The Role Of Activation Of Nrf2-ARE Pathway In The Protection Of Curcumin Against Methylmercury-induced Neurotoxicity In Primary Astrocytes

[Background and Objective] Mercury is a global pollutant and,methylmercury(MeHg)is the most toxic in all forms of mercury.Notably,MeHg has the most serious damaging effect on the nervous system.Currently,the toxic mechanisms of methylmercury have not been clarified.And there is no effective medicine for methylmercury poisoning yet.Therefore,it is particularly important to seek drugs antagonizing methylmercury.Curcumin(CUR)is a natural product extracted from the ginger plant.A large number of studies have shown that curcumin has antioxidant,anti-inflammatory,anti-tumor,anti-atherosclerosis,scavenging free radicals and other protective effects.Also,it has been reported that curcumin can complex with heavy metal to form compounds with low toxicity,playing an important role in inhibiting metal toxicity.The present study aims to investigate whether curcumin could protect the astrocytes from methylmercury-induced neurotoxicity and its possible protective mechanism,which could provide the theoretical basis for trentment of methylmercury poisoning.[Methods] The postnatal rat cortical astrocytes were primarily cultured and divided into different groups: control group,MeHg(5 ?mol/L)group,CUR(2,5,10,20 ?mol/L)group,CUR pretreatment(2,5,10,20 ?mol/L)+ Me Hg(5 ?mol/L)group.The cell viability and cytotoxicity were assessed by MTT reduction and LDH leakage,respectively.The levels of glutathione(GSH)and catalase(CAT)were tested with spectrophotometric method.The total protein,nuclear protein and cytoplasm protein levels of Nrf2 were detected with Western blotting.Nuclear Nrf2 translocation was evaluated by immunofluorescence staining.Western blotting was also used to detect expressions of heme oxygenase 1(HO-1)and quinone oxidoreductase(NQO1),the downstream genes of Nrf2.The cell viability of CUR pretreatment group was also evaluated by MTT assay after Nrf2-targeted siRNA transfection.After the treatment of extensive protein kinase C(PKC)inhibitor(Ro 31-8220)and specific PKC?-targeted inhibitor(Rottlerin),the expression of PKC and Nrf2 protein was detected by Western blotting,and the protective effect of CUR was assessed by MTT assay.[Results] Compared with the control group,MeHg significantly decreased the cell viability and enhanced the LDH leakage.Compared with the MeHg alone group,the pretreatment of CUR at concentrations of 5,10,20 ?mol/L significantly increased cell viability,decreased the LDH leakage,raised GSH and CAT levels.Meanwhile,CUR pretreatment significantly increased the total protein levels and promoted nuclear translocation of Nrf2,resulting in higher nuclear protein levels of Nrf2.The expression of NQO1 and HO-1,downstream genes of Nrf2-ARE were also enhanced.Protection of CUR pretreatment were significantly decreased after knockdown of Nrf2 with siRNA.PKC and Nrf2 protein levels were simultaneously decreased after treatment of Ro 31-8220 and Rottlerin.Protection of CUR pretreatment was significantly decreased after treatment of Ro 31-8220 and Rottlerin in high concentration of Me Hg(7.5 ?mol/L),though no significant change at 5 ?mol/L MeHg.[Conclusion] Curcumin exerted neuroprotection effects against MeHg-induced cytotoxicity in primary astrocytes via activation of Nrf2-ARE pathway,and PKC was involved in the activation of Nrf2 by curcumin.