平喘方对哮喘模型小鼠气道平滑肌内TLR4mRNA和蛋白及相关炎症因子水平的影响

Effect of Pingchuan Formula on Levels of TLR4mRNA and Protein and Inflammatory Cytokines in Airway Smooth Muscle of Asthmatic Mice

目的：通过研究平喘方对哮喘模型小鼠支气管平滑肌Toll样受体4（Toll-likereceptors,TLRs） mRNA及蛋白和肺泡灌洗液（bronchoalveolar lavage fluid,BA LF）中白介素6（Interleukin 6,IL-6）、白介素10（Interleukin 10,IL-10）、白介素17（Interleukin 17,IL-17）因子水平的影响,进一步探索平喘方治疗哮喘和干预气道炎症的作用机制。方法：对40只BALB/c雄性小鼠用卵蛋白（ovalbumin,OVA）和氢氧化铝腹腔注射致敏并雾化吸入激发,建立小鼠支气管哮喘模型。40只小鼠随机分为空白对照组、哮喘模型组、地塞米松组、平喘方组4组,每组10只。给药期间记录并观察各组小鼠基本情况,给药28天后取材,运用逆转录PCR法（Realtime-PCR）及蛋白质印迹法法（Western Blot）检测哮喘模型小鼠支气管平滑肌TLR4的mRNA和蛋白水平表达,酶联免疫吸附法（Elisa）检测BALF中IL-6、IL-17、IL-10的因子水平。结果：治疗28d后,哮喘模型组TLR4mRNA水平高于明显高于其他各组（P 〈0. 01）;空白组水平与平喘方组mRNA水平无差异（P〉 0. 05）。哮喘模型组TLR4蛋白WB灰度值高于平喘方组（P 〈0. 05）;平喘方组TLR4蛋白WB水平高于地塞米松组（P 〈0. 05）。哮喘模型组IL-6、IL-17因子水平明显高于其他组（P 〈0. 01）;平喘方组IL-6、IL-17因子水平明显高于地塞米松组（P 〈0. 01）。哮喘模型组IL-10因子水平明显低于其他组（P 〈0. 01）;空白组IL-10水平明显高于其他组（P 〈0. 01）;平喘方组IL-10与地塞米松组因子水平无差异（P〉 0. 05）。结论：平喘方可以减少气道平滑肌内TLR4mRNA的表达,以降低TLR4蛋白水平,进而影响肺内炎症因子IL-6、IL-17、IL-10的分泌,缓解哮喘模型小鼠的气道炎症。

Objective： Through the study pof Pingchuan Formula on asthma mice models bronchial smooth muscle Toll like receptor 4（Toll-likereceptors,TLRs） mRNA and protein in bronchoalveolar lavage fluid（bronchoalveolar and lavage fluid,BA LF）,levels of interleukin 6（IL-6）,interleukin 10（IL-10） and interleukin 17（IL-17）,to further explore the mechanism of Pingchuan Formula in the treatment of asthma and airway inflammation intervention. Methods： Forty male BALB/c mice with ovalbumin（ovalbumin,OVA） and aluminum hydroxide by intraperitoneal injection of sensitized and aerosol inhalation were established the model of asthma. Forty mice were randomly divided into control group,asthma model group,dexamethasone group and asthma group,10 rats in each group. During the administration of record and the basic situations of the mice were observed. After administered 28 days,reverse transcription PCR（Realtime-PCR） and protein India Trace method（Western Blot） were used to detect bronchial smooth muscle TLR4 and mRNA protein expression and enzyme linked immunosorbent assay（ELISA） was used to detect levels of IL-6,IL-17 and IL-10 in BALF. Results： After 28 d treatment,the asthma model group＇ s level of TLR4 mRNA was significantly higher than those of the other groups（P〈0.01）. The blank control group and Pingchuan Formula group＇s mRNA levels had no difference（P〈0.05）. The asthma model group＇s TLR4 protein WB gray value was higher than that of Pingchuan Formula group（P〈0.05）. Pingchuan Formula group＇ s TLR4 protein level of WB was higher than that of dexamethasone group（P〈0.05）. The asthma model group＇s IL-6 and IL-17 levels were significantly higher than those of the other groups（P〈0.01）. Pingchuan Formula group＇ s IL-6 and IL-17 levels were significantly higher than that of dexamethasone group（P〈0.01）. The asthma model group＇s IL-10 level was significantly lower than that of the other groups（P〈0.01）. The level of IL-10 group was significantly higher than that of the other groups（P〈0.01）. Pingchuan Formula group and dexamethasone group＇ s IL-10 level had no difference（P〈0.05）. Conclusion： Pingchuan Formula can reduce the expression of TLR4 mRNA in airway smooth muscle in order to reduce the levels of TLR4 protein,thereby affecting the pulmonary inflammatory factors IL-6,IL-17 and IL-10 and alleviating the airway inflammation of asthmatic mice.