Activation Of NRF2/ARE By Isosilybin Alleviates A?_25-35-indu Ced Oxidative Stress Injury In HT-22 Cells

Objective: To observe the effect of isosilybin on NRF2 / ARE signaling pathway in vitro and used by A?25-35 in mouse hippocampal cell line HT-22.Methods: MTT assay was used to detect the time-dose toxicity of Isosilybin to HT-22 cells and the time-dose toxicity of A?25-35 to HT-22 cells.In the range of non-toxic dose,MTT assay was used to detect the effective dose range and the optimal time of A?25-35.To investigate the effect of Isosilybin on A?25-35 induced MDA and LDH cells injury.To investigate the effect of Isosilybin on the antioxidant activity of T-AOC cells induced by A?25-35.The effect of Isosilybin on the level of endogenous ROS in HT-22 cells induced by A?25-35 was examined and the relationship between ROS and oxidative damage was examined by ROS specific inhibitor NAC.Western blotting was used to detect the effect of Isosilybin and A?25-35 on HO-1,AKR1C2 and GST protein expression in HT-22 cells.The effect of Isosilybin and A?25-35 on the expression of HO-1,AKR1C2 and GST mRNA in HT-22 cells was detected by qPCR.Construction of ARE luciferase reporter plasmid p G3L-ARE was transfected into HT-22 cells to detect the effect of Isosilybin or NRF2 specific agonist tBHQ on p G3L-ARE reporter gene expression activity fluorescence intensity.The effects of T-BHQ incubation on the changes of ROS,MAD,LDH and T-AOC in HT-22 cells were examined.Western blotting was used to detect the time and dose activation of Isosilybin on NRF2 in HT-22 cells.The cytoplasm of HT-22 was isolated and the distribution of NRF2 in the nucleus and cytoplasm was detected by Western blotting.Construction of h NRF2 siRNA for NRF2 expression was transfected into HT-22 cells to detect the expression of NRF2 in Isosilybin.The expression of NRF2 in Isosilybin was detected by co-transfection of h NRF2 siRNA and ARE luciferase reporter plasmid p G3L-ARE into HT-22 cells.The effects of A?25-35 on ROS,MAD,LDH and T-AOC in HT-22 cells after incubation with Isosilybin were detected by co-transfection of h NRF2 siRNA and p G3L-ARE into HT-22 cells.Results: Isosilybin was cytotoxic to HT-22 when greater than 20 ?M,and 10 ?M Isosilybin had no time-dependent toxicity to HT-22 cells.When A?25-35 is greater than 2 ?M,the inh- ibitory effect on HT-22 cells is obvious,so 2 ?M A?25-35 is used as the model concentration.Isosilybin preincubation 4 h is the best time to reverse the toxicity of A?25-35.Isosilybin can dose-dependently alleviate the toxicity of A?25-35-induced HT-22 cells,and thus 2,4,10?M Isosilybin as the therapeutic concentration of low,medium and high dose groups.Isosilybin can slow down the leakage of MDA and LDH induced by A?25-35 and increase the level of T-AOC.Isosilybin can effectively inhibit the accumulation of ROS induced by A?25-35 in HT-22,and NAC can effectively inhibit the damage of A?25-35-induced cell damage and antioxidant capacity.Isinilybin could induce HO-1,AKR1C2,GST protein and mRNA expression in a dose-dependent manner,while A?25-35 stimulated the levels of HO-1,AKR1C2 and GST.Isosilybin and t-BHQ can significantly improve the expression of luciferase in ARE reporter plasmids.T-BHQ significantly downregulated A?25-35-induced ROS accumulation and relieved MAD,LDH leakage and T-AOC reduction.Isosilybin can significant ly induce NRF2 expression in a dose-dependent manner.Isosilybin can push NRF2 into the nucleus.HNRF2 siRNA inhibited the activation of ARE luciferase reporter plasmids induced by Isosilybin and inhibited the expression of HO-1,AKR1C2 and GSH proteins in A RE-driven genes.In addition,h NRF2 siRNA can effectively block Isosilybin to relieve A?25-35-induced ROS accumulation,MDA,LDH release and T-AOC decline.Conclusion: 1)Isosilybin can relieve the toxicity and oxidative stress injury of HT-22 induced by A?25-35 in a dose-dependent manner.2)Isosilybin alleviates A?25-35-induced oxidative damage in cells by ROS.3)Isosilybin relieves ROS-mediated A?25-35-induced oxidative stress injury by inducing ARE-driven gene activation.4)Isosilybin stimulates ROS-mediated A?25-35-induced oxidative stress injury by NRF2-dependent activation of ARE-driven genes.