Y-27632为嘧啶衍生物,是一种近年来合成的特异性Rho相关螺旋卷曲蛋白激酶(Rho associated coiled-coil forming protein kinase,R O C K)抑制剂,能通过调控R O C K介导的多种生物效应从而抑制肝纤维化的形成.研究发现,转化生长因子β1(t...Y-27632为嘧啶衍生物,是一种近年来合成的特异性Rho相关螺旋卷曲蛋白激酶(Rho associated coiled-coil forming protein kinase,R O C K)抑制剂,能通过调控R O C K介导的多种生物效应从而抑制肝纤维化的形成.研究发现,转化生长因子β1(transforming growth factorβ1,TGF-β1)/结缔组织生长因子(connective tissue growth factor,CTGF)通路参与肝纤维化的形成,TGF-β1诱导其下游效应分子CTGF表达,促使细胞外基质生成增多,导致肝纤维化.Y-27632具有抑制TGF-β1和CTGF表达的作用,本文从ROCK抑制剂Y-27632对TGF-β1/CTGF通路的影响来阐述Y-27632的抗纤维化作用,借此更深入的了解Y-27632的作用靶点,为肝纤维化的靶向治疗提供理论依据.展开更多
Rho-associated kinase(ROCK) is a key regulatory protein involved in inflammatory secretion in microglia in the central nervous system.Our previous studies showed that ROCK inhibition enhances phagocytic activity in ...Rho-associated kinase(ROCK) is a key regulatory protein involved in inflammatory secretion in microglia in the central nervous system.Our previous studies showed that ROCK inhibition enhances phagocytic activity in microglia through the extracellular signal-regulated kinase(ERK) signaling pathway,but its effect on microglial migration was unknown.Therefore,in this study,we investigated the effects of the ROCK inhibitors Y27632 and fasudil on the migratory activity of primary cultured microglia isolated from the spinal cord,and we examined the underlying mechanisms.The microglia were treated with Y27632,fasudil and/or the ERK inhibitor U0126.Cellular morphology was observed by immunofluorescence.Transwell chambers were used to assess cell migration.ERK levels were measured by incell western blot assay.Y27632 and fasudil increased microglial migration,and the microglia were irregularly shaped and had many small processes.These inhibitors also upregulated the levels of phosphorylated ERK protein.The ERK inhibitor U0126 suppressed these effects of Y27632 and fasudil.These findings suggest that the ROCK inhibitors Y27632 and fasudil promote microglial migration in the spinal cord through the ERK signaling pathway.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
文摘Y-27632为嘧啶衍生物,是一种近年来合成的特异性Rho相关螺旋卷曲蛋白激酶(Rho associated coiled-coil forming protein kinase,R O C K)抑制剂,能通过调控R O C K介导的多种生物效应从而抑制肝纤维化的形成.研究发现,转化生长因子β1(transforming growth factorβ1,TGF-β1)/结缔组织生长因子(connective tissue growth factor,CTGF)通路参与肝纤维化的形成,TGF-β1诱导其下游效应分子CTGF表达,促使细胞外基质生成增多,导致肝纤维化.Y-27632具有抑制TGF-β1和CTGF表达的作用,本文从ROCK抑制剂Y-27632对TGF-β1/CTGF通路的影响来阐述Y-27632的抗纤维化作用,借此更深入的了解Y-27632的作用靶点,为肝纤维化的靶向治疗提供理论依据.
基金supported by the National Natural Science Foundation of China,No.81471200,81771341
文摘Rho-associated kinase(ROCK) is a key regulatory protein involved in inflammatory secretion in microglia in the central nervous system.Our previous studies showed that ROCK inhibition enhances phagocytic activity in microglia through the extracellular signal-regulated kinase(ERK) signaling pathway,but its effect on microglial migration was unknown.Therefore,in this study,we investigated the effects of the ROCK inhibitors Y27632 and fasudil on the migratory activity of primary cultured microglia isolated from the spinal cord,and we examined the underlying mechanisms.The microglia were treated with Y27632,fasudil and/or the ERK inhibitor U0126.Cellular morphology was observed by immunofluorescence.Transwell chambers were used to assess cell migration.ERK levels were measured by incell western blot assay.Y27632 and fasudil increased microglial migration,and the microglia were irregularly shaped and had many small processes.These inhibitors also upregulated the levels of phosphorylated ERK protein.The ERK inhibitor U0126 suppressed these effects of Y27632 and fasudil.These findings suggest that the ROCK inhibitors Y27632 and fasudil promote microglial migration in the spinal cord through the ERK signaling pathway.
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.