目的探讨Rho激酶(ROCK)在氢气改善离体脓毒症肠屏障功能中的作用。方法常规培养人结肠上皮细胞Caco-2,分为6组(n=3):对照组(C组)、富氢培养基组(H组)、脂多糖(LPS)处理组(L组)、富氢培养基+LPS组(HL组)、Rho激酶抑制剂Y-27632组(Y组)、Y...目的探讨Rho激酶(ROCK)在氢气改善离体脓毒症肠屏障功能中的作用。方法常规培养人结肠上皮细胞Caco-2,分为6组(n=3):对照组(C组)、富氢培养基组(H组)、脂多糖(LPS)处理组(L组)、富氢培养基+LPS组(HL组)、Rho激酶抑制剂Y-27632组(Y组)、Y-27632+LPS组(YL组)。H组给予0.6 mmol/L富氢培养基;LPS和Y-27632的处理浓度分别为50 mg/L、25μmol/L。建立Transwell小室模型,定期检测跨上皮电阻值(TEER值),当TEER值达到800Ω·cm^2后给予处理,于6、12、24 h检测TEER值,24 h时检测FITC-右旋糖酐通过率。细胞接种于6孔板,融合达80%~90%后给予处理,实时聚合酶链式反应技术检测闭锁小带蛋白1(ZO-1)mRNA和ROCK mRNA表达情况;蛋白免疫印迹技术检测ZO-1蛋白和ROCK蛋白表达水平。结果与C组比较,H组12、24 h TEER值升高(P<0.05),FITC-右旋糖酐通过率、ZO-1蛋白和ROCK蛋白表达水平差异无统计学意义;Y组6、12、24 h TEER值升高(P<0.05),FITC-右旋糖酐通过率差异无统计学意义,ZO-1 mRNA表达增加,ROCK mRNA表达减少(均P<0.05);L组6、12、24 h TEER值降低,FITC-右旋糖酐通过率增高,ZO-1 mRNA和蛋白表达均下降,ROCK mRNA和蛋白表达均增加(P<0.05)。与L组比较,6、12、24 h YL组TEER值增高,FITC-右旋糖酐通过率降低,ZO-1 mRNA表达增加,ROCK mRNA表达降低(均P<0.05)。与L组比较,HL组6、12、24 h TEER值增高,FITC-右旋糖酐通过率降低,各时间点ZO-1蛋白表达上升,ROCK蛋白表达下降(均P<0.05)。结论氢气可保护脓毒症肠屏障功能,改善肠上皮屏障完整性和通透性,增加肠细胞间紧密连接蛋白表达,这些保护机制可能与氢气抑制LPS诱导的ROCK过度表达有关。展开更多
目的:观察Rho激酶抑制剂法舒地尔(Fasudil)对人高侵袭潜能HCC细胞株3(human high metastatic liver cancer cells 3,HCCLM3)侵袭转移的影响,并且探讨其作用的机制。方法:应用100 mol/L Fasudil作用于HCCLM3细胞,采用肌动蛋白微丝荧光染...目的:观察Rho激酶抑制剂法舒地尔(Fasudil)对人高侵袭潜能HCC细胞株3(human high metastatic liver cancer cells 3,HCCLM3)侵袭转移的影响,并且探讨其作用的机制。方法:应用100 mol/L Fasudil作用于HCCLM3细胞,采用肌动蛋白微丝荧光染色和侵袭小室实验观察HCCLM3细胞的运动侵袭能力。HCCLM3细胞经过处理后分为阴性对照组、Fasudil作用组、BTBD7干扰组,通过Western印迹检测BTB/POZ结构域蛋白7(BR-C,ttk and bab/pox virus domain containing 7,BTBD7)、Ras同系物家族成员C(ras homolog family member C,Rho C)、Rho关联卷曲螺旋蛋白激酶2(Rhoassociated,coiled-coil containing protein kinase 2,ROCK2)、MMP2和MMP9蛋白表达水平,酶谱分析法检测MMP2和MMP9活性水平。BTBD7干扰组作为阳性对照。结果:Fasudil处理后HCCLM3侵袭运动能力下降,BTBD7,Rho C,ROCK2蛋白表达下调,MMP2和MMP9活性降低,与阴性对照组比较差异有统计学意义(均P<0.01)。结论:Fasudil具有干预BTBD7-ROCK2信号通路、抑制HCC侵袭转移的重要作用。展开更多
In this study, PC12 Adh cells and Neuro-2a cells were treated with Rho-associated kinase inhibitors （Y27632 and Fasudil）, a cyclooxygenase-1 selective inhibitor （SC560）, and a cyclooxygenase-2 inhibitor （NS398）....In this study, PC12 Adh cells and Neuro-2a cells were treated with Rho-associated kinase inhibitors （Y27632 and Fasudil）, a cyclooxygenase-1 selective inhibitor （SC560）, and a cyclooxygenase-2 inhibitor （NS398）. We found that these cells became tolerant to Rho-associated kinase inhibitors, as neurite outgrowth induced by these inhibitors diminished following more than 3 days of exposure in either cell line. The proteins cyclooxygenase-2 and cytosolic prostaglandin E synthetase were upregulated at day 3. NS398 decreased the tolerance to neurite outgrowth induction in both cell lines, whereas SC560 had almost no effect. These findings indicate that cells become tolerant to neurite outgrowth induced by Rho-associated kinase inhibitors, this is at least partly associated with upregulation of proteins involved in the cyclooxygenase-2 pathway, and cyclooxygenases-2 inhibition prevents this tolerance.展开更多
目的检测IgA肾病(immunoglobulin A nephropathy,IgAN)患者血清Rho相关卷曲螺旋形成蛋白激酶2(rho associated coiled coil containing protein kinase 2,ROCK2)、视黄醇结合蛋白4(retinol-binding protein 4,RBP4)表达水平,分析二者在...目的检测IgA肾病(immunoglobulin A nephropathy,IgAN)患者血清Rho相关卷曲螺旋形成蛋白激酶2(rho associated coiled coil containing protein kinase 2,ROCK2)、视黄醇结合蛋白4(retinol-binding protein 4,RBP4)表达水平,分析二者在评估患者病情及预测预后中的价值。方法选取2016年6月至2019年6月98例中国贵航集团302医院已确诊收治的IgAN患者为IgAN组,其中轻度IgAN 36例、中度IgAN 32例、重度IgAN 30例;同期选择在中国贵航集团302医院体检的健康者100名为对照组。酶联免疫吸附法检测血清中ROCK2、RBP4水平;采用Kaplan-Meier生存曲线分析ROCK2、RBP4表达水平与IgAN患者预后的关系;Pearson相关性分析ROCK2与RBP4表达相关性;受试者工作特征曲线(receiver operating characteristic curve,ROC)分析ROCK2、RBP4对IgAN的诊断价值;比例风险回归模型法分析IgAN患者预后的影响因素。结果IgAN组血清中ROCK2、RBP4表达水平分别为(24.25±5.31)μg/L、(59.70±9.43)μg/L,显著高于对照组的(15.57±4.16)μg/L、(32.14±7.82)μg/L,差异具有统计学意义(t=12.818、22.404,均P<0.001);轻度、中度、重度IgAN患者血清中ROCK2水平分别为(18.69±4.36)μg/L、(23.75±5.31)μg/L、(31.46±5.62)μg/L,RBP4表达水平分别为(45.37±7.86)μg/L、(62.48±8.12)μg/L、(73.94±8.65)μg/L。随着病情的加重,IgAN患者血清ROCK2、RBP4表达水平逐渐升高,差异具有统计学意义(F=51.854、102.234,均P<0.05);IgAN患者血清中ROCK2、RBP4表达水平与Lee氏分级、IgA/C3比值、病情程度有关(P<0.05);ROCK2、RBP4高表达组患者预后生存率分别为60.00%、74.19%,均显著低于低表达组的89.71%、91.67%,差异具有统计学意义(log rankχ^(2)=4.674,P=0.031);ROC曲线结果显示,血清ROCK2诊断IgAN的曲线下面积(area under curve,AUC)为0.889,敏感度为80.61%,特异性为80.00%,截断值为18.79μg/L;RBP4诊断IgAN的AUC为0.986,敏感度为96.94%,特异性为95.00%,截断值为41.31μg/L;二者联合检测的AUC为0.997,敏感度为98.98%,特异性为94.86%。Pearson相关性分析显示,IgAN患者血清ROCK2与RBP4表达水平呈显著正相关(r=0.701,P<0.001);多因素比例风险回归模型法分析表明,IgA/C3(OR=2.683,95%CI:1.060~6.794)、ROCK2(OR=1.831,95%CI:1.027~3.264)、RBP4(OR=1.517,95%CI:1.104~2.084)均是IgAN患者预后生存的影响因素(均P<0.05)。结论ROCK2、RBP4在IgAN患者血清中高表达,二者与IgAN患者的临床病理特征、病情严重程度及预后关系密切,ROCK2、RBP4表达水平对IgAN的诊断具有一定的价值。展开更多
目的:观察芪白平肺胶囊对慢性阻塞性肺疾病(COPD)痰瘀阻肺证模型大鼠肺功能、形态学改变以及对于血清Rock-1(Rho关联卷曲蛋白激酶1)、Rho关联卷曲蛋白激酶2(Rock-2)表达的影响。方法:健康SD雄性大鼠50只,随机分组为正常对照组、模型组...目的:观察芪白平肺胶囊对慢性阻塞性肺疾病(COPD)痰瘀阻肺证模型大鼠肺功能、形态学改变以及对于血清Rock-1(Rho关联卷曲蛋白激酶1)、Rho关联卷曲蛋白激酶2(Rock-2)表达的影响。方法:健康SD雄性大鼠50只,随机分组为正常对照组、模型组、芪白平肺胶囊低剂量组、芪白平肺胶囊高剂量组、法舒地尔组,复制COPD痰瘀阻肺证大鼠模型,每日造模结束后予大鼠灌胃。模型组、正常组每天生理盐水灌胃10 m L/kg,芪白平肺胶囊低剂量组、芪白平肺胶囊高剂量组每天给药量分别为1000 mg/kg、250 mg/kg(含芪白平肺胶囊50 mg/m L),法舒地尔每天给药量为2 m L/kg(含法舒地尔15mg/m L),每只大鼠每日灌胃1次,直至造模结束,测定大鼠肺功能、观察肺血管重构的病理改变,ELISA检测模型大鼠血清Rock-1、Rock-2的表达。结果:模型组肺小动脉管壁变厚、管腔变小,血清中Rock-1、Rock-2较正常组含量显著升高,(P<0.01);FEV0.3、FEV0.3/FVC、PEF均明显降低,差异有统计学意义(P<0.01);各治疗组与模型组比较肺小动脉管壁变薄,管腔增大,血清中Rock-1、Rock-2表达水平均有不同程度下降(P<0.01-0.05)。结论:芪白平肺胶囊对COPD大鼠的肺组织具有保护作用,可有效的防止痰瘀阻肺证慢阻肺模型大鼠肺功能的下降,降低COPD痰瘀阻肺证模型大鼠血清Rock-1、Rock-2表达水平。展开更多
BACKGROUND: The increased β-arrestin-2 and its combination with G-protein-coupled receptors (GPCRs) lead to GPCRs desensitization. The latter may be responsible for decreased contractile reactivity in the mesenteric ...BACKGROUND: The increased β-arrestin-2 and its combination with G-protein-coupled receptors (GPCRs) lead to GPCRs desensitization. The latter may be responsible for decreased contractile reactivity in the mesenteric arteries of cirrhotic patients and rats. The present study is to investigate the machinery changes of α-adrenergic receptors and G proteins and their roles in the contractility of mesenteric arteries of cirrhotic patients and animal models. METHODS: Patients with cirrhosis due to hepatitis B and cirrhotic rats induced by CCl 4 were studied. Mesenteric artery contractility in response to norepinephrine was determined by a vessel perfusion system. The contractile effect of G protein-coupled receptor kinase-2 (GRK-2) inhibitor on the mesenteric artery was evaluated. The protein expression of the α 1 adrenergic receptor, G proteins, β-arrestin-2, GRK-2 as well as the activity of Rho associated coiled-coil forming protein kinase-1 (ROCK-1) were measured by Western blot. In addition, the interaction of α 1 adrenergic receptor with β-arrestin-2 was assessed by co-immunoprecipitation. RESULTS: The portal vein pressure of cirrhotic patients and rats was significantly higher than that of controls. The doseresponse curve to norepinephrine in mesenteric arteriole was shifted to the right, and EC 50 was significantly increased in cirrhotic patients and rats. There were no significant differences in the expressions of the α 1 adrenergic receptor and G proteins in the cirrhotic group compared with the controls. However, the protein expressions of GRK-2 and β-arrestin-2 were significantly elevated in cirrhotic patients and rats compared with those of the controls. The interaction of the α 1 adrenergic receptor and β-arrestin-2 was significantly aggravated. This interaction was significantly reversed by GRK-2 inhibitor. Both the protein expression and activity of ROCK-1 were significantly decreased in the mesenteric artery in patients with cirrhosis compared with those of the controls, and this phenomenon was not shown in the cirrhotic rats. Norepinephrine significantly increased the activity of ROCK-1 in normal rats but not in cirrhotic ones. Norepinephrine significantly increased ROCK-1 activity in cirrhotic rats when GRK-2 inhibitor was used. CONCLUSIONS: β-arrestin-2 expression and its interaction with GPCRs are significantly upregulated in the mesenteric arteries in patients and rats with cirrhosis. These upregulations result in GPCR desensitization, G-protein dysfunction and ROCK inhibition. These may explain the decreased contractility of the mesenteric artery in response to vasoconstrictors.展开更多
Objective: To investigate the role of interleukin-1 (IL-1) receptor associated kinase-2 (IRAK-2) in IL1 and TNF-stimulated nuclear factor-kappa B (NF-kappa B ) activation. Metbods: The 293 cell was trans fectedwith an...Objective: To investigate the role of interleukin-1 (IL-1) receptor associated kinase-2 (IRAK-2) in IL1 and TNF-stimulated nuclear factor-kappa B (NF-kappa B ) activation. Metbods: The 293 cell was trans fectedwith antisense IRAK-2 oligonucleotide (IRAK-2 ODN) followed by stimulating the cell with IL-1 or tumor necrosis factor (TNF), and then the levels of NF-kappa B activation was analyzed by sandwich enzyme-linked immunosorbent assay (ELISA ). Result: Pre-transfecting with antisense IRAK-2 ODN could remarkably decreasethe levels of NF-kappa B activation stimulated by IL-1 in time- and concentration-dependent manner, it can not attenuate the one stimulated by TNF. Conclusion: The responses of IL-1 and TNF-stimulated NF-kappa B activation to antisense IRAK-2 oligonucleotids were different. IRAK-2 plays a key role in the IL-1 signaling events leading to NF kappa B activation.展开更多
文摘目的探讨Rho激酶(ROCK)在氢气改善离体脓毒症肠屏障功能中的作用。方法常规培养人结肠上皮细胞Caco-2,分为6组(n=3):对照组(C组)、富氢培养基组(H组)、脂多糖(LPS)处理组(L组)、富氢培养基+LPS组(HL组)、Rho激酶抑制剂Y-27632组(Y组)、Y-27632+LPS组(YL组)。H组给予0.6 mmol/L富氢培养基;LPS和Y-27632的处理浓度分别为50 mg/L、25μmol/L。建立Transwell小室模型,定期检测跨上皮电阻值(TEER值),当TEER值达到800Ω·cm^2后给予处理,于6、12、24 h检测TEER值,24 h时检测FITC-右旋糖酐通过率。细胞接种于6孔板,融合达80%~90%后给予处理,实时聚合酶链式反应技术检测闭锁小带蛋白1(ZO-1)mRNA和ROCK mRNA表达情况;蛋白免疫印迹技术检测ZO-1蛋白和ROCK蛋白表达水平。结果与C组比较,H组12、24 h TEER值升高(P<0.05),FITC-右旋糖酐通过率、ZO-1蛋白和ROCK蛋白表达水平差异无统计学意义;Y组6、12、24 h TEER值升高(P<0.05),FITC-右旋糖酐通过率差异无统计学意义,ZO-1 mRNA表达增加,ROCK mRNA表达减少(均P<0.05);L组6、12、24 h TEER值降低,FITC-右旋糖酐通过率增高,ZO-1 mRNA和蛋白表达均下降,ROCK mRNA和蛋白表达均增加(P<0.05)。与L组比较,6、12、24 h YL组TEER值增高,FITC-右旋糖酐通过率降低,ZO-1 mRNA表达增加,ROCK mRNA表达降低(均P<0.05)。与L组比较,HL组6、12、24 h TEER值增高,FITC-右旋糖酐通过率降低,各时间点ZO-1蛋白表达上升,ROCK蛋白表达下降(均P<0.05)。结论氢气可保护脓毒症肠屏障功能,改善肠上皮屏障完整性和通透性,增加肠细胞间紧密连接蛋白表达,这些保护机制可能与氢气抑制LPS诱导的ROCK过度表达有关。
文摘目的:观察Rho激酶抑制剂法舒地尔(Fasudil)对人高侵袭潜能HCC细胞株3(human high metastatic liver cancer cells 3,HCCLM3)侵袭转移的影响,并且探讨其作用的机制。方法:应用100 mol/L Fasudil作用于HCCLM3细胞,采用肌动蛋白微丝荧光染色和侵袭小室实验观察HCCLM3细胞的运动侵袭能力。HCCLM3细胞经过处理后分为阴性对照组、Fasudil作用组、BTBD7干扰组,通过Western印迹检测BTB/POZ结构域蛋白7(BR-C,ttk and bab/pox virus domain containing 7,BTBD7)、Ras同系物家族成员C(ras homolog family member C,Rho C)、Rho关联卷曲螺旋蛋白激酶2(Rhoassociated,coiled-coil containing protein kinase 2,ROCK2)、MMP2和MMP9蛋白表达水平,酶谱分析法检测MMP2和MMP9活性水平。BTBD7干扰组作为阳性对照。结果:Fasudil处理后HCCLM3侵袭运动能力下降,BTBD7,Rho C,ROCK2蛋白表达下调,MMP2和MMP9活性降低,与阴性对照组比较差异有统计学意义(均P<0.01)。结论:Fasudil具有干预BTBD7-ROCK2信号通路、抑制HCC侵袭转移的重要作用。
基金supported by Yunnan Provincial Science and Technology Department, No.2009CD079the National Natural Science Foundation ofChina, No.81060109.
文摘In this study, PC12 Adh cells and Neuro-2a cells were treated with Rho-associated kinase inhibitors （Y27632 and Fasudil）, a cyclooxygenase-1 selective inhibitor （SC560）, and a cyclooxygenase-2 inhibitor （NS398）. We found that these cells became tolerant to Rho-associated kinase inhibitors, as neurite outgrowth induced by these inhibitors diminished following more than 3 days of exposure in either cell line. The proteins cyclooxygenase-2 and cytosolic prostaglandin E synthetase were upregulated at day 3. NS398 decreased the tolerance to neurite outgrowth induction in both cell lines, whereas SC560 had almost no effect. These findings indicate that cells become tolerant to neurite outgrowth induced by Rho-associated kinase inhibitors, this is at least partly associated with upregulation of proteins involved in the cyclooxygenase-2 pathway, and cyclooxygenases-2 inhibition prevents this tolerance.
文摘目的:观察芪白平肺胶囊对慢性阻塞性肺疾病(COPD)痰瘀阻肺证模型大鼠肺功能、形态学改变以及对于血清Rock-1(Rho关联卷曲蛋白激酶1)、Rho关联卷曲蛋白激酶2(Rock-2)表达的影响。方法:健康SD雄性大鼠50只,随机分组为正常对照组、模型组、芪白平肺胶囊低剂量组、芪白平肺胶囊高剂量组、法舒地尔组,复制COPD痰瘀阻肺证大鼠模型,每日造模结束后予大鼠灌胃。模型组、正常组每天生理盐水灌胃10 m L/kg,芪白平肺胶囊低剂量组、芪白平肺胶囊高剂量组每天给药量分别为1000 mg/kg、250 mg/kg(含芪白平肺胶囊50 mg/m L),法舒地尔每天给药量为2 m L/kg(含法舒地尔15mg/m L),每只大鼠每日灌胃1次,直至造模结束,测定大鼠肺功能、观察肺血管重构的病理改变,ELISA检测模型大鼠血清Rock-1、Rock-2的表达。结果:模型组肺小动脉管壁变厚、管腔变小,血清中Rock-1、Rock-2较正常组含量显著升高,(P<0.01);FEV0.3、FEV0.3/FVC、PEF均明显降低,差异有统计学意义(P<0.01);各治疗组与模型组比较肺小动脉管壁变薄,管腔增大,血清中Rock-1、Rock-2表达水平均有不同程度下降(P<0.01-0.05)。结论:芪白平肺胶囊对COPD大鼠的肺组织具有保护作用,可有效的防止痰瘀阻肺证慢阻肺模型大鼠肺功能的下降,降低COPD痰瘀阻肺证模型大鼠血清Rock-1、Rock-2表达水平。
基金supported by a grant from the National Natural Science Foundation of China (30972920)
文摘BACKGROUND: The increased β-arrestin-2 and its combination with G-protein-coupled receptors (GPCRs) lead to GPCRs desensitization. The latter may be responsible for decreased contractile reactivity in the mesenteric arteries of cirrhotic patients and rats. The present study is to investigate the machinery changes of α-adrenergic receptors and G proteins and their roles in the contractility of mesenteric arteries of cirrhotic patients and animal models. METHODS: Patients with cirrhosis due to hepatitis B and cirrhotic rats induced by CCl 4 were studied. Mesenteric artery contractility in response to norepinephrine was determined by a vessel perfusion system. The contractile effect of G protein-coupled receptor kinase-2 (GRK-2) inhibitor on the mesenteric artery was evaluated. The protein expression of the α 1 adrenergic receptor, G proteins, β-arrestin-2, GRK-2 as well as the activity of Rho associated coiled-coil forming protein kinase-1 (ROCK-1) were measured by Western blot. In addition, the interaction of α 1 adrenergic receptor with β-arrestin-2 was assessed by co-immunoprecipitation. RESULTS: The portal vein pressure of cirrhotic patients and rats was significantly higher than that of controls. The doseresponse curve to norepinephrine in mesenteric arteriole was shifted to the right, and EC 50 was significantly increased in cirrhotic patients and rats. There were no significant differences in the expressions of the α 1 adrenergic receptor and G proteins in the cirrhotic group compared with the controls. However, the protein expressions of GRK-2 and β-arrestin-2 were significantly elevated in cirrhotic patients and rats compared with those of the controls. The interaction of the α 1 adrenergic receptor and β-arrestin-2 was significantly aggravated. This interaction was significantly reversed by GRK-2 inhibitor. Both the protein expression and activity of ROCK-1 were significantly decreased in the mesenteric artery in patients with cirrhosis compared with those of the controls, and this phenomenon was not shown in the cirrhotic rats. Norepinephrine significantly increased the activity of ROCK-1 in normal rats but not in cirrhotic ones. Norepinephrine significantly increased ROCK-1 activity in cirrhotic rats when GRK-2 inhibitor was used. CONCLUSIONS: β-arrestin-2 expression and its interaction with GPCRs are significantly upregulated in the mesenteric arteries in patients and rats with cirrhosis. These upregulations result in GPCR desensitization, G-protein dysfunction and ROCK inhibition. These may explain the decreased contractility of the mesenteric artery in response to vasoconstrictors.
文摘Objective: To investigate the role of interleukin-1 (IL-1) receptor associated kinase-2 (IRAK-2) in IL1 and TNF-stimulated nuclear factor-kappa B (NF-kappa B ) activation. Metbods: The 293 cell was trans fectedwith antisense IRAK-2 oligonucleotide (IRAK-2 ODN) followed by stimulating the cell with IL-1 or tumor necrosis factor (TNF), and then the levels of NF-kappa B activation was analyzed by sandwich enzyme-linked immunosorbent assay (ELISA ). Result: Pre-transfecting with antisense IRAK-2 ODN could remarkably decreasethe levels of NF-kappa B activation stimulated by IL-1 in time- and concentration-dependent manner, it can not attenuate the one stimulated by TNF. Conclusion: The responses of IL-1 and TNF-stimulated NF-kappa B activation to antisense IRAK-2 oligonucleotids were different. IRAK-2 plays a key role in the IL-1 signaling events leading to NF kappa B activation.
