地塞米松对兔胆道成纤维细胞P311/TGF-β1/α-SMA通路表达的影响

Effect of dexamethasone on P311/TGF-β1/α-SMA signaling pathway in rabbit biliary fibroblasts

摘要:

目的：研究地塞米松(dexamethasone，DEX）对兔良性胆道狭窄（benign biliary stricture，BBS）模型胆管成纤维细胞（model fibroblast，MF）中P311/转化生长因子－β1（transforming growth factor-β1，TGF-β1）/α-平滑肌动蛋白（α-smooth muscle actin，α-SMA）通路表达的影响。方法：取2只家兔正常胆管培养正常胆管成纤维细胞（normal bile duct fibroblasts，NF），对2只家兔采用切开再吻合方法建立BBS模型并获取MF，进行细胞鉴定后分组为：NF、MF、MF+不同浓度的DEX（0.02，0.1及0.5 mg/mL）组，各组给予对应浓度的DEX干预48 h，CCK-8细胞计数法测定各组细胞增殖水平；qRT-PCR检测各组细胞P311、TGF-β1及α-SMA 基因mRNA表达水平；Western blot检测各组细胞TGF-β1及α-SMA蛋白表达水平。结果：①各组细胞A值：NF为0.331±0.002，MF组为0.533±0.005，MF+DEX 0.02组为0.487±0.003，MF+DEX 0.1组为0.434±0.004，MF+DEX 0.5组为0.381±0.004。②各组细胞P311 mRNA值：NF组为1.000 0±0.024 8，MF组为4.146 3±0.037 1，MF+DEX 0.02组为3.789 9±0.056 8，MF+DEX 0.1组为3.566 1±0.148 1，MF+DEX 0.5组为2.945 8±0.165 4。③各组细胞TGF-β1 mRNA值NF组为1.000 0±0.034 6，MF组为2.647 9±0.048 5，MF+DEX 0.02组为1.929 7±0.054 8，MF+DEX 0.1组为1.678 2±0.080 2，MF+DEX 0.5组为1.676 2±0.036 9。④各组细胞α-SMA mRNA值：NF组为1.000 0±0.056 0，MF组为4.025 7±0.065 9，MF+DEX 0.02组为3.644 9±0.196 4，MF+DEX 0.1组为2.712 9±0.102 4，MF+DEX 0.5组为2.320 7±0.031 2。⑤各组细胞TGF-β1蛋白值：NF组为0.999 6±0.046 3，MF组为2.096 3±0.029 3，MF+DEX 0.02组为1.781 8±0.040 4，MF+DEX 0.1组为1.531 4±0.032 5，MF+DEX 0.5组为1.384 0±0.035 7。⑥各组细胞α-SMA蛋白值：NF组为1.000 8±0.051 4，MF组为3.231 4±0.116 0，MF+DEX 0.02组为2.875 3±0.078 0，MF+DEX 0.1组为2.262 8±0.059 8，MF+DEX 0.5组为1.940 8±0.093 7。在DEX干预48 h后，MF增殖明显受到抑制，细胞P311、TGF-β1及α-SMA基因mRNA及蛋白表达明显下调（均P<0.05，0.1～0.5 mg/mL），呈现浓度依耐性。结论： DEX抑制BBS形成的作用机制之一可能与它对MF中P311/TGF-β1/α-SMA通路的调控有关。

Abstract:

Objective：To investigate effects of dexamethasone（DEX） on P311/transforming growth factor-β1（TGF-β1）/α-smooth muscle actin（α-SMA） signaling pathway in benign biliary stricture（BBS） model fibroblast（MF）. Methods：Two normal rabbits were treated with normal bile duct fibroblasts（NF），BBS model was established via discission and anastomosis of two rabbits and MF was acquired；then they were divided into five groups：NF，MF，MF plus different concentration of DEX（0.02，0.1 and 0.5 mg/mL） groups. After respectively incubated with 48 h，cell proliferation was investigated by CCK-8；Relative mRNA expression of P311，TGF-β1 and α-SMA were assessed by real-time PCR；Relative protein expression of TGF-β1 and α-SMA were investigated by western blot. Results：①optical density（A） in each group：0.331±0.002 in the NF group，0.533±0.005 in the MF group，0.487±0.003 in the MF+DEX 0.02 group，0.434±0.004 in the MF+DEX 0.1 group，0.381±0.004 in the MF+DEX 0.5 group；②P311 mRNA in each group：1.000 0±0.024 8 in the NF group，4.146 3±0.037 1 in the MF group，3.789 9±0.056 8 in the MF+DEX 0.02 group，3.566 1±0.148 1 in the MF+DEX 0.1 group，2.945 8±0.654 0 in the MF+DEX 0.5 group；③TGF-β1 mRNA in each group：1.000 0±0.034 6 in the NF group，2.647 9±0.048 5 in MF group，1.929 7±0.054 8 in the MF+DEX 0.02 group，1.678 2±0.080 2 in MF+DEX 0.1 group and 1.676 2±0.036 9 in the MF+DEX 0.5 group；④cells α-SMA mRNA in each group：1.000 0±0.056 0 in the NF group，4.025 7±0.065 9 in the MF group，3.644 9±0.196 4 in the MF+DEX 0.02 group，2.712 9±0.102 4 in the MF+DEX 0.1 group and 2.320 7±0.031 2 in the MF+DEX 0.5 group；⑤TGF-β1 protein values in each group：0.999 6±0.046 3 in the NF group，2.096 3±0.029 3 in the MF group，1.781 8±0.040 4 in the MF+DEX 0.02 group，1.531 4±0.032 5 in the MF+DEX 0.1 group，and 1.384 0±0.035 7 in the MF+DEX 0.5 group；⑥The number of α-SMA protein in each group：1.000 8±0.051 4 in the NF group，3.2314±0.1160 in the MF group，2.875 3±0.078 0 in the MF+DEX 0.02 group，2.262 8±0.059 8 in the MF+DEX 0.1 group and 1.940 8±0.093 7 in the MF+DEX 0.5 group. After 48 h of DEX intervention，cell proliferation was significantly inhibited and both the mRNA and protein expression of P311，TGF-β1 and α-SMA（all P<0.05，0.1~0.5 mg/mL） were significantly regulated down，with a dose-dependent manner. Conclusion：Mechanism of DEX on anti-BBS may be because it is relevant to modulation of P311/TGF-β1/α-SMA signaling pathway.