Ac-SDKP经由Epac信号抑制矽肺肌成纤维细胞分化的作用及机制
Inhibition Effect and Related Mechanism of Ac-SDKP on Myofibroblast Differentiation via Epac Signalin Lung Silicosis
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摘要:
目的 观察N-乙酰基-丝氨酰-天门冬氨酰-赖氨酰-脯氨酸(N-acetyl-seryl-aspartly-lysyl-proline,Ac-SDKP)能否通过对环腺苷酸活化的交换蛋白(exchange protein activated by cAMP,Epac)信号的活化,抑制矽肺大鼠及促血管生成素Ⅱ(Ang Ⅱ)诱导的肌成纤维细胞分化。 方法 构建矽肺大鼠模型,分为对照组(4、8周组),矽肺模型组(4、8周组),Ac-SDKP抗纤维化治疗组及Ac-SDKP预防治疗组;采用Ang Ⅱ诱导人胚肺成纤维细胞MRC-5向肌成纤维细胞分化,并予以Ac-SDKP和Epac特异性活化剂8-Me-cAMP(8-pCPT-2'-O-Me-cAMP)预处理。HE及Masson染色观察肺组织病理形态。免疫组织(细胞)化学染色法观察α-平滑肌肌动蛋白(α-smooth mucle actin,α-SMA)的定位,Western blot法检测I型胶原、α-SMA和Epac蛋白的表达。 结果 在矽肺模型组中,出现明显矽结节,结节内可见α-SMA标记的肌成纤维细胞阳性表达;Western blot法检测矽肺组织中I型胶原、α-SMA表达上调,而Epac1表达下调;给予Ac-SDKP抗纤维化治疗或预防治疗能够抑制该变化。给予Ang Ⅱ诱导,可见MRC-5细胞胞浆内出现明显的α-SMA阳性显色;Westernblot法中可见α-SMA和I型胶原蛋白表达上调;而予以8-Me-cAMP或Ac-SDKP预处理,能够上调Epac1,抑制Ang Ⅱ诱导的α-SMA和I型胶原蛋白表达的上调。 结论 Ac-SDKP能够通过对Epac信号的活化,在体内外抑制矽肺大鼠肌成纤维细胞分化和胶原沉积,从而发挥抗矽肺纤维化的作用。 Abstract:Objective To explore the inhibition effect of N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) on myofibroblast differentiation via Epac (exchange protein directly activated by cAMP) signal in silicotic rat lung fibrosis and in MRC-5 human fetal lung fibroblasts induced by angiotensin (Ang) Ⅱ. Methods SiO2 powders were douched in the trachea of rat to establish the silicosis model (four-week and eight-week silicosis groups) and Ac-SDKP were administered in post-(Ac-SDKP intervention group) or pre-manner (Ac-SDKP treatment group).MRC-5 human fetal lung fibroblasts were induced to myofibroblast by Ang Ⅱ,and pre-treated with Ac-SDKP and 8-Me-cAMP (8-pCPT-2'-O-Me-cAMP).The pathological morphologic evidence was observed by HE and Masson staining.The expression of α-smooth muscle actin (α-SMA) was located by immunohistochemistry.The protein expressions of collagen type I (Col I),α-SMA,and Epac were measure by Western blot. Results Myofibroblast differentiation indicated by α-SMA positivewas observed in silicosis nodules in the silicosis groups.Up-regulated expression of Col I and α-SMA and down-regulated expressionof Epac1 protein were also detected by Western blot assay.Ac-SDKP intervention or treatment showed an anti-fibrotic effect in vivo and strongly attenuated the above induced changes.The positive stain of α-SMA was observed in MRC-5 cells induced by Ang Ⅱ and accompanied with the up-regulation of Col I and α-SMA.Pre-treatment with 8-Me-cAMP or Ac-SDKP promoted expression of Epac1 and attenuated Ang Ⅱ induced up-regulation of Col I and α-SMA. Conclusion Ac-SDKP could inhibit the myofibroblast differentiation and collagen deposition via Epac signal activation in silicotic rat lung fibrosis and in MRC-5 cells induced by Ang Ⅱgroup
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