中文摘要：

5-（1H-吲哚-3-基甲基）-3-甲基-2-硫酮-4-咪唑烷酮（Necrostatin-1,Nec-1）是一种能够特异的、有效抑制细胞程序性凋亡的小分子物质。为了探讨Nec-1对卡介苗（Bacillus Calmette-Guérin,BCG）诱导的小鼠（Mus musculus）巨噬细胞RAW264.7凋亡的调控作用,本研究采用3-（4,5-二甲基噻唑-2）-2,5-二苯基四氮唑溴盐（3-（4,5）-dimethylthiahiazo（-z-y1）-3,5-di-phenytetrazoliumromide,MTT）比色法检测细胞存活率,Annexin V和碘化丙啶（propidine iodide,PI）双染法检测细胞凋亡率,JC-1染色法检测细胞线粒体膜电位水平,采用分光光度法检测细胞内Caspase-3的酶活性,q RT-PCR和Western blot法检测凋亡相关基因的m RNA和蛋白表达水平。结果表明,Nec-1可提高被BCG感染巨噬细胞的存活率,降低其凋亡率,通过降低线粒体膜电位水平、上调抑凋亡基因Bcl-2的表达同时下调RIP1、RIP3和BAX基因的表达水平,从而有效降低了Caspase-3的蛋白表达量及酶活性。本研究表明Nec-1可通过提高线粒体膜电位水平、并下调促凋亡蛋白的表达量,从而抑制被BCG感染后巨噬细胞的凋亡,这将有助于进一步研究结核分枝杆菌（Mycobacterium tuberculosis）与巨噬细胞间的相互作用,对于揭示结核病的致病机理具有重要意义。

英文摘要：

Necrostatin-1（Nec-1） is a small molecule inhibitor of necroptosis which specifically target the receptor- interacting protein 1（RIP1） kinase, and has been extensively used in disease models to investigate the role of RIP1 in cell death and inflammation. RIP1 is a crucial adaptor kinase involved in the activation of NF- κB, production of reactive oxygen species（ROS） and phosphorylation of mitogen activated protein kinases（MAPKs）, which all play important roles in apoptotic signaling. It has been proved that the vaccine strain Mycobacterium bovis BCG can elicit robust protective immunity and induce apoptosis of macrophages.However, the effect of Nec- 1 on the apoptosis of macrophages induced by BCG remains unknown. In this study, we investigated the role of Nec-1 on apoptosis induced by BCG in a macrophage cell line RAW264.7.Cell viability was assayed by MTT（3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide） assay. The cell apoptosis rate was detected by Annexin V- FITC/PI double staining. Mitochondrial membrane potential（MMP） was assessed by Jc-1 dye. Caspase-3 activity in the cell extract sampled was measured by Caspase-3Colorimetric Assay Kit. Furthermore, the m RNA level of RIP1, RIP3, Bax and Bcl- 2 were tested with RTPCR, and the expression level of Bax, Bcl-2, Caspase-3, RIP1 and RIP3 were analyzed by western blotting.The results showed that Nec- 1 significantly increase MMP and reduce apoptosis rate of RAW264.7 infected with BCG. RT- PCR and western blotting confirmed that the m RNA and protein expression of Bcl- 2 was increased, while m RNA and protein levels of RIP1, RIP3 and BAX was decreased. We also found that expression and activity of caspase-3 was decreased. Taken together, these findings indicated that Nec-1 could inhibit BCG-induced macrophage apoptosis by increasing the level of Bcl-2 and decreasing expression of the pro- apoptotic protein BAX, RIP1 and RIP3. This finding may thus provide an insight into the underlying mechanism of alveolar macrophage cell