The Mechanism Of Resveratrol Reducing Oxidative Damage And Apoptosis Of HUVECs Induced By LPS

Objective: To study the role of resveratrol in oxidative stress of Human umbilical vein endothelial cells(HUVECs),and to investigate whether resveratrol(Resv)can regulate the expression of PCSK9 by increasing the synthesis of free radical scavenging enzymes,which leads to the reduction of ROS synthesis in cells,and down-regulating the expression of PCSK9,thereby reducing the oxidative damage and apoptosis of HUVECs,and finally achieving its antioxidant activity.Method: HUVECs were cultured in vitro,and treated with different concentrations(10,20,30,40,60,100)μg/mL LPS for 24 h,MTT method was used to screen the appropriate concentration of LPS.After pretreatment with different concentrations(5,10,20,30,40,60,100)μmol/L Resv for 24 h,HUVECs were treated with appropriate concentration of LPS for 24 h,suitable treatment concentration of resveratrol was obtained by MTT.The enzyme activity assay kit and reactive oxygen species assay kit were adopted to measured the activities of free radical scavenging enzymes CAT,GPX and SOD and the content of ROS in HUVECs,respectively.The expression of PCSK9,p38 MAPK and its phosphorylation were detected by Western blotting.The HUVECs apoptosis was analyzed by Hoechst 33342 staining and Annexin V-FITC/PI double staining.The apoptosis-related proteins Bax,Bcl-2 and caspase-3 and cleaved caspase-3 were determined by Western-blotting.Results:1.Cell viability of HUVECs treated with 10 μg/mL LPS for 24 h did not change significantly,and that of cells treated with 20,30,60,100 μg/mL LPS for 24 h was significantly reduced(P<0.05),which Compared with control group.The results indicated that the suitable treatment concentration of LPS on HUVECs was 20 μg/mL.After pretreatment with different concentrations of resveratrol for 24 h,HUVECs were treated with 20 μg/mL LPS for 24 h,MTT assay showed that cell viability of HUVECs pretreated with 30 μmol/L resveratrol was significantly higher than that of LPS group(P<0.05).The results showed that the optimun concentration of resveratrol could be divided into three groups: low 5 μmol/L,medium 20 μmol/L,and high 30 μmol/L.2.Hoechst 33342 staining showed that there were many apoptotic cells in the LPS group compared with the control group.When it compared with LPS group,the number of apoptotic cells in the 5 μmol/L Resv,20 μmol/L Resv,and 30 μmol/L Resv groups gradually decreased.3.Annexin V-FITC/PI double staining showed that,compared with Control group,about 16.1% of cells in LPS group showed apoptosis,and compared with LPS group,The apoptosis of 5 μM Resv,20 μmol/L Resv and 30μmol/L Resv group was significantly decreased with 5.65%,5.27% and 4.72%,respectively(P<0.05).4.The activity assay of CAT,GSH-PX and SOD showed that,compared with control group,20 μg/mL LPS treatment could significantly reduce the activity of CAT,SOD and GPX in HUVECs(P<0.05);Resveratrol of all three concentration groups could increase significantly SOD,CAT and GPX activities in LPS-induced HUVECs compared with LPS group(P<0.05).ROS detection showed that,compared with the control group,the ROS content in LPS group increased significantly(P<0.05).While,compared with LPS group,the ROS content decreased significantly in 5 μmol/L Resv,20 μmol/L Resv,and 30 μmol/L Resv groups(P<0.05).5.Western blotting analysis showed that the expression of PCSK9 protein in LPS group was significantly increased compared with control group(P<0.05).The expression of PCSK9 moderately decreased in 5μmol/L Resv and 20 μmol/L Resv group,when it compared with LPS group.While the PCSK9 protein in the 30 μmol/L Resv group was decreased significantly(P<0.05).6.The relative expression of p-p38 MAPK refered to p38 MAPK protein in LPS group was increased significantly compared with control group.When it compared with LPS group,the expression of p-p38 MAPK protein in 5 μmol/L Resv group was slightly decreased.The expression of p-p38 MAPK protein in 20 μmol/L Resv and 30 μmol/L Resv group was decreased significantly(P<0.05).7.When it compared with Control group,analyzed by Western blot,the expression of caspase-3 protein slightly increased,and the expression of cleaved caspase-3 protein and Bax protein increased significantly(P<0.05)in LPS group.When it compared with LPS group,in 5 μmol/L Resv group,the expression of Bax protein and Bcl-2 protein were increased significantly(P<0.05);in 20μM Resv group,the expression of caspase-3 protein and cleaved caspase-3 decreased slightly,the expression of Bax protein was markedly decreased(P<0.05),and the expression of Bcl-2 protein was signally increased(P<0.05);in 30μmol/L Resv group,the expression of caspase-3 protein was decrease dindistinctively,and cleaved caspase-3 and Bax protein expression decreased significantly(P<0.05),while Bcl-2 protein expression observably increased(P<0.05).Conclusion: Resveratrol may down-regulate the expression of PCSK9 through p38 MAPK pathway to decrease the synthesis of free radical scavenging enzymes such as CAT,SOD and GPX in HUVECs,which results in the reduction of ROS synthesis in cells.Meanwhile,by p38 MAPK pathway resveratrol could enhance the expression of anti-apoptotic gene Bcl-2,and inhibit the expression of pro-apoptotic genes cleaved caspase-3 and Bax.Through the above mechanisms resveratrol could reduces oxidative damage and apoptosis of HUVECs,and finally achieving its antioxidant activity..