Evodiamine exerts its inhibitory effects on the H2O2-induced inflammatory response and cell apoptosis in ARPE-19 cells through the upregulation of SIRT1 expression
Objective It is to investigate the effects of evodiamine(Evo)on the inflammatory response and apoptosis of human retinal pigment epithelial cells(ARPE-19)under H2O2 stimulation,and to elucidate the role of Sirtuin 1(SIRT1)and its related mechanisms.Methods ARPE-19 cells were treated with different concentrations of H2O2(0,25,50,100,200,and 400 μmol/L)and different concentrations of Evo(0,2.5,5.0,10.0,20.0,and 40.0 μmol/L),respectively,and the optimal action concentrations of H2O2 and Evo were screened by CCK-8 method.ARPE-19 cells were co-treated with H2O2(200 μmol/L)and different concentrations of Evo(2.5,5,10,20 μmol/L),and the expression of SIRT1 pro-tein in the cells was detected by Western blot.ARPE-19 cells were divided into 5 groups based on the different treatments:DMSO treated group(control group),200 μmol/L H2O2 treated group(H2O2 group),200 μmol/L H2O2+Evo 10 μmol/L treated group,200 μmol/L H2O2+Evo 20 μmol/L treated group,and H2O2+Evo 20 μmol/L+Sirtuin inhibitor Sirtinol group(H2O2+Evo 20 μmol/L+Sirtinol group).After 24 h of treatment,the levels of inflammatory factors TNF-α,IL-1β and IL-6 in the supernatants of ARPE-19 cells in each group were detected by ELISA,the apoptosis rate of ARPE-19 cells in each group was detected by Annexin V-FITC/PI staining,and the protein expressions of NF-κB p65,COX-2,Bcl-2,Bax,Cleaved Caspase-3,Caspase-3,and phospholipid Inositol 3-kinase(PI3K),p-PI3K,protein kinase B(Akt),p-Akt were detected by Western blot method.Results The inhibition of 200 μmol/L of H2O2 on the ARPE-19 cells was relatively stable.0,2.5,5.0,10.0,and 20.0 μmol/L of Evo had no significant effect on ARPE-19 cell viability(all P>0.05),and 40 μmol/L of Evo could significantly decreased ARPE-19 cell viability(P<0.05).The relative expression of SIRT1 protein in ARPE-19 cells was significantly lower in the H2O2 group and H2O2+Evo groups than that in the control group(all P<0.05);the relative expression of SIRT1 protein was significantly higher in the H2O2+Evo 5 μmol/L group,H2O2+Evo 10 μmol/L group and H2O2+Evo 20 μmol/L group than that in the H2O2 group(all P<0.05),and the increases were more significant in the H2O2+Evo 10 μmol/L group and H2O2+Evo 20 μmol/L group(all P<0.05).Compared with the control group,the levels of TNF-α,IL-1β,IL-6 and the relative expressions of COX-2,NF-κB p65,Bax proteins in the cells and the ratios of Cleaved Caspase-3/Caspase-3,p-PI3K/PI3K,p-Akt/Akt were significantly increased(all P<0.05),while the relative expressions of Bcl-2 protein were all significantly decreased in the H2O2 group,H2O2+Evo 10 μmol/L group,H2O2+Evo 20 μmol/L group and H2O2+Evo 20 μmol/L+Sirtinol group(all P<0.05);compared with the H2O2 group,the levels of TNF-α,IL-1β,IL-6 and the relative expressions of COX-2,NF-κB p65,Bax proteins in the cells and the ratios of Cleaved Caspase-3/Caspase-3,p-PI3K/PI3K,p-Akt/Akt were significantly decreased(all P<0.05),while the relative expressions of Bcl-2 protein were all significantly increased in the H2O2+Evo 10 μmol/L group,H2O2+Evo 20 μmol/L group and H2O2+Evo 20 μmol/L+Sirtinol group(all P<0.05);the levels of TNF-α,IL-1β,IL-6 and the relative expressions of COX-2,NF-κB p65,Bax proteins in the cells and the ratios of Cleaved Caspase-3/Caspase-3,p-PI3K/PI3K,p-Akt/Akt were significantly higher,while the relative expression of Bcl-2 protein was all significantly lower in the H2O2+Evo 20 μmol/L+Sirtinol group than those in the H2O2+Evo 20 μmol/L group(all P<0.05),the differences in the indexes were not significant different compared with the H2O2 group and H2O2+Evo 10 μmol/L group(all P>0.05).Conclusion Evo can inhibit the NF-κB pathway,mitochondria-mediated apoptotic pathway,and PI3K/Akt pathway in vitro by up-regulating the expression of SIRT1,thus to attenuate the inflammatory response and decrease ap-optosis in ARPE-19 cells stimulated by H2O2.
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