Activation Of Nrf2 Signature By Icariin Protects Against 6-OHDA-Induced Neurotoxicity

Objective:To observe whether Icariin(ICA)protects against neuronal damage induced by 6-hydroxydopamine(6-OHDA)through Nrf2 mechanism.Methods:(1)PC12 cells were seeded in a 96-well plate at 1×10~5/well.6-OHDA/ICA were added for 24 hours,MTT assay was used to detect 6-OHDA and ICA concentrations.(2)After 6-OHDA and ICA concentrations were determined,cells were randomly divided into control,6-OHDA,6-OHDA+ICA(low concentration),6-OHDA+ICA(high conentration).After 24 hours of ICA pretreatment,6-OHDA was added for another 24hours.Flow cytometry was used to detect intracellular ROS levels and early apoptosis rate.SOD level was measured by SOD kit.Oxidation-related Keap1,Nrf2,HO-1,NQO1,GCLC and apoptosis-related proteins Bax,Bcl-2,Caspase 3,and Cytochrome C protein levels were detected by western blotting.(3)PC12 cells were infected with Nrf2-siRNA(40 nmol/L),respectively.The Nrf2silencing/inhibitoryefficiencywasdetectedbyimmunoblottingandNrf2immunofluorescent staining.The content of intracellular ROS and early apoptosis rate were detected by flow cytometry.The level of SOD was measured by SOD kit.The levels of oxidative proteins Keap1,Nuclear Nrf2,HO-1,NQO1,GCLC and apoptosis-related proteins Bax,Bcl-2,Caspase 3 and Cytochrome C were detected by western blotting.Results:(1)The MTT results indicated that the neurotoxic effects of 6-OHDA from the concentration of 100?M were shown.Compared with the control cultures,the ROS and early apoptosis rate in the model was significantly higher,the SOD expression was significantly decreased,the expression of Bax/Bcl-2 and Caspase 3 and Cytochrome C were significantly increased.The expression of Nrf2 and its downstream proteins HO-1,NQO1 and GCLC increased.After ICA treatment,ROS was decreased and the SOD expression was increased significantly,the apoptotic protein Bax/Bcl-2,Caspase 3 and Cytochrome C were significantly increased.Keap1 expression was decreased,Nrf2 and its downstream proteins HO-1,NQO1,GCLC expressions were higher,compared with6-OHDA cultures.(2)After the addition of Nrf2-si RNA,ICA-mediated neuroprotection against6-OHDA-induced neurotoxicity was absent and the difference of changes of ROS,SOD,Nuclear Nrf2,HO-1,NQO1,GCLC,Bax,Bcl-2,Caspase 3,Cytochrome C and early apoptosis rate between the 6-OHDA and 6-OHDA+ICA cultures was not shown.Conclusion:ICA protected against 6-OHDA-induced neurotoxicity via the activation of Nrf2 pathways.