| Objective: Whether the antiaging effect of Ang1-7 on HUVECs is mediated by mitochondrion fission pathway,and whether it is mediated by p53 / Drp1 axis.Methods: 1 HUVECs were cultured,subcultured and seeded.When the cell density in the 6-well plate was over 80%,Ang? was added to establish the aging model of endothelial cells according to the experimental scheme;2 Experimental group: after a large number of preliminary experiments and literature review,it is determined that the optimal concentration of Ang? and Ang1-7 is 1?mol/L,as follows: Part I: To test whether Ang1-7 can alleviate the aging of endothelial cells induced by Ang ?(1)Control group;(2)Ang1-7 group: the cells were treated with 1?mol/L Ang1-7 complete culture medium for 48h;(3)Ang ? group: the cells were treated with 1?mol/L Ang? complete culture medium for 48h;(4)Ang?+Ang1-7group:1?mol/LAng1-7 was used for 30 minutes,and then the complete medium containing 1?mol/L Ang? was added for 48 hours.Check the aging condition of each group: observe and count the SA-?-gal positive cells in each group,detect the level of total ROS in cells with mitochondrial active oxygen kit,and detect the expression of aging protein(p53,Drp1)with Western blotting.Part?: To detect whether Ang1-7 causes the increase of ROS in cells through p53 / Drp1 axis,which leads to the aging of endothelial cells(1)Control group;(2)Ang? group: the cells were treated with 1 ?mol/L Ang? complete culture medium for 48h;(3)Ang ?+Ang1-7group,1 ?mol/L Ang1-7 was used for intervention for 30 minutes,and then a complete medium containing 1?mol/L Ang? was added for 48 hours;(4)Drp1 inhibitor group(Ang ?+Mdivi-1):different concentrations of Mdivi-1(1 ?mol /L,5 ?mol / L,10 ?mol / L)were used to intervene for 30 minutes,and then the complete culture medium of Ang? with 1 ?mol/ L was added for 48 hours.At first,the expression of Drp1 was measured after the intervention of different concentrations of Mdivi-1,and the inhibition effect of Mdivi-1 on Drp1 in endothelial cells was evaluated,and the concentration of Mdivi-1 in the follow-up experiment was determined.Then,the senescence of cells in each group was observed: SA-?-gal positive cells in each group were observed and counted,the level of total ROS in cells was detected by mitochondrial active oxygen kit.Western blotting was used to detect the protein expression of p53 and Drp1.Results: 1 To evaluate the effect of Ang1-7 on endothelial cell senescence:(1)Compared with control group,SA-?-gal positive cells,ROS level,relative expression of p53 and Drp1 protein were significantly increased in Ang ? treated group(P < 0.001);(2)Compared with Ang? group,SA-?-gal positive cells decreased(P < 0.01),ROS level,relative expression of p53 and Drp1 protein decreased in Ang?+Ang1-7 group(P < 0.001);(3)Compared with control group,there was no significant difference in the number of SA-?-gal positive cells,ROS level,p53 and Drp1 protein expression in the Ang1-7 group(P > 0.05).2 To detect whether p53/Drp1 axis can mediate endothelial cell aging(1)The changes of the expression of Drp1 in different concentrations of Mdivi-1 after intervention: compared with the control group,there was no significant difference in the relative expression of Drp1 in the 1 ?mol/L Mdivi-1 group(P >0.05);the relative expression of Drp1 in the 5 and 10 ? mol/L Mdivi-1 groups was significantly reduced(P < 0.01);compared with the 1 ?mol/L Mdivi-1 group,the relative expression of Drp1 in the 5 and 10 ?mol/L Mdivi-1 groups was significantly reduced(P < 0.05).According to Mdivi-1 direction,in the concentration range of 1-10 ?mol/L,with the increase of Mdivi-1 concentration,the relative expression of Drp1 protein decreased gradually,so the concentration of Mdivi-1 was 5 ?mol/L for subsequent experiments.(2)To detect the effect of Mdivi-1 on ROS level in Ang?-induced cell senescence: according to analysis combined with the image and the above experimental results: compared with the control group,the ROS of Ang? treatment group increased significantly,and that of Mdivi-1 group decreased;compared with Ang? group,the ROS of Ang?+Ang1-7 group and Mdivi-1 group decreased significantly.(3)Western blotting analysis of the expression of Drp1: compared with control group,the relative expression of Drp1 in Ang? group was significantly increased,while that in Mdivi-1 group was decreased;Compared with Ang? group,in Ang?+Ang1-7 group,the relative expression of Drp1 protein decreased,and that in Mdivi-1 group decreased significantly.Conclusion: 1Ang1-7 can alleviate the aging of HUVECs induced by Ang ?;2During the aging of HUVECs induced by Ang ?,Ang1-7 can affect the mitochondrion fission pathway through p53/Drp1 axis,reduce the level of ROS in cells,reduce the oxidative stress level of endothelial cells and resist aging. |
PDF Full Text Request