miR-146a-3p靶向TLR4减轻脂多糖诱导的小鼠急性肺损伤

miR-146a-3p alleviates LPS-induced acute lung injury in mice by targeting TLR4

目的研究微小RNA(miR)-146a-3p对脂多糖(LPS)诱导的小鼠急性肺损伤(ALI)和炎症反应的作用及机制。方法将32只BALB/c小鼠随机分为假手术组、ALI组、ALI+agomiR-阴性对照(NC)组以及ALI+miR-146a-3p激动剂组(ALI+agomiR-146a-3p组),每组8只。通过气管将5 mg/kg LPS滴入肺中以建立ALI模型,假手术组缓慢滴注等量生理盐水。ALI+agomiR-146a-3p组/NC组小鼠尾静脉注射8 mg/kg agomiR-146a-3p或agomiR-NC,1次/d,持续3 d。假手术组和模型组给予尾静脉注射等量生理盐水。24 h后处死并收集肺组织。RT-qPCR检测肺组织中miR-146a-3p和Toll样受体4(TLR4)mRNA的表达,蛋白印迹检测肺组织中TLR4、活化的半胱氨酸天冬氨酸蛋白酶-3(caspase-3)、Bcl-2相关X蛋白(Bax)、B细胞淋巴瘤-2(Bcl-2)蛋白的表达水平,苏木精–伊红染色观察肺组织病理变化,原位末端转移酶标记法检测肺组织细胞凋亡情况,酶联免疫吸附试验(ELISA)检测肺组织白细胞介素(IL)-1β、IL-6和肿瘤坏死因子-α(TNF-α)表达水平。双荧光素酶报告系统验证MRC-5细胞中miR-146a-3p和TLR4的靶向关系。将人正常肺细胞MRC-5分为对照组、LPS组、LPS+miR-146a-3p mimic组、LPS+pcDNA3.1(pc)-TLR4组、LPS+miR-146a-3p mimic+pc-TLR4组。将100 nmol/L的miR-146a-3p mimic、pc-TLR4质粒分别或联合转染进入MRC-5细胞24 h后,用1 000 ng/mL的LPS或生理盐水处理72 h。流式细胞术检测细胞凋亡率,蛋白印迹检测TLR4、活化的caspase-3、Bax、Bcl-2蛋白的表达水平,ELISA检测IL-1β、IL-6和TNF-α水平。结果与ALI组相比,ALI+agomiR-146a-3p组小鼠肺组织中miR-146a-3p表达上调,而TLR4mRNA和蛋白表达均下调,肺组织细胞凋亡率减少,活化的caspase-3和Bax蛋白表达下调,而Bcl-2蛋白表达上调,肺组织中TNF-α、IL-6和IL-1β含量减少,差异均有统计学意义(P<0.05)。双荧光素酶报告基因实验证实miR-146a-3p通过靶向TLR4 3’UTR序列调控其转录(P<0.05)。与LPS组相比,LPS+miR-146a-3p mimic组MRC-5细胞中TLR4蛋白表达下调,细胞凋亡减少,活化的caspase-3和Bax蛋白表达下调,TNF-α、IL-6和IL-1β含量减少,差异均有统计学意义(P<0.05)。过表达TLR4可逆转miR-146a-3p mimic过表达对LPS诱导的MRC-5细胞凋亡和炎症反应的作用(P<0.05)。结论 miR-146a-3p通过靶向下调TLR4缓解LPS诱导的小鼠ALI。

Objective To investigate the effect and mechanism of microRNA(miR)-146 a-3 p on acute lung injury(ALI) and inflammation induced by lipopolysaccharide(LPS) in mice.Methods Thirty-two BALB/c mice were randomly divided into sham group,ALI group,ALI+agomiR-negative control(NC) group,ALI+miR-146 a-3 p agonist(agomiR-146 a-3 p) group,with 8 mice in each group.The ALI model was established by instilling 5 mg/kg LPS into the lungs through the trachea,and the same amount of saline was instilled slowly in the sham group.The mice in the ALI+agomiR-146 a-3 p group/NC group were injected with 8 mg/kg agomiR-146 a-3 p or agomiR-NC respectively through the tail vein,once a day,for 3 days.The sham group and the model group were given the same amount of normal saline injection through the tail vein.After 24 hours,they were sacrificed and lung tissues were collected.The expressions of miR-146 a-3 p and toll-like receptor 4(TLR4) mRNA in lung tissue were detected by RT-qPCR,the expression levels of TLR4,cleaved caspase-3,Bcl-2 related X protein(Bax),B cell lymphoma-2(Bcl-2) protein in lung tissue were detected by Western blot.The changes of lung pathology were observed by hematoxylin-eosin staining.The apoptosis of lung tissue was detected by TdT-mediated dUTP nick-end labeling.The expression levels of IL-1β,IL-6 and TNF-α in lung tissue were detected by enzyme-linked immunosorbent assay(ELISA).The dual luciferase reporting system verified the targeting relationship between miR-146 a-3 p and TLR4 in MRC-5 cells.MRC-5 cells were divided into control group,LPS group,LPS+miR-146 a-3 p mimic group,LPS+pcDNA3.1(pc)-TLR4 group,LPS+miR-146 a-3 p mimic+pc-TLR4 group.100 nmol/L miR-146 a-3 p mimic and pc-TLR4 plasmids were transfected into MRC-5 cells separately or jointly for 24 hours,and then treated with 1 000 ng/mL LPS or normal saline for 72 hours.The apoptosis rate was detected by flow cytometry.The expression levels of TLR4,cleaved caspase-3,Bax,and Bcl-2 proteins were detected by Western blot.The levels of IL-1β,IL-6 and TNF-α were detected by ELISA.Results Compared with the ALI group,the expression of miR-146 a-3 p was up-regulated,the expressions of TLR4 mRNA and protein were down-regulated,the apoptotic rate was decreased,the expressions of cleaved caspase-3 and Bax protein was down-regulated,the expression of Bcl-2 protein was up-regulated,and the levels of TNF-α,IL-6 and IL-1β in lung tissue were decreased in the lung tissues of the ALI+agomiR-146 a-3 p group(P<0.05).Dual-luciferase reporter assay confirmed that miR-146 a-3 p regulates transcription by targeting TLR4 3’UTR sequence(P<0.05).Compared with the LPS group,the expression of TLR4 protein in MRC-5 cells of the LPS+miR-146 a-3 p mimic group was down-regulated,the apoptosis was reduced,the expressions of cleaved caspase-3 and Bax protein were down-regulated,and the levels of TNF-α,IL-6 and IL-1β in lung tissue were decreased(P<0.05).Overexpression of TLR4 reversed the effect of miR-146 a-3 p mimic overexpression on LPS-induced apoptosis and inflammation of MRC-5 cells(P<0.05).Conclusion miR-146 a-3 p alleviates LPS-induced ALI in mice by downregulating TLR4.