异钩藤碱上调miR-192-5p对TNF-α诱导的人支气管上皮细胞凋亡和炎症因子释放的作用及其机制  

Effect of isorhynchophylline on apoptosis and release of inflammatory factors in human bronchial epithelial cells induced by TNF-αvia up-regulating miR-192-5p and its mechanism

作　　者：侯从岭[1] 芦晓帆 雷小婷[1] 李彬[1] 赵润杨[1] HOU Congling;LU Xiaofan;LEI Xiaoting;LI Bin;ZHAO Runyang(Department of Pulmonary Diseases,Henan Provincial Hospital of Traditional Chinese Medicine,Zhengzhou 450002,China)

机构地区：[1]河南省中医院肺病科,河南郑州450002

出　　处：《吉林大学学报（医学版）》2021年第3期595-607,共13页Journal of Jilin University：Medicine Edition

基　　金：河南省中医药管理局国家中医临床研究基地科研专项(2019JDZX049);河南省科技厅攻关项目(192102310423)。

摘　　要：目的:探讨异钩藤碱(IRN)对肿瘤坏死因子α(TNF-α)诱导的人支气管上皮细胞(16HBE)凋亡和炎症因子释放的影响,并阐明其可能的作用机制。方法:采用不同浓度[(0(对照组)、2.5、5.0、10.0、20.0、30.0和40.0μmol·L^(-1)]IRN处理16HBE细胞24 h后,采用20 mg·L^(-1)TNF-α处理细胞18 h。将16HBE细胞分为对照组、TNF-α组(20 mg·L^(-1))、IRN组(20μmol·L^(-1))、TNF-α+IRN组(20 mg·L^(-1)TNF-α和20μmol·L^(-1)IRN)、TNF-α+IRN+miR-192-5p inhibitor组(20 mg·L^(-1)TNF-α、20μmol·L^(-1)IRN和50 nmol·L^(-1)miR-192-5p inhibitor)以及TNF-α+IRN+pcDNA3.1 CXCR5组[20 mg·L^(-1)TNF-α、20μmol·L^(-1)IRN和2μmol·L^(-1)pcDNA3.1-C-X-C趋化因子受体5(CXCR5)]。采用CCK-8法检测各组16HBE细胞活性,实时荧光定量PCR(RT-qPCR)法检测各组16HBE细胞中miR-192-5p和CXCR5 mRNA表达水平,Western blotting法检测各组16HBE细胞中白细胞介素1β(IL-1β)、单核细胞趋化蛋白1(MCP-1)、Bcl-2、Cleaved-Caspase3、CXCR5以及磷酸化的p38、c-Jun氨基末端激酶(JNK)、c-Jun和核因子-κB(NF-κB)p65蛋白表达水平,ELISA法检测各组16HBE细胞上清液中IL-1β和MCP-1水平,流式细胞术检测各组16HBE细胞凋亡率,TargetScan7.1网站预测靶基因并通过双荧光素酶报告基因实验验证miR-192-5p与CXCR5之间的靶向结合关系。结果:与对照组比较,不同浓度TNF-α组细胞活性明显降低(P<0.05);与对照组组比较,5.0、10.0、20.0和30.0μmol·L^(-1)IRN组细胞活性升高(P<0.05)。与对照组比较,TNF-α组16HBE细胞中miR-192-5p和Bcl-2蛋白表达水平明显降低(P<0.05),IL-1β、MCP-1、Cleaved-Caspase-3、p-p38、p-JNK、p-c-Jun和p-NF-κBp65蛋白表达水平,细胞凋亡率以及上清液中IL-1β和MCP-1水平明显升高(P<0.05);与TNF-α组比较,TNF-α+IRN组16HBE细胞中miR-192-5p和Bcl-2蛋白表达水平明显升高(P<0.05),IL-1β、MCP-1、Cleaved-Caspase-3、p-p38、p-JNK、p-c-Jun和p-NF-κBp65蛋白表达水平,细胞凋亡率以及上清液中IL-1β和MCP-1水�Objective:To observe the effect of isorhynchophylline(IRN)on TNF-α-induced the apoptosis and the release of inflammatory factors in the human bronchial epithelial(16HBE)cells,and to explore its possible mechanism.Methods:After treated with different concentrations[0(control group),2.5,5.0,10.0,20.0,30.0 and 40.0μmol·L^(-1)]of IRN for 24 h,the 16HBE cells were cultured with20 mg·L^(-1) TNF-αfor 18 h.The 16HBE cells were divided into control group,TNF-αgroup(20 mg·L^(-1)),IRN group(20μmol·L^(-1)),TNF-α+IRN group(20 mg·L^(-1) TNF-αand 20μmol·L^(-1) IRN),TNF-α+IRN+miR-192-5p inhibitor group(20 mg·L^(-1) TNF-α,20μmol·L^(-1) IRN and 50 nmol·L^(-1) miR-192-5p inhibitor)and TNF-α+IRN+pcDNA3.1 CXCR5 group[20 mg·L^(-1) TNF-α,20μmol·L^(-1) IRN and2μmol·L^(-1) pcDNA3.1-C-X-C chomokine receptor type 5(CXCR5)].CCK-8 method was used to detect the viabilities of 16HBE cells in various groups;Real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of miR-192-5p and CXCR5 mRNA in the 16HBE cells in various groups;Western blotting method was used to detect the expression levels of interleukins-1β(IL-1β)and monocyte chemoattactant protein-1(MCP-1),B cell lymphoma-2(Bcl-2),Cleaved-Caspase 3,CXCR5,and phosphorylated p38(p-P38),c-Jun N-terminal kinase(JNK),c-Jun,and nuclear factor-κB(NF-κB)p65 proteins in the 16HBE cells in various groups;ELISA method was used to detect the levels of IL-1βand MCP-1 in supernatant of the 16HBE cells in various groups.TargetScan7.1 website was used to predict the target genes and the targeted binding association between miR-192-5p and CXCR5 was verified by dual-luciferase reporter gene assay.Results:Compared with control group,the viability of the16HBE cells in TNF-αgroup was significantly decreased(P<0.05);compared with control group,the viabilities of 16HBE cells in 5.0,10.0,20.0,and 30.0μmol·L^(-1) IRN groups were increased(P<0.05).Compared with control group,the expression levels of miR-192-5p and Bcl-2 proteins in the 16HBE c

关 键 词：异钩藤碱 慢性阻塞性肺疾病 支气管上皮细胞 miR-192-5p C-X-C趋化因子受体5 

分 类 号：Q71[生物学—分子生物学]