Curcumin Attenuates BPA-induced Insulin Resistance In HepG2 Cells Through Suppression Of JNK/p38 Pathways

Objectives:First,we demonstrated that bisphenol A(BPA)could induce insulin resistance in HepG2 cells,and then we explored the role of MAPK and NF-?B pathways in this process.Furthermore,we investigated whether curcumin could have preventative effect on BPA-induced insulin resistance,and researched the mechanism.We wish we can make a significant contribution to treat diabetesMethods:The effects of BPA and curcumin on cell viability of HepG2 cells were examined by MTT assay.The effects of BPA and curcumin on glucose consumption of HepG2 cells were examined by glucose assay kit.Lipid peroxidation was examined by measurement of malondialdehyde(MDA).The concentrations of TNF-? and IL-6 in culture were measured using cytokine-specific ELISA kits.The proteins related to insulin signal transduction pathway was measured by Western blot.The activation of MAPK and NF-?B pathways were analyzed by Western blot.Results:1.BPA induces insulin resistance in HepG2 cellsWe showed that treatment with 100 nM BPA for 5 days significantly reduced glucose consumption of the HepG2 cells.Moreover,BPA significantly decreased IR and AKT phosphorylation and increasef IRS 1 phosphorylation,especially in 100nM.These data suggested that BPA could induce insulin resistance in these cells.2.BPA promotes inflammation and oxidative stress in HepG2 cellsIt was shown that BPA significantly increased the level of MDA in supernat of BPA-induced HepG2 cells.ELISA assays showed that the levels of pro-inflammatory cytokines(TNF-? and IL-6)were significantly elevated by BPA treatment.In addition,BPA markedly increased the expression of COX2 as compared to the control HepG2 cells.These results indicated that BPA promoted inflammation and oxidative stress in HepG2 cells.3.BPA activates MAPK and NF-?B pathwaysWe measured the levels of p-JNK,p-p3 8,p-ERK,p-IKK?,I?B? and p-p65 in BPA-treated HepG2 cells.Phosphorylated levels of JNK,p38 and ERK were observed in BPA treated cells in comparison with the control cells.Meanwhile,BPA induced phosphorylation of IKK?,p-p65 and decreased I?B-?.These results indicated that BPA treatment activated MAPKs and NF-?B pathways in HepG2 cells.4.JNK/p38 activation mediates BPA-induced insulin resistance in HepG2 cellsTreatments of JNK inhibitor(SP600125)and p38 inhibitor(SB203580),but not ERK inhibitor(U0126),effectively prevented the decrease of glucose consumption in HepG2 cells induced by BPA.It was also shown that treatments with JNK and p38 inhibitors,but rather ERK inhibitor,diminished BPA-induced alterations of p-IR,p-IRS1 and p-AKT.Treatment of NF-?B inhibitor(PDTC)had no effect on BPA-induced changes of glucose consumption and p-IR,p-IRS1 and p-AKT in HepG2 cells.These results suggested the role of JNK/p38 activation in BPA-induced insulin resistance and curcumin protection in HepG2 cells.5.Curcumin attenuates BPA-Induced insulin resistance in HepG2 CellsWe investigated the effects of 1?M,2.5?M and 5?M of curcumin on BPA-treated HepG2 cells.It was shown that glucose consumption was significantly reduced in HepG2 cell when treated with 100 nM BPA,and the reduction in glucose consumption induced by BPA was effectively prevented by curcumin treatment.Moreover,BPA-triggered alterations in the expression of p-IR,p-IRS1,and p-AKT were attenuated with curcumin.These results illustrated that curcumin ameliorated the insulin resistance induced by BPA.6.Curcumin diminishes BPA-induced Inflammation and oxidative stress in HepG2 CellsOur results showed that curcumin effectively prevented BPA-induced MDA production in HepG2 cells.Meanwhile,BPA-elicited proinflammatory cytokines TNF-? and IL-6 were significantly reduced by curcumin.BPA-induced expression of COX2 was also decreased with curcumin treatment.These results revealed that curcumin diminished BPA-induced inflammatory response and oxidative stress in HepG2 cells.7.Curcumin inhibits BPA-triggered activation of MAPKs and NF-?B pathwaysIt was found that curcumin significantly decreased the activation of JNK and p38,but rather ERK.Furthermore,curcumin treatment effectively reduced levels of p-IKK?,p-I?Ba and p-p65.These results suggested that curcumin suppressed BPA-induced JNK/p38 MAPKs and NF-?B activation in HepG2 cells.8.Curcumin attenuates BPA-Induced insulin resistance,inflammation and oxidative stress in a p38/JNK-dependent wayWe added anisomycin,a specific activator of JNK and p38,to the medium,to investigate the protection of curcumin under this condition.It was found that anisomycin cotreatment with curcumin increased the phosphorylation level of p38 and JNK,whereas incubation with curcumin alone had no effect on the phosphorylation levels of either p38 or JNK MAPK.Compared to BPA group,curcumin significantly increased the glucose consumption and attenuated the BPA-induced MDA,TNF-?,IL-6 and COX2 production.However,anisomycin reversed the protective effect observed with curcumin.These results suggested that JNK/p38 MAPK,anisomycin abolished the protective effect of curcumin on the insulin resistance induced by BPA.Conclusions:In conclusion,the present data showed that JNK/p38 pathways-mediated BPA induction of insulin resistance was effectively attenuated by curcumin treatment.Our finding suggested that curcumin may serve as a promising candidate for the intervention of BPA-induced insulin resistance.