耐甲氧西林金黄色葡萄球菌诱导巨噬细胞焦亡的机制研究

Mechanisms of pyroptosis induced by meticillin-resistant Staphylococcus aureus in macrophages

目的观察耐甲氧西林金黄色葡萄球菌(MRSA)对小鼠骨髓来源巨噬细胞(BMDM)焦亡的影响,探究MRSA诱导巨噬细胞焦亡的作用机制。方法采用脂多糖(LPS,100 ng/ml)与尼日利亚菌素(nigericin,Ng,10 mmol/L)或三磷酸腺苷(ATP,3 mmol/L)联用或MRSA单独刺激的方式构建细胞焦亡模型;通过细胞形态学观察以及乳酸脱氢酶(LDH)检测,评价细胞膜完整性以及细胞毒性;采用ELISA法测定细胞培养液中白细胞介素(IL)-1β、IL-6和肿瘤坏死因子(TNF)-α等炎症因子的表达水平;采用Western印迹法检测细胞培养上清以及细胞内胱天蛋白酶(caspase)-1和IL-1β蛋白表达水平;采用慢病毒短发夹RNA(shRNA)干扰BMDM内相关炎症小体感受器分子表达,检测MRSA诱导细胞焦亡的信号通路。结果LPS分别与Ng或ATP联用时能破坏BMDM细胞膜完整性,上调炎症因子IL-1β、IL-6和TNF-α的水平(P<0.01),以及caspase-1和IL-1β成熟形表达水平(P<0.01);MRSA单独刺激能产生细胞毒性,诱导炎症因子IL-1β、IL-6和TNF-α的水平显著升高(P<0.01),并提高caspase-1和IL-1β成熟形在培养基中释放水平(P<0.01);在BMDM细胞内敲除NLRP3或NLRC4基因表达后,MRSA诱导的IL-1β表达量下降(P<0.01)。结论MRSA可能通过NLRP3炎症小体或NLRC4炎症小体诱导BMDM细胞焦亡。

Objective To evaluate the effect of meticillin-resistant Staphylococcus aureus(MRSA)on the pyroptosis of bone-marrow derived macrophages(BMDM)and investigate the mechanism of MRSA-induced pyroptosis in macrophages.Methods The BMDM were triggered by the combination of lipopolysaccharide(LPS,100 ng/ml)with adenosine-triphosphate(ATP,3 mmol/L)or nigericin(Ng,10 mmol/L)or treated with MRSA(multiplicity of infection 200,MOI 200)alone to induce pyroptosis in vitro.The cell morphology examination and lactate dehydrogenase(LDH)assay were applied to evaluate the cytotoxicity.The expression of pro-inflammatory cytokines,the interleukin(IL)-1β,IL-6 and tumor-necrosis factor(TNF)-αwas detected by ELISA.The cleaved caspase-1 and mature IL-1βin the cells and released into the supernatant were detected by Western blotting.The signal pathway for the induction of pyroptosis by MRSA was investigated via the transfection of lentivirus-mediated short-hairpin RNA(shRNA)into the BMDM.Results The treatment of BMDM with LPS/ATP,LPS/nigericin or MRSA alone caused cytotoxicity and up-regulated the expression of pro-inflammatory cytokines,IL-1β,IL-6 and TNF-α,as well as the caspase-1 activation and mature Zl-1β(P<0.01).After silencing NLRP3 or NLRC4,the expression of IL-1βinduced by MRSA was significantly lessened(P<0.01).Conclusion MRSA could induce BMDM pyroptosis probably via activating NLRP3 inflammasome or NLRC4 inflammasome.