Mechanism Of MiR-200b Mediated MTOR On Oxidative Stress Injury And Autophagy Of Diabetic Vascular Endothelium

ObjectiveThe study aims to explore the changes of mir-200 b in the process of oxidative stress injury and autophagy in diabetic vascular endothelium.Bioinformatics software predicted that miR-200 b may be involved in the regulation of mTOR signaling pathway.The molecular mechanism of miR-200 b in the regulation of oxidative stress injury and autophagy was explored with the purpose of finding new therapeutic targets for diabetic vascular diseases.Methods1.25 db/db male rats aged 10 weeks were selected as the diabetes model group,and 25 wild male rats aged the same week were selected as the blank group.The body weight and blood glucose of the two groups were continuously monitored from the 10 th week of age.The random blood glucose < 16.7mmol/L in the model group was excluded.Serum samples were collected at 14 weeks of age,and oxidative stress related indexes such as AGEs,SOD and MDA were detected by Elisa;HE staining was used to observe the morphological and structural changes of the mouse thoracic aorta.RT-qPCR was used to detect the expression of miR-200 b in blood vessels.The expressions of mTOR,LC3-II and p62 in aortic vessels were detected by Western-blot.2.In order to construct a cell model of diabetic vascular disease,Eahy vascular endothelial cells were treated with AGEs at different concentrations(0,50,100,200,400 g/L)for 3h,6h,12 h,and 24 h,respectively.The optimal dose and time of AGEs intervention were obtained by detecting the cell drug-sensitive proliferation and oxidative stress levels such as ROS,SOD and MDA under different conditions.On the basis of the above research,the experiment was divided into blank group,AGEs group,AGEs+PBS group,AGEs+ miR-200 b inhibitor group,AGEs+ROS inhibitor group.The corresponding reagent was used to intervene for 48 h.The contents of vascular endothelial ROS,SOD and MDA were determined by cell flow technique and Elisa.RT-qPCR was used to detect the relative expression of vascular endothelial miR-200 b in each experimental group.Besides,the expressions of mTOR,LC3-II and p62 in endothelial cells were detected by western-blot.Results1.Compared with the blank group,the blood glucose and body weight in diabetic model group were significantly increased(P < 0.001).HE staining showed that atherosclerotic changes occurred locally in the thoracic aorta of mice in the model group.Abnormal vascular smooth cell were obvious hyperplasia and disorder in arrangement and some endothelial cells were exfoliated.However,no significant pathological changes were found in the blood vessels of mice in the blank group.The vascular smooth muscle in this group was orderly and the endothelial cells were intact.The contents of AGEs and MDA in serum of the model group were significantly higher than those of the blank group,while the content of SOD was lower than that of the blank group.The expressions of miR-200 b and LC3-II in the aorta of mice in the model group were significantly higher than those in the blank group,while the expression of mTOR was lower than that in the blank group.The above differences were statistically significant(P < 0.05).2.According to the results of drug sensitivity proliferation and the changes in oxidative stress levels such as ROS,SOD and MDA,the optimal experimental effect was determined when the AGEs intervention concentration was 200 ?g/L and the intervention time was 6h.The detection of oxidative stress related indicators confirmed that ROS and MDA levels in endothelial cells in AGEs group were significantly increased compared with the blank group,while SOD levels were decreased.Compared with the AGEs+PBS group,the levels of ROS and MDA in endothelial cells of the miR-200 b inhibitor group and the ROS inhibitor group were decreased but the expression levels of SOD was markedly elevated.There was no significant difference in the expression of oxidative stress between AGEs group and AGEs+PBS group(P>0.05).The results of RT-qPCR showed that the expression of miR-200 b in vascular endothelial cells in the AGEs group was significantly higher than that in the blank group,but the expression was not statistically different from that in the AGEs+PBS group.The expressions of miR-200 b in the miR-200 b inhibitor group and the ROS inhibitor group were significantly lower than those in the AGEs+PBS group.Compared with the blank group,the expression of mTOR protein decreased after AGEs group intervention,but the expression of LC3-II and p62 increased.There were no significant differences in the expressions of the three protein between AGEs group and AGEs+PBS group(P>0.05).Compared with the AGEs+PBS group,mTOR expression was increased in the AGEs+miR-200 b inhibitor group and the ROS inhibitor group,while the expression of LC3-II and p62 was decreased.The above differences were statistically significant(P < 0.05).Conclusion1.miR-200 b is upregulated during oxidative stress injury and autophagy of vascular endothelial cells in type 2 diabetes mellitus.2.Vascular endothelial oxidative stress injury induced by AGEs can promote the occurrence of autophagy in endothelial cells3.miR-200 b is involved in the regulation of vascular endothelial autophagy induced by AGEs by inhibiting the expression of mTOR.