SET8通过Keap1-Nrf2/HO-1信号通路抑制LPS诱导的BEAS-2B细胞炎症和氧化应激  

SET8 inhibited inflammation and oxidative stress of LPS-induced BEAS-2B via Keap1-Nrf2/HO-1 signaling pathway

作　　者：杨易帆 卢翔 王莉 陈向军 马媛 王光辉 薄丽艳 方芳 YANG Yifan;LU Xiang;WANG Li;CHEN Xiangjun;MA Yuan;WANG Guanghui;BO Liyan;FANG Fang(Department of Respiratory and Critical Care Medicine,Xi'an Chest Hospital,Xi’an 710061,China;Department of Respiratory Medicine,Northern War Zone General Hospital,Shenyang 110016,China)

机构地区：[1]西安市胸科医院呼吸与危重症医学科,陕西西安710061 [2]北部战区总医院呼吸内科,辽宁沈阳110016

出　　处：《安徽医学》2025年第3期291-298,共8页Anhui Medical Journal

基　　金：国家自然科学基金项目(编号:81800076)。

摘　　要：目的探究赖氨酸甲基转移酶8(SET8)在急性肺损伤(ALI)中的作用和可能的机制。方法购得人正常肺上皮细胞(BEAS-2B)随机分为正常组(不做任何处理)、脂多糖(LPS)组(5 mg/L LPS处理24 h)、LPS+pc-NC组(转染pc-DNA3.1质粒+LPS处理)、LPS+pc-SET8组(转染pcDNA3.1-SET8质粒+LPS处理)、LPS+pc-SET8+ML385组[转染pcDNA3.1-SET8质粒+LPS和核因子E2相关因子2(Nrf2)抑制剂ML385处理]和LPS+pc-SET8+SnPP组[转染pcDNA3.1-SET8+LPS和血红素氧合酶1(HO-1)抑制剂锡原卟啉IX(SnPP)处理]。实时荧光定量聚合酶链反应(RT-qPCR)分析SET8在LPS诱导的BEAS-2B细胞中的表达水平。细胞计数试剂盒8(CCK-8)法检测各组细胞活力。酶联免疫吸附法(ELISA)试剂盒检测各组细胞上清液中白细胞介素-1β(IL-1β)、IL-6和肿瘤坏死因子α(TNF-α)水平。采用超氧化物歧化酶(SOD)试剂盒、谷胱甘肽过氧化物酶(GSH-Px)试剂盒和丙二醛(MDA)试剂盒检测细胞中SOD、GSH-Px和MDA水平。2',7'-二氯荧光素二乙酸酯(DCFH-DA)荧光探针检测细胞中活性氧(ROS)。蛋白免疫印迹(WB)检测SET8、KELCH样ECH关联蛋白1(Keap1)、Nrf2和HO-1蛋白表达水平。结果与0 mg/L LPS组相比,SET8在不同浓度LPS诱导的BEAS-2B细胞中均表达下调(P<0.01)。与LPS+pc-NC组比较,LPS+pc-SET8组细胞上清液中IL-1β、IL-6和TNF-α水平降低(P<0.01),ROS和MDA水平降低(P<0.01),而SOD和GSH-Px水平明显升高(P<0.01)。此外,LPS+pc-SET8组中Keap1蛋白表达水平明显降低(P<0.01),Nrf2和HO-1蛋白表达明显增加,细胞核中Nrf2蛋白表达减少(P<0.01)。与LPS+pc-SET8组比较,LPS+pc-SET8+ML385和LPS+pc-SET8+SnPP组炎症因子IL-1β、IL-6和TNF-α水平明显升高(P<0.01),ROS和MDA水平显著升高(P<0.01),而SOD和GSH-Px水平明显降低(P<0.01)。结论SET8通过Keap1-Nrf2/HO-1信号通路抑制LPS诱导的BEAS-2B细胞炎症响应和氧化应激,对LPS诱导的BEAS-2B的细胞损伤有保护作用。Objective To explore the role and potential mechanism of lysine methyltransferase 8(SET8)in acute lung injury(ALI).Methods Human normal lung epithelial cells(BEAS-2B)were divided into normal group(no treatment),lipopolysaccharide(LPS)group(5 mg/L LPS treatment 24 h),LPS+pc-NC group(transfected pc-DNA3.1 plasmid+LPS treatment),LPS+pc-SET8 group(transfected pc-DNA3.1-SET8 plasmid+LPS treatment),LPS+pc-SET8+ML385 group(transfected pcDNA3.1-SET8 plasmid+LPS and nuclear factor E2-related factor 2(Nrf2)inhibitor ML385 treatment)and LPS+pc-SET8+tin protoporphyrinⅨ(SnPP)group(transfected pcDNA3.1-SET8+LPS and heme oxygenase-1(HO-1)inhibitor SnPP treatment).Real-time quantitative PCR was used to analyze the expression level of SET8 in LPS-induced BEAS-2B cells.Cell counting kit 8(CCK-8)was used to measure cell viability.Enzyme-linked immunosorbent assay(ELISA)kit was employed to detect the levels of interleukin-1β(IL-1β),IL-6 and tumor necrosis factor-α(TNF-α)in supernatant of cells in each group.Superoxide dismutase(SOD)kit,glutathione peroxidase(GSH-Px)kit and malondialdehyde(MDA)were used to detect the levels of SOD,GSH-Px and MDA in cells.2’7’-Dichlorofluorescin diacetate(DCFH-DA)fluorescent probe was used to analyze the level of reactive oxygen species(ROS)in cells.Western blotting was utilized to determine the protein expression levels of SET8,Kelch-like ECH associated protein 1(Keap1),Nrf2 and HO-1.Results Compared with the 0 mg/L LPS group,the expression of SET8 was down-regulated in BEAS-2B cells induced by different concentrations of LPS(P<0.01).Compared with LPS+pc-NC group,the levels of IL-1β,IL-6 and TNF-αin cell supernatant were reduced(P<0.01),ROS and MDA levels also decreased(P<0.01),and the levels of SOD and GSH-Px significant increased(P<0.01)in LPS+pc-SET8 group.In addition,the Keap1 protein level was reduced,and the Nrf2 and HO-1 protein levels significantly in‐creased and the Nrf2 protein level decreased in nucleus(P<0.01)in LPS+pc-SET8 group.Compared with LPS+pc-SET8 group,the levels of

关 键 词：赖氨酸甲基转移酶8 急性肺损伤 炎症 氧化应激 

分 类 号：R28[医药卫生—中药学]