清络饮调控TGF-β1/Smads信号传导通路干预大鼠特发性肺纤维化急性加重

Intervention of Qingluoyin in acute exacerbation of idiopathic pulmonary fibrosis rats by regulating the TGF-β1/Smads signal transduction pathway

目的:从TGF-β1/Smads信号传导通路入手,探讨基于络病理论所拟复方清络饮干预特发性肺纤维化急性加重(AE-IPF)的机制。方法:56只雄性Wistar大鼠采用首次经气管注射博来霉素,二次腹腔注射脂多糖方法进行AE-IPF造模,并将大鼠分为阴性对照组(Control组)、缓解期模型组(IPF组)、急性加重期模型组(AE-IPF组)、中药低剂量组(QLY-L组)、中药中剂量组(QLY-M组)、中药高剂量组(QLY-H组)、激素组(PNS组),激素组予醋酸泼尼松(3.125 mg/mL)灌胃,中药组分别予低、中、高剂量清络饮(0.259、0.518、1.036 g/mL)灌胃。运用肺组织活检、动脉血气分析、羟脯氨酸(HYP)测定方法评价大鼠肺氧气交换能力和肺纤维化程度,并运用免疫组化、Western Blot、Quantitative RT-PCR等方法对大鼠肺组织TGF-β1、TGFβRⅠ、TGFβRⅡ、Smad2、Smad4、Smad7蛋白及mRNA表达进行检测,分析清络饮调控TGF-β1/Smads信号传导通路干预大鼠AE-IPF的过程。结果:QLY-L组、QLY-M组、QLY-H组较AE-IPF组大鼠肺组织质地较软、瘀斑瘀点较少,肺组织水肿情况较轻,与PNS组表现基本一致;动脉血气分析显示,QLY-L组、QLY-M组、QLY-H组与PNS组相近,其中QLY-M组、QLY-H组较AE-IPF组显著升高(P<0.05);HYP含量测定结果显示,QLY-M组与PNS组相近,较AE-IPF组显著降低(P<0.01);QLY-M组和QLY-H组在TGF-β1、TGFβRⅠ、Smad2、Smad4、Smad7蛋白和mRNA表达上与AE-IPF组存在显著性差异(P<0.01,P<0.05)。结论:清络饮在AE-IPF的治疗中具有一定疗效,其可通过调控TGF-β1/Smads信号传导通路,抑制AE-IPF的进展,中剂量和高剂量可作为临床应用的参考剂量。

Objective:To be conducted from TGF-β1/Smads signal transduction pathway,to elaborate the mechanism of the intervention of compound Qingluoyin in AE-IPF based on collateral disease theory.Methods:A total of 56 male Wistar rats were injected bleomycin through air tube for the first time and lipopolysaccharide for the second time to make AE-IPF model.The rats were divided into negative control group(Control group),remission model group(IPF group),acute exacerbation model group(AE-IPF group),low dose group(QLY-L group),medium dose group(QLY-M group),high dose group(QLY-H group)and hormone group(PNS group).The hormone group was given Prednisone acetate(3.125 mg/mL)by gavage,and the Chinese medicine group was given low,medium,high doses of Chinese herbal compound Qingluoyin(0.259,0.518,1.036 g/mL).Pulmonary tissue biopsy,arterial blood gas analysis and hydroxyproline determination were used to evaluate the pulmonary oxygen exchange capacity and the degree of pulmonary fibrosis in rats,and immunohistochemistry,Western Blot and quantitative RT-PCR were used to detect TGF-β1,TGFβR I,TGFβRII,Smad2,Smad4,Smad7 in rat lung tissue,to analyze TGF-β1/Smads signal transduction pathway intervenes the process of acute exacerbation of pulmonary fibrosis in rats.Results:The lung tissue texture of rats in the QLY-L,QLY-M,and QLY-H groups was softer,with fewer ecchymosis and ecchymosis compared to those in the AE-IPF groups.The edema of the lung tissue was lighter,which was basically consistent with the performance of the PNS group.Arterial blood gas analysis showed that the QLY-L,QLY-M and QLY-H groups were similar to the PNS group,but increased in the QLY-M and QLY-H group compared to the AE-IPF group(P<0.05).The results of HYP content determination showed that the QLY-M group was similar to the PNS group,but decreased compared to the AE-IPF group(P<0.01).There were significant differences in TGF-β1,TGFβR I,Smad2,Smad4,and Smad7 protein and mRNA expression between the QLY-M group and QLY-H group compared to that in the AE-IPF group(P<0.01,P<0.05).Conclusion:Qingluoyin has a certain curative effect in the treatment of AE-IPF.It can inhibit the progression of AEIPF by regulating TGF-β1/Smads signaling pathway.The medium and high doses can be used as reference doses for clinical application.