吴茱萸碱对H_(2)O_(2)诱导ARPE-19细胞的炎症反应、细胞凋亡和SIRT1表达的影响

Evodiamine exerts its inhibitory effects on the H_(2)O_(2)-induced inflammatory response and cell apoptosis in ARPE-19 cells through the upregulation of SIRT1 expression

目的 探究吴茱萸碱对H_(2)O_(2)刺激下人视网膜色素上皮细胞ARPE-19的炎症反应和细胞凋亡的影响,阐明去乙酰化酶1(SIRT1)在其中的作用和相关机制。方法 分别采用不同浓度的H_(2)O_(2)(0,25,50,100,200,400μmol/L)和不同浓度的吴茱萸碱(0,2.5,5.0,10.0,20.0,40.0μmol/L)处理ARPE-19细胞,CCK-8法筛选H_(2)O_(2)和吴茱萸碱的最佳作用浓度。使用H_(2)O_(2)(200μmol/L)与不同浓度的吴茱萸碱(2.5,5,10,20μmol/L)联合处理ARPE-19细胞,Western blot法检测细胞中SIRT1蛋白表达情况。按处理方式的不同将ARPE-19细胞分为二甲基亚砜处理的对照组、200μmol/L H_(2)O_(2)处理组(H_(2)O_(2)组)、200μmol/L H_(2)O_(2)与不同浓度吴茱萸碱处理组(H_(2)O_(2)+吴茱萸碱10μmol/L组、H_(2)O_(2)+吴茱萸碱20μmol/L组)及100μmol/L的SIRT1抑制剂Sirtinol拮抗组(H_(2)O_(2)+吴茱萸碱20μmol/L+Sirtinol组),处理24 h后,ELISA法检测各组ARPE-19细胞上清液中炎症因子肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)和白细胞介素-6(IL-6)水平,Annexin V-FITC/PI染色检测各组ARPE-19细胞的凋亡率,Western blot法检测各组ARPE-19细胞中核因子-κB p65(NF-κB p65)、环氧化酶-2(COX-2)、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、裂解型半胱氨酸天冬氨酸蛋白酶3(Cleaved Caspase-3)、Caspase-3、磷脂酰肌醇3-激酶(PI3K)、磷酸化PI3K(p-PI3K)、蛋白激酶B(Akt)、磷酸化Akt(p-Akt)蛋白表达情况。结果 200μmol/L的H_(2)O_(2)对ARPE-19细胞的生长抑制相对稳定。0,2.5,5.0,10.0,20.0μmol/L的吴茱萸碱对ARPE-19细胞活力无明显影响(P均>0.05),40μmol/L吴茱萸碱可明显降低ARPE-19细胞活力(P均<0.05)。H_(2)O_(2)组和H_(2)O_(2)+吴茱萸碱各组ARPE-19细胞中SIRT1蛋白相对表达量均明显低于对照组(P均<0.05);H_(2)O_(2)+吴茱萸碱5μmol/L组、H_(2)O_(2)+吴茱萸碱10μmol/L组和H_(2)O_(2)+吴茱萸碱20μmol/L组中SIRT1蛋白相对表达量均明显高于H_(2)O_(2)组(P均<0.05),且H_(2)O_(2)+吴茱萸碱10μmol/L组和H_(2)O_(2)+吴茱萸碱20μmol/L组升高更明显。与对照组比较,H_(2)O_(2)组、H_(2)O_(2)+吴茱萸碱10μmol/L组、H_(2)O_(2)+吴茱萸碱20μmol/L组和H_(2)O_(2)+吴茱萸碱20μmol/L+Sirtinol组细胞中TNF-α、IL-1β、IL-6水平和COX-2、NF-κB p65、Bax蛋白相对表达量及Cleaved Caspase-3/Caspase-3、p-PI3K/PI3K、p-Akt/Akt比值均明显升高(P均<0.05), Bcl-2蛋白相对表达量均明显降低(P均<0.05);与H_(2)O_(2)组比较,H_(2)O_(2)+吴茱萸碱10μmol/L组、H_(2)O_(2)+吴茱萸碱20μmol/L组中TNF-α、IL-1β、IL-6水平和COX-2、NF-κB p65、Bax蛋白相对表达量及Cleaved Caspase-3/Caspase-3、p-PI3K/PI3K、p-Akt/Akt比值均明显降低(P均<0.05),Bcl-2蛋白相对表达量均明显升高(P均<0.05);H_(2)O_(2)+吴茱萸碱20μmol/L+Sirtinol组中TNF-α、IL-1β、IL-6水平和COX-2、NF-κB p65、Bax蛋白相对表达量及Cleaved Caspase-3/Caspase-3、p-PI3K/PI3K、p-Akt/Akt比值均明显高于H_(2)O_(2)+吴茱萸碱20μmol/L组(P均<0.05), Bcl-2蛋白相对表达量明显低于H_(2)O_(2)+吴茱萸碱20μmol/L组(P<0.05),各指标与H_(2)O_(2)组、H_(2)O_(2)+吴茱萸碱10μmol/L组比较差异均无统计学意义(P均>0.05)。结论 吴茱萸碱可在体外通过上调SIRT1表达来抑制NF-κB通路、线粒体介导的凋亡通路和PI3K/Akt通路,从而减轻H_(2)O_(2)刺激下ARPE-19细胞的炎症反应,减少细胞凋亡。

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 H_(2)O_(2) stimulation,and to elucidate the role of Sirtuin 1(SIRT1)and its related mechanisms.Methods ARPE-19 cells were treated with different concentrations of H_(2)O_(2)(0,25,50,100,200,and 400μmol/L)and different concentrations of Evo(0,2.5,5.0,10.0,20.0,and 40.0μmol/L),respectively,and the optimal action concentrations of H_(2)O_(2) and Evo were screened by CCK-8 method.ARPE-19 cells were co-treated with H_(2)O_(2)(200μmol/L)and different concentrations of Evo(2.5,5,10,20μmol/L),and the expression of SIRT1 protein 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),200μmol/L H_(2)O_(2) treated group(H_(2)O_(2) group),200μmol/L H_(2)O_(2)+Evo 10μmol/L treated group,200μmol/L H_(2)O_(2)+Evo 20μmol/L treated group,and H_(2)O_(2)+Evo 20μmol/L+Sirtuin inhibitor Sirtinol group(H_(2)O_(2)+Evo 20μmol/L+Sirtinol group).After 24 h of treatment,the levels of inflammatory factors TNF-α,IL-1βand 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 200μmol/L of H_(2)O_(2) on the ARPE-19 cells was relatively stable.0,2.5,5.0,10.0,and 20.0μmol/L of Evo had no significant effect on ARPE-19 cell viability(all P>0.05),and 40μmol/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 H_(2)O_(2) group and H_(2)O_(2)+Evo groups than that in the control group(all P<0.05);the relative expression of SIRT1 protein was significantly higher in the H_(2)O_(2)+Evo 5μmol/L group,H_(2)O_(2)+Evo 10μmol/L group and H_(2)O_(2)+Evo 20μmol/L group than that in the H_(2)O_(2) group(all P<0.05),and the increases were more significant in the H_(2)O_(2)+Evo 10μmol/L group and H_(2)O_(2)+Evo 20μmol/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 H_(2)O_(2) group,H_(2)O_(2)+Evo 10μmol/L group,H_(2)O_(2)+Evo 20μmol/L group and H_(2)O_(2)+Evo 20μmol/L+Sirtinol group(all P<0.05);compared with the H_(2)O_(2) 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 H_(2)O_(2)+Evo 10μmol/L group,H_(2)O_(2)+Evo 20μmol/L group and H_(2)O_(2)+Evo 20μmol/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 H_(2)O_(2)+Evo 20μmol/L+Sirtinol group than those in the H_(2)O_(2)+Evo 20μmol/L group(all P<0.05),the differences in the indexes were not significant different compared with the H_(2)O_(2) group and H_(2)O_(2)+Evo 10μmol/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 apoptosis in ARPE-19 cells stimulated by H_(2)O_(2).