Compound Astragalus and Salvia miltiorrhiza extract inhibits cell proliferation, invasion and collagen synthesis in keloid fibroblasts by mediating transforming growth factor-β/Smad pathway
Compound Astragalus and Salvia miltiorrhiza extract inhibits cell proliferation, invasion and collagen synthesis in keloid fibroblasts by mediating transforming growth factor-β/Smad pathway
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DOI：
10.1111/j.1365-2133.2011.10674.x
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发表时间：
2012-03-01
影响因子：
10.3
通讯作者：
Zhang, X.
中科院分区：
文献类型：
--
作者：
He, S.;Yang, Y.;Zhang, X.
背景 转化生长因子 (TGF)-β/Smad 通路在疤痕疙瘩的发展中发挥着关键作用。我们之前已经证明复方黄芪丹参提取物（CASE）通过介导TGF-β/Smad通路抑制肝纤维化并降低HepG2细胞的侵袭能力。因此我们推测CASE也可能通过介导TGF-β/Smad信号通路发挥抗瘢痕疙瘩纤维化作用。 目的 探讨CASE对瘢痕疙瘩成纤维细胞增殖、侵袭和胶原合成的影响，探讨CASE对TGF-β/Smad信号通路的影响，阐明其作用机制。 评估了CASE对瘢痕疙瘩成纤维细胞的影响。 MTT法检测细胞增殖情况；利用Transwell侵袭室观察细胞侵袭；通过H-3-脯氨酸掺入测定来测量瘢痕疙瘩成纤维细胞中的胶原合成。分别通过蛋白质印迹和免疫荧光研究了TGF-β1诱导的蛋白质表达及其在瘢痕疙瘩成纤维细胞中的细胞内定位。采用实时逆转录聚合酶链式反应测定纤溶酶原激活剂抑制剂-1（PAI-1）转录活性。结果CASE显着抑制新生牛血清诱导的瘢痕疙瘩成纤维细胞增殖以及TGF-β1诱导的胶原合成和细胞侵袭，而对正常成纤维细胞作用较弱。 CASE处理显着降低了Smad2/3的磷酸化，并且与C末端区域磷酸化（pSmad2C和pSmad3C）相比，CASE对接头区域磷酸化（pSmad2L和pSmad3L）表现出更强的抑制作用。此外，CASE 阻断 Smad2/3/4 复合物的形成及其核转位，但以剂量依赖性方式上调 Smad7 表达。 CASE处理也抑制了PAI-1 mRNA和蛋白水平。结论这些结果表明CASE对瘢痕疙瘩成纤维细胞的细胞增殖、侵袭和胶原合成具有抑制作用，其作用机制可能涉及TGF-β/Smad通路。
Background The transforming growth factor (TGF)-beta/Smad pathway plays a key role in keloid development. We have previously demonstrated that compound Astragalus and Salvia miltiorrhiza extract (CASE) inhibits liver fibrosis and reduces invasion capacity of HepG2 cells by mediating the TGF-beta/Smad pathway. We therefore hypothesize that CASE may also exert antifibrotic effects in keloids by mediating the TGF-beta/Smad pathway.Objectives To investigate the effects of CASE on cell proliferation, invasion and collagen synthesis in keloid fibroblasts, and to explore the effects of CASE on the TGF-beta/Smad signal pathway in order to elucidate its mechanisms of action.Methods The inhibitory effects of CASE on keloid fibroblasts were evaluated. Cell proliferation was studied by MTT assay; cell invasion was observed utilizing Transwell invasion chambers; and collagen synthesis in keloid fibroblasts was measured by H-3-proline incorporation assay. Expression of proteins induced by TGF-beta 1 and their intracellular localization in keloid fibroblasts were investigated by Western blot and immunofluorescence, respectively. Plasminogen activator inhibitor-1 (PAI-1) transcriptional activity was measured by real-time reverse transcription-polymerase chain reaction.Results CASE significantly inhibited cell proliferation induced by newborn bovine serum as well as collagen synthesis and cell invasion induced by TGF-beta 1 in keloid fibroblasts, while it showed weak effects on normal fibroblasts. The phosphorylation of Smad2/3 was markedly reduced by CASE treatment, while CASE exhibited stronger inhibitory effects on linker region phosphorylation (pSmad2L and pSmad3L) compared with effects on C-terminal region phosphorylation (pSmad2C and pSmad3C). In addition, CASE blocked formation of Smad2/3/4 complexes and their nuclear translocation, but upregulated Smad7 expression in a dose-dependent manner. PAI-1 mRNA and protein levels were also suppressed by CASE treatment.Conclusions These results suggest that CASE exhibits inhibitory effects on cell proliferation, invasion and collagen synthesis in keloid fibroblasts, and its mechanisms of action may involve the TGF-beta/Smad pathway.