摘要
目的探讨过氧化物酶体增殖物激活受体α（PPARα）对脂多糖刺激巨噬细胞引发炎症反应的影响及其调节机制。方法通过小鼠的股骨分离骨髓干细胞，经过分化培养获得骨髓来源的巨噬细胞，再通过脂多糖刺激巨噬细胞诱导细胞炎症因子表达的细胞模型。利用PPARα选择性激动州Wy44643干预炎症细胞模型，3-甲基腺嘌呤（3-methyladenine，3-MA）和Atg7小干扰RNA（Atg7 siRNA）分别作为自噬抑制剂干预自噬。通过实时定量PCR方法检测炎症细胞因子肿瘤坏死因子α（tumor necrosis factor α，TNF-α）、白细胞介素-1β（interleukin-1β，IL-1β）、IL-6和自噬相关基因Atg5、Atg7、Beclin-1的mRNA表达水平；通过免疫印迹技术检测自噬相关蛋白Atg5、Ats7、Beclin-1和LC3的表达；质粒GFP-LC3转染并观察自噬点的形成。结果研究表明，不同浓度的PPARct选择性激动剂wy14643（10μmol／L、25μmol／L和50μmol／L）预处理LPS刺激的巨噬细胞，与单独LPS刺激巨噬细胞相比，细胞炎症因子TNF-α、IL-1β、IL-6表达下降，并具有削量依赖性。此外，不同浓度Wy14643作用于巨噬细胞，也同时促进自噬相关蛋白Atg5、Atg7、Beclin-1的mRNA水平表达升高；Western blot结果显示自噬相关蛋白Atg5、Atg7、Beclin-1和LC3表达增加；荧光显微镜观察显示巨噬细胞中自噬点的形成增加。在Wy14643预处理的基础上，3-MA和Atg7siRNA分别抑制细胞自噬，结果显示炎症细胞因子TNF-α、IL-1β、IL-6的mRNA表达水平发生了逆转，重新表达升高。结论PPARα激活可抑制LPS刺激巨噬细胞诱导的促炎细胞因子的表达，并且PPARα通过促进细胞自噬而发挥抗炎作用。因此，PPARα-自噬分子途径是调控LPS诱导的巨噬细胞炎症反应的信号通路之一。
Objective To investigate the effects of peroxisome proliferator-activated receptor α （PPARα） on macrophage-mediated inflammatory responses with the interference of lipopolysaccharide and the possible mechanism. Methods The bone marrow stem cells were isolated from the femora of mice. The granulocyte-macrophage colony stimulating factor （GM-CSF） was used to stimulate the in vitro differentiation from bone marrow stem ceils into primary macrophages. An in vitro model with cultured cells expressing in- flammatory cytokines was established by treating the primary macrophages with lipopolysaccharide （LPS）. A specific chemical agonist, Wy-14643, was used to activate PPARα. Antophagy inhibitors including 3-methyladenine （3-MA） and small interfering RNA against Atg7 （Atg7 siRNA） were used to inhibit the autophagy. Western blot assay was performed to detect the expression of autophagy-related proteins （ Atg5, Atg7, Beclin-1 and LC3）. The transcriptional levels of TNF-α, IL-1β, IL-6, Atg5, Atg7 and Beclin-1 were analyzed by qRT-PCR. Results Compared with the macrophages treated with LPS alone, those pretreated with various conc, entrations of Wy-14643 （ 10 μmol/L, 25 μmol/L and 50 μmol/L） showed inhibited ex- pression of proinflammatory cytokines （TNF-α, IL-1β and IL-6） and enhanced expression of autophagy-relat- ed proteins （ Atg5, Atg7 and Beclin-1 ） at mRNA level in a dose-dependent manner. The expression of auto- phagy-related proteins （ AtgS, Atg7, Beclin-1 and LC3） by macrophages was promoted with the pretreatment of Wy-14643 as indicated by Western blot assay. The transcriptional levels of TNF-α, IL-1 βand IL-6 were increased in Wy - 14643 pretreated - inaerophages after stimulation with 3-MA or Atg7 siRNA . Conclusion PPARa suppressed the maerophage-mediated inflammatmT responses by promoting autophagy, suggesting that the PPARα-autophagy pathway might be one of the signaling pathways regulating LPS induced-inflammatory responses.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2015年第6期431-435,共5页
Chinese Journal of Microbiology and Immunology
基金
国家十二·五科技重大专项（2012ZX10002004-006,2012ZX10004904-003-001,2013ZX10002002-006-001）
国家自然科学基金项目（81270532）
首都特色临床应用研究（Z121107001012167）
王宝恩肝纤维化研究基金项目（CFHPC20131031）
北京市卫生系统高层次卫生技术人才培养计划（2013-3-075）
关键词
过氧化物酶体增殖物激活受体Α
细胞自噬
炎症反应
巨噬细胞
脂多糖
Peroxisome proliferator-activated receptor α
Autophagy
Inilammatory response
Maerophages
Lipopolysaecharide
