α7烟碱型乙酰胆碱受体激动剂对TGF-β1介导的血管外膜成纤维细胞表型转化影响的体外研究

Effect of α7 nicotinic acetylcholine receptor activation on transforming growth factor β1-induced phenotypic transformation of adventitia fibroblasts studied in vitro

目的 :探讨α7烟碱型乙酰胆碱受体(α7 nicotinic acetylcholine receptor,α7 nAchR)与血管外膜成纤维细胞(adventitial fibroblasts, AF)表型转化间的关系。方法:选取雄性Sprague-Dawley(SD)大鼠,用组织切块法培养其胸主动脉外膜AF。为评估转化生长因子β1(transforming growth factor-β1, TGF-β1)刺激AF表型转化的作用,将体外培养的AF分为空白对照组A、TGF-β1处理24 h组和TGF-β1处理48 h组3组,处理结束后分别应用实时定量PCR和免疫印迹试验,检测相α7 nAchR基因和蛋白的表达以及α-肌动蛋白(αsmooth muscle actin,α-SMA)和Ⅰ型胶原的表达情况。为进一步探讨α7 nAchR的激活能否抑制TGF-β1刺激表型转化及其分子机制,另将体外培养的AF分为空白对照组B、TGF-β1处理48 h组及TGF-β1处理48 h合并α7 n AchR激活剂PNU-282987处理组3组,处理48 h后用免疫印迹法检测α-SMA和Ⅰ型胶原的表达及细胞外调节蛋白激酶1/2 (extracellular regulated protein kinases1/2, Erk1/2)磷酸化情况。结果 :与空白对照A相比,TGF-β1可使α7 n AchR的基因和蛋白表达明显降低(P<0.05),同时TGF-β1可使α-SMA和Ⅰ型胶原的表达明显增加(P<0.05)。α7 nAchR激动剂PNU-282987可抑制TGF-β1刺激的AF中α-SMA和Ⅰ型胶原的表达,亦可抑制TGF-β1刺激的Erk1/2磷酸化。结论:α7 nAchR的抑制参与TGF-β1介导的血管AF表型转化,其机制可能与Erk1/2磷酸化有关。

Objective: To investigate the correlation between α7 nicotinic acetylcholine receptor(α7 nAchR) and phenotypic transformation of adventitial fibroblasts(AF). Methods: AF were isolated and cultured by using thoracic aorta of male Sprague-Dawley(SD) rats. For evaluating the effect of transforming growth factor-β1(TGF-β1) on phenotypic transformation of AF, cultured AF were divided into three groups: blank control group A, group with 24 h TGF-β1 treatment, and group with 48 h TGF-β1 treatment. After treatment, real-time quantitative PCR and western blotting were used to detect the mRNA level and protein level of α7 nAchR, and the expression of a smooth muscle actin(α-SMA) and collagen I. To further explore whether the activation of α7 nAchR can inhibit TGF-β1-induced phenotypic transformation and the underlying molecular mechanism, AF cultured in vitro were divided into three groups: blank control group B,group with 48 h TGF-β1 treatment, and group with 48 h TGF-β1 +PNU-282987(an α7 nAchR activator) treatment. After48 h treatment, the expression of α-SMA and collagen I and the phosphorylation of extracellular regulated protein kinase1/2(Erk1/2) were detected by immunoblotting. Results: Compared with the blank control group A, TGF-β1 significantly decreased the mRNA level and protein level of α7 n AchR(P<0.05), whereas TGF-β1 induced significant increase of aSMA and collagen I(P<0.05). PNU-282987, an α7 nAchR agonist, inhibited TGF-β1-induced expression of a-SMA and collagen type I. Meanwhile, PNU-282987 also significantly inhibited TGF-β1-induced Erk1/2 phosphorylation.Conclusions: The inhibition of α7 n AchR is involved in TGF-β1-induced phenotypic transformation in AF, and the mechanism may be related to Erk1/2 phosphorylation.