Protective Effect And Mechanism Of Resveratrol On Lipopolysaccharide-induced Damage Of H9c2 Cell

Objective To investigate the pathways of LPS-induced H9c2 cell damage and the effect and relevant mechanism of Resveratrol on lipopolysaccharide induced H9c2 cell injury.Methods 1.Randomly to divide the H9c2 cells into Contral group(Con),Resveratrol 5 ?M group(R5),Resveratrol 10 ?M group(R10),Resveratrol 20 ?M group(R20),Resveratrol 50?M group(R50).Con group received no treatment,H9c2 cells from group R5,R10,R20,R50 were treated with Resveratrol for 24 hours.MTT were used to detect cell proliferation.2.To divide the H9c2 cells into Contral group(Con),lipopolysaccharide group(LPS),lipopolysaccharide +Resveratrol 5 ?M group(L+R5),lipopolysaccharide +Resveratrol 10?M group(L+R10),lipopolysaccharide +Resveratrol 20 ?M group(L+R20),lipopolysaccharide +Resveratrol 50 ?M group(L+R50).Con group received no treatment,H9c2 cells from group L+R5,L+R10,L+R20,L+R50 were pretreated with Resveratrol for 24 hours.then,cells from group LPS,L+R5,L+R10,L+R20,L+R50 were incubated with LPS for 12 hours.MTT were used to detect cell proliferation.3.H9c2 cells were cultured in six-well plates for 24 hours in 37 ? 5% CO2 Incubator,LPS(10?g/mL)was used to stimulate H9c2 cells for 0,5,10,20,30 and 60 min to evaluate the effect of LPS at different time points on the p-P38,P38,p-JNK1/2,JNK1/2,p-ERK1/2,ERK1/2,I?B?,p-p65,p65 and ?-actin.4.To divide the H9c2 cells into Contral group(Con),lipopolysaccharide group(LPS),lipopolysaccharide +Resveratrol 5 ?M group(L+R5),lipopolysaccharide +Resveratrol 10 ?M group(L+R10),lipopolysaccharide +Resveratrol 20 ?M group(L+R20),lipopolysaccharide +Resveratrol 50 ?M group(L+R50).Con group received no treatment,H9c2 cells from group L+R5,L+R10,L+R20,L+R50 were pretreated with Resveratrol for 24 hours.then,cells from group LPS,L+R5,L+R10,L+R20,L+R50 were incubated with LPS for 20 min.The protein level of p-P38,P38,p-JNK1/2,JNK1/2,p-ERK1/2,ERK1/2,I?B?,p-p65,p65,?-actin was measured by Western blot respectively.5.The H9c2 cells were randomly divided into Con group,LPS group,RSV group,L + R5 group;wherein the RSV group,L + R5 group were pre-treated with 5?M of RSV for 24 hours,then LPS group and L + R5 group were co-treated with 10?g / mL of LPS for 12 hours.Oxygen species reactive(ROS)levels were detected by Immunofluorescence method.Flow cytometry were used to detect cell death.Detected by ELISA in the cytoplasm of TNF-?expression.Result 1)Little effect of low concentrations of resveratrol on H9c2 cells,but higher concentrations(50?M)of resveratrol significantly inhibited H9c2 cell proliferation(P<0.05).2)Compared with the control group,the stimulation of LPS on H9c2 cells for 12 hours produce significant inhibition of cell proliferation,while pre-processed H9c2 cell with 5?M of resveratrol significantly reduced inhibition of LPS on H9c2 cell proliferation(P <0.05).3)After the H9c2 cells were treated with LPS for different time,it was found after LPS stimulation 5min,the expression levels of p-P38,p-JNK1/2,p-ERK1/2,p-p65 started to rise,while the I?B? expression level began to drop.After stimulation 20 min,the phosphorylation levels P38,JNK1/2,ERK1/2,p65 increased and reached peak levels(P <0.01),while I?B? decreased to the lowest(P <0.01).The p-P38,p-JNK1/2,p-ERK1/2,p-p65 expression levels began to decline,I?B? expression levels begin to rise after treatment 30 min.4)After treatment H9c2 cardiomyocytes with different concentrations of RSV for 24 hours,then treated with LPS 20 min,5?M RSV can be significantly reduced p-P38,p-JNK1/2,p-ERK1/2,p-p65 expression(P < 0.01)and raised I?B? expression(P <0.01),but 50?M RSV significantly upregulated p-P38,p-JNK1/2,p-ERK1/2,p-p65 expression(P <0.01),and reduced the expression of I?B?(P <0.01).5)Compared with the control group,the generation of ROS in the LPS group in H9c2 significantly increased(P <0.01);and after 5?M concentration of RSV pretreatment can significantly reduce the amount of ROS generation(P <0.01).6)Using flow cytometry to detecte different groups cell apoptosis,the number of H9c2 myocardial apoptosis of LPS treatment significantly increased(P <0.05),but after 5?M concentration of RSV pretreatment significantly reduced LPS-induced H9c2 myocardial apoptosis(P <0.01).7)Compared with Con group,LPS-induced TNF-? was significantly up-regulated(P <0.05),whereas pretreatment with 5?M concentrations of RSV,the expression of TNF-? were significantly decreased(P <0.05).Conclusion 1)LPS induced H9c2 cell damage by up-regulating p-P38,p-JNK1/2,p-ERK1/2,p-p65 expression and down-regulating I?B? expression.2)Resveratrol may exert its cytoprotection effects on LPS-induced H9c2 cells via down-regulating the activation of p-P38,p-JNK1/2,p-ERK1/2,p-p65 and up-regulating the activation of I?B?.