Lu Yang, Wang Xin-Min, Yang Pu, Han Ling, Wang Ying-Zi, Zheng Zhi-Hong, Wu Fang, Zhang Wan-Jiang, Zhang Le
Department of Pathophysiology/the Key Laboratories for Xinjiang Endemic and Ethnic Diseases Department of Urinary Surgery, The First Affiliated Hospital, Medical College of Shihezi University, Shihezi, Xinjiang, China.
Medicine (Baltimore). 2018 Aug;97(35):e12125. doi: 10.1097/MD.0000000000012125.
Apoptosis and inflammation have been shown to play an important role in the mechanisms involved in the pathogenesis of Mycobacterium tuberculosis (MTB) infection. When macrophages undergo apoptosis and polarization, gap junctions (GJs) may be needed to provide conditions for their functions. Connexin 43 (Cx43) and connexin 37 (Cx37) are the main connexins in macrophages that participate in the formation of GJ channels.
An H37Rv infection RAW264.7 macrophage model was established to investigate the associate between connexins and host macrophage immune defense response after MTB infection. First, Real-time Polymerase Chian Reaction (RT-PCR) was used to detect the mRNA expression of Cx43 and Cx37. Cx43 protein expression and location was detected by western blotting and immunofluorescence. Confocal microscope was used to assay the gap junctional intercellular communication (GJIC). Then, electron microscope used to observe the morphology of macrophages. Finally, RAW264.7 macrophage apoptosis and mitochondrial membrane potential was detected by flow cytometry, and the expression of inflammation factors such as CD86, CD206, and IL-6, IL-10, TNF-α, and TGF-β were detected by Real-time PCR and enzyme-linked-immunosorbent serologic assay (ELISA).
H37Rv infection significantly promoted host macrophage Cx43 mRNA and protein expression (increased 1.6-fold and 0.3-fold respectively), and enhanced host macrophage GJIC. When host macrophage cell-to-cell communication induced by H37Rv infection, the apoptosis rate and inflammatory factors expression also increased.
The results confirm that H37Rv infection can obviously induce host macrophage Cx43 expression and enhance GJIC, which may implicated in host macrophage inflammatory reaction, to regulate the release of inflammatory factors and/or initiate apoptosis to activate host immune defense response.
细胞凋亡和炎症在结核分枝杆菌（MTB）感染发病机制所涉及的机制中发挥重要作用。当巨噬细胞发生凋亡和极化时，可能需要间隙连接（GJs）为其功能提供条件。连接蛋白43（Cx43）和连接蛋白37（Cx37）是巨噬细胞中参与形成GJ通道的主要连接蛋白。
建立H37Rv感染RAW264.7巨噬细胞模型，以研究MTB感染后连接蛋白与宿主巨噬细胞免疫防御反应之间的关联。首先，采用实时聚合酶链反应（RT-PCR）检测Cx43和Cx37的mRNA表达。通过蛋白质印迹法和免疫荧光法检测Cx43蛋白的表达和定位。使用共聚焦显微镜检测间隙连接细胞间通讯（GJIC）。然后，用电子显微镜观察巨噬细胞的形态。最后，通过流式细胞术检测RAW264.7巨噬细胞凋亡和线粒体膜电位，并通过实时PCR和酶联免疫吸附测定（ELISA）检测CD86、CD206以及IL-6、IL-10、TNF-α和TGF-β等炎症因子的表达。
H37Rv感染显著促进宿主巨噬细胞Cx43 mRNA和蛋白表达（分别增加1.6倍和0.3倍），并增强宿主巨噬细胞GJIC。当H37Rv感染诱导宿主巨噬细胞间通讯时，凋亡率和炎症因子表达也增加。
结果证实，H37Rv感染可明显诱导宿主巨噬细胞Cx43表达并增强GJIC，这可能与宿主巨噬细胞炎症反应有关，以调节炎症因子的释放和/或引发凋亡来激活宿主免疫防御反应。
