结核分枝杆菌介导TWIK2-NLRP3通路参与小鼠肺泡巨噬细胞损伤的研究  

Effect of TWIK2-NLRP3 pathway mediated by Mycobacterium tuberculosis on alveolar macrophage injury in mice

作　　者：何莹[1] 韩鑫[2] 蔺咏梅[1] 皇甫彤 陆长春 罗居东 HE Ying;HAN Xin;LIN Yong-mei(Department of Infectious Diseases,3201 Hospital Affiliated to Xi'an Jiaotong University,Hanzhong Shaanxi 723000,China;Department of General Surgery,Hanzhong People's Hospital,Hanzhong Shaanxi 723000,China.)

机构地区：[1]西安交通大学附属三二〇一医院感染性疾病科,陕西汉中723000 [2]汉中市人民医院普外科,陕西汉中723000 [3]常州市第二人民医院肿瘤科,江苏常州213000

出　　处：《临床和实验医学杂志》2021年第3期229-233,共5页Journal of Clinical and Experimental Medicine

基　　金：江苏省社会发展项目(编号:BE2018643)。

摘　　要：目的分析结核分枝杆菌(MTB)介导TWIK2-NLRP3通路参与小鼠肺泡巨噬细胞损伤。方法将40只昆明小鼠按照随机数字表法分为卡介苗组和H37Rv组,每组20只。2组分别经尾静脉给予一次性注射MTB活菌卡介苗和H37Rv菌悬液制备小鼠感染模型,剂量均0.3 mL。2组小鼠于感染后1、3、7、14 d脱颈处死,采用磷酸盐缓冲液对气管、肺泡进行灌洗,收集的感染小鼠肺泡巨噬细胞以激光共聚焦显微镜技术、MTT、流式细胞术、酶联免疫吸附实验(ELISA)、实时聚合酶链式反应(RT-PCR)及蛋白质印迹(Western blotting)等技术对2组小鼠不同时间点巨噬细胞的吞噬能力、凋亡率、乳酸脱氢酶(LDH)表达量、炎性因子含量、K+浓度、钾离子通道蛋白(TWIK2)、炎症小体(NLRP3)及凋亡相关斑点样蛋白(ASC)的mRNA和蛋白表达量进行检测、对比。结果感染后1、3、7、14 d,2组小鼠肺泡巨噬细胞吞噬菌种的量、凋亡率以及LDH表达量均随时间显著增加,K+浓度则随时间显著降低,且均在在感染后7 d达到最高或最低。感染7 d后,与卡介苗组相比,H37Rv组小鼠中TWIK2、NLRP3及ASC mRNA和蛋白的相对表达量以及炎性因子白细胞介素-1β(IL-1β)、白细胞介素-18(IL-18)和肿瘤坏死因子-α(TNF-α)含量均显著升高,差异有统计学意义(P<0.05)。结论MTB和卡介苗菌株均介导TWIK2-NLRP3通路参与小鼠肺泡巨噬细胞损伤。小鼠体内注入适量MTB可被肺泡巨噬细胞吞噬,MTB可促进肺泡巨噬细胞的凋亡与损伤,其作用机制可能与TWIK2的激活密切相关。Objective To analyze the effect of TWIK2-NLRP3 pathway mediated by Mycobacterium tuberculosis(MTB)on alveolar macrophage injury in mice.Methods Forty Kunming mice were divided into BCG group and H37Rv group with 20 mice in each group by random number table.The two groups were given one-time injection of live MTB Bacillus Calmette-Guerin vaccine and H37Rv bacterial suspension via tail vein to prepare mouse infection models,each with a dose of 0.3 mL.The mice in each group were decapitated at 1,3,7 and 14 days after infection.The trachea and alveoli were lavaged with phosphate buffer solution buffer.The phagocytic capacity,apoptosis rate,lactate dehydrogenase expression(LDH),inflammatory factor expression,K+concentration,potassium channel protein(TWIK2),inflammatory bodies(NLRP3)and apoptosis related spot like proteins(ASC)were detected and compared by laser confocal microscopy,MTT,flow cytometry,enzyme-linked immunosorbent assay(ELISA),real-time polymerase chain reaction(RT-PCR)and Western blotting.Results After 1,3,7,14 days of infection,the amount of phagocytosis of alveolar macrophages in the two groups of mice,the proportion of apoptosis,and the expression of LDH increased significantly over time.The K+concentration decreased significantly over time,and all reached the highest or lowest on the 7th day after infection.After 7 days of infection,compared with the BCG vaccine group,the relative expression of TWIK2,NLRP3 and ASC mRNA and protein and the levels of inflammatory factors IL-1β,IL-18 and TNF-αin the H37Rv group increased significantly,the differences were statistically significant(P<0.05).Conclusion Both MTB and Bacillus Calmette Guerin(BCG)mediated twik2-nlrp3 pathway in alveolar macrophage injury in mice.MTB can be phagocytosed by alveolar macrophages by injecting a proper amount of MTB into mice.MTB can promote the apoptosis and injury of alveolar macrophages,and its mechanism may be closely related to the activation of TWIK2.

关 键 词：小鼠 结核分枝杆菌 乳酸脱氢酶 凋亡相关斑点样蛋白 吞噬能力 

分 类 号：R52[医药卫生—内科学]