肺炎支原体脂质相关膜蛋白激活THP-1单核细胞系产生醌氧化还原酶1抑制IL-8的分泌

Quinine oxidoreductase 1 inhibits the secretion of IL-8 in Mycoplasma pneumoniae lipid-associated membrane proteins stimulated the THP-1 monocyte line

目的探讨肺炎支原体(Mycoplasma pneumoniae,Mp)脂质相关膜蛋白(lipid-associated membrane proteins,LAMPs)刺激人单核细胞系THP-1细胞表达醌氧化还原酶1(quinine oxidoreductase 1,NQO-1)的作用及NQO-1对LAMPs诱导IL-8分泌的影响,初步了解机体对Mp感染所致炎症反应的调节及机制。方法大量培养Mp,提取LAMPs,CCK8试验检测LAMPs的细胞毒性。用不同浓度LAMPs分别作用THP-1细胞不同时间,Western blot检测NQO-1蛋白水平。用特异性siRNA沉默THP-1细胞NF-E2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)后,Western blot分析LAMPs对NQO-1蛋白表达的影响。用NQO-1抑制剂Diminutol(Dim)预处理THP-1细胞后,ELISA检测LAMPs刺激后IL-8的分泌水平。结果提取的LAMPs对THP-1细胞无毒性;NQO-1蛋白表达水平与LAMPs刺激呈剂量依赖性和时间依赖性,5.0μg/ml和7.5μg/ml LAMPs刺激12 h后THP-1细胞产生的NQO-1蛋白浓度最高。沉默Nrf2表达后,LAMPs诱导THP-1细胞产生的NQO-1蛋白显著减少。Dim抑制NQO-1后,LAMPs刺激细胞分泌的IL-8增多。结论Mp LAMPs经Nrf2诱导单核细胞表达的NQO-1可抑制IL-8的分泌。

Objective To investigate the effects and regulation mechanism of lipid-associated membrane proteins(LAMPs)derived from Mycoplasma pneumoniae(Mp)on the expression of quinine oxidoreductase 1(NQO-1)in human monocyte cell line THP-1 cells,and to know the effect of NQO-1 to interleukin 8 secretion in LAMPs stimulated cells,so as to better understand the regulation mechanism upon Mp infection.Methods Mp were cultivated and the precipitate was collected to extract LAMPs.The cytotoxicity of LAMPs to THP-1 cells was analyzed by using CCK8 test.THP-1 cells were cultured in vitro with different concentrations of LAMPs for different times,and the expression of NQO-1 protein was detected by Western blot.Nrf2 siRNA was used to investigate the role of Nrf2 in NQO-1 expression in LAMPs induced cells,and NQO-1 inhibitor Diminutol was performed to test whether they blocked interleukin 8(IL-8)secretion when treated with LAMPs in THP-1 cells.Results LAMPs extracted from Mp had no cytotoxicity to THP-1 cells.The expression of NQO-1 protein in LAMPs-stimulated THP-1 cells showed a dose-dependent and time-dependent manner.The production of NQO-1 protein reached peaks when treated with 5.0μg/ml or 7.5μg/ml of LAMPs for 12 h.Silencing of Nrf2 by siRNA significantly decreased NQO-1 production,and blocking NQO-1 by Dim increased the level of IL-8 in LAMPs-stimulated cells.Conclusions LAMPs derived from Mp induced the expression of NQO-1 protein in THP-1 cells via Nrf2,and NQO-1 can inhibit IL-8 secretion in LAMPs stimulated monocytes.