miR-133靶向NLRP3对小鼠Kupffer细胞炎性活化的影响  

Effect of miR-133 targeting NLRP3 on inflammatory activation of mouse Kupffer cells

作　　者：徐秀亮[1] 杨江华[2] 盛皓宇 梁曼曼[2] 陈聪[1] 鲁稻 江启贵 XU Xiu-liang;YANG Jiang-hua;SHENG Hao-yu;LIANG Man-man;CHEN Cong;LU Dao;JIANG Qi-gui(Department of Infectious Diseases,The People's Hospital of Chizhou,Chizhou 247000,China;Department of Infectious Diseases,Yijishan Hospital of Wannan Medical College,Wuhu 241001,China)

机构地区：[1]池州市人民医院感染科,安徽池州247000 [2]皖南医学院附属弋矶山医院感染病科,安徽芜湖241001

出　　处：《中国病理生理杂志》2020年第5期854-859,共6页Chinese Journal of Pathophysiology

基　　金：2014年度安徽高校省级自然科学研究项目资助(No.KJ2014A271)。

摘　　要：目的:探究微小RNA-133(miR-133)靶向核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)对小鼠库普弗(Kupffer)细胞(KCs)炎症活化的影响。方法:从小鼠肝脏中分离KCs并鉴定。鉴定成功后用1 mg/L脂多糖(LPS)诱导KCs,并分别转染miR-133 inhibitor和miR-133 mimic。采用RT-qPCR检测细胞中miR-133和NLRP3的mRNA水平;ELISA法检测细胞培养液中白细胞介素1β(IL-1β)和肿瘤坏死因子α(TNF-α)水平;Western blot实验检测细胞中NLRP3、含胱天蛋白酶募集结构域的凋亡相关斑点样蛋白(ASC)和胱天蛋白酶1(caspase-1)蛋白水平;TargetScan查找NLRP3 mRNA的3’UTR与miR-133的结合位点,并经双萤光素酶报告基因检测试剂盒鉴定。结果:72 h时KCs体积较24 h时大,边界清晰,形态基本稳定;碳素墨水实验观察到细胞中有大量黑色颗粒,证明该细胞有较强的吞噬能力,为KCs。1 mg/L LPS诱导后KCs中miR-133水平降低,NLRP3 mRNA和蛋白、caspase-1蛋白及细胞培养液中IL-1β和TNF-α水平升高(P<0.05)。转染miR-133 inhibitor后细胞中miR-133水平降低,细胞中NLRP3mRNA和蛋白、caspase-1蛋白及细胞培养液中IL-1β和TNF-α水平升高(P<0.05);转染miR-133 mimic后细胞中miR-133水平升高,细胞中NLRP3 mRNA和蛋白、caspase-1蛋白及细胞培养液中IL-1β和TNF-α水平降低(P<0.05)。TargetScan分析显示,NLRP3 mRNA的3’UTR含有miR-133序列保守碱基。转染miR-133 mimic的细胞萤光素酶相对活性下降(P<0.05)。结论:miR-133可通过靶向NLRP3减轻小鼠KCs炎症,实现对KCs的保护。AIM:To explore the effect of microRNA-133(miR-133)targeting nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)on the inflammatory activation of Kupffer cells(KCs).METHODS:The KCs were isolated from mouse liver and identified.After successful identification,1 mg/L lipopolysaccharide(LPS)was used to induce the KCs transfected with miR-133 inhibitor or miR-133 mimic.The mRNA expression levels of miR-133 and NLRP3 were detected by RT-qPCR.The levels of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in cell culture medium were measured by ELISA.The protein levels of NLRP3,apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC)and caspase-1 were determined by Western blot.TargetScan was used to find the binding site of miR-133 and 3’UTR of NLRP3 mRNA,and the target relationship was identified by dual-luciferase reporter detection kit.RESULTS:The volume of KCs at 72 h was larger than that at 24 h,with clear boundary and stable shape.The result of carbon ink experiment showed that a large number of black particles were observed in the cells,which proved that the cells had strong phagocytic capacity and were KCs.After the KCs was induced by LPS at 1 mg/L,the level of miR-133 was decreased,while the expression of NLRP3 at mRNA and protein levels,the caspase-1 protein in the cells,and the levels of IL-1β and TNF-α in cell culture medium were increased(P<0.05).After transfection with miR-133 inhibitor,the level of miR-133 in the cells was decreased,while the expression of NLRP3 at mRNA and protein levels,the caspase-1 protein in the cells,and the levels of IL-1β and TNF-α in cell culture medium were increased(P<0.05).After transfection with miR-133 mimic,the level of miR-133 in the cells was increased,while the expression of NLRP3 at mRNA and protein levels,the caspase-1 protein in the cells,and the levels of IL-1β and TNF-α in cell culture medium were decreased(P<0.05).TargetScan analysis showed that the 3’UTR of NLRP3 mRNA contained the conservative b

关 键 词：微小RNA-133 核苷酸结合寡聚化结构域样受体蛋白3 库普弗细胞 炎症 

分 类 号：R363.2[医药卫生—病理学] R329.25[医药卫生—基础医学]