Oleanolic Acid Can Protect Human Umbilical Vein Endothelial Cells From Oxidative Damage By TBX20 / PPARγ / PON2 Signal Pathway

Objective Repeat the oxidative damage model of human umbilical vein endothelial cells(HUVECs) which is induced by oxygenized low density lipoprotein(ox-LDL), to study the protective effect of oleanolic acid(OA)for this model by TBX20/ PPARγ / PON2 signal pathway. Methods CCK-8 screened out the appropriate concentration of ox-LDL to injury HUVECs, the cytoxicity、pre-protection time and concentration of OA. This experiment was desigened by the following groups: Normal control group, the model group made by ox-LDL, the different concentration of oleanolic acid(10,20,40μmol/L);We took the enzymatic chemical methods to detect catalase(CAT), glutathione glutathione peroxidase(GSH-PX), nitric oxide(NO), total nitric oxide synthase(NOS) activity or content in different groups; Electron spin resonance(ESR) technique was used to detect the free radical of ROS and nitric oxide(NO) in cells; Real-time PCR and Western bolt were used respectively to detect mRNA and protein level(TBX20 、 PPARγ 、 PON2) in these groups;Liposomal siRNA transientl transfection and adding an inhibitor were used to silence the protein expression in cells, and then we detected the protein expression by Western blot. Results According to the IC50, we adopted ox-LDL(100μg/ml) to injury the HUVECs. CCK-8 results showed that:Compared with the model group, the cell survival rates in group pre-treatmented with OA for 16 h were significantly higher. What’s more, the higher concentration, the more obviously increased on cell viability. The enzymatic chemical analysis showed that: The CAT, GSH-PX, NOS activity and NO content in model group was significantly lower than the normal group, but when we gived different concentrations of OA 16 h in advanced, the cells in these groups can dose-dependently increase these indices compared with the model group. The ESR detection showed that the ROS in model group was hugly increased while NO content was decreased; Then we pre-treatmented each group with OA for 16 h, the ROS fress radical droped, at the same time NO content rised compared with the model group. The results of detecting by RT-PCR and Western blot showed that these mRNA and protein levels includingT-bx20, PPARγ, PON2 in model group lessened obviously in model group, while these indices were up in experimental group. The ransfection efficiency of HUVECs which was transfected by T-bx-si RNA can be up to 90%; The protein level of TBX20、PPARγ、PON2 in transfected group was lower than the no-silence group; When we disposed the group with the inhibitor of PPARγ, PPARγ and PON2 protein levels decreased, while the TBX20 protein level had no variation. Conclusion The ox-LDL(100 μg/mL) can accelerate the oxidative damage of HUVECs by inhibiting TBX20 /PPARγ / PON2 signaling pathway; While OA can protect HUVECs from ox-LDL-induced damage through this pathway.