ALK5抑制剂对转化生长因子-β1(TGF-β1)致视网膜色素上皮细胞纤维化的干预作用

Effect of activated receptor kinase 5 inhibitor on transforming growth factor-beta1-induced retinal pigment epithelial cell fibrosis

目的探讨转化生长因子-β1(transforming growth factor-beta1,TGF-β1)对人视网膜色素上皮(retinal pigment epithelial,RPE)细胞向成纤维细胞转化的影响,及活化素受体样激酶5(activated receptor kinase 5,ALK5)抑制剂(SB-431542)对这一影响的干预作用。方法体外培养人RPE细胞,随机分为对照组、TGF-β1处理组、TGF-β1+SB-431542处理组、SB-431542处理组。对照组不做任何处理,其余三组分别用10μg·L-1 TGF-β1、10μg·L-1 TGF-β1+30μmol·L-1 SB-431542、30μmol·L-1 SB-431542处理细胞24 h。MTT法检测各组RPE细胞增殖率;划痕实验观察各组处理后0 h、12 h、24 h RPE细胞迁移情况;免疫荧光法检测各组细胞ALK5蛋白的分布和表达;Western blot法检测各组ALK5、血管内皮生长因子(vascular endothelial growth factor,VEGF)、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、Ⅰ型胶原(collagen typeⅠ,ColⅠ)以及纤维粘连素(fibronectin,FN)蛋白的表达。结果与对照组相比,TGF-β1作用于RPE细胞24 h后,可改变RPE细胞表型,使其呈成纤维细胞样外观,并明显促进RPE细胞增殖和迁移。10.0 g·L-1 TGF-β1作用24 h后RPE细胞增殖率为对照组的(1.33±0.08)倍,30μmol·L-1 SB-431542作用24 h后RPE细胞增殖率为10.0 g·L-1 TGF-β1处理组的(67.77±6.78)%。经TGF-β1作用24 h后RPE细胞内ALK5、VEGF、α-SMA、ColⅠ及FN蛋白的表达显著上调,分别是对照组的(3.69±0.37)倍、(1.76±0.05)倍、(2.58±0.18)倍、(1.86±0.11)倍、(1.74±0.08)倍;SB-431542可逆转TGF-β1诱导的ALK5、VEGF、α-SMA、ColⅠ及FN蛋白表达上调,分别是TGF-β1处理组的(67.73±5.15)%、(71.71±3.50)%、(79.87±0.05)%、(63.59±3.16)%、(83.07±2.31)%,差异均有统计学意义(均为P<0.05)。结论 TGF-β1可促进RPE细胞增殖、迁移,向成纤维细胞转化,并能显著上调RPE细胞内ALK5、VEGF、α-SMA、ColⅠ及FN蛋白的表达;SB-431542可通过抑制TGF-β1的作用而抑制RPE细胞的纤维化。

Objective To investigate the transformation effect of transforming growth factor beta1(TGF-β1) of human retinal pigment epithelium(hRPE) cells into fibroblasts and the intervention effect of activated receptor kinase 5(ALK5) inhibitor(SB-431542) of on them.Methods hRPE cells were cultured in vitro and randomly divided into four groups:control group,TGF-β1 treatment group,TGF-β1+SB-431542 treatment group,SB-431542 treatment group.In the control group,no treatment was performed.TGF-β1 treatment group,TGF-β1+SB-431542 treatment group,SB-431542 treatment group were treated with a concentration of 10 μg·L-1 TGF-β1,10 μg·L-1 TGF-β1+30 μmol·L-1 SB-431542 and 30 μmol·L-1 SB-431542 for 24 hours.The proliferation rates of hRPE cells were detected by MTT assay in each group.The migration of RPE cells was observed in each group at 0 h,12 h and 24 h after treatment by scratch test.The distribution and expression of ALK5 protein were detected by immunofluorescence assay in each group.The expression of ALK5,vascular endothelial growth factor(VEGF),α-smooth muscle actin(α-SMA),collagen type Ⅰ(Col Ⅰ),and fibronectin(FN) proteins in each group was performed by Western blot.Results Compared with the control group,10 μg·L-1 TGF-β1 changed the appearance of RPE cells,which showed a fibroblast-like appearance after 24 h of action on RPE cells,and significantly promoted the proliferation and migration of RPE cells.In addition,the proliferation rate of RPE cells was(1.33±0.08) times that of the control group.After treatment with 30 μmol·L-1 SB-431542 for 24 h,the proliferation rate of RPE cells was(67.77±6.78)% in the 10 μg·L-1 TGF-β1 treated group.After treatment with TGF-β1 for 24 h,protein expressions of ALK5,VEGF,α-SMA,Col Ⅰ and FN increased significantly,which was(3.69±0.37)times,(1.76±0.05)times,(2.58±0.18)times,(1.86±0.11)times,(1.74±0.08)times,respectively that of the control group.SB-431542 reversed the upregulated expression of ALK5,VEGF,α-SMA,Col Ⅰ and FN proteins induced by TGF-β1,which presented(67.73±5.15)%,(71.71±3.50)%,(79.87±0.05)%,(63.59±3.16)%,(83.07±2.31)%,respectively,compared with TGF-β1 treatment group,with statistically significant differences(all P<0.05).Conclusion TGF-β1 can promote RPE cells migration,fibroblasts transformation,and significantly increase protein expressions of ALK5,VEGF,α-SMA,Col Ⅰ and FN of RPE cells;SB-431542 can inhibit the fibrosis of RPE cells by inhibiting the effects of TGF-β1.