PPARγ促进miR-16表达抑制脓毒症炎症反应的作用研究

Peroxisome proliferators-activated receptor-γ inhibits inflammatory response by upregulating miR-16 in sepsis

目的探讨PPARγ上调miR-16的机制及其抑制脓毒症炎症反应的作用。方法经Real time RT-PCR检测脓毒症患者和健康者的外周血单核细胞PPARγ和miR-16的表达,分析其相关性;分别用PPARγ激动剂RGZ、PPARγsiRNA或miR-16抑制剂(antagomir-16)处理THP-1和RAW246.7,经real time RT-PCR和Western blot检测miR-16及其靶基因IKKα的表达;细胞转染含miR-16启动子的报告基因质粒,经PPARγ激动剂RGZ或拮抗剂GW9662处理后,检测细胞报告基因活性;细胞经PPARγ激动剂RGZ处理,再经LPS处理,ELISA检测炎症因子TNFα和IL-6的表达;LPS诱导的脓毒症小鼠经PPARγ激动剂RGZ,或经antagomir-16预处理后,再经PPARγ激动剂RGZ处理小鼠,real time RT-PCR检测小鼠外周血单核细胞中miR-16的表达,ELISA检测血清炎症因子TNFα和IL-6的表达。结果脓毒症患者外周血单核细胞中PPARγ与miR-16的表达均降低且二者的表达呈显著负相关(P<0.05);PPARγ通过促进miR-16的启动子活性上调miR-16的表达,进而抑制miR-16靶分子IKKα的表达(P<0.05);PPARγ上调miR-16后显著抑制炎症细胞产生TNFα和IL-6(P<0.05);PPARγ上调miR-16抑制脓毒症小鼠血清TNFα和IL-6的表达(P<0.05)。结论激动剂活化的PPARγ上调miR-16进而抑制细胞炎症因子表达及脓毒症小鼠炎症反应。

ObjectiveTo explore the mechanism by which peroxisome proliferatorsactivated receptorγ （PPARγ） upregulates miR-16 and inhibits inflammatory response in sepsis. MethodsRealtime PCR was used to investigate the expression of PPARγ and miR-16 in peripheral blood monocytes of patients with sepsis and healthy subjects, and the correlation between PPARγ and miR-16 was analyzed. THP-1 and RAW246.7 cells were treated with the PPARγ agonist RGZ, PPARγ siRNA or a mir16 inhibitor （antagomir16）, and the changes in the expressions of miR-16 and its target gene IKKα were detected using realtime PCR and Western blotting. The cells were transfected with luciferase reporter gene plasmid containing the miR-16 promoter region followed by treatment with RGZ or the PPARγ antagonist GW9662, and luciferase reporter assay was performed to detect the changes in the reporter gene activity. In cells treated with RGZ followed then by LPS, the expression levels of the inflammatory factors tumor necrosis factor α （TNFα） and interleukin6 （IL-6） were detected using ELISA. In a mouse model of LPSinduced sepsis, following treatment with RGZ with or without antagomir16 pretreatment, the expression of miR-16 in peripheral blood monocytes was detected with realtime PCR, and the expression of TNFα and IL-6 were determined using ELISA. ResultsThe expressions of PPARγ and miR-16 in peripheral blood monocytes were significantly reduced in patients with sepsis （P〈0.05）. In THP-1 and RAW246.7 cells, the activation of PPARγ obviously increased the expression of miR-16 by enhancing the transcriptional activity of miR16 promoter and consequently inhibited the expression of IKKα, the target gene of miR-16 （P〈0.05）. PPARγ inhibited production of TNFα and IL-6 in the inflammatory cells by increasing the expression of miR-16 （P〈0.05）. Treatment with PPARγ significantly decreased the serum levels of TNFα and IL-6 in mouse models of LPSinduced sepsis （P〈0.05）. ConclusionActivation of PPARγ inhibits the expression of inflammatory cytokines in inflammatory cells and suppresses inflammatory response in septic mice by upregulating miR-16.