[目的]金黄色葡萄球菌是一种革兰阳性共生细菌，是一种胞内菌，其利用坏死性凋亡在人和动物宿主中引起多种疾病。本研究旨在了解混合系列蛋白激酶样结构域(mixed lineage kinase domain like protein，MLKL)是否参与调控金黄色葡萄球菌感染所致炎性反应，为防控金黄色葡萄球菌感染所致疾病提供新思路与理论依据。[方法]通过转录组测序分析Pam2CSK4和金黄色葡萄球菌SA113刺激前后小鼠巨噬细胞中基因的变化;通过分子对接技术分析Pam2CSK4与MLKL的对接方式及位点;通过实时荧光定量PCR对差异表达基因进行验证;通过Western blotting检测MLKL，p65，p38和ERK的磷酸化水平;通过ELISA方法检测促炎性细胞因子(肿瘤坏死因子-α(TNF-α)和白细胞介素-1β(IL-1β))、趋化因子(RANTES)和抗炎因子(IL-10)的分泌水平。[结果]转录组分析结果表明，Pam2CSK4和金黄色葡萄球菌SA113刺激巨噬细胞后可导致坏死性凋亡相关基因表达，且MLKL基因相关度较高(P＜0。05)。分子对接预测结果显示，Pam2CSK4与MLKL蛋白结合良好，并与ARG-304、ARG-301、GLN-331和LYS-333氨基酸残基形成氢键。实时荧光定量PCR和Western blotting结果显示，与未刺激组相比，Pam2CSK4和金黄色葡萄球菌SA113刺激可诱导小鼠巨噬细胞中MLKL的磷酸化和基因表达水平极显著上调(P＜0。01)。表明MLKL参与金黄色葡萄球菌感染过程。通过 Western blotting检测MLKL对NF-κB和MAPK信号通路激活的影响，结果显示，与C57BL/6J小鼠相比，在金黄色葡萄球菌SA113刺激30和60 min后，MLKL-/-小鼠巨噬细胞中p65磷酸化水平显著或极显著上调(P＜0。05;P＜0。01);而在Pam2CSK4刺激30和60 min后，MLKL-/-小鼠巨噬细胞中p65磷酸化水平极显著下调(P＜0。01);在Pam2CSK4刺激15、30和60 min后，MLKL-/-小鼠巨噬细胞中ERK磷酸化水平极显著下调(P＜0。01);Pam2CSK4和金黄色葡萄球菌SA113刺激60 min后，MLKL-/-小鼠巨噬细胞中p38磷酸化水平显著或极显著下调(P＜0。05;P＜0。01)。与C57BL/6J小鼠相比，Pam2CSK4刺激后，MLKL-/-小鼠巨噬细胞上清液中TNF-α和RANTES分泌水平均极显著升高(P＜0。01);SA113刺激后，MLKL-/-小鼠巨噬细胞上清液中TNF-α、IL-1β和RANTES分泌水平均极显著升高(P＜0。01)，1L-10分泌水平极显著降低(P＜0。01)。[结论]MLKL对金黄色葡萄球菌诱导的炎症介质分泌及炎症信号通路激活具有调控作用。
Effects of MLKL on Secretion of Inflammatory Mediators and Activation of Inflammatory Signaling Pathway Induced by Staphylococcus aureus
[Objective]Staphylococcus aureus(S.aureus),a Gram-positive facultative anaerobic bacterium,was an intracellular pathogen capable of causing various diseases in both humans and animal hosts through the utilization of necrotic apoptosis.This study was aimed to investigate whether the mixed lineage kinase domain-like protein(MLKL)was involved in regulating the inflammatory response induced by S.aureus infection,and provide new ideas and theoretical basis for the prevention and control of diseases caused by S.aureus infection.[Method]In this study,genetic change in mouse macrophages before and after stimulation with Pam2CSK4 and S.aureus SA113 were analyzed by transcriptome sequencing.The docking modes and sites of Pam2CSK4 and MLKL were analyzed by molecular docking technique.The differentially expressed genes were verified by Real-time quantitative PCR.The phosphorylation levels of MLKL,p65,p38 and ERK were detected by Western blotting.The secretion levels of pro-inflammatory cytokines(tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)),chemokine(RANTES)and anti-inflammatory factor(IL-10)were detected by ELISA.[Result]The results of transcriptional analysis showed that stimulation with Pam2CSK4 and S.aureus SA1 13 induced the expression of necroptosis-related genes in macrophages,with a high correlation in MLKL gene(P＜0.05).Molecular docking prediction results showed that Pam2CSK4 was well bound to MLKL protein and formed hydrogen bonds with ARG-304,ARG-301,GLN-331 and LYS-333 amino acids.Real-time quantitative PCR and Western blotting results showed that compared to unstimulated group,the stimulation with Pam2CSK4 and S.aureus SA113 led to an extremely significant upregulation in the phosphorylation and gene expression levels of MLKL in mouse macrophages(P＜0.01).These findings suggested the involvement of MLKL in the S.aureus infection process.The impact of MLKL on the activation of the NF-κB and MAPK signaling pathways was examined using Western blotting.The results indicated that compared with C57BL/6J mice,MLKL-/-mouse macrophages exhibited a significant or extremely significant increase in p65 phosphorylation after S.aureus stimulation for 30 and 60 min(P＜0.05 or P＜0.01),while an extremely significant decrease was observed after Pam2CSK4 stimulation for 30 and 60 min(P＜0.01).After 15,30 and 60 min of Pam2CSK4 stimulation,the phosphorylation level of ERK in MLKL-/-mouse macrophages was extremely significantly downregulated(P＜0.01).After 60 min of stimulation with Pam2CSK4 and S.aureus SA113,the phosphorylation level of p38 in MLKL/-mouse macrophages was significantly or extremely significantly downregulated(P＜0.05 or P＜0.01).Compared with C57BL/6J mice,after Pam2CSK4 stimulation,the secretion levels of TNF-α and RANTES in the supernatant of MLKL/-mice macrophages were extremely significantly increased(P＜0.01).After SA113 stimulation,the secretion levels of TNF-α,IL-1β and RANTES in the supernatant of MLKL-/-mouse macrophages were extremely significantly increased(P＜0.01),while the secretion level of IL-10 was extremely significantly decreased(P＜0.01).[Conclusion]These results suggested that MLKL played a regulatory role in the secretion of inflammatory mediators and the activation of inflammatory signaling pathways induced by S.aureus.
Staphylococcus aureusmixed lineage kinase domain like protein(MLKL)necroptosistranscriptomicsinflammatory response
万方数据
维普
