问号钩端螺旋体经线粒体相关信号通路诱导巨噬细胞凋亡的研究

Leptospira interrogans inducing apoptosis of macrophages through mitochondria-associated signaling pathway

目的确定线粒体相关信号转导途径在问号钩端螺旋体（简称钩体）诱导小鼠单核一巨噬细胞凋亡过程中的作用。方法建立问号钩体黄疸出血群赖株诱导小鼠单核．巨噬样细胞株J774A．1凋亡模型。采用透射电镜观察感染细胞线粒体病变情况，JC-1染色法检测感染细胞线粒体膜电位变化，荧光探针DCFH-DA检测感染细胞内活性氧（ROS）水平。采用试剂盒检测感染细胞caspase．8和caspase-9活性变化。流式细胞术检测感染细胞凋亡情况以及caspase阻断剂阻断凋亡的效果。采用Westernblot检测线粒体内和胞质中的细胞色素C（CytC）以及凋亡诱导因子（AIF）、核酸内切酶G（EndoG）和Smac水平。应用免疫荧光染色法检测AIF和EndoG从细胞质至核内的转位。结果问号钩体赖株可诱导J774A．1细胞凋亡。感染细胞的线粒体有明显病变，线粒体膜电位降低且胞内活性氧水平升高。感染细胞caspase-8活化，caspase-9则否，但caspase阻断剂不能完全阻断细胞凋亡。感染细胞AIF和EndoG从线粒体释放至胞质并转位至细胞核内。未检测到感染细胞胞质内CytC水平升高及Smac的释放。结论线粒体可通过非caspase途径的AIF和EndoG参与问号钩体诱导单核-巨噬细胞凋亡的过程。

Objective To determine mitochondria-associated signaling pathway on cell apoptosis in Leptospira interrogans infected murine mononuclear-macrophages. Methods A cell apoptotic model of L. interrogan serogroup Ieterohaemorrhagiae strain Lai inducing apoptosis of murine mononuclear-maerophage line （ J774A. 1 ） was established. Pathological changes of mitochondria in the infected cells were observed under transmission electron microscope. Mitoehondrial potential and reactive oxygen species（ROS） levels in the infected cells were detected using JC-1 staining method and DCFH-DA fluorescent probe, respectively, and caspase-8/-9 activities in the infected cells were measured using commercial kits. and the apoptosis block effects of caspase inhibitors were determined by flow eytometry. Apoptosis in the infected cells and apoptosis-bloeking effect by caspase inhibitors were detected by flow eytometry. Western blot assay was adopted to examine the levels of cytochrome C （ CytC）, AIF, EndoG and Smac in the mitochondria and cytoplasm. The transposition of either AIF or EndoG from the cytoplasm into cell nucleus was determined by immunoflu- orescenee staining test. Results L. interrogans strain Lai could induce J774A. 1 cell apoptosis. In the infected cells, visible mitoehondrial injury, declined mitochondrial membrane potential and elevated ROS level were presented. The activity of caspase-8 but not of caspase-9 was significantly increased. The caspase inhibitors could not completely block the cell apoptosis. Both the AIF and EndoG was released from the mitoehondria and subsequently transferred from the cytoplasm into the nucleus in the infected cells. However, elevation of CytC level and Smae release in cytoplasm of the infected cells could not be found. Conclusion L. interrogans can induce apoptosis in the infected mononuclear-maerophage via AIF and EndoG belonging to easpase-independent pathway.