Icartin Protects Dopaminergic Neurons From 6-OHDA Induced Injury By Alleviating Oxidative Stress

Objective:To investigate the protective effect of icaritin(ICT)on 6-hydroxydopamine(6-OHDA)-induced MN9D cell injury,and to explore whether ICT can alleviate 6-OHDA induced cell injury by inhibiting oxidative stress,so as to further explore the possible mechanism of ICT inhibiting oxidative stress.Methods:(1)MN9D cells were seeded into 96-well plates(1×10~5/well),after 24 hours,the cells were treated with 6-OHDA,ICT and vitamin C(Vitamin C as a positive antioxidant control)for 24 hours,and the concentrations of 6-OHDA,ICT and vitamin C were detected by CCK-8.(2)After detection the concentrations of 6-OHDA,ICT and vitamin C by CCK-8,MN9D cells were randomly divided into blank group,6-OHDA group,6-OHDA+vitamin C,6-OHDA+ICT low dose group and 6-OHDA+ICT high dose group.After pre-treatment with ICT and vitamin C for 1 hour,the incubation was carried out for 24 hours after adding 6-OHDA,the damage of MN9D cells was detected by CCK-8,TH protein was detected by immunocytochemistry staining,the content of ROS was determined by flow cytometry,the activity of SOD was detected by SOD kit,and the expression of a-syn,TH,oxidative stress related proteins Nrf2 and HO-1 were detected by Western blot.Results:In MN9D cells,CCK-8 showed that 6-OHDA at a concentration of 50μM has cytotoxic effect,compared with the blank group,the production of ROS in 6-OHDA group was significantly increased,SOD activity and TH protein expression were significantly decreased,however,the expression ofα-syn,Nrf2 and downstream protein HO-1 were increased.After treatment with ICT and vitamin C respectively,ROS production was alleviated,SOD activity was improved and TH protein expression was increased,the expression ofα-syn was significantly reduced,and the expression of Nrf2 and its downstream protein HO-1 was further prompted,the protective effect of ICT treatment was more significant than that of vitamin C treatment.Conclusion:ICT is capable of protecting dopaminergic neurons from 6-OHDA-induced injury by inhibiting oxidative stress,which may be relevant to the effect of the expression of oxidative stress-related proteins Nrf2 and HO-1.