两种亚型肠易激综合征中氨基酸神经递质及其受体变化

Contents of amino acid neurotransmitters and expression of γ-aminobutyric acid receptor in two subtypes of irritable bowel syndrome

目的:探讨脑局部微透析-高效液相色谱(high performance liquid chromatography,HPLC)法测定谷氨酸(glutamate,Glu)、甘氨酸(glycine,Gly)、γ-氨基丁酸(γ-aminobutyric acid,γ-GABA)、牛磺酸(taurine,Tau)含量的可行性,便秘主导型肠易激综合征[irritable bowel syndrome(IBS)with constipation,IBS-C]和腹泻主导型肠易激综合征(IBS with diarrhea,IBS-D)大鼠脑组织中氨基酸类神经递质含量及γ-氨基丁酸受体A(γ-aminobutyric acid receptor A,G A B AAα1)蛋白的表达情况,初步探讨IBS-C、IBS-D的共性发病机制.方法:以体外相对回收率和体内相对损失率为考察指标,分析Glu、Gly、Tau和γ-GABA的脑局部微透析采样可行性,建立荧光高效液相色谱分析方法;采用冰水灌胃法建立IBS-C动物模型,番泻叶灌胃结合束缚刺激法建立IBS-D动物模型,并检测各组大鼠脑内氨基酸类神经递质含量;采用Western blot法测定各组大鼠脑组织中GABAAα1蛋白的表达情况.结果:脑局部微透析-HPLC法可用于脑内Glu、γ-GABA的含量测定,体外相对回收率分别约为21.6%、24.5%,体内相对损失率分别约为41.8%、32.5%;与正常组大鼠相比,IBS-C组和IBS-D组大鼠脑内Glu/γ-GABA比值均显著性升高(P<0.05),GABAAα1蛋白表达显著增加(P<0.05);与IBS-C组大鼠比较,IBS-D组大鼠脑内GABAAα1受体蛋白表达显著增加(P<0.05).结论:IBS-C、IBS-D的发病可能与脑内兴奋性氨基酸Glu和抑制性氨基酸γ-GABA含量比值、GABAAα1蛋白表达量有关,存在共性基础.

AIM： To explore the feasibility of using in vivo cerebral microdialysis and high performance liquid chromatography（HPLC） to determine the contents of glutamate（Glu）, glycine（Gly）, γ-aminobutyric acid（γ-GABA） and taurine（Tau）, and to observe the protein expression of γ-aminobutyric acid receptor A（GABAAα1） in irritable bowel syndrome with diarrhea（IBS-D） and irritable bowel syndrome with constipation（IBS-C） to investigate the preliminary pathogenesis of these two subtypes. METHODS： Using in vitro relative recovery and in vivo relative loss ratio as monitoring indicators, the feasibility of using cerebral microdialysis and HPLC to determine the contents of Glu, Gly, Tau and γ-GABA was first explored. IBS-C was induced by cold water administration and IBS-D by folium sennae combined with restraint. Cerebral microdialysis and HPLC were then used to determine the contents of Glu, Gly, Tau and γ-GABA in the rat brain. Western blot was used to detect the expression of GABAAα1 protein in brain tissue. RESULTS： In vivo cerebral microdialysis and HPLC were feasible for determining the contents of Glu and γ-GABA; the in vitro relative recovery rates were 21.6% and 24.5%, respectively; in vivo relative loss rates were 41.8% and 32.5%. Compared with rats in the normal group, the ratio of Glu/γ-GABA in the IBS-C and IBS-D groups significantly increased（P 〈0.05）. GABAAα1 protein expression in both IBS-C and IBS-D groups were significantly higher than that in the normal group（P〈 0.05）. In addition, GABAAα1 protein expression in the IBS-D group was significantly higher than that in the IBS-C group（P〈 0.05）.CONCLUSION： The pathogenesis of IBS-C and IBS-D may be related with Glu/γ-GABA ratio and GABAα1 in the brain.