平喘方对哮喘小鼠HMGB1/TLR4/NF⁃κB通路及IKKs、TGF-β1的影响

Effects of Pingchuan Recipe on HMGB1/TLR4/NF⁃κB pathway, IKKs and TGF⁃β1 in asthmatic mice

目的研究平喘方对哮喘小鼠高迁移率族蛋白B1(HMGB1)/Toll样受体4(TLR4)/核因子-κB(NF-κB)及气道重塑相关因子IκB激酶家族(IKK_(s))、转化生长因子-β1(TGF-β1)的影响。方法将32只雄性Balb/c小鼠随机分为空白组、模型组、地米组、平喘方组,每组8只。采用卵清白蛋白(OVA)联合氢氧化铝腹腔注射和5%OVA雾化吸入激发的方法建立哮喘模型,当日雾化结束1 h后灌胃给药14 d。各组于最后一次给药后收集小鼠血清、肺泡灌洗液(BALF)及肺组织。采用HE染色法观察小鼠肺组织病理改变,免疫组化染色法观察HMGB1、TLR4、NF-κB表达,ELISA法检测BALF及外周血中HMGB1、TLR4、TGF-β1、IKK_(s)含量,RT-PCR检测肺组织中HMGB1、TLR4、NF⁃κB及IKK_(s) mRNA表达,Western blot法检测肺组织中HMGB1、TLR4蛋白表达。结果与空白组比较,模型组气道重塑严重,HMGB1、TLR4及NF-κB表达明显增强,HMGB1、TLR4、TGF-β1及IKK_(s)含量升高(P<0.05),HMGB1、TLR4、NF⁃κB及IKK_(s) mRNA表达升高(P<0.05),HMGB1、TLR4蛋白表达升高(P<0.05)。与模型组比较,平喘方组气道重塑减轻,HMGB1、TLR4、TGF-β1及IKK_(s)含量均明显降低(P<0.05),HMGB1、TLR4、NF⁃κB及IKK_(s) mRNA表达降低(P<0.05),HMGB1、TLR4蛋白表达减少(P<0.05)。结论平喘方能降低HMGB1、TLR4、NF⁃κB及IKK_(s) mRNA表达,降低IKK_(s)和TGF-β1分泌,减少HMGB1、TLR4蛋白表达,减轻哮喘模型小鼠肺组织炎症,改善肺组织病理改变。

Objective To study the effects of Pingchuan Recipe on high mobility group protein B1/Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB),airway remodeling related factors IκB kinase family(IKK_(s))and transforming growth factor-β1(TGF-β1)in asthmatic mice.Methods Thirty-two male Balb/c mice were randomly divided into 4 groups:blank group,model group,dexamethasone group,and Pingchuan Recipe group,with 8 mice in each group.The asthma model was established by intraperitoneal injection of ovalbumin(OVA)with aluminum hydroxide and aerosolized inhalation of 5%OVA.One hour after atomization on the same day,Pingchuan Recipe was administered by gavage for 14 days.Serum,bronchoalveolar lavage fluid(BALF)and lung tissues were collected from mice in each group after the last administration.HE staining was used to observe the histopathological changes in the lungs of mice.Immunohistochemical staining was used to observe the expression of HMGB1,TLR4 and NF-κB.The content of HMGB1,TLR4,TGF-β1 and IKK_(s) in BALF and peripheral blood was determined by ELISA.The expression of HMGB1,TLR4,NF-κB and IKK_(s) mRNA in lung tissues was detected by RT-PCR.The expression of HMGB1 and TLR4 proteins in lung tissues was detected by Western blot.Results Compared with the condition of the blank group,the model group had severe airway remodeling,increased expression of HMGB1,TLR4 and NF-κB,increased HMGB1,TLR4,TGF-β1 and IKK_(s) secretion(P<0.05),increased HMGB1,TLR4,NF⁃κB and IKK_(s) mRNA expression(P<0.05),and increased HMGB1 and TLR4 protein expression(P<0.05).Compared with the condition of the model group,Pingchuan Recipe group had alleviated airway remodeling,significantly lower HMGB1,TLR4,TGF-β1 and IKK_(s) secretion(P<0.05),reduced HMGB1,TLR4,NF⁃κB and IKK_(s) mRNA expression(P<0.05),and decreased HMGB1 and TLR4 protein expression(P<0.05).Conclusion Pingchuan Recipe can reduce HMGB1,TLR4,NF-κB and IKK_(s) mRNA expression,decrease IKK_(s) and TGF-β1 secretion,lower HMGB1 and TLR4 protein expression,alleviate the inflammation of lung tissues,and improve the pathological changes of lung tissues in asthmatic model mice.