The Role Of Exogenous Hydrogen Sulfide In Falmitic Acid Induced Inflammation In HEPG2 Hepatocytes

Objective To investigate whether exogenous H2 S can protect Hep G2 cells against inflammation induced by palmitic acid(PA).Methods1.The cytotoxicity of Na HS on Hep G2 cells was examined using MTT assay after 24 h treatment with Na HS at various concentrations(0,10,50,100,and 200 μM).2.Hep G2 cells were pre-treated with various concentrations of Na HS(0,10,50,100,and 200 μM)for 2h and then co-treated with or without PA(0.4 m M)for another 24 h.levels of TNF-α,IL-6 and IL-1β in cell culture supernatant were detected by ELISA assay.The protein expressions of TNF-α,IL-6,IL-1β,NLRP3,p-IKKα/β,Ik Bα,and P65 were determined by Western blot.3.Hep G2 cells were divide into 6 groups: 1)control group;2)PA group;3)PA+Bay 11-7082(Bay)group;4)Na HS group;5)PA+Na HS group;6)PA+Bay+Na HS group.The protein expressions of TNF-α,IL-6,IL-1β and NLRP3 were determined by Western blot.The NF-κB p65 translocation process was confirmed with immuno fluorescence.Results1.Cell viability was not significantly altered by 24 h treatment with Na HS at various concentrations(0,10,50,100,and 200 μM).2.PA significantly induced TNF-α,IL-6 and IL-1β release following incubation,while the cells dose-dependently produced lower levels of proinflammatory cytokines after been treated with Na HS at the concentration of 50μM.Western blot showed that Na HS significantly and dose-dependently inhibited the PA-induced expression of TNF-α,IL-6,IL-1β,NLRP3,phosphorylation of IKKα/β,P65 and degradation of Ik Bα.3.The expression of pro-inflammatory cytokines and NLRP3 inflammasome were dramatically suppressed by BAY or Na HS(50 μM).Upon PA stimulation,the p65 subunit of NF-κB was translocated into the nucleus.However,the optimum dose of BAY or Na HS(50 μM)dramatically suppressed this translocation.Notably,Na HS exerted a synergistic anti-inflammatory effect with BAY in PA-induced Hep G2 cells.ConclusionIn the presence of PA,Na HS observably inhibited the expression of pro-inflammatory cytokines,NLRP3,and suppressed the NF-κB signaling pathway in a dose-dependent manner.