基于TGF-β_(1)/Smad3信号通路探讨益气温阳护卫汤防治支气管哮喘的作用机制

Yiqi Wenyang Huwei Decoction in Treatment of Bronchial Asthma in Rats by Regulating TGF-β_(1)/Smad3 Signaling Pathway

目的:探讨益气温阳护卫汤(YWHD)调控转化生长因子(TGF)-β_(1)/Smad同源物3(Smad3)信号通路对支气管哮喘大鼠气道炎症的作用及机制。方法:60只雄性SD大鼠随机均分为正常组、模型组、地塞米松组及YWHD低、中、高剂量组,每组10只。支气管哮喘大鼠模型采用腹腔注射卵清蛋白(OVA)氢氧化铝混悬液1 mL致敏,2%OVA雾化吸入激发。每天雾化OVA前1 h,正常组给予生理盐水,地塞米松组给予3 mg·kg^(-1)药液,益气温阳护卫汤低、中、高剂量组分别以3、6、12 g·kg^(-1)灌服。观察大鼠一般情况及肺功能;收集大鼠肺泡灌洗液(BALF)和血清,计数肺泡灌洗液中炎性细胞,酶联免疫吸附测定法(ELISA)检测血清中免疫球蛋白E(IgE)和BALF中炎性因子水平;苏木素-伊红(HE)、马松(Masson)和碘酸雪夫(PAS)染色分别观察肺组织病理形态学变化、胶原沉积和气道黏液分泌;实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白免疫印迹法(Western blot)检测肺组织中TGF-β_(1)/Smad3相关mRNA和蛋白水平。结果:与正常组比较,模型组大鼠吸气总气道阻力(RL)升高而动态顺应性(Cdyn)明显降低(P<0.05,P<0.01);血清IgE水平、肺泡灌洗液中炎性细胞计数及其炎症因子水平显著增加(P<0.01);肺组织有大量炎性细胞浸润、胶原沉积和黏液分泌明显增多;肺组织TGF-β_(1)、α-平滑肌肌动蛋白(α-SMA)及Smad3磷酸化的表达水平显著增加(P<0.01)。与模型组比较,地塞米松组和益气温阳护卫汤高剂量组显著降低哮喘大鼠的气道阻力(P<0.01),改善其肺动态顺应性(P<0.05);血清IgE水平、肺泡灌洗液中炎性细胞计数和炎症因子水平显著降低(P<0.05);肺组织病理损伤得到改善;肺组织TGF-β_(1)、α-SMA及Smad3磷酸化的表达降低(P<0.01)。结论:益气温阳护卫汤降低支气管哮喘大鼠气道炎症,改善肺组织病理损伤及气道重塑,可能与TGF-β_(1)、α-SMA蛋白及Smad3磷酸化水平下调有关。

Objective:To investigate the effect and mechanism of Yiqi Wenyang Huwei decoction(YWHD)on airway inflammation in bronchial asthma(BA)rats based on transforming growth factor-β_(1)(TGF-β_(1))/SMAD family member 3(Smad3)signaling pathway.Method:Sixty male SD rats were randomly divided into a normal group,a model group,a dexamethasone(DEX)group,and low-,medium-,and high-dose YWHD groups,with 10 rats in each group.The BA model was induced by intraperitoneal injection of 1 mL of ovalbumin(OVA)-aluminum hydroxide suspension for sensitization,followed by nebulization with 2%OVA.One hour before daily OVA nebulization,the control group was treated with saline,the DEX group with DEX solution at 0.2 g·L^(-1),and the low-,medium-,and high-dose YWHD groups with YWHD at 1,2,4 g·mL^(-1),respectively.General conditions and lung function were observed.Bronchoalveolar lavage fluid(BALF)and serum were collected to count inflammatory cells in BALF and measure immunoglobulin E(IgE)levels in serum and inflammatory cytokines in BALF using enzyme-linked immunosorbent assay(ELISA).Pathological changes in lung tissues,collagen deposition,and airway mucus secretion were observed by hematoxylin-eosin(HE),Masson,and periodic acid-Schiff(PAS)staining.TGF-β_(1)/Smad3-related mRNA and protein levels in lung tissues were determined by Real-time fluorescent quantitative polymerase chain translation(Real-time PCR)and Western blot analysis.Result:Compared with the normal group,the model group showed increased total airway resistance(RL)and decreased dynamic compliance(Cdyn)(P<0.05,P<0.01),elevated serum IgE levels,increased inflammatory cell counts,and higher inflammatory cytokine levels in BALF(P<0.01).Additionally,there was significant inflammatory cell infiltration,collagen deposition,and mucus secretion in lung tissues.The levels of TGF-β_(1),α-smooth muscle actin(α-SMA),and Smad3 phosphorylation in lung tissues were significantly increased(P<0.01).Compared with the model group,the DEX group and high-dose YWHD group exhibited significantly reduced RL(P<0.01),improved lung dynamic compliance(P<0.05),and lower serum IgE levels,inflammatory cell counts,and inflammatory cytokine levels in BALF(P<0.05).Moreover,these treatments alleviated pathological damage in lung tissues and reduced the levels of TGF-β_(1),α-SMA,and Smad3 phosphorylation(P<0.01).Conclusion:YWHD reduces airway inflammation,improves pathological damage,and mitigates airway remodeling in bronchial asthma rats,possibly by downregulating TGF-β_(1),α-SMA protein levels,and Smad3 phosphorylation.