祛风扶阳汤对支气管哮喘大鼠TGF-β_(1)/Smad信号通路与Th17/Treg失衡的影响

Effect of decoction for dispelling wind and warming Yang on TGF-β_(1)/Smad signaling pathwayand Th17/Treg imbalance in asthmatic rats

目的观察祛风扶阳汤对支气管哮喘大鼠气道炎症、气道重塑、转化生长因子-β_(1)(TGF-β_(1))/Smad信号通路及辅助性T细胞17(Th17)/调节性T细胞(Treg)失衡的影响,探究其作用及机制。方法将45只SD大鼠随机分为空白组、哮喘组、地塞米松组、祛风扶阳汤中剂量组、祛风扶阳汤高剂量组,每组9只。除空白组外,其余组大鼠均采用卵蛋白腹腔注射和雾化吸入方法建立支气管哮喘模型。建模成功后,地塞米松组给予地塞米松0.001 g/kg灌胃,祛风扶阳汤中、高剂量组分别给予祛风扶阳汤9.95 g/kg和19.9 g/kg灌胃,空白组和哮喘组灌胃等量生理盐水,均1次/d,连续灌胃4周。记录各组大鼠末次灌胃后咳嗽次数,HE染色观察肺组织病理形态,ELISA法检测肺泡灌洗液中白细胞介素-6(IL-6)、白细胞介素-10(IL-10)、白细胞介素-17A(IL-17A)、TGF-β_(1)水平,Western blot法检测肺组织中TGF-β_(1)、α-平滑肌肌动蛋白(α-SMA)、Smad2/3、p-Smad2/3蛋白表达情况,流式细胞仪检测肺组织中IL-17A^(+)CD4^(+)、CD4^(+)CD25^(+)Foxp3^(+)Treg比例。结果与空白组比较,哮喘组大鼠咳嗽次数明显增加(P<0.05),肺泡灌洗液中IL-6、IL-17A、TGF-β_(1)水平和肺组织中TGF-β_(1)、α-SMA、p-Smad2/3蛋白相对表达量及IL-17A^(+)CD4^(+)比例均明显升高(P均<0.05),肺泡灌洗液中IL-10水平和肺组织中CD4^(+)CD25^(+)Foxp3^(+)Treg比例均明显降低(P均<0.05);肺组织胶原纤维沉积、炎性细胞浸润明显。与哮喘组比较,各药物组大鼠的咳嗽次数均明显减少(P均<0.05),地塞米松组和祛风扶阳汤高剂量组肺泡灌洗液中IL-6、IL-17A、TGF-β_(1)水平和肺组织中TGF-β_(1)、α-SMA、p-Smad2/3蛋白相对表达量及IL-17A^(+)CD4^(+)比例均明显降低(P均<0.05),地塞米松组和祛风扶阳汤高剂量组肺泡灌洗液中IL-10水平和祛风扶阳汤高剂量组肺组织中CD4^(+)CD25^(+)Foxp3^(+)Treg比例均明显升高(P均<0.05);各药物组肺组织损伤、胶原纤维沉积、炎性细胞浸润均减轻。结论祛风扶阳汤可以通过调节TGF-β_(1)/Smad信号通路以及Th17/Treg细胞平衡,从而减轻支气管哮喘大鼠气道炎症反应与气道重塑。

Objective It is to observe the effects of decoction for dispelling wind and warming Yang on airway inflammation and remodeling,and transforming growth factor-β_(1)(TGF-β_(1))/Smad signaling pathway and Th17/Treg imbalance,and to explore its action mechanism.Methods Forty SD rats were randomly divided into blank group,asthma group,dexamethasone group,medium dose and high dose groups of decoction for dispelling wind and warming Yang,with 9 rats in each group.The rats of all group except for the blankd group were used to establish models of bronchial asthma by intraperitoneal injection of and nebulized inhalation of ovalbumin.After successful modeling,the dexamethasone group was given dexamethasone 0.001 g/kg by gavage,the medium dose and high dose groups of decoction for dispelling wind and warming Yang were given decoction for dispelling wind and warming Yang 9.95 g/kg and 19.9 g/kg by gavage,respectively,and the blank group and asthma group were given the same amount of saline by gavage,all once daily,continuously gavaged for 4 weeks.The time of cough of rats in each group after the last gavage was recorded,the pathological morphology of lung tissue was observed by HE staining,the levels of interleukin-6(IL-6),interleukin-10(IL-10),interleukin-17A(IL-17A)and TGF-β_(1)in alveolar lavage fluid were detected by ELISA,the protein expressions of TGF-β_(1),α-smooth muscle actin(α-SMA),Smad2/3,p-Smad2/3 in lung tissue were detected by Western blot method,and the ratio of IL-17A^(+)CD4^(+),CD4^(+)CD25^(+)Foxp3^(+)Treg in lung tissue were detected by flow cytometry.Results Compared with the blank group,the time of cough of rats in the asthma group were significantly increased(P<0.05),the levels of IL-6,IL-17A,TGF-β_(1)in alveolar lavage fluid and the relative protein expressions of TGF-β_(1),α-SMA,p-Smad2/3 in lung tissue and the ratio of IL-17A^(+)CD4^(+)were significantly increased(all P<0.05),and the level of IL-10 in alveolar lavage fluid and the ratio of CD4^(+)CD25^(+)Foxp3^(+)Treg were significantly decreased(all P<0.05);collagen fiber deposition and inflammatory cell infiltration were obvious in the alveolar lavage fluid.Compared with the asthma group,the times of cough of rats in each treatment group were significantly reduced(all P<0.05),the levels of IL-6,IL-17A,TGF-β_(1)in alveolar lavage fluid and the relative protein expressions of TGF-β_(1),α-SMA,p-Smad2/3 and the ratio of IL-17A^(+)CD4^(+)in lung tissues of rats in the dexamethasone group and high-dose group of decoction for dispelling wind and warming Yang were all significantly reduced(all P<0.05),the levels of IL-10 in alveolar lavage fluid in the dexamethasone group and high dose group of decoction for dispelling wind and warming Yang and the ratio of CD4^(+)CD25^(+)Foxp3^(+)Treg in lung tissues of rats in the high-dose group were significantly increased(all P<0.05);the lung tissue injury,collagen fiber deposition,and inflammatory cell infiltration were alleviated in each treatment group.Conclusion Decoction for dispelling wind and warming Yang can attenuate airway inflammatory response and airway remodeling in rats with bronchial asthma via regulating the TGF-β_(1)/Smad signaling pathway as well as Th17/Treg cell balance.