目的探讨Rho激酶抑制剂Y-27623对心肌缺血再灌注损伤(MIRI)中细胞凋亡的影响,以及对丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)和凋亡相关蛋白表达水平的变化和意义.方法成年雄性SD大鼠60只,应用随机数字表法分为4组,...目的探讨Rho激酶抑制剂Y-27623对心肌缺血再灌注损伤(MIRI)中细胞凋亡的影响,以及对丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)和凋亡相关蛋白表达水平的变化和意义.方法成年雄性SD大鼠60只,应用随机数字表法分为4组,每组15只:正常对照组(Sham组)、缺血再灌注组(ischemia-reperfusion,I/R组)、Dil组(Diltiazem,地尔硫卓组)和Y-27632组.Dil组每日给予地尔硫卓(10 mg/kg)灌胃,Y-27632组每日给予Y-27632(5 mg/kg),其余两组给予等体积清水.给药5天后Sham组只穿线,不结扎冠状动脉左前降支,I/R组、Dil组和Y-27632组均建立MIRI模型.TUNEL法检测各组心肌凋亡,计算心肌细胞凋亡指数(AI),western blotting法检测心肌组织中MAPK信号传导途径相关蛋白(p-JNK/ERK/P38)和凋亡相关蛋白(Bcl-2、Bax、Caspase-3和Caspase-9)的表达.结果相对于Sham组,I/R组AI明显增加(35.15±3.12比1.15±0.12,P<0.01),MAPK信号传导途径(p-JNK:0.792±0.071比0.344±0.055;p-ERK:0.809±0.087比0.273±0.055;p-P38:0.781±0.049比0.158±0.071)和心肌促凋亡相关蛋白(Bax:1.127±0.060比0.47±0.054;Caspase-3:0.843±0.092比0.289±0.068;Caspase-9:0.831±0.068比0.273±0.048)表达显著增加(P均<0.05),心肌抗凋亡蛋白Bcl-2表达显著减少(0.255±0.060比0.809±0.056,P<0.05);相对于I/R组,Y-27632治疗组AI明显下降(20.05±1.75比35.15±3.12,P<0.01),与Dil治疗组差异无统计学意义(20.05±1.75比22.12±2.01,P>0.05),Y-27632治疗组MAPK信号传导途径相关蛋白(p-JNK:0.443±0.027比0.792±0.344;p-ERK:0.284±0.038比0.809±0.087;p-P38:0.301±0.049比0.781±0.049)和Bax(0.807±0.072比1.127±0.060)、Caspase-3(0.329±0.040比0.843±0.092)和Caspase-9(0.454±0.048比0.831±0.068)表达均显著降低(P<0.05),Bcl-2表达显著增加(0.582±0.048比0.255±0.060,P<0.05),与Dil治疗组差异无统计学意义(p-JNK:0.443±0.027比0.552±0.044;p-ERK:0.284±0.038比0.273±0.049;p-P38:0.301±0.049比0.339±0.038;Bax:0.807±0.072比0.645±0.108;Caspase-3:0.329±0.040比0.378±0.096;Caspase-9:0.454±0.048比0.434±0.032;Bcl-2:0.582±0.048比0.614±0.06,P均>0.05).结论Y-27632通过抑制JNK/ERK/P38的磷酸化,抑制Bax、Caspase-3和Caspase-9的表达,加强Bcl-2的表达,减少心肌细胞的凋亡,从而减轻心肌缺血再灌注损伤.展开更多
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
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 induction.展开更多
AIM:To investigate the effect of Y-27632 on the survival and neurite outgrowth of the cultured retinal neurocytes. METHODS:After the postnatal day 2-3, Sprague-Dawley retinal neurocytes were cultured for 48 hours, the...AIM:To investigate the effect of Y-27632 on the survival and neurite outgrowth of the cultured retinal neurocytes. METHODS:After the postnatal day 2-3, Sprague-Dawley retinal neurocytes were cultured for 48 hours, the culture media was replaced with serum-free media (control group) and serum-free media contained 30μmol/L Y-27632 (Y-27632 group), and the cells were continually cultured another 48 hours. The cultured retinal neurocytes were identified with anti-neuron specific enolase (NSE) immunocytochemistry. The survival state of those cells was estimated by MTT assay, and the neurite outgrowth of those cells was evaluated by the computerized image-analysis system. RESULTS:Compared with the control group, the absorbance values of cells survival in Y -27632 group increased 12.90% and 33.33% respectively after 72 and 96 hours culture. Y-27632 had no significant effect on the diameter of cultured retinal neurocytes. Compared with the control group, Y-27632 induced a stable improvement of neurite outgrowth of retinal neurocytes after 72 and 96 hours culture (P =0.001). CONCLUSION:Y-27632 could promote the survival and neurite outgrowth of the early postnatal cultured retinal neurocytes.展开更多
Rho-associated protein kinase is an essential regulator of cytoskeletal dynamics during the process of neurite extension. However, whether Rho kinase regulates microtubule remodeling or the distri- bution of adhesive ...Rho-associated protein kinase is an essential regulator of cytoskeletal dynamics during the process of neurite extension. However, whether Rho kinase regulates microtubule remodeling or the distri- bution of adhesive proteins to mediate neurite outgrowth remains unclear. By specifically modulat- ing Rho kinase activity with pharmacological agents, we studied the morpho-dynamics of neurite outgrowth. We found that lysophosphatidic acid, an activator of Rho kinase, inhibited neurite out- growth, which could be reversed by Y-27632, an inhibitor of Rho kinase. Meanwhile, reorganization of microtubules was noticed during these processes, as indicated by their significant changes in the soma and growth cone. In addition, exposure to lysophosphatidic acid led to a decreased mem- brane distribution of vinculin, a focal adhesion protein in neurons, whereas Y-27632 recruited vin- culin to the membrane. Taken together, our data suggest that Rho kinase regulates rat hippocampal neurite growth and microtubule formation via a mechanism associated with the redistribution of vinculin.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 induction.
基金National Natural Science Foundation of China (No. 81070728 and No. 81000373)
文摘AIM:To investigate the effect of Y-27632 on the survival and neurite outgrowth of the cultured retinal neurocytes. METHODS:After the postnatal day 2-3, Sprague-Dawley retinal neurocytes were cultured for 48 hours, the culture media was replaced with serum-free media (control group) and serum-free media contained 30μmol/L Y-27632 (Y-27632 group), and the cells were continually cultured another 48 hours. The cultured retinal neurocytes were identified with anti-neuron specific enolase (NSE) immunocytochemistry. The survival state of those cells was estimated by MTT assay, and the neurite outgrowth of those cells was evaluated by the computerized image-analysis system. RESULTS:Compared with the control group, the absorbance values of cells survival in Y -27632 group increased 12.90% and 33.33% respectively after 72 and 96 hours culture. Y-27632 had no significant effect on the diameter of cultured retinal neurocytes. Compared with the control group, Y-27632 induced a stable improvement of neurite outgrowth of retinal neurocytes after 72 and 96 hours culture (P =0.001). CONCLUSION:Y-27632 could promote the survival and neurite outgrowth of the early postnatal cultured retinal neurocytes.
基金supported by the National Natural Science Foundation of China,No.31170941the Fundamental Research Funds for the Central Universities,No.21612424the Science and Technology Planning Project of Guangdong Province,No.2010B031600102
文摘Rho-associated protein kinase is an essential regulator of cytoskeletal dynamics during the process of neurite extension. However, whether Rho kinase regulates microtubule remodeling or the distri- bution of adhesive proteins to mediate neurite outgrowth remains unclear. By specifically modulat- ing Rho kinase activity with pharmacological agents, we studied the morpho-dynamics of neurite outgrowth. We found that lysophosphatidic acid, an activator of Rho kinase, inhibited neurite out- growth, which could be reversed by Y-27632, an inhibitor of Rho kinase. Meanwhile, reorganization of microtubules was noticed during these processes, as indicated by their significant changes in the soma and growth cone. In addition, exposure to lysophosphatidic acid led to a decreased mem- brane distribution of vinculin, a focal adhesion protein in neurons, whereas Y-27632 recruited vin- culin to the membrane. Taken together, our data suggest that Rho kinase regulates rat hippocampal neurite growth and microtubule formation via a mechanism associated with the redistribution of vinculin.
