.Methods: The myocardial cells were divided into the group of control(0 μmol/L free fatty acids)and high free fatty acid(HFFA,400 μmol/L free fatty acids).The expression of LACTB was knockdown down by its specific siRNA.On this basis,the HFFA group was further divided into control,LACTB siRNA and Scra siRNA group.Furthermore,the LACTB siRNA group was divided into the group of vehicle and NAC(5mmol/L NAC pretreatment for 6 h).Meanwhile,HFFA group was divided into vehicle and Acipimox(15mmol/L treatment for 18 h).Finally,the Acipimox was divided into control,LACTB siRNA and Scra siRNA group.1.The expression level of LACTB was detected by quantitative Real-time PCR and Western blot.2.The expression of LACTB was knocked down by siRNA.3.Cell apoptosis was tested by flow cytometry.4.The changes of ROS were detected by DHE staining and ELISA kit.Results: 1.To investigate the effects of HFFA on myocardial cells and LACTB expression,the methods of flow cytometry,DHE staining,ROS ELISA,qRT-PCR,and western blot were used.As a result,compared with control,the apoptosis rate(P=0.0035)and the generation of ROS(P=0.0003)in HFFA group were significantly increased.The elevated expression of LACTB was detected by qRT-PCR,which was consistently with western blot.2.To identify whether LACTB plays a role in the apoptosis of myocardial cells induced by HFFA,the methods of flow cytometry,DHE staining,ROS ELISA,qRT-PCR,and western blot were used.We found that,compared with control and Scra siRNA,the mRNA(LACTB siRNA vs Control:P=0.0001;LACTB siRNA vs Scra siRNA:P=0.0006)and protein level(LACTB siRNA vs Control:P=0.0024;LACTB siRNA vs Scra siRNA:P=0.0031)of LACTB were both significantly decreased in the group of LACTB siRNA.Conversely,the cell apoptosis(LACTB siRNA vs Control:P=0.0010;LACTB siRNA vs Scra siRNA:P=0.0009)and ROS level(LACTB siRNA vs Control:P=0.0013;LACTB siRNA vs Scra siRNA:P=0.0012)in LACTB siRNA group were obvious increased.3.In order to verify whether oxidative stress is the internal mechanism of myocardial injury under HFFA condition,the methods of flow cytometry and DHE staining were used in our study.We demonstrated that,compared with vehicle,the cell apoptosis and ROS level were decreased.4.To investigate the effects of Acipimox on myocardial cells and LACTB expression,the methods of flow cytometry,DHE staining,ROS ELISA,qRT-PCR,and western blot were used.Compared with vehicle,the mRNA(P=0.0018)and protein(P=0.0063)expression of LACTB were both increased the apoptosis rate(P=0.0293)and the generation of ROS(P=0.0015)in Acipimox group were significantly decreased.5.To test whether Acipimox plays a role in HFFA induced myocardial cell injury by modulating LACTB,the methods of flow cytometry,and ROS ELISA were used.As a result,compared with control and Scra siRNA,the apoptosis rate(LACTB siRNA vs Control:P =0.0011;LACTB siRNA vs Scra siRNA:P=0.0016)increased and the generation of ROS increased(LACTB siRNA vs Control:P=0.0003;LACTB siRNA vs Scra siRNA:P=0.00027)in LACTB siRNA group.Conclusion: 1.LACTB downregulation increased myocardial apoptosis in the condition of high free fatty acids,while oxidative stress is the key mechanisms.2.Acipimox could increse the expression of LACTB,and LACTB is a key for acipimox exert protective effect.