维生素D诱导自噬对巨噬细胞清除结核分枝杆菌的作用

Role of vitamin D-induced autophagy in macrophages against Mycobacterium tuberculosis

目的:探讨维生素D诱导巨噬细胞产生自噬并清除巨噬细胞内结核分枝杆菌(Mtb)的作用。方法:使用豆蔻酰佛波醇乙酯(PMA)诱导U937细胞分化使之具有吞噬能力,分化后的U937细胞随机分为阴性对照组、维生素D组、自噬抑制剂(3-MA)+维生素D组、阳性对照(雷帕霉素)组,以Mtb感染U937细胞6 h。感染后第4天,利用半定量RT-PCR检测自噬相关基因ATG5、Beclin-1及LL-37、LC3B mRNA的表达,流式细胞术检测LC3B-Ⅱ+和/或结核分枝杆菌抗原85A+(Ag85A+)的细胞。结果:与对照组相比,维生素D组ATG5、Beclin-1、LL-37、LC3B mRNA的表达增强(P<0.01),LC3B-Ⅱ+-细胞增多,Ag85A+-细胞减少,且LC3B-Ⅱ+-Ag85A--细胞增加(维生素D组38.0%比阴性对照组1.08%)。与维生素D组相比,在自噬抑制剂3-MA+维生素D组中,不但ATG5、Beclin-1、LC3B的mRNA表达受到抑制,LL-37的mRNA表达较维生素D组减少,而且3-MA抑制了细胞LC3B-Ⅱ的表达,同时抑制了1,25(OH)2D3对LC3B-Ⅱ+-Ag85A--细胞增加的作用。结论:维生素D能够诱导巨噬细胞产生自噬作用,并进一步有助于巨噬细胞清除结核分枝杆菌。

Objective：To investigate the role of vitamin D-induced autophagy in macrophages against Mycobacterium tuberculosis（ Mtb）. Methods： Induce U937 cells to differentiate into macrophages with phagocytic capacity by phorbol 12-myristate 13-acetate（PMA）. The cells were randomly divided into negative control group,vitamin D group,autophagy inhibitor（3-MA） ＋ vitamin D group,positive control（ rapamycin） group. Infect all groups with Mtb for 6 hours. In the 4th day after infection,the mRNA expressions of autophagy-related genes ATG5,Beclin-1 and LL-37,LC3 B were determined by semi-quantitative RT-PCR. LC3B-Ⅱ＋and / or Mycobacterium tuberculosis antigen 85A＋（Ag85A＋）-cells were counted by flow cytometry. Results： Compared with the control groups,the mRNA expressions of ATG5,Beclin-1,LL-37 and LC3 B were enhanced（ P〈0. 01）,the numbers of LC3B-Ⅱ＋-cells increased,the numbers of Ag85A＋-cells decreased,and the numbers of LC3B-Ⅱ＋-Ag85A--cells increased（ vitamin D group 38. 0% vs negative control group1. 08%）. Compared with the vitamin D group,the mRNA expressions of ATG5,Beclin-1 and LC3 B were suppressed in the autophagy inhibitor（3-MA） ＋ Vitamin D group,the mRNA expressions of LL-37 were reduced,and 3-MA inhibited the expression of LC3B-Ⅱin cells with inhibition of LC3B-Ⅱ＋-Ag85A--cells increase as well. Conclusion： Vitamin D can induce macrophage autophagy and further contribute to scavenging Mycobacterium tuberculosis.