目的:研究内皮素-1(ET-1)诱导心肌肥大的机制及对抗的药物。方法:在培养新生大鼠心肌细胞中,采用L-型钙通道阻滞剂拉西地平(larc id ip ine)和MN9202、钙激活氯通道阻断剂尼氟灭酸(n iflum ic ac id,NFA)、蛋白激酶C(prote in k inase C...目的:研究内皮素-1(ET-1)诱导心肌肥大的机制及对抗的药物。方法:在培养新生大鼠心肌细胞中,采用L-型钙通道阻滞剂拉西地平(larc id ip ine)和MN9202、钙激活氯通道阻断剂尼氟灭酸(n iflum ic ac id,NFA)、蛋白激酶C(prote in k inase C,PKC)通路的阻断剂白屈菜季氨碱(chelerythrine,che)和ERK通路阻断剂PD98059(PD)观察内皮素-1在诱导心肌蛋白质合成中的影响。结果:对照组(DMEM)蛋白质含量为273±20μg/m l,ET-1组为312±30μg/m l,较对照组升高14%。ET-1+NFA组、ET-1+che组、ET-1+MN9202组、ET-1+larc id ip ine组、ET-1+PD98059组分别为280±10μg/m l、283±10μg/m l、285±27μg/m l、275±22μg/m l、293±33μg/m l;与ET-1组比较分别降低10%、9%、8.6%、13.1%、6.1%。结论:ET-1刺激引起的心肌细胞蛋白合成与钙激活氯通道和L-型钙通道有关,PKC和ERK通路在ET-1诱导心肌肥大的信号转导通路中起重要作用。展开更多
Objective： To explore the role that ceramide plays in the activation of mitogen-activated protein kinases （MAPKs） during cerebral ischemia and reperfusion. Methods： Rats were subjected to ischemia by the fourvesse...Objective： To explore the role that ceramide plays in the activation of mitogen-activated protein kinases （MAPKs） during cerebral ischemia and reperfusion. Methods： Rats were subjected to ischemia by the fourvessel occlusion （4-VO） method. The sphingomyelinase inhibitor TPCK was administered to the CA1 subregion of the rat hippocampus before inducing ischemia. Western blot was used to examine the activity of extracellular- signal regulated kinase （ERK） and c-Jun N-terminal protein kinase （JNK） using antibodies against ERK, JNK and diphosphorylated ERK and JNK. Results： At lh reperfusion post-ischemia, JNK reached its peak activity while ERK was undergoing a sharp inactivation （P 〈 0.05）. The level of diphosphorylated JNK was significantly reduced but the sharp inactivation of ERK was visibly reversed （P 〈 0.05） by the sphingomyelinase inhibitor. Conclusion： The ceramide signaling pathway is up-regulated through sphingomyelin hydrolysis in brain ischemia, promoting JNK activation and suppressing ERK activation, culminating in the ischemic lesion.展开更多
Objective： To study the effect of salvianolic acid B （SAB） and curcumin, the extracts of Solvie Miltiorrhize and Curcume Longe, on the proliferation and activation of hepatic stellate cell （HSC）, and the extracel...Objective： To study the effect of salvianolic acid B （SAB） and curcumin, the extracts of Solvie Miltiorrhize and Curcume Longe, on the proliferation and activation of hepatic stellate cell （HSC）, and the extracellular signal regulated kinase （ERK） expression in it. Methods： Rat＇s HSC-T6 were cultured and treated by SAB or curcumin. The inhibitory effect on cell proliferation was determined by 3-（4,5-dimthyl-2- 2thiazoly）-2,5-diphenyl-2H-tetrazolium bromide （MTT） colorimetry, and the expression levels of a smooth actin （a-SMA）, collagen type Ⅰ , and ERK were determined by Western blot. Results： SAB and curcumin inhibited the proliferation and activation of rat＇s HSC-T6 in dose-dependent fashion and significantly reduced the expression level of a-SMA （ P〈0.01 ）. Curcumin significantly reduced the expression of collagen type Ⅰ （P〈0.05）. Both SAB and curcumin showed insignificant effect on the ERK expression level, but they could significantly reduce the level of phosphorylated-ERK expression, showing significant difference as compared with that in the control group （ P〈0.01 and P〈0.05 respectively）. Conclusion： SAB and curcumin could significantly inhibit the proliferation, activation of HSC, and the production of type Ⅰ collagen in HSC, the mechanism may be associated with their inhibition on ERK phosphorylation.展开更多
AIM:To investigate the role of hepatocyte growth factor(HGF) in cholangiocarcinoma(CCA) cell invasiveness and the mechanisms underlying such cellular responses. METHODS:Effects of HGF on cell invasion and motility wer...AIM:To investigate the role of hepatocyte growth factor(HGF) in cholangiocarcinoma(CCA) cell invasiveness and the mechanisms underlying such cellular responses. METHODS:Effects of HGF on cell invasion and motility were investigated in two human CCA cell lines,HuCCA-1 and KKU-M213,using Transwell in vitro assay.Levels of proteins of interest and their phosphorylated forms were determined by Western blotting.Localization of E-cadherin was analyzed by immunofluorescence staining and visualized under confocal microscope. Activities of matrix degrading enzymes were determined by zymography. RESULTS:Both CCA cell lines expressed higher Met levels than the H69 immortalized cholangiocyte cell line.HGF induced invasion and motility of the cell lines and altered E-cadherin from membrane to cytoplasm localization,but did not affect the levels of secreted matrix metalloproteinase(MMP) -2,MMP-9 andurokinase plasminogen activator,key matrix degrading enzymes involved in cell invasion.Concomitantly,HGF stimulated Akt and extracellular signal-regulated kinase(ERK) 1/2 phosphorylation but with slightly different kinetic profiles in the two cell lines.Inhibition of the phosphoinositide 3-kinase(PI3K) /Akt pathway by the PI3K inhibitor,LY294002,markedly suppressed HGFstimulated invasion of both CCA cell lines,and inhibition of the ERK pathway by U0126 suppressed HGF-induced invasion of the KKU-M213 cell line but had a moderate effect on HuCCA-1 cells. CONCLUSION:These data indicate that HGF promotes CCA cell invasiveness through dys-localization of E-cadherin and induction of cell motility by distinct signaling pathways depending on cell line type.展开更多
Background RhoA/ Rho kinase （ROCK） pathway is involved in pulmonary arterial hypertension （PAH） and pulmonary artery smooth muscle cell （PASMC） proliferation. Inhibition of ROCK has been proposed as a treatment ...Background RhoA/ Rho kinase （ROCK） pathway is involved in pulmonary arterial hypertension （PAH） and pulmonary artery smooth muscle cell （PASMC） proliferation. Inhibition of ROCK has been proposed as a treatment for PAH. But the mechanism of RhoA/ROCK pathway and its downstream signaling in proliferation of human PASMCs is unclear. We investigated the effect of fasudil, a selective ROCK inhibitor, on platelet-derived growth factor （PDGF） induced human PASMC proliferation, and the possible association between RhoA/ROCK and extracellular signal-regulated kinase （ERK）,p27KiP1.Methods Human PASMCs were cultured with the stimulation of 10 ng/ml PDGF, and different concentrations of fasudil were added before the addition of mitogen. Cell viability and cell cycle were determined with MTT and flow cytometry respectively. ROCK activity, ERK activity and protein expression of proliferating cell nuclear angigen （PCNA） and p27Kip1 were measured by immunoblotting.Results By MTT assay, PDGF significantly increased the OD value that represented human PASMC proliferation, and pretreatment with fasudil significantly reversed this effect in a dose-dependent manner. After PDGF stimulation, the percentage of cells in S phase increased dramatically from 15.6% to 24.3%, while the percentage in G0/G1 phase was reduced from 80.6% to 59%. And pretreatment with fasudil reversed the cell cycle effect of PDGF significantly in a dose-dependent manner. PDGF markedly induced ROCK activity and ERK activity with a peak at 15 minutes, which were significantly inhibited by fasudil. In addition, fasudil significantly inhibited PDGF-induced PCNA expression and fasudil also upregulated p27Kip1 expression in human PASMCs, which decreased after PDGF stimulation.Conclusion RhoA/ROCK is vital for PDFG-induced human PASMC proliferation, and fasudil effectively inhibited PDGF-induced human PASMC proliferation by up-regulation of p27Kip1, which may be associated with inhibition of ERK activity.展开更多
文摘目的:研究内皮素-1(ET-1)诱导心肌肥大的机制及对抗的药物。方法:在培养新生大鼠心肌细胞中,采用L-型钙通道阻滞剂拉西地平(larc id ip ine)和MN9202、钙激活氯通道阻断剂尼氟灭酸(n iflum ic ac id,NFA)、蛋白激酶C(prote in k inase C,PKC)通路的阻断剂白屈菜季氨碱(chelerythrine,che)和ERK通路阻断剂PD98059(PD)观察内皮素-1在诱导心肌蛋白质合成中的影响。结果:对照组(DMEM)蛋白质含量为273±20μg/m l,ET-1组为312±30μg/m l,较对照组升高14%。ET-1+NFA组、ET-1+che组、ET-1+MN9202组、ET-1+larc id ip ine组、ET-1+PD98059组分别为280±10μg/m l、283±10μg/m l、285±27μg/m l、275±22μg/m l、293±33μg/m l;与ET-1组比较分别降低10%、9%、8.6%、13.1%、6.1%。结论:ET-1刺激引起的心肌细胞蛋白合成与钙激活氯通道和L-型钙通道有关,PKC和ERK通路在ET-1诱导心肌肥大的信号转导通路中起重要作用。
基金supported by grants from the National Natural Science Foundation of China (No.30871200)the Practice and Innovation Training Program for Students in Colleges and Universities of Jiangsu Province (NO.20090370)
文摘Objective： To explore the role that ceramide plays in the activation of mitogen-activated protein kinases （MAPKs） during cerebral ischemia and reperfusion. Methods： Rats were subjected to ischemia by the fourvessel occlusion （4-VO） method. The sphingomyelinase inhibitor TPCK was administered to the CA1 subregion of the rat hippocampus before inducing ischemia. Western blot was used to examine the activity of extracellular- signal regulated kinase （ERK） and c-Jun N-terminal protein kinase （JNK） using antibodies against ERK, JNK and diphosphorylated ERK and JNK. Results： At lh reperfusion post-ischemia, JNK reached its peak activity while ERK was undergoing a sharp inactivation （P 〈 0.05）. The level of diphosphorylated JNK was significantly reduced but the sharp inactivation of ERK was visibly reversed （P 〈 0.05） by the sphingomyelinase inhibitor. Conclusion： The ceramide signaling pathway is up-regulated through sphingomyelin hydrolysis in brain ischemia, promoting JNK activation and suppressing ERK activation, culminating in the ischemic lesion.
基金Supported by the National Natural Science Foundation of China (No. 30300458)
文摘Objective： To study the effect of salvianolic acid B （SAB） and curcumin, the extracts of Solvie Miltiorrhize and Curcume Longe, on the proliferation and activation of hepatic stellate cell （HSC）, and the extracellular signal regulated kinase （ERK） expression in it. Methods： Rat＇s HSC-T6 were cultured and treated by SAB or curcumin. The inhibitory effect on cell proliferation was determined by 3-（4,5-dimthyl-2- 2thiazoly）-2,5-diphenyl-2H-tetrazolium bromide （MTT） colorimetry, and the expression levels of a smooth actin （a-SMA）, collagen type Ⅰ , and ERK were determined by Western blot. Results： SAB and curcumin inhibited the proliferation and activation of rat＇s HSC-T6 in dose-dependent fashion and significantly reduced the expression level of a-SMA （ P〈0.01 ）. Curcumin significantly reduced the expression of collagen type Ⅰ （P〈0.05）. Both SAB and curcumin showed insignificant effect on the ERK expression level, but they could significantly reduce the level of phosphorylated-ERK expression, showing significant difference as compared with that in the control group （ P〈0.01 and P〈0.05 respectively）. Conclusion： SAB and curcumin could significantly inhibit the proliferation, activation of HSC, and the production of type Ⅰ collagen in HSC, the mechanism may be associated with their inhibition on ERK phosphorylation.
基金Supported by Mahidol University,Thailand and Thailand Research Fund(Suthiphongchai T)Strategic Consortia for Capacity Building of University Faculties and Staff Scholarship,Commission on Higher Education,Ministry of Education,Thailand(Menakongka A)
文摘AIM:To investigate the role of hepatocyte growth factor(HGF) in cholangiocarcinoma(CCA) cell invasiveness and the mechanisms underlying such cellular responses. METHODS:Effects of HGF on cell invasion and motility were investigated in two human CCA cell lines,HuCCA-1 and KKU-M213,using Transwell in vitro assay.Levels of proteins of interest and their phosphorylated forms were determined by Western blotting.Localization of E-cadherin was analyzed by immunofluorescence staining and visualized under confocal microscope. Activities of matrix degrading enzymes were determined by zymography. RESULTS:Both CCA cell lines expressed higher Met levels than the H69 immortalized cholangiocyte cell line.HGF induced invasion and motility of the cell lines and altered E-cadherin from membrane to cytoplasm localization,but did not affect the levels of secreted matrix metalloproteinase(MMP) -2,MMP-9 andurokinase plasminogen activator,key matrix degrading enzymes involved in cell invasion.Concomitantly,HGF stimulated Akt and extracellular signal-regulated kinase(ERK) 1/2 phosphorylation but with slightly different kinetic profiles in the two cell lines.Inhibition of the phosphoinositide 3-kinase(PI3K) /Akt pathway by the PI3K inhibitor,LY294002,markedly suppressed HGFstimulated invasion of both CCA cell lines,and inhibition of the ERK pathway by U0126 suppressed HGF-induced invasion of the KKU-M213 cell line but had a moderate effect on HuCCA-1 cells. CONCLUSION:These data indicate that HGF promotes CCA cell invasiveness through dys-localization of E-cadherin and induction of cell motility by distinct signaling pathways depending on cell line type.
基金This work was supported by the National Basic Research Development Program of China (No. 2007CB512005)the National NaturalScience Foundation of China (No. 30770785)+1 种基金the Cultivation Fund of the Key Scientific and Technical Innovation Project of Ministry ofEducation, China (No. 705045)the Research Fund for the Doctoral Program (No. 20050698012).
基金This study was supported by grants from the National Natural Science Foundation of China （No. 30972958）, Beijing Natural Science Foundation （No. 7112046）, Beijing Municipal Education Commission （No. PXM2011_014226 07 000060）.
文摘Background RhoA/ Rho kinase （ROCK） pathway is involved in pulmonary arterial hypertension （PAH） and pulmonary artery smooth muscle cell （PASMC） proliferation. Inhibition of ROCK has been proposed as a treatment for PAH. But the mechanism of RhoA/ROCK pathway and its downstream signaling in proliferation of human PASMCs is unclear. We investigated the effect of fasudil, a selective ROCK inhibitor, on platelet-derived growth factor （PDGF） induced human PASMC proliferation, and the possible association between RhoA/ROCK and extracellular signal-regulated kinase （ERK）,p27KiP1.Methods Human PASMCs were cultured with the stimulation of 10 ng/ml PDGF, and different concentrations of fasudil were added before the addition of mitogen. Cell viability and cell cycle were determined with MTT and flow cytometry respectively. ROCK activity, ERK activity and protein expression of proliferating cell nuclear angigen （PCNA） and p27Kip1 were measured by immunoblotting.Results By MTT assay, PDGF significantly increased the OD value that represented human PASMC proliferation, and pretreatment with fasudil significantly reversed this effect in a dose-dependent manner. After PDGF stimulation, the percentage of cells in S phase increased dramatically from 15.6% to 24.3%, while the percentage in G0/G1 phase was reduced from 80.6% to 59%. And pretreatment with fasudil reversed the cell cycle effect of PDGF significantly in a dose-dependent manner. PDGF markedly induced ROCK activity and ERK activity with a peak at 15 minutes, which were significantly inhibited by fasudil. In addition, fasudil significantly inhibited PDGF-induced PCNA expression and fasudil also upregulated p27Kip1 expression in human PASMCs, which decreased after PDGF stimulation.Conclusion RhoA/ROCK is vital for PDFG-induced human PASMC proliferation, and fasudil effectively inhibited PDGF-induced human PASMC proliferation by up-regulation of p27Kip1, which may be associated with inhibition of ERK activity.
