Icariin Regulates Nrf2 Signaling To Inhibit LPS-induced Microglial Inflammation

Objective: To investigate the anti-neuroinflammatory effects of ICA and elucidate whether ICA-mediated actions were dependent on Nrf2 signaling activation.Methods:(1)BV2 cells were randomly divided into control,ICA(0.1 ?M)alone,LPS(1 ?g / ml),LPS + ICA(0.01 ?M),and LPS + ICA(0.1 ?M).BV2 cells were treated with ICA(0.01 and 0.1 ?M)for 30 min and then incubated with LPS(1 ?g/ml)for 24 h.Cell viability was evaluated by MTT assay.Activation of microglia and Nrf2 were detected by immunofluorescence.The m RNA expressions of Nrf2,HO-1 and NQO1 were determined by real-time RT-PCR at 2h,6h and 24 h.The protein expressions of Iba-1,nuclear factor erythroid-2 related factor 2(Nrf2),heme oxygenase-1(HO-1)and nicotinamide adenine dinucleotide phosphate: quinine oxidoreductase-1(NQO1)were detected by western blotting.The production of NO,interleukin(IL)-18 and IL-1? was detected by ELISA and Griess kit,respectively.(2)The BV-2 cell line was pretreated with different concentrations of ICA and then stimulated by LPS,while some of them were pretreated with Nrf2-si RNA or Zn PP simultaneously.The silencing efficiency of Nrf2 and the inhibition efficiency of HO-1 were detected by western blotting.Activation of BV-2 cells and Nrf2 were detected by immunofluorescence.The m RNA expressions of Nrf2,HO-1 and NQO1 were determined by RT-PCR at 2h,6h and 24 h.The protein expressions of Iba-1,Nrf2,HO-1 and NQO1 were detected by western blotting.The production of NO,IL-18 and IL-1?,was detected by ELISA and Griess kit,respectively.Results:(1)There is no significant difference among the control,ICA(0.1 ?M)alone,LPS(1 ?g/ml),and LPS+ICA(0.01 and 0.1 ?M)treatment was indicated.ICA suppressed LPSinduced microglial activation.ICA decreased LPS-induced production of NO,IL-1? and IL-18 in culture medium.LPS led to an apparent increase in m RNA expressions of Nrf2 at 2 h,HO-1 and NQO1 at 6 h in BV2 cells.ICA increased the LPS-induced elevated transcripts of Nrf2 at 2 h,HO-1 and NQO1 at 6 h.Moreover,immunofluorescence staining assay indicated that ICA elicited Nrf2 activation in BV2 cells.ICA induced an increase in the translocation of Nrf2 from cytoplasm to nucleus.Also,ICA enhanced the protein expressions of HO-1 and NQO1.(2)A marked deletion of Nrf2 protein in Nrf2 si RNA-transfected BV2 cells was demonstrated by western blotting.MTT assay revealed that Nrf2-si RNA(50 n M)had no cytotoxicity on BV2 cells.ICA increased LPS-elevated transcripts of HO-1 and NQO1 at 6 h.However,the m RNA expressions of HO-1 and NQO1 were decreased after Nrf2-si RNA application.Consistent with m RNA detection,ICA enhanced the protein expressions of HO-1 and NQO1,which could be reduced by Nrf2-si RNA treatment.ICA inhibited LPS-induced microglial activation and these inhibitory effects were abolished by Nrf2-si RNA.In addition,Nrf2-si RNA eliminated ICA-decreased production of NO,IL-1? and IL-18 in the culture medium.(3)Zn PP decreased HO-1 protein expression,while Zn PP had no neurotoxicity on BV2 cells.Zn PP had no significant effects on ICA-induced Nrf2 activation.ICA suppressed LPSinduced microglial activation,which could be neutralized by Zn PP treatment.Also,Zn PP weakened ICA-reduced pro-inflammatory factors release.Conclusion: ICA inhibited microglia-mediated neuroinflammation via the activation of Nrf2 signaling.