解脲脲原体脂质相关膜蛋白通过激活核因子κB调控诱导性一氧化氮合酶在小鼠巨噬细胞中的表达

为探讨解脲脲原体(Uu)的脂质相关膜蛋白(LAMPs)诱导小鼠巨噬细胞表达诱导性一氧化氮合酶(iNOS)的分子机制,从解脲脲原体提取的脂质相关膜蛋白,刺激小鼠巨噬细胞,以RT_PCR、Western blot等方法分析iNOS的表达及NO的产生;用细胞免疫化学、间接免疫荧光及Western blot等方法检测核因子κB(NF_κB)的激活,另外检测了NF_κB的特异性抑制剂二硫代氨基甲酸吡咯烷(PDTC)和蛋白酶抑制剂放线菌酮(CHX)对iNOS的表达及NF_κB激活的影响。结果表明,解脲脲原体的LAMPs通过激活NF_κB诱导小鼠巨噬细胞表达iNOS的mRNA和蛋白,且能以时间和剂量依赖方式刺激小鼠巨噬细胞产生NO,NF_κB的抑制剂PDTC或蛋白酶抑制剂放线菌酮(CHX),可抑制NF_κB的激活及iNOS的表达。由于解脲脲原体的脂质相关膜蛋白通过激活NF_κB诱导小鼠巨噬细胞表达iNOS和产生NO,因而可能是一个重要的致病因素。

Expression of inducible nitric oxide synthase was regulated by activating nuclear factor κB in mouse macrophages stimulated with Ureaplasma urealyticum lipid-associated membrane proteins

The aim was to investigate the molecular mechanisms responsible for the inducible nitric oxide synthase (iNOS) gene expression stimulated by lipid associated membrane proteins (LAMPs) of Ureaplasma urealyticum (Uu). Mouse macrophages were stimulated by Ureaplasma urealyticum LAMPs to analyze the production of nitric oxide (NO) and the expression of iNOS detected by RT-PCR and Western blot. The activation of NF-kappaB was examined in mouse macrophages treated with LAMPs by indirect immunofluorescence (IFA), immunocytochemistry and Western blot. The effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB and of cycloheximide (CHX), a protein synthase inhibitor, on the expression of iNOS and on the activation of NF-kappaB were also investigated in mouse macrophages treated with LAMPs. Results showed Ureaplasma urealyticum LAMPs stimulated mouse macrophages to express iNOS and thus produce NO in dose- and time-dependent manners by activating nuclear factor kappaB. The activation of NF-kappaB and the expression of iNOS were inhibited by LAMPs combination with PDTC or CHX. In conclusion, these findings suggested Ureaplasma urealyticum may be an important pathogenic factor due to the ability of LAMPs to stimulate the expression of iNOS, which is probably medicated by the activation of NF-kappaB.