Study On The Mechanism Of MiR-34b/miR-23a Targeting Keap1/ULK1 In Regulating Oxidative Damage Induced By Cerebral Ischemia-reperfusion

Objective: To investigate the molecular mechanism of mi R-34b/mi R-23 a targeting Keap1/ULK1 in regulating oxidative stress in rats with cerebral ischemia-reperfusion.Methods:(1)The expression of mi R-34b/mi R-23 a in the core tissue of I/R rats with cerebral infarction was detected by QPCR.Mi R-34b/mi R-23 a was overexpressed in the brain tissue of MCAO rats by lentivirus technology.After injury,Berdron score and TTC staining were used to detect the nerve injury and cerebral infarction volume.The total SOD,NO and Mn SOD content were determined by chemical colorimetry.The content of 3-NT was detected by ELISA.(2)The expression of Keap1/ULK1 in core tissues of I/R rat cerebral infarction was detected by QPCR and Western blot.Furthermore,the regulation of Keap1/ULK1 e--------UTR by dual luciferase assay.(3)Oxidative damage of cells was induced by H2O2,and the KEAP1 was reduced or increased,mir-34 b and Keap1 were overexpressed,mir-23 a and ULK1 were overexpressed,the content of total SOD,NO and Mn SOD was determined by chemical colorimetric,and the content of 3-NT was detected by ELISA.Results:(1)Compared with the sham operation group,the expression of mi R-34 b in the core tissue of I/R rats was significantly decreased,and decreased at 1,4,24 h after reperfusion.The expression of mir-23 a was the lowest in 1h after reperfusion,and the number of 4h and 24 h recovered obviously.Compared with the control lentivirus group,the neurological injury score and cerebral infarction volume of the overexpressing mi R-34b/mi R-23 a group were significantly decreased.The contents of 3-NT and NO were decreased while the total of SOD and Mn SOD were significantly increased.(2)Compared with the sham operation group,the expression of Keap1 in the core tissue of I/R rats was decreased,and the expression of Nrf2 and HO-1 increased.The expression of ULK1 was increased at 1h and 4h after reperfusion and decreased at 24 h.Compared with the control group,the expression of Keap1 was decreased and the expression of Nrf2 and HO-1 was increased in the overexpressed mi R-34 b group.And the expression of ULK1 was decreased in the overexpressed mi R-23 a group.In the wild-type transfection group of Keap1-3-UTR/ULK1-3-UTR,the mi R-34b/mi R-23 a mimic could inhibit the fluorescence activity,and the inhibitor could promote the fluorescence activity;Keap1-3-UTR/ULK1-3-UTR mutant transfection group,there was no significant difference in fluorescence activity between the groups.(3)Compared with the control group,the levels of 3-NT and NO in the Keap1 overexpression group were increased while the total of SOD and Mn SOD were decreased significantly,and the results were opposite in Keap1 interference group.Mi R-34 b overexpression significantly reduced NO and 3-NT levels compared with the vector group,and total SOD and Mn SOD levels were significantly increased.Keap1 overexpression colud significantly restored the effect of mi R-34b;mi R-23 a mimic group significantly decreased 3-NT and NO content,and total SOD and Mn SOD levels increased significantly,and ULK1 overexpression cloud significantly restored the effect of mi R-23 a.Conclusion:(1)Mi R-34 b and mi R-23 a are protective factors of cerebral infarction in I/R rats.(2)Mir-34b/mir-23 a can specifically down-regulate Keap1/ULK1 to inhibit oxidative stress injury in cerebral ischemia reperfusion.