AIM: To investigate the interfering effect of Y-27632, a ROCK-I selective inhibitor, on the signal transduction pathway of transforming growth factor-beta 1 (TGF-beta 1) in ocular Tenon capsule fibroblasts (OTFS) in v...AIM: To investigate the interfering effect of Y-27632, a ROCK-I selective inhibitor, on the signal transduction pathway of transforming growth factor-beta 1 (TGF-beta 1) in ocular Tenon capsule fibroblasts (OTFS) in vitro. METHODS: After OTFS from passages 4 to 6 47 vitro were induced by TGF-beta 1 and then treated by Y-27632, the changes of the OTFS cell cycles were analyzed via flow cytometry, and the proteins expression of the alpha -smooth muscular actin (alpha -SMA), connective tissue growth factor (CTGF), collagen I were calculated by Western blot. After OTFS treated by the different concentrations of Y-27632, the expression levels of the alpha -SMA, CTGF and collagen I mRNA were assayed by RT-PCR. RESULTS: Y-27632 had no markedly effect on the OTFS cell cycles. After treated by TGF-beta 1, OTFS in G1 period significantly increased. The cell cycles distribution by both TGF-beta 1 and Y-27632 had no remarkable difference from that in control group. Y-27632 significantly inhibited the proteins expressions of both alpha -SMA and CTGF, while to some extent inhibited that of collagen I. TGF-beta 1 significantly promoted the proteins expressions of alpha -SMA, CTGF and collagen I. After OTFS treated by both TGF-beta 1 and Y-27632, of alpha -SMA, the protein expression was similar with that in control group (P=0.066>0.05), but the protein expression of CTGF or collagen I, respectively, was significantly different from that in control group (P=0.000<0.01). The differences of expressions of the alpha -SMA, CTGF and collagen I mRNA in 30, 150, 750 mu mol/L Y-27632 group were statistically significant, compared with those in control group, respectively (alpha -SMA, P=0.002, 0.000, 0.000; CTGF, P=0.014, 0.002, 0.001; collagen I,P=0.003, 0.002, 0.000). CONCLUSION: Blocking the Rho/ROCK signaling pathway by using of Y-27632 could inhibit the cellular proliferation and the expression of both CTGF and alpha -SMA whatever OTFS induced by TGF-beta 1 or not. Y-27632 suppressed the expression of collagen I mRNA without inducti展开更多
AIM：To research the effect of Y-27632,a selective Rhoassociated coiled-coil kinase（ROCK） inhibitor,on TGF-β1/Smad2,3 signal transduction in ocular Tenon＇s capsule fi broblasts（OTFs）.METHODS：Primary ocular Teno...AIM：To research the effect of Y-27632,a selective Rhoassociated coiled-coil kinase（ROCK） inhibitor,on TGF-β1/Smad2,3 signal transduction in ocular Tenon＇s capsule fi broblasts（OTFs）.METHODS：Primary ocular Tenon＇s capsule fibroblasts had been cultured in vitro.The effect of Y27632 on proliferation of OTF stimulated by lysophosphatidic acid（LPA） was evaluated by MTT colorimetric assay so as to sift out the proper concentrations range of Y-27632 for the next experiment.Real time-polymerase chain reactor（RT-PCR） was to analyze the changes of Smad2 and Smad3 genes of cells affected by Y-27632,though unaffected by transforming growth factorbeta1（TGF-β1）.Proteins of Smad2,Smad3,phosphorylated Smad2（Ser245/250/255）,and phosphorylated Smad3（Ser423/425/203） were respectively quantifi ed by Western blot after OTFs were successively incubated by TGF-β1 and Y-27632.Meanwhile,α-smooth muscular actin（α-SMA） protein was also quantified after the small intervening gene fragments of human Smad2 and Smad3 were designed,synthesized,and then transfected to OTFs.RESULTS：Y-27632 signifi cantly inhibited OTFs proliferation stimulated by LPA.Also Y-27632 signifi cantly suppressed the expressions of Smad2 m RNA,Smad2,3 proteins expressions,Smad3 phosphorylation at the carboxylic terminals of Ser423/425/203 which had been radically promoted by TGF-β1.Si RNA-Smad2,3 suppressed α-SMA expressions,but less effectively than Y-27632.CONCLUSION：The inhibition of ROCK signaling may be a potential therapeutic candidate for the treatment of the fi ltration channel fi brosis.展开更多
基金Shaanxi Province Science and Technology Gongguan Program, China (No.2011-K14-02-03)
文摘AIM: To investigate the interfering effect of Y-27632, a ROCK-I selective inhibitor, on the signal transduction pathway of transforming growth factor-beta 1 (TGF-beta 1) in ocular Tenon capsule fibroblasts (OTFS) in vitro. METHODS: After OTFS from passages 4 to 6 47 vitro were induced by TGF-beta 1 and then treated by Y-27632, the changes of the OTFS cell cycles were analyzed via flow cytometry, and the proteins expression of the alpha -smooth muscular actin (alpha -SMA), connective tissue growth factor (CTGF), collagen I were calculated by Western blot. After OTFS treated by the different concentrations of Y-27632, the expression levels of the alpha -SMA, CTGF and collagen I mRNA were assayed by RT-PCR. RESULTS: Y-27632 had no markedly effect on the OTFS cell cycles. After treated by TGF-beta 1, OTFS in G1 period significantly increased. The cell cycles distribution by both TGF-beta 1 and Y-27632 had no remarkable difference from that in control group. Y-27632 significantly inhibited the proteins expressions of both alpha -SMA and CTGF, while to some extent inhibited that of collagen I. TGF-beta 1 significantly promoted the proteins expressions of alpha -SMA, CTGF and collagen I. After OTFS treated by both TGF-beta 1 and Y-27632, of alpha -SMA, the protein expression was similar with that in control group (P=0.066>0.05), but the protein expression of CTGF or collagen I, respectively, was significantly different from that in control group (P=0.000<0.01). The differences of expressions of the alpha -SMA, CTGF and collagen I mRNA in 30, 150, 750 mu mol/L Y-27632 group were statistically significant, compared with those in control group, respectively (alpha -SMA, P=0.002, 0.000, 0.000; CTGF, P=0.014, 0.002, 0.001; collagen I,P=0.003, 0.002, 0.000). CONCLUSION: Blocking the Rho/ROCK signaling pathway by using of Y-27632 could inhibit the cellular proliferation and the expression of both CTGF and alpha -SMA whatever OTFS induced by TGF-beta 1 or not. Y-27632 suppressed the expression of collagen I mRNA without inducti
基金Supported by Scientific and Technological Project of Shaanxi Province,China(No.2016SF-010)
文摘AIM：To research the effect of Y-27632,a selective Rhoassociated coiled-coil kinase（ROCK） inhibitor,on TGF-β1/Smad2,3 signal transduction in ocular Tenon＇s capsule fi broblasts（OTFs）.METHODS：Primary ocular Tenon＇s capsule fibroblasts had been cultured in vitro.The effect of Y27632 on proliferation of OTF stimulated by lysophosphatidic acid（LPA） was evaluated by MTT colorimetric assay so as to sift out the proper concentrations range of Y-27632 for the next experiment.Real time-polymerase chain reactor（RT-PCR） was to analyze the changes of Smad2 and Smad3 genes of cells affected by Y-27632,though unaffected by transforming growth factorbeta1（TGF-β1）.Proteins of Smad2,Smad3,phosphorylated Smad2（Ser245/250/255）,and phosphorylated Smad3（Ser423/425/203） were respectively quantifi ed by Western blot after OTFs were successively incubated by TGF-β1 and Y-27632.Meanwhile,α-smooth muscular actin（α-SMA） protein was also quantified after the small intervening gene fragments of human Smad2 and Smad3 were designed,synthesized,and then transfected to OTFs.RESULTS：Y-27632 signifi cantly inhibited OTFs proliferation stimulated by LPA.Also Y-27632 signifi cantly suppressed the expressions of Smad2 m RNA,Smad2,3 proteins expressions,Smad3 phosphorylation at the carboxylic terminals of Ser423/425/203 which had been radically promoted by TGF-β1.Si RNA-Smad2,3 suppressed α-SMA expressions,but less effectively than Y-27632.CONCLUSION：The inhibition of ROCK signaling may be a potential therapeutic candidate for the treatment of the fi ltration channel fi brosis.
