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
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摘要
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.
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.
引文
[1] CUNNINGHAM M A,TARANTOLA R M,FOLK J C,SOHN E H,BOLDT H C,GRAFF J A,et al.Proliferative vitreoretinopathy may be a risk factor in combined macular hole retinal detachment cases[J].Retina,2013,33(3):579-585.
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[3] ZHANG J,YUAN G,DONG M,ZHANG T,HUA G,ZHOU Q,et al.Notch signaling modulates proliferative vitreoretinopathy via regulating retinal pigment epithelial-to-mesenchymal transition[J].Histochem Cell Biol,2017,147(3):367-375.
[4] BRENMOEHL J,MILLER S N,HOFMANN C,VOGL D,FALK W,SCHOLMERICH J,et al.Transforming growth factor-beta 1 induces intestinal myofibroblast differentiation and modulates their migration[J].World J Gastroenterol,2009,15(12):1431-1442.
[5] LI M,LI H,JIANG P,LIU X,XU D,WANG F.Investigating the pathological processes of rhegmatogenous retinal detachment and proliferative vitreoretinopathy with metabolomics analysis[J].Mol Biosyst,2014,10(5):1055-1062.
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[7] DVASHI Z,GOLDBERG M,ADIR O,SHAPIRA M,POLLACK A.TGF-beta1 induced transdifferentiation of RPE cells is mediated by TAK1[J].PLoS One,2015,10(4):e122229.
[8] XIAO Y Q.The role of activin receptor-like kinase 5 inhibitor in the inhibition of scar formation after filtering surgery[D].Shanghai:Fudan University,2007.肖以钦.ALK5抑制剂对兔滤过性手术后滤过道瘢痕化的抑制及其机制的研究[D].上海:复旦大学,2007.
[9] LAPING N J,GRYGIELKO E,MATHUR A,BUTTER S,BOMBERGER J,TWEED C,et al.Inhibition of transforming growth factor (TGF)-beta1-induced extracellular matrix with a novel inhibitor of the TGF-beta type I receptor kinase activity:SB-431542[J].Mol Pharmacol,2002,62(1):58-64.
[10] HOLLBORN M,KOHEN L,WIEDEMANN P,ENZMANN V.The influence of pro-inflammatory cytokines on human retinal pigment epithelium cell receptors[J].Graefes Arch Clin Exp Ophthalmol,2001,239(4):294-301.
[11] CHEN Z.LYTAK1 inhibits the proliferation,migration and interstitialization of human retinal pigment epithelial cells[D].Chongqing:Chongqing Medical University,2016.陈臻.LYTAK1抑制人视网膜色素上皮细胞增殖、迁移和间质化机制的研究[D].重庆:重庆医科大学,2016.
[12] LU Y,HUANG Y Y,CAI J P,CHENG J W,WEI R L.Effects of SB431542 and TGF-β1 on α-SMA and CTGF gene expression in orbital fibroblast of patients with thyroid-associated ophthalmolopathy[J].Rec Adv Ophthalmol,2015,35(3):210-213.陆燕,黄媛媛,蔡季平,程金伟,魏锐利.SB431542和TGF-β1对甲状腺相关性眼病患者眼眶成纤维细胞α-SMA及CTGF基因表达的影响[J].眼科新进展,2015,35(3):210-213.
[13] OSBORNE N N,CAZEVIEILLE C,WOOD J P,NASH M S,PERGANDE G,BLOCK F,et al.Flupirtine,a nonopioid centrally acting analgesic,acts as an NMDA antagonist[J].Gen Pharmacol,1998,30(3):255-263.
[14] BARAK A,MORSE L S,GOLDKORN T.Ceramide:a potential mediator of apoptosis in human retinal pigment epithelial cells[J].Invest Ophthalmol Vis Sci,2001,42(1):247-254.
[15] LEE S C,KWON O W,SEONG G J,KIM S H,AHN J E,KAY E D.Epitheliomesenchymal transdifferentiation of cultured RPE cells[J].Ophthalmic Res,2001,33(2):80-86.
[16] WANG J Z.The role of endothelin in proliferative vitreoretinopathy[D].Xi’an:Fourth Military Medical University,2005.王建洲.内皮素在增生性玻璃体视网膜病变中作用的研究[D].西安:第四军医大学,2005.
[17] AIT-OUFELLA H,TALEB S,MALLAT Z,TEDGUI A.Recent advances on the role of cytokines in atherosclerosis[J].Arterioscler Thromb Vasc Biol,2011,31(5):969-979.
[18] GUO W,SHAN B,KLINGSBERG R C,QIN X,LASKY J A.Abrogation of TGF-beta1-induced fibroblast-myofibroblast differentiation by histone deacetylase inhibition[J].Am J Physiol Lung Cell Mol Physiol,2009,297(5):864-870.
[19] INMAN G J,NICOLAS F J,CALLAHAN J F,HARLING J D,GASTER L M,REITH A D,et al.SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4,ALK5,and ALK7[J].Mol Pharmacol,2002,62(1):65-74.
[20] AHN J Y,KIM M H,LIM M J,PARK S,LEE S L,YUN Y S,et al.The inhibitory effect of ginsan on TGF-beta mediated fibrotic process[J].J Cell Physiol,2011,226(5):1241-1247.
[21] MEDINA C,SANTOS-MARTINEZ M J,SANTANA A,PAZ-CABRERA M C,JOHNSTON M J,MOURELLE M,et al.Transforming growth factor-beta type 1 receptor (ALK5) and Smad proteins mediate TIMP-1 and collagen synthesis in experimental intestinal fibrosis[J].J Pathol,2011,224(4):461-472.
[22] CHOI K,LEE K,RYU S W,IM M,KOOK K H,CHOI C.Pirfenidone inhibits transforming growth factor-beta1-induced fibrogenesis by blocking nuclear translocation of Smads in human retinal pigment epithelial cell line ARPE-19[J].Mol Vis,2012,18(104-07):1010-1020.
[23] STODTLER M,MIETZ H,WIEDEMANN P,HEIMANN K.Immunohistochemistry of anterior proliferative vitreoretinopathy.Report of 11 cases[J].Int Ophthalmol,1994,18(6):323-328.
[24] HIGASHIYAMA H,YOSHIMOTO D,KAISE T,MATSUBARA S,FUJIWARA M,KIKKAWA H,et al.Inhibition of activin receptor-like kinase 5 attenuates Bleomycin-induced pulmonary fibrosis[J].Exp Mol Pathol,2007,83(1):39-46.
[2] NEROEV V V,KISELEVA T N,ZAITSEVA O V,KRUZHKOVA G V,KUZNETSOVA I S.Ocular and orbital blood flow in patients with proliferative vitreoretinopathy associated with rhegmatogenous retinal detachment[J].Vestn Oftalmol,2012,128(5):6-9.
[3] ZHANG J,YUAN G,DONG M,ZHANG T,HUA G,ZHOU Q,et al.Notch signaling modulates proliferative vitreoretinopathy via regulating retinal pigment epithelial-to-mesenchymal transition[J].Histochem Cell Biol,2017,147(3):367-375.
[4] BRENMOEHL J,MILLER S N,HOFMANN C,VOGL D,FALK W,SCHOLMERICH J,et al.Transforming growth factor-beta 1 induces intestinal myofibroblast differentiation and modulates their migration[J].World J Gastroenterol,2009,15(12):1431-1442.
[5] LI M,LI H,JIANG P,LIU X,XU D,WANG F.Investigating the pathological processes of rhegmatogenous retinal detachment and proliferative vitreoretinopathy with metabolomics analysis[J].Mol Biosyst,2014,10(5):1055-1062.
[6] FEIST R J,KING J L,MORRIS R,WITHERSPOON C D,GUIDRY C.Myofibroblast and extracellular matrix origins in proliferative vitreoretinopathy[J].Graefes Arch Clin Exp Ophthalmol,2014,252(2):347-357.
[7] DVASHI Z,GOLDBERG M,ADIR O,SHAPIRA M,POLLACK A.TGF-beta1 induced transdifferentiation of RPE cells is mediated by TAK1[J].PLoS One,2015,10(4):e122229.
[8] XIAO Y Q.The role of activin receptor-like kinase 5 inhibitor in the inhibition of scar formation after filtering surgery[D].Shanghai:Fudan University,2007.肖以钦.ALK5抑制剂对兔滤过性手术后滤过道瘢痕化的抑制及其机制的研究[D].上海:复旦大学,2007.
[9] LAPING N J,GRYGIELKO E,MATHUR A,BUTTER S,BOMBERGER J,TWEED C,et al.Inhibition of transforming growth factor (TGF)-beta1-induced extracellular matrix with a novel inhibitor of the TGF-beta type I receptor kinase activity:SB-431542[J].Mol Pharmacol,2002,62(1):58-64.
[10] HOLLBORN M,KOHEN L,WIEDEMANN P,ENZMANN V.The influence of pro-inflammatory cytokines on human retinal pigment epithelium cell receptors[J].Graefes Arch Clin Exp Ophthalmol,2001,239(4):294-301.
[11] CHEN Z.LYTAK1 inhibits the proliferation,migration and interstitialization of human retinal pigment epithelial cells[D].Chongqing:Chongqing Medical University,2016.陈臻.LYTAK1抑制人视网膜色素上皮细胞增殖、迁移和间质化机制的研究[D].重庆:重庆医科大学,2016.
[12] LU Y,HUANG Y Y,CAI J P,CHENG J W,WEI R L.Effects of SB431542 and TGF-β1 on α-SMA and CTGF gene expression in orbital fibroblast of patients with thyroid-associated ophthalmolopathy[J].Rec Adv Ophthalmol,2015,35(3):210-213.陆燕,黄媛媛,蔡季平,程金伟,魏锐利.SB431542和TGF-β1对甲状腺相关性眼病患者眼眶成纤维细胞α-SMA及CTGF基因表达的影响[J].眼科新进展,2015,35(3):210-213.
[13] OSBORNE N N,CAZEVIEILLE C,WOOD J P,NASH M S,PERGANDE G,BLOCK F,et al.Flupirtine,a nonopioid centrally acting analgesic,acts as an NMDA antagonist[J].Gen Pharmacol,1998,30(3):255-263.
[14] BARAK A,MORSE L S,GOLDKORN T.Ceramide:a potential mediator of apoptosis in human retinal pigment epithelial cells[J].Invest Ophthalmol Vis Sci,2001,42(1):247-254.
[15] LEE S C,KWON O W,SEONG G J,KIM S H,AHN J E,KAY E D.Epitheliomesenchymal transdifferentiation of cultured RPE cells[J].Ophthalmic Res,2001,33(2):80-86.
[16] WANG J Z.The role of endothelin in proliferative vitreoretinopathy[D].Xi’an:Fourth Military Medical University,2005.王建洲.内皮素在增生性玻璃体视网膜病变中作用的研究[D].西安:第四军医大学,2005.
[17] AIT-OUFELLA H,TALEB S,MALLAT Z,TEDGUI A.Recent advances on the role of cytokines in atherosclerosis[J].Arterioscler Thromb Vasc Biol,2011,31(5):969-979.
[18] GUO W,SHAN B,KLINGSBERG R C,QIN X,LASKY J A.Abrogation of TGF-beta1-induced fibroblast-myofibroblast differentiation by histone deacetylase inhibition[J].Am J Physiol Lung Cell Mol Physiol,2009,297(5):864-870.
[19] INMAN G J,NICOLAS F J,CALLAHAN J F,HARLING J D,GASTER L M,REITH A D,et al.SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4,ALK5,and ALK7[J].Mol Pharmacol,2002,62(1):65-74.
[20] AHN J Y,KIM M H,LIM M J,PARK S,LEE S L,YUN Y S,et al.The inhibitory effect of ginsan on TGF-beta mediated fibrotic process[J].J Cell Physiol,2011,226(5):1241-1247.
[21] MEDINA C,SANTOS-MARTINEZ M J,SANTANA A,PAZ-CABRERA M C,JOHNSTON M J,MOURELLE M,et al.Transforming growth factor-beta type 1 receptor (ALK5) and Smad proteins mediate TIMP-1 and collagen synthesis in experimental intestinal fibrosis[J].J Pathol,2011,224(4):461-472.
[22] CHOI K,LEE K,RYU S W,IM M,KOOK K H,CHOI C.Pirfenidone inhibits transforming growth factor-beta1-induced fibrogenesis by blocking nuclear translocation of Smads in human retinal pigment epithelial cell line ARPE-19[J].Mol Vis,2012,18(104-07):1010-1020.
[23] STODTLER M,MIETZ H,WIEDEMANN P,HEIMANN K.Immunohistochemistry of anterior proliferative vitreoretinopathy.Report of 11 cases[J].Int Ophthalmol,1994,18(6):323-328.
[24] HIGASHIYAMA H,YOSHIMOTO D,KAISE T,MATSUBARA S,FUJIWARA M,KIKKAWA H,et al.Inhibition of activin receptor-like kinase 5 attenuates Bleomycin-induced pulmonary fibrosis[J].Exp Mol Pathol,2007,83(1):39-46.
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