Huang Chien-Da, Hsiung Te-Chih, Ho Shu-Chuan, Lee Kang-Yun, Chan Yao-Fei, Kuo Li-Wei, Lin Shu-Min, Wang Chun-Hua, Lin Horng-Chyuan, Kuo Han-Pin
Department of Thoracic Medicine, Chang Gung Memorial Hospital at Linkou, Chang Gung University College of Medicine, Taoyuan, Taiwan.
Biomed J. 2014 Jul-Aug;37(4):191-8. doi: 10.4103/2319-4170.132890.
Modification of human airway smooth muscle (ASM) function by proinflammatory cytokines has been regarded as a potential mechanism underlying bronchial hyperresponsiveness in asthma. Human ASM cells express intercellular adhesion molecule (ICAM)-1 in response to cytokines. Synthetic ligands for peroxisome proliferator-activated receptor (PPAR)γ reportedly possess anti-inflammatory and immunomodulatory properties. In this study, we examined whether ciglitazone, a synthetic PPARγ ligand, can modulate the basal and tumor necrosis factor (TNF)α-induced ICAM1 gene expression in human ASM cells.
Human ASM cells were treated with TNFα. ICAM-1 expression was assessed by flow cytometry and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. PPARγ activity was inhibited by target-specific small interfering (si) RNA targeting PPARγ and GW9662, a PPARγ antagonist. Activity of nuclear factor (NF)-κB was assessed by using immunoblot analysis, immune-confocal images, and electrophoretic mobility shift assay (EMSA).
By flow cytometry, ciglitazone alone had no effect on ICAM-1 expression in ASM cells, but inhibited ICAM-1 expression in response to TNFα (10 ng/ml) in a dose-dependent manner (1-10 μM). It also inhibited TNFα-induced ICAM1 gene expression by RT-PCR analysis. Knockdown of PPARγ gene by target-specific siRNA targeting PPARγ enhanced ICAM-1 expression and the inhibitory effect of ciglitazone on TNFα-induced ICAM-1 expression was reversed by PPARγ siRNA and GW9662. SN-50 (10 μg/ml), an inhibitor for nuclear translocation of NF-κB, inhibited TNFα-induced ICAM-1 expression. Ciglitazone did not prevent TNFα-induced degradation of the cytosolic inhibitor of NF-κB (IκB), but inhibited the nuclear translocation of p65 induced by TNFα and suppressed the NF-κB/DNA binding activity.
These findings suggest that ciglitazone inhibits TNFα-induced ICAM1 gene expression in human ASM cells through the ligand-dependent PPARγ activation and NF-κB-dependent pathway.
促炎细胞因子对人气道平滑肌（ASM）功能的改变被认为是哮喘患者支气管高反应性的潜在机制。人ASM细胞在细胞因子作用下表达细胞间黏附分子（ICAM）-1。据报道，过氧化物酶体增殖物激活受体（PPAR）γ的合成配体具有抗炎和免疫调节特性。在本研究中，我们检测了合成PPARγ配体吡格列酮是否能调节人ASM细胞中基础和肿瘤坏死因子（TNF）α诱导的ICAM1基因表达。
用人TNFα处理人ASM细胞。通过流式细胞术和逆转录聚合酶链反应（RT-PCR）分析评估ICAM-1表达。用靶向PPARγ的特异性小干扰（si）RNA和PPARγ拮抗剂GW9662抑制PPARγ活性。通过免疫印迹分析、免疫共聚焦图像和电泳迁移率变动分析（EMSA）评估核因子（NF）-κB的活性。
流式细胞术检测显示，吡格列酮单独作用对ASM细胞中ICAM-1表达无影响，但能剂量依赖性地（1-10μM）抑制TNFα（10ng/ml）诱导的ICAM-1表达。RT-PCR分析也显示其抑制TNFα诱导的ICAM1基因表达。用靶向PPARγ的特异性siRNA敲低PPARγ基因可增强ICAM-1表达，且PPARγ siRNA和GW9662可逆转吡格列酮对TNFα诱导的ICAM-1表达的抑制作用。NF-κB核转位抑制剂SN-50（10μg/ml）抑制TNFα诱导的ICAM-1表达。吡格列酮不能阻止TNFα诱导的NF-κB胞质抑制剂（IκB）降解，但抑制TNFα诱导的p65核转位并抑制NF-κB/DNA结合活性。
这些发现表明，吡格列酮通过配体依赖性PPARγ激活和NF-κB依赖性途径抑制人ASM细胞中TNFα诱导的ICAM1基因表达。
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