M3型乙酰胆碱受体对血管平滑肌细胞Ⅰ型胶原蛋白的表达及机制

Effects of m3 muscarinic acetylcholine receptor on collagen type Ⅰ expression and associated mechanism in vascular smooth muscle cells

目的 探讨M3型乙酰胆碱受体（M3R）对血管平滑肌细胞Ⅰ型胶原蛋白（COL-Ⅰ）表达影响及其机制.方法 体外培养A7r5大鼠主动脉平滑肌细胞株,Western blot法测定COL-Ⅰ、细胞外信号调节激酶1/2 （ERK1/2）、磷酸化ERK1/2 （p-ERK1/2）和转化生长因子-β1 （TGF-β1）表达.结果 乙酰胆碱（Ach 1.0×10^-7、1.0×10^-6、1.0×10^-5 mol/L,2h）呈浓度依赖性促进COL-Ⅰ的表达（P＜0.05）,M3R特异性阻断剂4-DAMP抑制Ach（1.0×10^-6mol/L）介导COL-Ⅰ表达（1.302±0.181比1.892±0.292,P＜0.05）;M3R促进p-ERK1/2表达上调（165％,P＜0.05）,ERK1/2特异性抑制剂U0126抑制M3R介导的COL-Ⅰ表达（1.421±0.176比1.912±0.190,P＜0.05）;M3R促进TGF-β1的表达（194％,P＜0.05）,TGF-β1特异性抑制剂SB431542抑制M3R介导的p-ERK1/2表达上调（1.121 ±0.059比1.875±0.078,P＜0.05）.结论 M3R通过激活TGF-β1-ERK1/2信号通路促进COL-Ⅰ的表达。

Objective To investigate the effects of m3 muscarinic acetylcholine receptor （M3R） on collagen type Ⅰ （COL-Ⅰ） expression and its related mechanisms.Methods In cultured vascular smooth muscle cells A7r5,Western blotting was used to examine the protein expression of COL-Ⅰ,extracellular signal regulated kinase 1 and 2 （ERK1/2）,phosphorylated-ERK1/2 （p-ERK1/2） and transforming growth factor-β1 （TGF-β1）.Results M3R agonist acetycholine （1.0 × 10^-9,1.0 x 10^-8,1.0 x 10^-7 mol/L） up-regulated COL-Ⅰ expression in A7r5 （P 〈 0.05）,and the effect could be attenuated by M3R selective antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide （4-DAMP）（1.302±0.181 vs.1.892 ±0.292,P 〈0.05）.Furthermore,the up-regulation of COL-Ⅰ expression by M3R appeared to be mediated by an upregulation in phosphorylation of ERK1/2 （1.764 ± 0.067 vs.1.115± 0.053,P 〈 0.05）.In addition,M3R enhanced the TGF-β1 expression （1.894 ± 0.064 vs.0.972 ± 0.037,P 〈 0.05） and TGF-β1 antagonist SB431542 inhibited M3R-induced p-ERK1/2 up-regulation （1.121 ±0.059 vs.1.875 ± 0.078,P〈0.05）.Conclusion These results suggest that M3R promotes COL-I expression in A7r5 via TGF-β1-ERK1/2 signaling pathway.