Dietary Flavonoid Genistein Induces Nrf2 And Phase Ⅱ Detoxification Gene Expression And Protects Against Oxidative Stress In Caco-2 Cells

Objective:Flavonoids have well-known anti-oxidant,anti-inflammatory,and anti-cancer activities.Isoflavone genistein is considered a potent anti-oxidant agent against oxidative stress.Although several mechanisms have been proposed,a clear anti-oxidant mechanism of genistein is still remained to be answered.Methods:(1)The Caco-2 cells were treated with various concentrations of genistein(2.5 μM,5 μM,10 μM)for 6 h.The protein expressions of Heme Oxygenase-1(HO-1),glutamate-L-cysteine ligase catalytic subunit(GCLC)and NF-E2-related factor-2(Nrf2)were determined by western blotting.The mRNA expression levels of HO-1,GCLC and Nrf2 were detected by RT-PCR analysis.(2)The Cells treated with 10 μM genistein for 0,2,4,6,8 and 24 h.The expression of HO-1,GCLC and Nrf2 was measured by western blotting and RT-PCR analysis.(3)The Caco-2 cells were pretreated with genistein(2.5 μM,5 μM,10 μM)for 3 h,and then treated with H2O2(10 mM)for another 3 h.MTT method was used to detect cell viability.(4)The Caco-2 cells were pretreated with 20μM ZnPP and 200 μM BSO for 2 h and then treated with 10 μM genistein for 3 h.Next,cells were further exposed to 10 mM H2O2 for 3 h to evaluate cell viability.(5)The Caco-2 cells were pretreated with genistein(10 μM)for 3 h,and then treated with H2O2(10 mM)for another 3 h.The protein and mRNA expressions of HO-1,GCLC and Nrf2 were measured.(6)The Caco-2 cells were transfected with non-targeting control siRNA or siNrf2 for 6 h,followed by treatment with genistein(10 μM)for an additional 42 h.The protein expressions of HO-1,GCLC and Nrf2 were measured by western blotting.(7)The Caco-2 cells were treated with various concentrations of genistein(2.5 μM,5 μM,10μM)for 6 h.The protein expressions of ERK1/2,p-ERK1/2,PKC,p-PKC,P38,p-P38,JNK,p-JNK,AKT,p-AKT were determined by western blotting.(8)The Caco-2 cells were respectively pretreated with 20μM PD98059,20 μM GF109203X,20μM SB203580,20μM SP600125,or 20 μM LY294002 for 1 h and then treated with 10μM genistein for 3 h.Next,cells were further exposed to 10 mM H2O2 for 3 h.MTT method was used to detect cell viability.Results:In this study,we focused on the concerted effects on expression of nuclear factor erythroid 2-related factor 2(Nrf2)and phase II enzyme pathway components.In Caco-2 cells,treatment with genistein markedly attenuated H2O2-induced peroxide formation;this amelioration was reversed by buthionine sulfoximine(glutamate-cysteine ligase catalytic subunit(GCLC)inhibitor)and zinc protoporphyrin(heme oxygenase(HO-1)inhibitor).Genistein dose-dependently increased HO-1 and GCLC mRNA and protein expression.Genistein treatment activated the extracellular signal-related protein kinases 1 and 2(ERK1/2)and the protein kinase C(PKC)signaling pathway;therefore increased Nrf2 mRNA and protein expression.The roles of the ERK1/2 and PKC signaling pathway were determined using PD98059(ERK1/2 inhibitor)and GF109203X(PKC inhibitor)and RNA interference directed against Nrf2.Both inhibitors and siNrf2 abolished genistein-induced HO-1 and GCLC protein expression.These results suggest the involvement of ERK1/2,PKC,and Nrf2 in inducing HO-1 and GCLC by genistein.Conclusions:Our studies show that genistein up-regulated HO-1 and GCLC expressions through the EKR1/2 and PKC/Nrf2 pathways during oxidative stress.