Tanshinone?A Regulates Autophagy And Protects Ox-LDL-induced Endothelial Cell Damage And Its Mechanism

Purpose:By establishing an human umbilical vein endothelial cells(EA.hy926)oxidative stress injury model induced by oxidative low density lipoprotein(ox-LDL),we observed the effect of tanshinone ?A on EA.hy926 cells injured by oxidative stress through the regulation of PI3K/Akt/m TOR signaling pathway to mediate the autophagy and explored its molecular mechanism.Material and method:The first part: Effect of tanshinone II A on autophagy of ox-LDL-induced EA.hy926 and its time-effect relationship.1.The effect of different concentrations of ox-LDL and tanshinone ?A on the proliferation of EA.Hy926 cells was observed by MTT assay,and the optimal concentrations of ox-LDL and tanshinone ?A in the intervention of EA.Hy926 cells were screened.2.We investigated the effect of tanshinone ?A on oxidative stress injury induced by ox-LDL in EA.hy926 cells.Colorimetric method was used to detect MDA content and SOD activity in oxidative stress injury.Chemical staining probe was used to observe oxidative stress and ROS staining.3.The expression of MAP1-LC3 protein in cells at different time points was detected by fluorescence immunocytochemistry and Western blot.The effect of tanshinone II A on autophagy of ox-LDL-induced EA.Hy926 was observed.The second part is the molecular mechanism of Tanshinone II A regulating PI3K/Akt/m TOR signaling pathway mediating autophagy on endothelial cells from Oxidative Stress Injury The PI3K/Akt/m TOR pathway inhibitor LY294002 was used to culture EA.hy926 cells in vitro and randomly divided into the normal group,the model group,the ox-LDL+Tanshinone ?A group,the ox-LDL+tanshinone ?A+LY294002 group,the tanshinone ?A group,and the LY294002 group.Colorimetric assay was used to detect the MDA content and SOD activity in oxidative stress.The autophagy was detected by fluorescence immunoassay and Naolive3 D cell explorer.Western blot was used to detect the expression of autophagy-related protein and PI3K/Akt/m TOR pathway protein.Based on PI3K/Akt/m TOR signaling pathway,the protective effect and mechanism of tanshinone II A-mediated autophagy on oxidative stress injury induced by ox-LDL in endothelial cells were investigated.Results:1.100?g / ml ox-LDL can induce obvious oxidative stress injury in EA.Hy926 cells 24 hours,and Tanshinone ?A can reduce the oxidative stress injury induced by ox-LDL in EA.Hy926 cells.LC3 / DAPI staining found that LC3 autophagosomes of Tanshinone ?A group increased at 6h,12 h and 24 h compared with model group and reached the peak at 12 h.Western blotting showed that LC3-II/I protein expression was significantly increased at 12 H and 24 H,but the expression of LC3-II/I at 12 H was the most obvious.2.Compared with the normal group,MDA content in the model group increased,SOD activity decreased,and LC3-II/Iprotein content increased(P <0.01);Tanshinone?A decreased MDA content,and SOD activity,LC3-II/Iprotein content increased(P <0.01).After adding PI3 K inhibitor LY294002,MDA content increased,SOD activity decreased,and LC3-II/I protein content decreased.3.Compared with the normal group,the expression of PI3 K,P-Akt and P-m TOR in the model group decreased significantly(P <0.05 or P <0.01).Compared with the model group,the expression of PI3 K,P-Akt and P-m TOR in the model group decreased significantly(P <0.05 or P <0.01).Compared with ox-LDL + Tanshinone?A group,the expression of PI3 K,P-Akt,P-m TOR protein in PI3 K inhibitor LY294002 decreased significantly(P <0.05 or P <0.01).Conclusion:1.Tanshinone ?A had significant inhibitory effects on the injury,such as reduction of viability and enhanced oxidative stress in ox-LDL-induced EA.hy926 cell.2.Tanshinone II A had an effect on the autophagy activity of ox-LDL-induced EA.hy926 oxidative stress cell model and had a time-effect relationship.3.Tanshinone ?A could promote the autophagic activity of EA.hy926 cells induced by ox-LDL,and the enhancement of autophagy activity was one of the mechanisms of tanshinone ?A on ox-LDL-induced EA.hy926 cell injury.4.Tanshinone ?A may promote autophagy by regulating the PI3K/Akt/m TOR signaling pathway and protect against oxidative stress injury induced by ox-LDL in EA.Hy926 cells.