Ursolic Acid Protects HUVECs Against Oxidized-LDL Induced Oxidative Damage By Autophagy Activation Through The SIRT1/Atg5 Pathway

Objective: Our study reproduced the ox-LDL(100 μg/ml) induced human umbilical vein endothelial cells(HUVECs) oxidative damage model. Then we investigated the protective role of ursolic acid(UA)by activating autophagy via the Sirt1/Atg5 signaling pathway. Methods: HUVECs were subjected to following treatments: control group, model group, UA groups, model+UA groups, Vitamin E group,3-MA group and EX-527 group. The content of H2O2, MDA, NO, NOS and SOD were assessed with colormetric method. Transmission electron microscope(TEM) was used to observe the ultrastructure of the HUVECs. Electron spin resonance(ESR) was utilized for the first time to detect the intracellular NO and ROS in HUVECs. The protein level of Sirt1, LC3 B, p62, Atg5 and ac-Atg5 were investigated with western blotting and immunoprecipitation(IP). Results: After exposure to ox-LDL, the cell viability of HUVECs was remarkably decreased, meanwhile, the level of SOD, NOS and NO decreased while the content of MDA and H2O2 increased. 5-20 μM UA pretreatment for 16 h significantly reversed the detrimental alterations dose dependently(P<0.05). TEM results demonstrated condensed cytoplasm and vacuoles in model group cells as compared to control group.Significant amount of autophagosome were observed in UA treated cells. The western blotting results indicated that UA upregulated the Sirt1 expression level and LC3BII/I ratio while it decreased the expression level of p62. ESR data displayed that after 24 h treatment with ox-LDL, ROS generation was increased while NO synthesis was decreased in HUVECs. 5-20 μM UA pretreatment can alleviate these damage, which could be blunted by 3-MA and EX-527. Moreover, in current study, EX-527 significantly down-regulated UA induced autophagy(P<0.01). IP results showed that UA decreased the expression of acetylated Atg5 protein, while the Sirt1 inhibitor EX-527 attenuated this effect(P<0.01), indicating that Sirt1 regulated autophagy by deacetylating autophagy-related genes.Conclusion: 100 μg/ml ox-LDL treatment for 24 h resulted in oxidative damage in HUVECs. While5-20 μM UA dose-dependently improved cell viability and oxidative damage. We established ESR method for the quantification of NO and ROS levels in HUVECs and demonstrated the antioxidant effect of UA is mediated via the STRT1/Atg5 induced autophagy pathway.