呼吸道合胞病毒感染后IL-17、 hCLCA1表达变化及罗格列酮的抑制效应

摘要

目的 研究呼吸道合胞病毒(RSV)感染A549细胞后过氧化物酶体增殖物激活受体γ(PPARγ)、IL-17及人钙激活氯离子通道辅助蛋白1(hCLCA1) mRNA和蛋白表达变化及罗格列酮和IL-17抗体的干预作用.方法 将传代细胞随机分6组:罗格列酮/RSV组、罗格列酮/GW9662/RSV组、DMSO/RSV组、IL-17抗体/RSV组、GW9662/RSV组、细胞对照组.DMSO/RSV组予0.02％ DMSO预处理0.5h,罗格列酮/RSV组予罗格列酮(10μmol/L)预处理0.5h,罗格列酮/GW9662/RSV组在应用罗格列酮前先以GW9662(10μmol/L)预处理0.5h,IL-17抗体/RSV组在RSV感染前予IL-17抗体预处理2h.各组培养6、12、24h收获细胞及上清液待测.应用实时荧光定量RT-PCR检测PPARγ、IL-17、hCLCA1 mRNA表达,Western blot法检测hCLCA1蛋白水平,ELISA检测IL-17蛋白表达.结果 与细胞对照组相比,DMSO/RSV组PPARγ、IL-17、hCLCA1 mRNA和蛋白表达在3个时间点均明显升高,随时间增加表达量增加,24h达高峰,与12h比较,差异有统计学意义(P均＜0.05);在同一时间点上罗格列酮/RSV组、IL-17抗体/RSV组IL-17、hCLCA1 mRNA和蛋白均明显低于DMSO/RSV组,与罗格列酮/GW9662/RSV组相比,罗格列酮/RSV组IL-17、hCLCA1 mRNA和蛋白表达均明显降低.罗格列酮及IL-17抗体对IL-17 mRNA和蛋白的抑制作用在干预后6h最强,与12、24h相比较,差异有统计学意义(P＜0.05).结论 罗格列酮可抑制RSV感染后IL-17、hCLCA1 mRNA及蛋白表达增加,机制可能为上调PPARγ基因的表达,使PPARγ活性增强,在转录水平下调IL-17表达,从而抑制气道黏液高分泌.%Objective To observe the expressions of peroxisome proliferator activatedγ (PPARγ),IL-17 and calcium activated family member 1 (hCLCA1) mRNA and protein in A549 cell infected with respiratory syncytial virus(RSV),and to explore the effect of rosiglitazone and anti-IL-17 Ab.Methods A549 cells were seeded in 6-well culture plates and randomly divided into six groups.DMSO/RSV group received medium containing 0.02％ DMSO 30min prior to RSV infection.Rosiglitazone/RSV group received rosiglitazone (101μmol/L) 30 min prior to RSV infection.GW9662/rosiglitazone/RSV group was first exposed to GW9662 (10μmol/L)for 30 min,prior to rosiglitazone and RSV infection.Anti-IL-17 Ab (0.15μmol/L) was administered to anti-IL-17 Ab/RSV group 2 hour prior to RSV infection.Supernatants were harvested at 6h,12h and 24h post-RSV infection,retrospectively.The expression of PPARγ,IL-17,hCLCA1 mRNA were measured by real-time quantitative PCR.Protein levels of hCLCA1 and IL-17 were measured by Western blot and ELISA,retrospectively.Results The levels of PPARγ,IL-17and hCLCA1 mRNA and protein were higher in DMSO/RSV group than those in cell control group (P ＜ 0.05) at three time points.The expression of IL-17,hCLCA1 mRNA and protein were lower both in rosiglitazone/RSV group and anti-IL-17 Ab/RSV group than those in DMSO/RSV group at the same time (P ＜0.05).While,both IL-17,hCLCA1 protein and mRNA expression in GW9662/Rosiglitazone/RSV group were similar to that in DMSO/RSV group (P＜ 0.05).Compared with that at 12h and 24h,rosiglitazone and anti-IL-17 Ab had the strongest inhibition effect onthe expression of IL-17 mRNA and protein at 6h (P ＜ 0.05).Conclusion Rosiglitazone can inhibit RSV-induced expression of IL-17,hCLCA1 and may inhibit airway mucus hypersecretion by increasing the activity of PPARγ.