Nerve growth factor（NGF） promotes axonal growth in PC12 cells primarily by regulating the RTK-RAS-MEK-ERK pathway. Panaxydol, a polyacetylene isolated from Panax notoginseng, can mimic the effects of NGF. Panaxydol ...Nerve growth factor（NGF） promotes axonal growth in PC12 cells primarily by regulating the RTK-RAS-MEK-ERK pathway. Panaxydol, a polyacetylene isolated from Panax notoginseng, can mimic the effects of NGF. Panaxydol promotes neurite outgrowth in PC12 cells, but its molecular mechanism remains unclear. Indeed, although alkynol compounds such as panaxydol can increase intracellular cyclic adenosine 3′,5′-monophosphate（cAMP） levels and the ERK inhibitor U0126 inhibits alkynol-induced axonal growth, how pathways downstream of cAMP activate ERK have not been investigated. This study observed the molecular mechanism of panaxydol-, NGF-and forskolin-induced PC12 cell axon growth using specific signaling pathway inhibitors. The results demonstrated that although the RTK inhibitor SU5416 obviously inhibited the growth-promoting effect of NGF, it could not inhibit the promoting effect of panaxydol on axonal growth of PC12 cells. The adenylate cyclase inhibitor SQ22536 and cAMP-dependent protein kinase inhibitor RpcAMPS could suppress the promoting effect of forskolin and panaxydol on axonal growth. The ERK inhibitor U0126 inhibited axonal growth induced by all three factors. However, the PKA inhibitor H89 inhibited the promoting effect of forskolin on axonal growth but could not suppress the promoting effect of panaxydol. A western blot assay was used to determine the effects of stimulating factors and inhibitors on ERK phosphorylation levels. The results revealed that NGF activates the ERK pathway through tyrosine receptors to induce axonal growth of PC12 cells. In contrast, panaxydol and forskolin increased cellular cAMP levels and were inhibited by adenylyl cyclase inhibitors. The protein kinase A inhibitor H89 completely inhibited forskolin-induced axonal outgrowth and ERK phosphorylation, but could not inhibit panaxydol-induced axonal growth and ERK phosphorylation. These results indicated that panaxydol promoted axonal growth of PC12 cells through different pathways downstream of cAMP. Considering that exc展开更多
目的探讨环磷酸腺苷活化交换蛋白1（Epac1）对大鼠内脏高敏感的调控作用及其机制。方法选择雄性SD大鼠45只,随机分为对照组、模型组、CE3F4组、每组15只。模型组、CE3F4组建立内脏高敏感模型,对照组不建模。标准环境下饲养至出生后第8周...目的探讨环磷酸腺苷活化交换蛋白1（Epac1）对大鼠内脏高敏感的调控作用及其机制。方法选择雄性SD大鼠45只,随机分为对照组、模型组、CE3F4组、每组15只。模型组、CE3F4组建立内脏高敏感模型,对照组不建模。标准环境下饲养至出生后第8周,对照组、模型组鞘内注射生理盐水25μL,CE3F4组鞘内注射0.2nmol/μL CE3F4溶液25μL。鞘内注射第4天采用球囊扩张法扩张结直肠,分别于20、40、60、80 mm Hg压力下行腹壁收缩反射（AWR）评分,同时测量疼痛感觉阈值、最大容量感觉阈值时的压力。应用qRT-PCR及蛋白印迹法检测支配结肠的L5~S1节段背根神经节（DRG）Epac1、蛋白激酶C（PKC）εmRNA和蛋白表达。结果与对照组比较,模型组40、60、80 mm Hg压力时AWR评分升高,疼痛感觉阈值与最大容量感觉阈值时的压力下降（P均〈0.05）,提示成功建立内脏高敏感模型。与模型组比较,CE3F4组在40、60 mm Hg压力时AWR评分显著下降（P均〈0.05）。模型组在疼痛感觉阈值、最大容量感觉阈值时的压力较对照组明显降低（P均〈0.05）,而CE3F4组较模型组明显升高（P均〈0.05）。与对照组比较,模型组DRG的Epac1、PKCεmRNA和蛋白表达均显著升高（P均〈0.05）。与模型组比较,CE3F4组Epac1 mRNA和蛋白表达无明显变化（P均〉0.05）,但PKCεmRNA和蛋白表达显著降低（P均〈0.05）。结论 Epac1可能参与大鼠内脏高敏感的调控,其机制与下游PKCε活化有关。展开更多
目的环磷酸腺苷交换蛋白1(the exchange protein directly activated by cAMP1,Epac1)可活化Ras样GTP酶,且与许多肿瘤发生发展相关,但在结直肠癌中鲜见报道。本研究探讨Epac1在结直肠癌中的表达特征及其临床意义。方法蛋白质印迹法检测...目的环磷酸腺苷交换蛋白1(the exchange protein directly activated by cAMP1,Epac1)可活化Ras样GTP酶,且与许多肿瘤发生发展相关,但在结直肠癌中鲜见报道。本研究探讨Epac1在结直肠癌中的表达特征及其临床意义。方法蛋白质印迹法检测结肠癌细胞株(Caco2、HT29和SW480)及正常结肠上皮黏膜细胞株(FHC)中Epac1蛋白表达;免疫组化(EnVision法)检测西南医科大学附属医院2009-03-01-2012-03-01行结直肠癌手术治疗的79例癌组织配对79例正常组织标本,以及22例腺瘤组织标本中Epac1的表达,分析Epac1表达与结直肠癌患者临床病理特征及预后相关性。结果 Caco2、HT29、SW480和FHC细胞中Epac1蛋白相对表达量分别为0.756±0.050、0.862±0.076、1.042±0.078和0.593±0.057,Caco2、HT29和SW480中Epac1蛋白表达高于FHC,差异有统计学意义,P<0.05;结直肠癌组织、正常组织和腺瘤组织中Epac1表达率分别为65.8%(52/79)、11.4%(9/79)和22.7%(5/22),差异有统计学意义,χ~2=52.491,P<0.001;Epac1高表达与肿瘤TNM分期(χ~2=4.579,P=0.032)、浸润深度(χ~2=6.706,P=0.010)和淋巴结转移(χ~2=5.157,P=0.023)有关,而与肿瘤大小、肿瘤部位和分化程度无关,均P>0.05;Epac1高表达与低表达患者5年总生存率分别为53.8%和77.8%(χ~2=4.495,P=0.034),5年无病生存率分别为43.1%和53.9%(χ~2=4.956,P=0.026),差异均有统计学意义。结论 Epac1在结直肠癌细胞及组织中表达上调,其异常高表达可能参与结直肠癌的发生发展过程,Epac1对结直肠癌预后评价有一定参考价值。展开更多
OBJECTIVE In this study we explored the role of Epac1-Rap1 pathway in the acute myocardial ischemia/reperfusion injury(MIRI) in vitro and in vivo.METHODS An acute myocardial ischemia/reperfusion injury model was estab...OBJECTIVE In this study we explored the role of Epac1-Rap1 pathway in the acute myocardial ischemia/reperfusion injury(MIRI) in vitro and in vivo.METHODS An acute myocardial ischemia/reperfusion injury model was established by the ligation of left anterior descending coronary.Myocardial architecture,fibers and apoptosis was evaluated by the Masson trichrome staining,Sirius red staining and TUNEL assay.H9c2 cells were subjected to hypoxia for 5 h followed by 1-h reoxygen.ation in vitro.Cell viability was measured by MTT assay and cellular injury was evaluated by measuring the release of lactate dehydrogenase(LDH).Western blot,real-time PCR and immunofluorescence were used to detect the expressions of Epac1 and relative downstream molecules.RESULTS Myocardial IR-induced cardiac apoptosis and accumulation of Epac1 and Rap1 in rat IR injury model.Direct Epac activation by 8-CPT(8-(4-chlorophenylthio)-2′-O-methyl-cAMP) exacerbated cardiomyocyte death and dysfunction following hypoxia-reoxygenation(H/R),selective activation of Epac in response to H/R was evident which enriched for cytosolic/membrane proteins and mRNA.Harmacological inhibitor of Epac(ESI-09) significantly ameliorated myocardial injury with the decline of Epac expression.Epac inhibitor and agonist studies also implicated the effect of Rap1,which is downstream of Epac in this pathway.The expression of Rap1 elevated when activated by Epac agonist and was blocked by Epac inhibitor.The same result was true for myocyte CaMK-II and intracellular calcium ions activation.Moreover,ESI-09 also increased ERK1/2 phosphorylation.CONCLUSION Our study reveal that Epac1/Rap1 signaling pathway is involved in the pathogenesis of myocardial I/R injury in rats,which provides evidence on the development of therapeutic strategies target this pathway for myocardial I/R injury.展开更多
基金supported partly by the National Natural Science Foundation of China,No.30873057,81171245a grant from the Key Basic Project of Shanghai Municipal Science and Technology Commission of China,No.08JC1413600,11JC1406600
文摘Nerve growth factor（NGF） promotes axonal growth in PC12 cells primarily by regulating the RTK-RAS-MEK-ERK pathway. Panaxydol, a polyacetylene isolated from Panax notoginseng, can mimic the effects of NGF. Panaxydol promotes neurite outgrowth in PC12 cells, but its molecular mechanism remains unclear. Indeed, although alkynol compounds such as panaxydol can increase intracellular cyclic adenosine 3′,5′-monophosphate（cAMP） levels and the ERK inhibitor U0126 inhibits alkynol-induced axonal growth, how pathways downstream of cAMP activate ERK have not been investigated. This study observed the molecular mechanism of panaxydol-, NGF-and forskolin-induced PC12 cell axon growth using specific signaling pathway inhibitors. The results demonstrated that although the RTK inhibitor SU5416 obviously inhibited the growth-promoting effect of NGF, it could not inhibit the promoting effect of panaxydol on axonal growth of PC12 cells. The adenylate cyclase inhibitor SQ22536 and cAMP-dependent protein kinase inhibitor RpcAMPS could suppress the promoting effect of forskolin and panaxydol on axonal growth. The ERK inhibitor U0126 inhibited axonal growth induced by all three factors. However, the PKA inhibitor H89 inhibited the promoting effect of forskolin on axonal growth but could not suppress the promoting effect of panaxydol. A western blot assay was used to determine the effects of stimulating factors and inhibitors on ERK phosphorylation levels. The results revealed that NGF activates the ERK pathway through tyrosine receptors to induce axonal growth of PC12 cells. In contrast, panaxydol and forskolin increased cellular cAMP levels and were inhibited by adenylyl cyclase inhibitors. The protein kinase A inhibitor H89 completely inhibited forskolin-induced axonal outgrowth and ERK phosphorylation, but could not inhibit panaxydol-induced axonal growth and ERK phosphorylation. These results indicated that panaxydol promoted axonal growth of PC12 cells through different pathways downstream of cAMP. Considering that exc
文摘目的探讨环磷酸腺苷活化交换蛋白1（Epac1）对大鼠内脏高敏感的调控作用及其机制。方法选择雄性SD大鼠45只,随机分为对照组、模型组、CE3F4组、每组15只。模型组、CE3F4组建立内脏高敏感模型,对照组不建模。标准环境下饲养至出生后第8周,对照组、模型组鞘内注射生理盐水25μL,CE3F4组鞘内注射0.2nmol/μL CE3F4溶液25μL。鞘内注射第4天采用球囊扩张法扩张结直肠,分别于20、40、60、80 mm Hg压力下行腹壁收缩反射（AWR）评分,同时测量疼痛感觉阈值、最大容量感觉阈值时的压力。应用qRT-PCR及蛋白印迹法检测支配结肠的L5~S1节段背根神经节（DRG）Epac1、蛋白激酶C（PKC）εmRNA和蛋白表达。结果与对照组比较,模型组40、60、80 mm Hg压力时AWR评分升高,疼痛感觉阈值与最大容量感觉阈值时的压力下降（P均〈0.05）,提示成功建立内脏高敏感模型。与模型组比较,CE3F4组在40、60 mm Hg压力时AWR评分显著下降（P均〈0.05）。模型组在疼痛感觉阈值、最大容量感觉阈值时的压力较对照组明显降低（P均〈0.05）,而CE3F4组较模型组明显升高（P均〈0.05）。与对照组比较,模型组DRG的Epac1、PKCεmRNA和蛋白表达均显著升高（P均〈0.05）。与模型组比较,CE3F4组Epac1 mRNA和蛋白表达无明显变化（P均〉0.05）,但PKCεmRNA和蛋白表达显著降低（P均〈0.05）。结论 Epac1可能参与大鼠内脏高敏感的调控,其机制与下游PKCε活化有关。
文摘目的环磷酸腺苷交换蛋白1(the exchange protein directly activated by cAMP1,Epac1)可活化Ras样GTP酶,且与许多肿瘤发生发展相关,但在结直肠癌中鲜见报道。本研究探讨Epac1在结直肠癌中的表达特征及其临床意义。方法蛋白质印迹法检测结肠癌细胞株(Caco2、HT29和SW480)及正常结肠上皮黏膜细胞株(FHC)中Epac1蛋白表达;免疫组化(EnVision法)检测西南医科大学附属医院2009-03-01-2012-03-01行结直肠癌手术治疗的79例癌组织配对79例正常组织标本,以及22例腺瘤组织标本中Epac1的表达,分析Epac1表达与结直肠癌患者临床病理特征及预后相关性。结果 Caco2、HT29、SW480和FHC细胞中Epac1蛋白相对表达量分别为0.756±0.050、0.862±0.076、1.042±0.078和0.593±0.057,Caco2、HT29和SW480中Epac1蛋白表达高于FHC,差异有统计学意义,P<0.05;结直肠癌组织、正常组织和腺瘤组织中Epac1表达率分别为65.8%(52/79)、11.4%(9/79)和22.7%(5/22),差异有统计学意义,χ~2=52.491,P<0.001;Epac1高表达与肿瘤TNM分期(χ~2=4.579,P=0.032)、浸润深度(χ~2=6.706,P=0.010)和淋巴结转移(χ~2=5.157,P=0.023)有关,而与肿瘤大小、肿瘤部位和分化程度无关,均P>0.05;Epac1高表达与低表达患者5年总生存率分别为53.8%和77.8%(χ~2=4.495,P=0.034),5年无病生存率分别为43.1%和53.9%(χ~2=4.956,P=0.026),差异均有统计学意义。结论 Epac1在结直肠癌细胞及组织中表达上调,其异常高表达可能参与结直肠癌的发生发展过程,Epac1对结直肠癌预后评价有一定参考价值。
基金This work supported by the National Natural Science Foundation of China (81470432) and Natural Science Foundation of Anhui Province Education Department (KJ2016A357).
文摘OBJECTIVE In this study we explored the role of Epac1-Rap1 pathway in the acute myocardial ischemia/reperfusion injury(MIRI) in vitro and in vivo.METHODS An acute myocardial ischemia/reperfusion injury model was established by the ligation of left anterior descending coronary.Myocardial architecture,fibers and apoptosis was evaluated by the Masson trichrome staining,Sirius red staining and TUNEL assay.H9c2 cells were subjected to hypoxia for 5 h followed by 1-h reoxygen.ation in vitro.Cell viability was measured by MTT assay and cellular injury was evaluated by measuring the release of lactate dehydrogenase(LDH).Western blot,real-time PCR and immunofluorescence were used to detect the expressions of Epac1 and relative downstream molecules.RESULTS Myocardial IR-induced cardiac apoptosis and accumulation of Epac1 and Rap1 in rat IR injury model.Direct Epac activation by 8-CPT(8-(4-chlorophenylthio)-2′-O-methyl-cAMP) exacerbated cardiomyocyte death and dysfunction following hypoxia-reoxygenation(H/R),selective activation of Epac in response to H/R was evident which enriched for cytosolic/membrane proteins and mRNA.Harmacological inhibitor of Epac(ESI-09) significantly ameliorated myocardial injury with the decline of Epac expression.Epac inhibitor and agonist studies also implicated the effect of Rap1,which is downstream of Epac in this pathway.The expression of Rap1 elevated when activated by Epac agonist and was blocked by Epac inhibitor.The same result was true for myocyte CaMK-II and intracellular calcium ions activation.Moreover,ESI-09 also increased ERK1/2 phosphorylation.CONCLUSION Our study reveal that Epac1/Rap1 signaling pathway is involved in the pathogenesis of myocardial I/R injury in rats,which provides evidence on the development of therapeutic strategies target this pathway for myocardial I/R injury.
