结核分枝杆菌感染对巨噬细胞糖酵解的影响和机制研究

doi:10.3969/j.issn.1673-8640.2019.07.016

摘要/Abstract

摘要：

目的分析结核分枝杆菌感染对巨噬细胞糖酵解的影响和潜在机制。方法建立人型结核分枝杆菌H37Rv感染人巨噬细胞系U937细胞的体外模型,分别于感染后0、30、60和90 min检测乳酸生成情况。根据不同处理方法将巨噬细胞U937分为感染组（结核分枝杆菌H37Rv感染60 min）、感染+SB203580[p38丝裂原活化蛋白激酶（MAPK）特异性抑制剂]组、感染+小干扰RNA（siRNA）阴性对照组、感染+siRNA-6-磷酸果糖激酶-2/果糖-2,6-二磷酸酶3（PFKFB3）组和空白对照组。采用实时定量聚合酶链反应（qRT-PCR）和免疫印迹法检测各组细胞中PFKFB3。磷酸化p38 MAPK（p-p38 MAPK）的表达及肿瘤坏死因子（TNF）-α、白细胞介素（IL）-1β、IL-6、乳酸的生成情况。结果结核分枝杆菌可诱导巨噬细胞中PFKFB3的表达,增加乳酸的生成量,感染60 min后乳酸水平达到最大值;给予SB203580干预,可抑制感染后p-p38 MAPK和PFKFB3的表达。沉默巨噬细胞内源性PFKFB3的表达可明显拮抗结核分枝杆菌感染诱导的巨噬细胞炎性因子增加和糖酵解增强。结论结核分枝杆菌感染巨噬细胞后,可通过p38 MAPK信号通路的活化上调PFKFB3的表达,并促进巨噬细胞糖酵解和炎性因子分泌。

关键词: 结核分枝杆菌, 感染, 巨噬细胞, 糖酵解

Abstract:

Objective To investigate the influence and underlying mechanism of Mycobacterium tuberculosis infection on aerobic glycolysis in macrophage. Methods U937 cells were infected with Mycobacterium tuberculosis H37Rv. At 0,30,60 and 90 min after infection,lactate production was determined. The routinely cultured U937 cells were classified into infection group（infected with Mycobacterium tuberculosis H37Rv for 60 min）,infection+SB203580 group [p38 mitogen-activated protein kinase（MAPK） inhibitor],infection+small interfering RNA（siRNA）-scramble group,infection+siRNA-6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3（PFKFB3） group and control group. Real-time quantitation polymerase chain reaction（qRT-PCR） and western blotting were used to determine PFKFB3. The expression of phosphorylated p38 MAPK（p-p38 MAPK） and the production of tumor necrosis factor（TNF）-α,interleukin（IL）-1β,IL-6 and lactate were determined in different groups. Results The expression of PFKFB3 and the production of lactate were increased in U937 cells induced by Mycobacterium tuberculosis H37Rv,both reaching the peak at 60 min. SB203580 could effectively inhibit the expressions of p-p38 MAPK and PFKFB3. However,silencing PFKFB3 expression abated the enhancement of inflammatory cytokine and aerobic glycolysis induced by Mycobacterium tuberculosis. Conclusions Infection with Mycobacterium tuberculosis up-regulates PFKFB3 expression and promotes aerobic glycolysis and inflammatory cytokine secretion via activating p38 MAPK signaling pathway.

Key words: Mycobacterium tuberculosis, Infection, Macrophage, Aerobic glycolysis

中图分类号:

R392.9

马磊, 梁龙龙. 结核分枝杆菌感染对巨噬细胞糖酵解的影响和机制研究[J]. 检验医学, 2019, 34(7): 643-647.

MA Lei, LIANG Longlong. Influence of Mycobacterium tuberculosis infection on aerobic glycolysis in macrophage and its underlying mechanism[J]. Laboratory Medicine, 2019, 34(7): 643-647.

图/表 5

图1 结核分枝杆菌感染巨噬细胞后各组乳酸生成量

图2 结核分枝杆菌感染巨噬细胞后各组PFKFB3表达量

图3 结核分枝杆菌激活p38 MAPK信号通路后巨噬细胞中PFKFB3的表达量

图4 各组细胞中TNF-α、IL-1β、IL-6分泌量比较

图5 各组细胞中乳酸生成量比较

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