Autophagy Modulates The Functions Of Human Umbilical Vein Endothelial Cells Exposed To Hydrogen Peroxide And It's Mechanism

Objectives:1.To investigate the effect of oxidative stress?OS?on the EndMT and proliferation and apoptosis of HUVECs.2.To observe changes of autophagy and Wnt/?-catenin signaling pathway in HUVECs under oxidative stress.3.To investigate modulation of autophagy and Wnt/?-catenin signaling pathway in the regulation of endothelial function under oxidative stress.Methods:1.To study the effect of oxidative stress on the endothelium differentiation and proliferation of HUVECs.The HUVECs were cultured with the following conditions:Control group:normal conditions?21%O₂,5%CO₂,74%N₂,37°C?cultured under HUVECs;Oxidative stress group:Under the normal condition,add the H₂O₂?200?M?.The corresponding HUVECs cultured for 5 days.Then perform the experiments as follows:?1?Detect the intracellular SOD activity and MDA content with spectrophotometry;?2?Using flow cytometry to detect the apoptotic rate;?3?Observe the morphologic changes of cells under optical microscope;?4?Using immunofluorescence staining to observe CD31 and SMA changes and CD31⁺/?-SMA⁺double positive cells under the fluorescence microscope;?5?Western blot analysis was performed to detect the relative protein expressions of CD31,?-SMA,FSP-1 in the HUVECs and comparison.2.To observe the changes of autophagy and Wnt/?-catenin signaling pathway in HUVECs under the condition of oxidative stress.The HUVECs were cultured with the following conditions.Contro groupl:normal conditions?21%O₂,5%CO₂,74%N₂,37°C?;Oxidative stress group:H₂O₂ induced conditions[H₂O₂?200?M?].HUVECs of the above groups were cultured for 5 days.Then perform the following experiment:Western blot analysis was used to measured the related expression of protein of Wnt/?-catenin signal pathway?Wnt3a,?-catenin,survivn?and autophagy?P62,LC3-II,LC3-I and Beclin-1?.3.Regulate the autophagy and Wnt/beta-catenin signaling pathway to modulate the function of HUVECs under oxidative stress.They were divided into 7 groups:i)Oxidative stress group:H₂O₂ induced conditions[H₂O₂?200?M?];ii)H₂O₂+Rapamycin?Rap??100nm?group:HUVECs in H₂O₂?200?M?for 3 days befor co-treatment with Rap for 2 days;?)The H₂O₂+6-amino methyl purine?3MA??5mm?group:HUVECs in H₂O₂?200?M?3 days befor co-treatment with 3MA for 2days;?)The H₂O₂+BIO?1?M?group:HUVECs in H₂O₂?200?M?3 days befor co-treatment with BIO for 2 days;?)The group H₂O₂+XAV939?1?M?:HUVECs in H₂O₂?200?M?3 days then treat with XAV939 for 2 days;?)H₂O₂+Rap+BIO group:H₂O₂?200?M?3 days then treatment with BIO 1 day befor co-treatment with Rap for2 days;?)H₂O₂+Rap+XAV939 group:H₂O₂?200?M?3 days then treatment with XAV939 1 day befor co-treatment with Rap for 2 days;Then perform the following experiments:?1?flow cytometry to detect the apoptotic rate;?2?Western Blot was used to detect the correlation expressioon of Wnt/b-catenin signaling pathway related protein?Wnt3a,?-catenin,survivn?,autophagy?P62,LC3-II,LC3-I and Beclin-1?and EndMT?CD31,?-SMA and FSP-1?.?3?Using the immunofluorescence staining to observe CD31 and?-SMA intensity change and CD31⁺/?-SMA⁺double positive cells under the fluorescence microscope,counting and analysis.Results:1.Effect of oxidative stress on EndMT and proliferation and apoptosis in HUVECs:?1?The SOD activity and MAD content of oxidative stress injury indicators were detected by spectrophotometry.The results showed that compared with the control group,the activity of SOD and the content of MAD increased significantly in H₂O₂ treatment group.?P<0.05?.?2?The apoptosis rate of each group detected by flow cytometry showed that compared with the control group,the apoptosis rate of the cells in the hydrogen peroxide treatment group increased significantly?P<0.05?.?3?Optical microscope showed that compared with the control group,the morphology of HUVECs in H₂O₂ group gradually changed,and the normal growth was paving or cobblestone like cells,and gradually grew in long spindle shape or spindle shape.?4?The results of double immunofluorescence staining showed that compared with the control group,there were obvious CD31⁺/?-SMA⁺double positive cells in groupH₂O₂ HUVECs.?5?Western Blot detection showed that oxidative stress promoted the expression of proteinCD31,?-SMA and FSP-1?P<0.05?2.Changes of autophagy and Wnt/?-catenin signaling pathway in HUVECs under oxidative stress:?1?Western Blot detection of autophagy and Wnt/?-catenin signaling pathway related protein results showed that compared with the control group,the expression of P62 and Beclin-1 protein in HUVECs was down regulated,and the expression of LC3-II/,LC3-I,Wnt3a,survivn and?-catenin protein was upregulated.3.Regulation of autophagy and Wnt/beta-catenin signaling pathway in the regulation of HUVECs function under oxidative stress:?1?Flow cytometry to detect the apoptotic rate showed that:compared with H₂O₂ group,H₂O₂+Rap group's apoptosis rate decreased significantly?P<0.05?,and the apoptosis rate in H₂O₂+3MA group was significantly increased;the apoptosis rate in H₂O₂+BIO group was lower than in group H₂O₂;The apoptosis rate in group H₂O₂+XAV939 was significantly higher than that in group H₂O₂+BIO and higher than that in group H₂O₂,and the apoptosis rate in group H₂O₂+Rap+XAV939 was lower than that in H₂O₂+XAV939group,but higher than that in H₂O₂+Rap group?P<0.05?.?2?Western Blot detection:Rap promoted the expression of associated protein LC3-II/LC3-I and Beclin-1,inhibit the expression of oxidative stress induced EndMT and Wnt/?-catenin signaling pathway related protein?Wnt3a,?-catenin,?-SMA,FSP-1?while 3MA group performance the opposite trend;compared with H₂O₂+Rap group,BIO promoted the expression of oxidative stress induced EndMT associated protein?-SMA and FSP-1,and promoted the induction of oxidative stress conditions of the Wnt/?-catenin signal pathway related protein Wnt3a,?-catenin expression;Rap and Rap+BIO inhibited the expression of oxidative stress induced EndMT related protein?-SMA and FSP-1,inhibition of Rap+BIO group was weaker than the Rap group?P<0.05?.?3?immunofluorescence staining results showed that compared with group H₂O₂,the intensity of?-SMA increased and the intensity of CD31 was weakened in both group BIO and 3MA,while group Rap and XAV939have opposite trend?P<0.05?;Compared with the BIO group,the intensity of?-SMA in the Rap+BIO group was weakened,and the intensity of CD31 was enhanced?P<0.05?.Conclusion:1.Oxidative stress conditions promoted EndMT and apoptosis in HUVECs.2.Oxidative stress activates the Wnt/?-catenin signaling pathway and autophagy in HUVECs.3.Autophagy regulates the function of HUVECs under the condition of oxidative stress by regulating the Wnt/?-catenin signaling pathway.