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

作　　者：周洋[1] 美丽巴努·玉素甫[1] ZHOU Yang;Meilibanu Yusufu(The Fifth Affiliated Hospital of Xinjiang Medical University,Urumqi 830011,Xinjiang,China)

机构地区：[1]新疆医科大学第五附属医院,新疆乌鲁木齐830011

出　　处：《现代中西医结合杂志》2024年第10期1330-1337,1343,共9页Modern Journal of Integrated Traditional Chinese and Western Medicine

基　　金：新疆维吾尔自治区卫生健康青年医学科技人才专项科研项目(WJWY-202039)。

摘　　要：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_(2Objective 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 signi

关 键 词：吴茱萸碱 去乙酰化酶1 ARPE-19细胞 炎症反应 细胞凋亡 

分 类 号：R285.5[医药卫生—中药学]