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

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/m L)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;q RT-PCR检测各组细胞P311、TGF-β1及α-SMA基因m RNA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 m RNA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 m RNA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 m RNA值:NF组为1.000 0±0.056 0,MF组为4.025 7±0.065 9,MF+DEX0.02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m RNA及蛋白表达明显下调(均P<0.05,0.1~0.5 mg/m L),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。

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： （1）optical 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;（2）P311 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,3.5661 ±0.148 1 in the MF＋DEX 0.1 group,2.945 8 ± 0.654 0 in the MF＋DEX 0.5 group; （3）TGF-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;（4）eells 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;（5）TGF-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; （6）The 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.