CAJ | 학술논문

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选择性激动剂Wy14643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 factorα，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、Atg7、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PPARα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，并具有剂量依赖性。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Atg7 siRNA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。
목적：탐토과양화물매체증식물격활수체α（ PPARα）대지다당자격거서세포인발염증반응적영향급기조절궤제。방법통과소서적고골분리골수간세포，경과분화배양획득골수래원적거서세포，재통과지다당자격거서세포유도세포염증인자표체적세포모형。이용PPARα선택성격동제Wy14643간예염증세포모형，3-갑기선표령（3-methyladenine，3-MA）화Atg7소간우RNA（Atg7 siRNA）분별작위자서억제제간예자서。통과실시정량PCR방법검측염증세포인자종류배사인자α（ tumor necrosis factorα，TNF-α）、백세포개소-1β（ interleukin-1β，IL-1β）、IL-6화자서상관기인Atg5、Atg7、Beclin-1적mRNA표체수평；통과면역인적기술검측자서상관단백Atg5、Atg7、Beclin-1화LC3적표체；질립GFP-LC3전염병관찰자서점적형성。결과연구표명，불동농도적PPARα선택성격동제Wy14643（10μmol／L、25μmol／L화50μmol／L）예처리LPS자격적거서세포，여단독LPS자격거서세포상비，세포염증인자TNF-α、IL-1β、IL-6표체하강，병구유제량의뢰성。차외，불동농도Wy14643작용우거서세포，야동시촉진자서상관단백Atg5、Atg7、Beclin-1적mRNA수평표체승고；Western blot결과현시자서상관단백Atg5、Atg7、Beclin-1화LC3표체증가；형광현미경관찰현시거서세포중자서점적형성증가。재Wy14643예처리적기출상，3-MA화Atg7 siRNA분별억제세포자서，결과현시염증세포인자TNF-α、IL-1β、IL-6적mRNA표체수평발생료역전，중신표체승고。결론 PPARα격활가억제LPS자격거서세포유도적촉염세포인자적표체，병차PPARα통과촉진세포자서이발휘항염작용。인차，PPARα-자서분자도경시조공LPS유도적거서세포염증반응적신호통로지일。
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 cells 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. Autophagy 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 concentrations of Wy-14643 (10 μmol/L, 25 μmol/L and 50 μmol/L) showed inhibited ex-pression of proinflammatory cytokines ( TNF-α,IL-1βand 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 (Atg5, 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-1βand IL-6 were increased in Wy-14643 pretreated-macrophages after stimulation with 3-MA or Atg7 siRNA .Conclusion PPARαsuppressed the macrophage-mediated inflammatory responses by promoting autophagy, suggesting that the PPARα-autophagy pathway might be one of the signaling pathways regulating LPS induced-inflamma-tory responses.