天龙喘咳灵对转化生长因子-β_1诱导的气道α-平滑肌肌动蛋白的影响及信号转导机制

The role of Tianlongchuankeling in modulation of the α-smooth muscle actin induced by rhTGF-β_1 and the involved signal transduction

目的观察中药天龙喘咳灵对转化生长因子-β1(TGF-β1)诱导的气道上皮下成纤维细胞(HPBFs)α-平滑肌肌动蛋白(α-SMA)表达的影响,探讨其可能的信号转导机制。方法2005—2006年,在广州市红十字会医院呼吸科于原代培养的HPBFs中,分别加入天龙喘咳灵合剂、天龙喘咳灵主要组分(淡附子、青天葵和法夏)的单味药预孵育2h,再加入TGF-β1(10μg/L)共孵育;刺激10min和30min,分别提取细胞总蛋白,Western免疫印迹方法检测磷酸化p38MAPK、ERK1/2和smad2的蛋白表达;刺激20h和72h,提取细胞总RNA及蛋白,检测α-SMA蛋白及mRNA的表达。结果天龙喘咳灵组和淡附子组α-SMA蛋白和mRNA的表达均较单纯TGF-β1刺激组减少(P<0·05),天龙喘咳灵组对α-SMA蛋白和mRNA表达量的抑制程度高于淡附子组;天龙喘咳灵组和淡附子组磷酸化ERK1/2的表达明显低于单纯TGF-β1刺激组,而青天葵和法夏两组与单纯TGF-β1组相似。各药物处理组对TGF-β1诱导的磷酸化p38和smad2的表达无明显影响。结论天龙喘咳灵抑制HPBFs的α-SMA表达,可能是通过降低TGF-β1诱导的ERK1/2磷酸化水平实现的,并且其中的组分淡附子可能起主要作用。

Objective To investigate the role of Chinese traditional medicine-Tianlongchuankeling in modulation of the transdifferentiation of sub-epithelial fibroblasts into myofibroblasts induced by rhTGF-β1 ,and the involved mechanism of the signal transduction. Methods Human primary bronchial sub-epithelial fibroblasts （HPBFs） were incubated with either Tianlongchuankeling or its main components of Qingtiankui, Danfuzi and Faxia respectively before being treated with rhTGF-β1 （10 μg/L）for different time. Total protein of cells were collected at different time[apse（ 10min,30min respectively）. Phosphorylated MAPKs for p38, ERK1/2, phosphorylated smad2 and the expression of α-SMA were detected by using Western blotting. Total RNA was extracted at 20 h and 72 h and subjected to RT-PCR for evaluation of the expression of α-SMA gene. Results The induction of α-SMA in HPBFs by TGF-β1 was down-regulated significantly in the cells treated with Tianlongchuankeling or Danfuzi. The phosphorylation of ERK1/2 under TGF-β1 stimulation was inhibited significantly in Tianlongchuankeling and Danfuzi group. No difference of the level in phosphorylation of p-38 and smad2 was found among groups. Conclusion Tianlongchuankeling has the capability to suppress the induction of α-SMA by TGF-β1 through down-regulation of the phosphorylation of ERK1/2 ,thereby preventing the transdifferentiation of sub-epithelial fibroblasts ＇into myofibroblasts, which may be responsible for the treatment of airway hyperresponsiveness in asthma. Danfuzi, one of main components of Tianlongchuankeling, may make a major contribution to this process.