MAPK信号通路在转化生长因子β1诱导心肌成纤维细胞趋化运动中的作用

Roles of MAPK signaling pathway on chemotaxis of cardiac fibroblasts induced by transforming growth factor-β

摘要:

目的 探讨丝裂原活化蛋白激酶(MAPK)信号转导通路在转化生长因子β1(TGF-β1)诱导心肌成纤维细胞趋化运动中可能的作用机制。方法 培养新生SD大鼠的心肌成纤维细胞,将细胞随机分为空白对照组、TGF-β1组、c-Jun氨基末端激酶(JNK)抑制因子(SP600125,10 μmol/L)组、P38MAPK抑制因子(SB203580,10 μmol/L)组、细胞外信号调节蛋白激酶(ERK)抑制因子(U0126,10 μmol/L)组,除空白对照组外,其余组均给与10 μg/L TGF-β1及对应抑制因子处理。MTT比色法测定心肌成纤维细胞增殖活性,Transwell小室检测心肌成纤维细胞运动能力,羟脯氨酸试剂盒检测胶原含量,酶联免疫吸附法(ELISA)检测心肌成纤维细胞中单核细胞趋化蛋白1(MCP-1)、纤溶酶原激活物抑制剂1(PAI-1)水平,Western blot检测成纤维细胞中α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原(Col-1)、基质金属蛋白酶9(MMP-9)表达水平。结果 TGF-β1可明显促进心肌成纤维细胞增殖活性、趋化运动能力及胶原含量,MAPK信号途径抑制因子干预后能够抑制这种增殖及趋化运动,并降低胶原含量(P＜0.05)；ELISA结果显示,MAPK信号途径抑制因子能够显著降低TGF-β1处理后导致的MCP-1、PAI-1水平升高(P＜0.05)；Western blot结果显示,MAPK信号途径抑制因子能够显著降低TGF-β1处理后导致的α-SMA、Col-1、MMP-9蛋白表达升高(P＜0.05)。结论 TGF-β可能通过激活MAPK信号通路促进心肌成纤维细胞的趋化运动,通过使用MAPK抑制剂能够一定程度的抑制心肌纤维化。

Abstract:

Aim To explore possible action mechanism of mitogen-activated protein kinase (MAPK) signal transduction pathway on chemotaxis of cardiac fibroblasts (CF) induced by transforming growth factor-β1 (TGF-β1). Methods The CF of neonatal SD rats were cultured. Cell were randomly divided into blank control group, TGF-β1 group, c-Jun N-terminal kinase (JNK) inhibitor (SP600125 10 μmol/L) group, P38MAPK inhibitor (SB203580 10 μmol/L) group and extracellular signal-regulated protein kinase (ERK) inhibitor (U0126 10 μmol/L) group. Except blank control group, the other groups were given 10 μg/L TGF-β1 and corresponding inhibitors. Methyl thiazolyl tetrazolium (MTT) colorimetric assay was applied to detect cell viability of CF. Transwell chamber was applied to detect motor ability of CF. The collagen content was detected by hydroxyproline kit. The enzyme-linked immunosorbent assay (ELISA) was applied to detect levels of monocyte chemotactic protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1) in CF. Western blot was applied to detect expression levels of α-smooth muscle actin (α-SMA), type Ⅰ collagen (Col-1) and matrix metalloproteinase-9 (MMP-9) in CF. Results TGF-β1 could significantly promote cell viability and chemotaxis of CF and collagen content. MAPK inhibitors could inhibit the proliferation and chemotaxis, and decrease collagen content (P＜0.05). ELISA results showed that MAPK signaling pathway inhibitors could significantly decrease increased MCP-1 and PAI-1 induced by TGF-β1 treatment (P＜0.05). Western blot results showed that MAPK signaling pathway inhibitors could significantly decrease increased expression levels of α-SMA, Col-1 and MMP-9 protein induced by TGF-β1 treatment (P＜0.05). Conclusion TGF-β1 may promote chemotaxis of CF by activating MAPK signaling pathway. The application of MAPK inhibitors can inhibit myocardial fibrosis to certain extent.