miR-204-5p通过调节TLR2的表达减轻哮喘小鼠的炎症反应和气道重塑

miR-204-5p alleviates inflammation and airway remodeling in asthmatic mice by regulating the expression of TLR2

目的:探讨miR-204-5p对哮喘小鼠的炎症反应和气道重塑的影响及其可能的分子机制。方法:40只7周龄的SPF级昆明雄性小鼠,挑选10只作为对照组,其余30只小鼠采用卵白蛋白(OVA)致敏,激发建立哮喘模型并随机分为哮喘组、表达上调组、表达下调组,每组10只。表达上调组小鼠尾静脉注射miR-204-5p激动剂干预,表达下调组尾静脉注射miR-204-5p拮抗剂干预。对照组及哮喘组小鼠以同种方式注射等量0.9%生理盐水。HE染色观察小鼠肺组织的病理变化,RT-qPCR检测各组小鼠miR-204-5p及TLR2 mRNA的表达水平,Western blot检测TLR2、肿瘤转换生长因子β1(TGF-β1)及NF-κB p65蛋白表达水平,ELISA法检测各组小鼠支气管肺泡灌洗液(BALF)中侧支气管中白细胞介素4(IL-4)、IL-5、IL-13等细胞炎症因子浓度。结果:对照组小鼠肺组织及支气管结构正常,肺泡轮廓清晰,哮喘组、上调组、下调组小鼠均出现气道壁增厚,细胞排列杂乱,炎性细胞浸润。与对照组相比,哮喘组小鼠miR-204-5p mRNA表达水平下降、TLR2 mRNA表达上升(P<0.05),哮喘组、表达上调组、表达下调组小鼠TLR2、TGF-β1、NF-κB p65、IL-4、IL-5、IL-13等细胞炎症因子的蛋白表达水平均上升(P<0.05);与哮喘组相比,表达上调组的miR-204-5p mRNA表达水平上升、TLR2 mRNA下降(P<0.05);TLR2、TGF-β1、NF-κB p65、IL-4、IL-5、IL-13等细胞炎症因子的蛋白表达水平均下降(P<0.05),表达下调组miR-204-5p mRNA表达水平下降、TLR2 mRNA表达上升(P<0.05),TLR2、TGF-β1、NF-κB p65、IL-4、IL-5、IL-13等细胞炎症因子的蛋白表达水平均上升(P<0.05)。结论:miR-204-5p可能通过调节TLR2的表达抑制哮喘小鼠由TGF-β1及NF-κB p65诱导的气道重塑,有效减轻小鼠的炎症反应,可作为预防哮喘气道重塑的潜在治疗靶点。

Objective:To investigate the effect of miR-204-5p on the inflammatory response and airway remodeling in asthmatic mice,and to study its potential molecular mechanism.Methods:40 seven-week-old SPF kunming white male mice were selected,and 10 mice were randomly selected as the control group.The other 30 mice were sensitized and stimulated by ovalbumin(OVA)to establish asthma model,and randomly divided into asthma group,up-regulated group and down-regulated group,with 10 mice in each group.miR-204-5p agonist was injected into the tail vein of the up-regulated group,and miR-204-5p antagonist was injected into the tail vein of the down-regulated group.Mice in control group and asthma group were injected with 0.9%normal saline in the same way.He staining was used to observe the pathological changes of lung tissues of mice.The expression levels of miR-204-5P and TLR2 mRNA were detected by RT-QPCR.The expression levels of TLR2,TGF-β1 and NF-κB P65 protein were detected by Western blot.The concentrations of IL-4,IL-5,IL-13 and other inflammatory cytokines in the middle bronchus of bronchoalveolar lavage fluid(BALF)were detected by ELISA.Results:The lung tissue and bronchial structure of mice in the control group were normal,and the alveolar contour was clear.The airway wall of mice in asthma group,up-regulation group and down-regulation group were thickened,and the cells were disorderly and inflammatory cells were infiltrated.Compared with the control group,the expression level of miR-204-5p mRNA in asthmatic mice decreased(P<0.05),the expression levels of TLR2 mRNA increased,the protein expression levels of inflammatory cytokines such as TLR2,TGF-β1 and NF-κB P65 protein,IL-4,IL-5 and IL-13 in asthma group,up-regulated group and down-regulated group were significantly increased(P<0.05).Compared with asthma group,miR-204-5p mRNA expression level was significantly increased in up-regulated group(P<0.05),the expression levels of TLR2 mRNA decreased,the protein expression levels of inflammatory cytokines such as TLR2,TGF-β1,NF-κB P65 protein,IL-4,IL-5,and IL-13 were significantly decreased(P<0.05).miR-204-5p mRNA expression level was significantly decreased in down-regulated group(P<0.05),the expression levels of TLR2 mRNA increased,the protein expression levels of inflammatory cytokines such as TLR2,TGF-β1,NF-κB P65 protein,IL-4,IL-5 and IL-13 were significantly increased(P<0.05).Conclusion:miR-204-5p may inhibit airway remodeling induced by TGF-β1 and NF-κB P65 in asthmatic mice by regulating the expression of TLR2,and effectively reduce the inflammatory response in mice,which may be a potential therapeutic target for preventing airway remodeling in asthma.