Genistein Protects Against Beta-amyloid-induced Neurotoxicity By Induction Of HO-1 Via Activation Of Nrf2 And PI3K

ObjectiveTo investigate the protective effect and the molecular mechanism of Genistein（Gen）on beta-Amyloid-induced neurotoxicity,and thus to provide experimental basis for the treatment and prevention of Alzheimer’s disease.MethodsThe neurocyte cell line SH-SY5Y was cultured in vitro and was pre-treatment with 0⁵0μmol/L of Gen followed by stimulation of20μmol/L of Aβ_25-35 for 0²4h.The proliferation of SH-SY5Y cells were detected by MTT;Expression of Heme oxygenase-1（HO-1）protein and mRNA were measured by Western blot and real-time PCR,respectively.The enzymic activity of HO-1 was detected by colorimetry.To confirmation of the requirement of ongoing transcription and translation in the induction of HO-1,cells were pre-incubated with 0.1¹0μmol/L of actinomycin D（Act D）or cycloheximide（CHX）for 1h and then stimulated with Gen and Aβ_25-35,expression of HO-1 was detected by Western blot.The total protein and the nuclear protein were extracted,western blot was used for the detection of the pholphorylated PI3K and the nuclear translocation of NF-E2-related factor 2（Nrf2）.The PI3K inhibitor LY294002 or the Nrf2 siRNA were used to analyze the involvement of PI3K and Nrf2 in HO-1 expression.Furthermore,cells were preincubated with the HO-1 inhibitor ZnPP or the agonist CoPP,MTT was applied to observe the protective role of HO-1 in beta-Amyloid-induced cytotoxicity.Results1.MTT results indicated there were no significantly changes toward the viability of SH-SY5Y cells after treatment of 0⁵0μmol/L of Gen.In contrast,treatment of 20μmol/L of Aβ_25-35 for 24h significantly decreased its viability.However,pre-treatment of 0⁵0μmol/L of Gen for 1.5h abrogated the neurotoxicity against Aβ_25-35.2.Real-time PCR indicated that the mRNA level was very low in the control group,and Aβ_25-35 treatment only slightly increased its expression.After treatment with different concentration of Gen,the HO-1 mRNA and protein level were significantly increased,as demonstrated by real-time PCR and western blot.Gen-induced HO-1 expression could be abolished by actinomycin D or cycloheximide.Furthermore,the enzymic activity of HO-1 in the control group was very low,and Aβ_25-35 treatment only slightly increase its activity,but the HO-1 activity was upregulated under the condition of Gen incubation.3.The phosphorylated level of p85αwas low in the control group.After Gen incubation,the phosphorylated p85αwas significantly increased.LY294002,an inhibitor of PI3K,could further abrogate the Gen-induced HO-1 expression.4.50μmol/L Gen incubation for 0¹20min could decrease the cytoplasmic Nrf2 in SH-SY5Y cells.In contrast,the Nrf2 in the nucleus was gradually increased,and Nrf2 siRNA treatment could significantly decrease Gen-induced HO-1 expression.5.ZnPP,an inhibitor of HO-1,significantly abrogated the protective effect of Gen,while the HO-1 agonist CoPP exhibited similar protective effect as Gen.Conclusion1.Genistein exhibits protective effect against Aβ_25-35 induced cytotoxicity in SH-SY5Y cells.2.Genistein abrogates Aβ_25-35 induced cytotoxicity could be by activation of PI3K/Akt and Nrf2,and then induction of HO-1.