Epac1对大鼠内脏高敏感的调控作用及其机制

Regulating effect of Epac1 on visceral hypersensitivity in rats and its mechanism

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。

Objective To investigate the regulating effect of Epacl on the visceral hypersensitivity in rats and its downstream mechanism. Methods Forty-five neonatal SD male rats were randomly divided into three groups： the control group, model group and CE3F4 group with 15 rats per group. Visceral hypersensitive model was established in the model group and CEBF4 group but not in the control group. At the age of 8 weeks, rats in the control group and model group were both injected by 25 μL normal saline intrathecally, while CE3F4 group was injected by 25 p,L solution of Epacl specific an- tagonist CE3F4 （0.2 nmol/μL）. Three days after injection, AWR scores, pain threshold and maximal tolerance threshold at graded colorectal distension pressures （20, 40, 60 and 80 mmHg） were examined. Real-time quantitative PCR and Western blotting were operated to investigate the mRNA and protein expression levels of Epacl and PKC8 in L5-S1 colonic- afferent dorsal root ganglions （DRGs）. Results Compared with the control group, AWR scores in model group were in- creased at 40, 60 and 80 mmHg, while the pain threshold and maximal tolerance threshold were decreased （ all P 〈 0.05）, which indicated the successful establishment of visceral hypersensitive model. Compared with the model group, AWR scores in the CE3F4 group were decreased at 40 and 60 mmHg, while the pain threshold and maximal tolerance threshold were increased （all P 〈 0.05 ）, meanwhile, the CE3F4 group was significantly higher than the model group （ all P 〈 0. 05）. Compared with the control group, the expression levels of Epacl and PKGsat mRNA and protein in DRGs of the model group were increased （all P 〈 0.05）. Compared with the model group, the expression levels of Epacl at mRNA and protein in DRGs of the CE3F4 group were not significantly different （all P 〉 0.05 ）, but the expression of PKCe at mRNA and protein levels in DRGs of the CE3F4 group were significantly decreased （all P 〈 0.05）. Conclusions Epacl partici-pates in the regulation of visceral hypersensitivity and the mechanism may be related with the downstream activation of PKCε.