Regulatory role and mechanism of the inhibition of the Mcl-1 pathway during apoptosis and polarization of H37Rv-infected macrophages.
Regulatory role and mechanism of the inhibition of the Mcl-1 pathway during apoptosis and polarization of H37Rv-infected macrophages.
复制标题
抑制Mcl-1通路在H37Rv感染巨噬细胞凋亡和极化过程中的调控作用及机制
DOI：
10.1097/md.0000000000022438
复制
发表时间：
2020-10-16
期刊：
影响因子：
1.6
通讯作者：
Zhang L
中科院分区：
文献类型：
--
作者：
Han L;Lu Y;Wang X;Zhang S;Wang Y;Wu F;Zhang W;Wang X;Zhang L
背景：髓样细胞白血病-1 （Mcl-1）在结核分枝杆菌（MTB）感染的清除中起重要作用。具有抗凋亡、保护吞噬病原体的巨噬细胞、阻止病原体清除的作用。同时，MAPK信号通路在结核感染过程中调控Mcl-1的表达发挥重要作用。在潜伏感染和活动性感染的情况下，MTB感染过程中巨噬细胞的凋亡和极化都有很大的影响，因此我们讨论Mcl-1对凋亡和极化的影响。然后，进一步探讨了其机理。方法：建立感染RAW264.7巨噬细胞模型，研究Mcl-1通路抑制对H37Rv感染细胞凋亡和极化的调控作用及机制。首先，采用western blotting和Real-Time Polymerase Chain Reaction （RT-PCR）对Mcl-1蛋白和mRNA进行鉴定。流式细胞术检测RAW264.7巨噬细胞凋亡。RT-PCR检测Bax、Caspase-3、Cyt-c、Bcl-2 mRNA表达。然后采用ELISA法检测炎症因子CD86、CD206、iNOS、Fizz1、IL-6、IL-10、TNF-α、TGF-β的表达水平。扫描电镜观察巨噬细胞表型。最后用western blotting检测Bax、Bcl-2和Bcl-xl的表达。使用JC-10试剂盒使用共聚焦显微镜分析线粒体膜电位。结果：本研究发现，抑制Mcl-1表达信号通路可导致不同毒力结核分枝杆菌的感染，以及Mcl-1蛋白和mRNA表达的变化。同时，巨噬细胞凋亡率也发生了变化，感染细胞出现了M1和M2两种表型状态。我们还发现线粒体通路被激活，其相关基因Bax、casepase3和Cyt-c表达增加，而Bcl-2表达减少，线粒体膜去极化功能发生改变。结论：我们发现Mcl-1影响结核分枝杆菌感染巨噬细胞的凋亡和极化，早期主要影响M1，晚期主要影响M2。此外，线粒体在这一过程中起着至关重要的作用。
Abstract Background: Myeloid cell leukemia-1 (Mcl-1) plays an important role in the clearance of Mycobacterium tuberculosis (MTB) infection. It has the effect of anti-apoptosis, protecting macrophages that have engulfed pathogens and preventing pathogen clearance. Meanwhile, the MAPK signaling pathway plays a significant role in regulating Mcl-1 expression during tuberculosis infection. In the case of latent infection and active infection, the apoptosis and polarization of macrophages have a great influence during MTB infection, so we discussed the effect of Mcl-1 on apoptosis and polarization. Then, further discussed its mechanism. Methods: An infected RAW264.7 macrophage model was established to investigate the regulatory role and mechanism of the Mcl-1 pathway inhibition during apoptosis and polarization of H37Rv infection. First, Mcl-1 protein and mRNA was identified by western blotting and Real-Time Polymerase Chain Reaction (RT-PCR). RAW264.7 macrophage apoptosis was detected by flow cytometry. RT-PCR was utilized to detect Bax, Caspase-3, Cyt-c and Bcl-2 mRNA expression. Next, Then the expression levels of inflammation factors CD86, CD206, iNOS, Fizz1, IL-6, IL-10, TNF-α, and TGF-β was detected by ELISA. SEM was used to observe macrophages phenotype. Finally, Bax, Bcl-2 and Bcl-xl the expression was detected by western blotting. Confocal microscopy was used to analyze mitochondrial membrane potential using the JC-10 kit. Results: In this study, we found that inhibiting the Mcl-1 expression signaling pathway led to infection by different virulence Mycobacterium tuberculosis, as well as changes in Mcl-1 protein and mRNA expression. Concomitantly macrophage apoptosis rate also changed, While, two phenotypic states of M1 and M2 appeared in the infected cells. We also found that the mitochondrial pathway was activated, the expression of its related genes Bax, casepase3, and Cyt-c, increased, whereas that of Bcl-2 decreased, and the mitochondrial membrane depolarization function was changed. Conclusions: We found that Mcl-1 affected the apoptosis and polarization of macrophages infected by Mycobacterium tuberculosis, mainly M1 in the early stage and M2 in the later stage. In addition, mitochondria played a crucial role in this process.