肠上皮γ-氨基丁酸能信号通路调节小肠液分泌及其作用机制
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摘要
第一部分：γ-氨基丁酸能信号通路在肠上皮表达及对小肠液分泌的调节
目的
γ-氨基丁酸(γaminobutyric acid,GABA)是中枢神经系统中最主要的抑制性神经递质之一,其在大脑和脊髓分布广泛,参与突触后神经元的快速抑制效应。在生理条件下,GABA由神经元内谷氨酸脱羧酶(GAD)催化谷氨酸形成。根据分子量的不同,GAD可分为两种,分别为：GAD65(65kDa)和GAD67(67kDa).GAD65主要分布在神经元轴突末梢内的囊泡膜上,参与囊泡释放的GABA的合成；GAD67则在神经元胞体内散在分布,其合成的GABA释放后与突触外的GABA受体结合发挥作用。神经元轴突末梢的GABA释放后快速激活突触后膜上的GABAA(γ-氨基丁酸A型)受体门控的氯离子通道,引起氯离子向细胞内流动,使突触后神经元细胞膜发生超极化,从而抑制神经元的兴奋性。GABA、GABA受体及其代谢酶等分子统称为GABA信号系统(GABAergic signal system)。除中枢神经系统之外,GABA能信号系统在外周组织也广泛表达,如肺、肝脏、脾、生殖系统等。GABAA受体作为氯离子通道,在肺上皮细胞腔面侧表达,并且被证实通过氯离子跨膜向细胞外转运调节气管和支气管电解质和粘液的分泌。氯离子向肠腔内的跨膜转运是肠道液体分泌的原动力,肠道内氯离子的聚集为水、钠向肠道转运提供渗透压和电化学驱动力基础。有报道称GABA能信号系统在胃癌和结肠癌细胞内高水平表达,但是其在正常消化道各部分的表达和生理作用尚不十分清楚。我们推测GABA能信号系统可能在肠粘膜上皮表达,并且有可能参与肠道液体分泌。因此本研究的目的是观察GABA能信号系统是否在小肠组织表达,尤其是在参与液体分泌的上皮细胞上的表达情况；探讨GABA能信号系统在肠粘膜上皮表达的生理意义。
方法
RT-PCR(反转录聚合酶链反应)
无菌条件下,将IEC-18(a cell line derived from the ileum of rat intestine)细胞种植于直径为10cm的圆形培养皿中；待细胞浓度增殖达到90%以上时,用4℃预冷的磷酸盐缓冲液(Phosphate buffered saline, PBS)冲洗细胞三次,加入Trizol试剂提取细胞RNA。
健康成年雄性Wistar大鼠脱颈牺牲后,小心取出大脑组织,用4℃预冷的PBS冲洗3次,剥离出新鲜大脑皮质,剪碎后,加入Trizol提取组织RNA。按照反转录试剂盒的说明,提取细胞和组织的cDNA;然后用GABAA受体各亚单位和GAD65/67的引物通过PCR扩增得到相应的DNA。再用DNA凝胶电泳观察各目的带表达情况。
免疫组织化学
石蜡切片染色步骤：将新鲜动物组织用4%多聚甲醛(PFA)固定液浸泡24小时后,将组织脱水并用石蜡包埋后切片(组织厚度：4-5μm),依次进行脱蜡、水化和抗原修复处理。用10%与二抗来源相同的血清室温条件下封闭切片1小时后,加入一抗,4℃环境下孵育过夜。PBS冲洗切片3次,每次5分钟,加入荧光标记的二抗室温避光孵育1小时。PBS冲洗3次,每次5分钟,75%甘油缓冲液封片,荧光显微镜观察拍片。
细胞免疫染色步骤：将IEC-18细胞种植在10%多聚赖氨酸(PDL)包被的盖玻片上,培养48小时,待细胞贴壁良好后,室温条件下,用4%多聚甲醛迅速固定10分钟,PBS冲洗三次,每次5分钟,其余步骤同石蜡切片染色。
Western Blot
将新鲜组织或细胞匀浆后,4℃离心(12000g)10分钟,取离心后的上清液进行蛋白定量。用10%十二烷基硫酸钠聚丙烯酰胺凝胶(sodium dodecylsulfate polyacrylamide gel electrophoresis, SDS-PAGE)下层胶和5%的上层分离胶电泳,湿转方法将蛋白转移至孔径为0.45微米的PVDF (Polyvinylidene Fluoride)膜上,5%脱脂牛奶室温封闭PVDF膜1小时,加入一抗覆盖PVDF膜,4℃孵育过夜。用1倍的TTBS冲洗PVDF膜3次,每次10分钟。室温下加入辣根过氧化酶标记的二抗孵育1小时。用1倍TTBS冲洗3次,每次10分钟,ECL显影。
膜片钳记录实验
将IEC-18细胞种植在10%多聚赖氨酸包被的盖玻片上,24小时后待细胞贴壁,开始全细胞记录。记录时用正常细胞外液(155mM NaC1、1.3mM CaC12,5.4mM KC1、25mM HEPES和33mM葡萄糖(pH7.4,渗透压315mosmol/kgH2O))常温循环灌流细胞,用700B放大器进行记录。记录电极内液为155mM KC1、15mM KOH、10mM HEPES、2mM MgC12、1mM CaC12、10mM EGTA和2mM Tetraethylammonium(pH7.35,渗透压315mosmol/kgH20) o电压钳记录时,将细胞膜电位钳制在-60mV。信号采集后用低通滤波器(1-2kH)进行滤波处理。
在体小肠液分泌实验
成年雄性BALB/c小鼠,体重在25-30克之间,实验前禁食,自由饮水。用2%戊巴比妥钠(45-50mg/kg)腹腔注射麻醉动物,固定四肢和头部,在恒温条件下(36℃-38℃)行开腹手术。在距离盲肠1-2cm处结扎回肠,沿向心端方向量取2cm回肠进行结扎,使之形成长约2cm的回肠襻,用直径为0.33mm的胰岛注射器向肠襻内注射100μ1的生理盐水或药物,确保无渗漏后依次关闭腹腔各层。待动物苏醒后给予鼠粮和饮水。5小时后,脱颈牺牲小鼠,取出回肠襻,量取回肠襻的长度和称取去除肠液前后的肠襻重量,进行统计分析。
统计分析
小肠液分泌试验中,用去除肠液前后重量差除以回肠襻的长度(g/cm)作为观察指标,比较对照组和处理组之间有无统计学差异。
所有数据采用均数±标准误(Mean±SEM)表示,用单因素方差分析进行统计学处理,以P<0.05作为显著性差异界值。
实验结果
1、RT-PCR结果显示,小肠上皮细胞内表达GABA的合成酶谷氨酸脱羧酶65和67(GAD65/67)以及GABAA受体各亚单位：并且,在大鼠、小鼠的小肠以及小肠上皮细胞株等组织细胞表达GABAA受体β2、ππ亚单位以及GAD65/67蛋白。
2、免疫荧光染色结果显示GABA和GAD65/67在小肠上皮细胞胞浆内表达,GABAA受体p2/β3和π单位分别在IEC-18细胞膜和小鼠、胎猪的小肠绒毛上皮细胞腔面侧表达。
3、人的回肠隐窝和绒毛上皮细胞内也表达谷氨酸脱羧酶65/67和GABAA受体π亚单位。
4、膜片钳全细胞记录显示,小肠上皮IEC-18细胞,其膜电位在-37±8mV；在约36%的细胞上记录到GABA诱导的内向电流,GABAA受体特异性激动剂—muscimol可模拟上述反应。电流钳记录结果显示muscimol导致上皮细胞膜去极化。
5、小鼠回肠襻液体分泌实验表明,GABA(100μM)和muscimol(10μM)均促进小肠液体的分泌，这种促分泌作用不能被TTX(1μM,河豚毒素)阻断；而GABAA受体特异性阻断剂--Gabazine (100μM)则明显降低GABA引起的小肠液分泌量。
结论
1, GABA能信号系统在小鼠、大鼠、猪和人的小肠上皮细胞功能性表达
2, GABA可通过GABAA受体参与小肠液体分泌。
第二部分：γ-氨基丁酸能信号通路在腹泻发生中的作用及其机制
目的
腹泻是临床上常见的消化系统症状,病因和发病机制多样,但大多数是由于病毒或细菌毒素侵犯肠道黏膜,破环肠道结构和肠道上皮细胞正常的分泌和吸收,导致肠道组织通透性增加和上皮细胞电解质和粘液分泌增多,引起腹泻。由于小肠上皮细胞腔面膜上的氯离子跨膜转运是肠道水、钠、钾、碳酸氢根等物质分泌的原动力,很多腹泻的发生机制都离不开肠道上皮细胞膜上氯离子通道的参与,如CFTR和CaCC两种氯离子通道与霍乱毒素和旅行者腹泻的发生有直接关系。因此,对肠上皮细胞上氯离子通道的研究以及开发通道特异性的药物为临床治疗腹泻提供重要的理论基础。我们发现GABA能信号系统在小肠上皮细胞表达,外源性GABAA受体激动剂引起小肠液的大量分泌。因此我们推测,GABAA受体作为氯离子通道受体,有可能参与腹泻的发生过程。本部分我们对GABA能信号系统在食物过敏原诱导的腹泻过程中的作用及其机制进行了详细探讨。
方法
免疫组织化学同第一部分
食物致敏原诱导的腹泻动物模型的建立
健康成年雄性BALB/c小鼠(20-25g),随机分为对照组、OVA (50mg, Ovalbumin,卵白蛋白)诱导模型组、OVA诱导和Gabazine (100μM)处理组、OVA诱导和Picrotoxin(100μM)处理组(非特异性GABAA受体阻断剂)。实验前禁食,自由饮水。所有动物在处理第一天均用硫酸铝佐剂稀释的OVA (1mg)腹腔内注射,致敏全部小鼠。从第七天开始,对照组给予生理盐水250μl灌胃,模型组和处理组给予OVA和相应药物溶于生理盐水中250μl灌胃,1小时后观察并记录动物粪便性状和排便反应。隔天灌胃一次,共灌胃10次后牺牲动物,收集动物回肠进行免疫组织化学染色和Western blot检测。
Western Blot
动物组织蛋白获取和检测步骤同第一部分。
IL-13处理后的IEC-18细胞,预冷的PBS冲洗3次,用裂解液破碎和收集细胞,4℃离心,取离心上清液蛋白定量,加入上样缓冲液后变性蛋白,进行SDS-PAGE电泳。湿转方法将蛋白转移至PVDF膜,5%脱脂牛奶室温封闭1小时,TTBS冲洗3次,每次10分钟,加入一抗,4℃环境下孵育过夜。TTBS冲洗3次,每次10分钟,加入辣根过氧化物酶标记的二抗,室温条件下孵育1小时,TTBS洗3次后,ECL显影。
统计分析
动物模型指标观察：灌胃后1小时观察动物粪便稀薄、透明、不成型视为腹泻模型成功,计数每次灌胃后模型成功动物的个数,依次累计,直至牺牲动物。以每次灌胃后各组动物腹泻发生率作为统计指标作图分析。
结果
1, OVA诱导的过敏性动物腹泻模型在第7次灌胃时达到100%成功；对照组动物无动物发生腹泻；用Gabazine和Picrotoxin处理的两组动物,明显降低小鼠腹泻的发生率并延缓腹泻的进程。
2,OVA诱导的腹泻动物回肠表达GAD67和GABAA受体β2/β3、π亚单位比对照组显著增高。GABAA受体阻断剂则降低动物肠道β2/β3蛋白的表达,而小肠上皮细胞和腔面膜上的谷氨酸脱羧酶65/67、π亚单位表达则增加。
3,IL-13作用4分钟和4.5分钟时最高。而GABAA受体β2/β3亚单位蛋白表
达量亦在IL-13处理后4分钟开始增高。
结论
1,γ-氨基丁酸能信号系统在过敏性动物腹泻发病过程中表达明显增加,特异性或非特异性阻断该系统后,动物经OVA诱导后腹泻的发病率明显降低,且发病进程减缓。
2,Y-氨基丁酸能信号系统在腹泻发病过程中的上调很可能是由PI3K-AKT-GABAA-R信号通路介导完成。
Part1GABAergic signal system is expressed in ileum epithelium and is involved in the regulation of fluid secretion
Objective
y-aminobutyric acid (GABA for short) is one of the most important inhibitory neurotransmitter in mammalian CNS which mediates the fast inhibition of neurons. Physiologically, GABA is synthesized by glutamate decarboxylase (GAD) from glutamate in neurons which are called GABAergic neurons. And there are two isoforms of GAD:GAD65and67identified by the different molecular weight. GAD65is found to sit in the GABA-contained vesicles near the axon terminals, and the GABA is released to the synaptic cleft acting on the GABAA receptors in postsynaptic membrane mediating fast inhibition in adult brain. While GAD67is distributed randomly in the cytosol, and GABA synthesized by GAD67is thought to act on GABA receptors located in extrasynaptic membrane. As for the release mechanism of cytosolic GABA remains obscure. GABAA receptors are chloride ion channels. GABA or its analogous activates GABAA receptors whose opening leads to chloride influx with membrane hyperpolarization as a consequent. In immature neurons or some types of neurons, GABAA receptor hyperpolarized cell membrane to excite cells. GABA, GABA receptors and GAD are termed as GABAergic signal system. Besides CNS, GABAergic signal system is found in many peripheral tissues and organs like lungs, reproductive system, kidney as well as gastrointestinal tract. GABA and GABAA receptors are reported to play important roles in regulating pulmonary epithelial fluid and mucus secretion. The system is found to be upregulated in gastric and colon carcinoma, but whether it is there in normal gastrointestinal tract is still unknown despite its physiological functions. We hypothesized that GABAergic signal system is expressed in human and animal intestine, which might contributes to the fluid and mucus secretion when activated.
Methods
RT-PCR
RNAs were extracted from IEC-18cell line and cortex of healthy male Wistar rat by Trizol. RT-PCR KIT was used to reproduce cDNA(complementary DNA). And GABAA receptor subunits and GAD DNA were amplified using their primers. The DNA bands were got by running DNA agar gel.
Immunohistochemistry
Paraffin embedded slice staining:fresh tissues were soaked in4%PFA (Paraformal dehyde) for24hours before paraffin embedding. Slice the tissue with4-5μm thickness. Followed by deparaffination, hydration and antigen repair. Then block the slice with5%serum for1hour at room temperature. Incubate the slices at4℃overnight with primary antibody after the serum blocking. Rinse slices3times with PBS the next day,5minutes per rinse. Incubate with fluorescence labeled secondary antibodies for1hour at room temperature. Rinse slices3times and5minutes per time. Seal the slice with75%glycerol buffer and take pictures with fluorescence microscope.
IEC-18cell staining:cells were planted on coverslips coated with10%Poly-L-Lysine. Fix cells with4%PFA for10minutes at room temperature, followed by PBS rinses for3times,5minutes per time. The rest procedures are the same as the paraffin embedded slice staining.
Western Blot Analysis
Fresh tissues and cells were homogenized, centrifuged at12000g for10min at4℃.Total proteins were fractionated on a5%to10%gradient SDS-PAGE (sodium d odecylsulfate polyacrylamide gel electrophoresis). They were transferred to0.45μm P olyvinylidene Fluoride (PVDF) membranes. Membranes were blocked in blocking bu ffer (5%non-fat dry milk, TTBS) for60min at room temperature, incubated with pri mary antibody at4℃overnight. After washing10minutes for three times, the PVDF membranes were incubated for1h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies followed by3times TTBS washing. Finally, immunoreactive proteins were detected by ECL (Electrochemiluminescence) plus.
Electrophysiology
IEC-18cells were incubated on PDL (Poly-D-Lysine) coated coverslips for24hours. Record cells using Axon700B amplifier. The cells were bathed in extracellular solution containing (in mmol/1):155NaCl,1.3CaC12,5.4KC1,25HEPES, and33glucose (pH7.4, osmolarity315mosmol/kgH2O) while the recording pipette was filled with intracellular solution containing (in mmol/1)155KC1,15KOH,10HEPES,2MgC12,1CaC12,10EGTA and2Tetraethylammonium (pH7.35, osmolarity:315mosmol/kgH2O).
In vivo intestinal fluid secretion experiment
Healthy male BALB/c mice (weight between25-30g) were used in this experiment. Animals were fast overnight while free drinking. Animals were anesthetized with2%pentobarbital sodium (45-50mg/kg intraperitoneal injection). Body temperature of mice was maintained between36and38℃during surgery using a heating pad. An abdominal incision (～1.5cm) was made to expose the small intestine, and a closed loop of empty ileum (20mm of length) proximal to the cecum was isolated by sutures. Ileum loops were injected with100μl normal saline (NS) alone or NS containing drugs. The abdominal incision was closed with sutures, and the animal was allowed to recover from anesthesia. Five hours later, sacrifice the mice, and ileum loops were isolated. The ileum loops were weighted after removal of mesentery and connective tissues. After the lumen fluid was released through a longitudinal incision, the weight and length of ileum loops were measured. The ratio of intestinal fluid weight to intestinal length was calculated.
Statistical Analysis
In the intestinal fluid secretion experiment, the relative values of intestinal fluid weight to intestinal length were used to analyze the difference between groups.
All the values in these experiments were presented as mean±SEM. One way analysis of variance (ANOVA) and the student's t-test was used to analyze. P<0.05was considered to be a significant difference.
Results
1, RT-PCR results showed GAD65/67and GABAA receptor subunits mRNA expression in intestinal epithelial cells. And GAD65/67,β2/β3and%subunits of GABAA receptor were found to be expressed in protein level using Western Blot technique.
2, Immunohistochemistry staining located GAD65/67to be in cytosol of epithelial cells, while β2/β3and π subunits of GABAA receptor sit in the apical side of intestinal villi.
3, GAD65/67and π subunits of GABAA receptor are also found in epithelial cells of human ileum.
4, Whole cell patch clamp of IEC-18cells showed an inward current triggered by1mM GAB A when holding cell membrane potential at-60mV. Muscimol, the selective GABAA receptor agonist, works the same as GAB A on IEC-18cells.5, In the intestinal fluid secretion experiment, GAB A (100μm) and Muscimol (10μm) showed similar increasing fluid accumulation in intestine, while the increased fluid secretion can only be partly blocked by TTX1μm, Tetrodotoxin). Gabazine, the selective GABAA receptor blocker, decreased intestinal fluid accumulation caused by GABA.
Conclusion
1, GABAergic signal system is functionally expressed in mouse, rat and human intestinal epithelial cells.
2, Exogenous GABAA receptor agonist and antagonist activate/deactivate GABAA receptor to regulate intestinal fluid secretion.
Part Ⅱ The role of GABAergic signal system in diarrhea and the underlying mechanisms
Objective
Diarrhea happens commonly in patients with digestive diseases, and there are kinds of mechanisms involved in diarrhea. Viruses and bacteria toxins invade intestinal mucous leading to increased permeability of the intestinal epithelium, and disturb secretion and absorption of the epithelia, thus leading to diarrhea. Chloride secretion from the apical membrane to lumen is the driving force for other ions and water efflux. The malfunction of chloride channel activity is always the direct reason for diarrhea. For example, Cholera and Travelers'diarrhea need the involvement of CFTR and CaCC channels. Both of CFTR and CaCC are chloride channels whose activating leads to water, electrolytes and mucus accumulation in intestinal lumen. It's important to discuss the roles that chloride channels. play in physiological and pathophysiological progress of intestine. Targeting chloride channels in intestinal epithelial membrane might be an alternative way to treat diarrhea.
GABAA receptors are found to be functionally expressed in intestinal epithelial cells according to our previous results. Since GABAA receptors are chloride channels too, it's necessary to explore the functional significance of GABAergic signal system. We aim to research the roles that GABAergic signal system plays in food allergen induced diarrhea and the underlying mechnisms.
Methods
Immunohistochemistry procedure is the same as part1.
Food allergen induced diarrhea animal model experiment protocol
Healthy male adult BALB/c mice (body weight between20-25g) were divided into four groups randomly:control group, OVA (50mg, Ovalbumin) induced diarrhea group, OVA mixed with Gabazine (selective GABAA receptor antagonist), OVA mixed with Picrotoxin (nonselective GABAA receptor blocker). All the animal were injected with OVA (1mg) diluted in aluminum potassium sulfate adjuvant to sensitize animals. At the7th day after sensitization, mice were fasted for4h. Mice in different groups were given NS (control), OVA (50mg), OVA mixed with gabazine (1.2mg/kg), OVA mixed with picrotoxin (1.2mg/kg) via intragastric administration. The properties of mouse stool were observed1h after intragastric administration of NS and drugs. The occurrence of diarrhea in mice was determined by comparing the property of stools before and after OVA or drug administration. Mice were sacrificed by cervical dislocation after10times of intragastric drug administration every other day. The ileum (0.5cm from cecum,2-3cm length) was excised from each mouse and was prepared for immunohistochemistry assay or Western blot.
Western Blot Analysis
Tissue protein samples obtaining and testing procedure is the same as Part1.
Cell protein analysis:IEC-18cells were rinsed with cold PBS3times after being treated with IL-13. Cell lysis was used to break cells followed by centrifuging at4℃. Protein is in the supernatant. Proteins were denatured at100℃for5minutes. Then they were allowed to go through the SDS-PAGE. Proteins were transferred to PVDF membrane. After blocking PVDF membrane with5%non-fat dry milk in TTBS for60min at room temperature, incubate membrane with primary antibody at4℃overnight. After washing10minutes for three times, the PVDF membranes were incubated for1h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies followed by3times TTBS washing. Finally, immunoreactive proteins were detected by ECL (Electrochemiluminescence) plus.
Statistical Analysis
The properties of mouse stool were observed1h after intragastric administration of NS and drugs. Animals with loose or watery stool were considered to be diarrhea. The diarrhea incidence rate of each intragastric administration (diarrhea animal number to the total number of each group) was taken as the statistical index.
Results
1, All the animals showed diarrhea symptom after the7th intragastric administration with OVA, while the NS group animals were of no diarrhea. Diarrhea incidence was significantly reduced when treating animals with OVA and GABAA receptor antagonists (Gabazine and Picrotoxin).
2, OVA treatment increased expression of GAD67, π and β2/β3subunits of GABAA receptor in mouse ileum. The increased expression of β2/β3subunits was reversed by GABAA receptor antagonists, Gabazine and Picrotoxin, while GAD65/67and π subunit were still in higher expression.
3, Phosphorylated-AKT473and phosphorylated-AKT308reached the highest level at
4and4.5minute after IL-13treatment on IEC-18cells. And GABAA receptor β2/β3subunits expression began to increase at4minute.
Conclusion
1, GABAergic signal system expression was significantly increased during the progress of allergic diarrhea, while GABAA receptors antagonists alleviated diarrhea symptoms and slowed down diarrhea incidence.
2, PI3K-AKT-GABAA receptor signal pathway might account for the upregulation of GABAergic signal system.
目的
γ-氨基丁酸(γaminobutyric acid,GABA)是中枢神经系统中最主要的抑制性神经递质之一,其在大脑和脊髓分布广泛,参与突触后神经元的快速抑制效应。在生理条件下,GABA由神经元内谷氨酸脱羧酶(GAD)催化谷氨酸形成。根据分子量的不同,GAD可分为两种,分别为：GAD65(65kDa)和GAD67(67kDa).GAD65主要分布在神经元轴突末梢内的囊泡膜上,参与囊泡释放的GABA的合成；GAD67则在神经元胞体内散在分布,其合成的GABA释放后与突触外的GABA受体结合发挥作用。神经元轴突末梢的GABA释放后快速激活突触后膜上的GABAA(γ-氨基丁酸A型)受体门控的氯离子通道,引起氯离子向细胞内流动,使突触后神经元细胞膜发生超极化,从而抑制神经元的兴奋性。GABA、GABA受体及其代谢酶等分子统称为GABA信号系统(GABAergic signal system)。除中枢神经系统之外,GABA能信号系统在外周组织也广泛表达,如肺、肝脏、脾、生殖系统等。GABAA受体作为氯离子通道,在肺上皮细胞腔面侧表达,并且被证实通过氯离子跨膜向细胞外转运调节气管和支气管电解质和粘液的分泌。氯离子向肠腔内的跨膜转运是肠道液体分泌的原动力,肠道内氯离子的聚集为水、钠向肠道转运提供渗透压和电化学驱动力基础。有报道称GABA能信号系统在胃癌和结肠癌细胞内高水平表达,但是其在正常消化道各部分的表达和生理作用尚不十分清楚。我们推测GABA能信号系统可能在肠粘膜上皮表达,并且有可能参与肠道液体分泌。因此本研究的目的是观察GABA能信号系统是否在小肠组织表达,尤其是在参与液体分泌的上皮细胞上的表达情况；探讨GABA能信号系统在肠粘膜上皮表达的生理意义。
方法
RT-PCR(反转录聚合酶链反应)
无菌条件下,将IEC-18(a cell line derived from the ileum of rat intestine)细胞种植于直径为10cm的圆形培养皿中；待细胞浓度增殖达到90%以上时,用4℃预冷的磷酸盐缓冲液(Phosphate buffered saline, PBS)冲洗细胞三次,加入Trizol试剂提取细胞RNA。
健康成年雄性Wistar大鼠脱颈牺牲后,小心取出大脑组织,用4℃预冷的PBS冲洗3次,剥离出新鲜大脑皮质,剪碎后,加入Trizol提取组织RNA。按照反转录试剂盒的说明,提取细胞和组织的cDNA;然后用GABAA受体各亚单位和GAD65/67的引物通过PCR扩增得到相应的DNA。再用DNA凝胶电泳观察各目的带表达情况。
免疫组织化学
石蜡切片染色步骤：将新鲜动物组织用4%多聚甲醛(PFA)固定液浸泡24小时后,将组织脱水并用石蜡包埋后切片(组织厚度：4-5μm),依次进行脱蜡、水化和抗原修复处理。用10%与二抗来源相同的血清室温条件下封闭切片1小时后,加入一抗,4℃环境下孵育过夜。PBS冲洗切片3次,每次5分钟,加入荧光标记的二抗室温避光孵育1小时。PBS冲洗3次,每次5分钟,75%甘油缓冲液封片,荧光显微镜观察拍片。
细胞免疫染色步骤：将IEC-18细胞种植在10%多聚赖氨酸(PDL)包被的盖玻片上,培养48小时,待细胞贴壁良好后,室温条件下,用4%多聚甲醛迅速固定10分钟,PBS冲洗三次,每次5分钟,其余步骤同石蜡切片染色。
Western Blot
将新鲜组织或细胞匀浆后,4℃离心(12000g)10分钟,取离心后的上清液进行蛋白定量。用10%十二烷基硫酸钠聚丙烯酰胺凝胶(sodium dodecylsulfate polyacrylamide gel electrophoresis, SDS-PAGE)下层胶和5%的上层分离胶电泳,湿转方法将蛋白转移至孔径为0.45微米的PVDF (Polyvinylidene Fluoride)膜上,5%脱脂牛奶室温封闭PVDF膜1小时,加入一抗覆盖PVDF膜,4℃孵育过夜。用1倍的TTBS冲洗PVDF膜3次,每次10分钟。室温下加入辣根过氧化酶标记的二抗孵育1小时。用1倍TTBS冲洗3次,每次10分钟,ECL显影。
膜片钳记录实验
将IEC-18细胞种植在10%多聚赖氨酸包被的盖玻片上,24小时后待细胞贴壁,开始全细胞记录。记录时用正常细胞外液(155mM NaC1、1.3mM CaC12,5.4mM KC1、25mM HEPES和33mM葡萄糖(pH7.4,渗透压315mosmol/kgH2O))常温循环灌流细胞,用700B放大器进行记录。记录电极内液为155mM KC1、15mM KOH、10mM HEPES、2mM MgC12、1mM CaC12、10mM EGTA和2mM Tetraethylammonium(pH7.35,渗透压315mosmol/kgH20) o电压钳记录时,将细胞膜电位钳制在-60mV。信号采集后用低通滤波器(1-2kH)进行滤波处理。
在体小肠液分泌实验
成年雄性BALB/c小鼠,体重在25-30克之间,实验前禁食,自由饮水。用2%戊巴比妥钠(45-50mg/kg)腹腔注射麻醉动物,固定四肢和头部,在恒温条件下(36℃-38℃)行开腹手术。在距离盲肠1-2cm处结扎回肠,沿向心端方向量取2cm回肠进行结扎,使之形成长约2cm的回肠襻,用直径为0.33mm的胰岛注射器向肠襻内注射100μ1的生理盐水或药物,确保无渗漏后依次关闭腹腔各层。待动物苏醒后给予鼠粮和饮水。5小时后,脱颈牺牲小鼠,取出回肠襻,量取回肠襻的长度和称取去除肠液前后的肠襻重量,进行统计分析。
统计分析
小肠液分泌试验中,用去除肠液前后重量差除以回肠襻的长度(g/cm)作为观察指标,比较对照组和处理组之间有无统计学差异。
所有数据采用均数±标准误(Mean±SEM)表示,用单因素方差分析进行统计学处理,以P<0.05作为显著性差异界值。
实验结果
1、RT-PCR结果显示,小肠上皮细胞内表达GABA的合成酶谷氨酸脱羧酶65和67(GAD65/67)以及GABAA受体各亚单位：并且,在大鼠、小鼠的小肠以及小肠上皮细胞株等组织细胞表达GABAA受体β2、ππ亚单位以及GAD65/67蛋白。
2、免疫荧光染色结果显示GABA和GAD65/67在小肠上皮细胞胞浆内表达,GABAA受体p2/β3和π单位分别在IEC-18细胞膜和小鼠、胎猪的小肠绒毛上皮细胞腔面侧表达。
3、人的回肠隐窝和绒毛上皮细胞内也表达谷氨酸脱羧酶65/67和GABAA受体π亚单位。
4、膜片钳全细胞记录显示,小肠上皮IEC-18细胞,其膜电位在-37±8mV；在约36%的细胞上记录到GABA诱导的内向电流,GABAA受体特异性激动剂—muscimol可模拟上述反应。电流钳记录结果显示muscimol导致上皮细胞膜去极化。
5、小鼠回肠襻液体分泌实验表明,GABA(100μM)和muscimol(10μM)均促进小肠液体的分泌，这种促分泌作用不能被TTX(1μM,河豚毒素)阻断；而GABAA受体特异性阻断剂--Gabazine (100μM)则明显降低GABA引起的小肠液分泌量。
结论
1, GABA能信号系统在小鼠、大鼠、猪和人的小肠上皮细胞功能性表达
2, GABA可通过GABAA受体参与小肠液体分泌。
第二部分：γ-氨基丁酸能信号通路在腹泻发生中的作用及其机制
目的
腹泻是临床上常见的消化系统症状,病因和发病机制多样,但大多数是由于病毒或细菌毒素侵犯肠道黏膜,破环肠道结构和肠道上皮细胞正常的分泌和吸收,导致肠道组织通透性增加和上皮细胞电解质和粘液分泌增多,引起腹泻。由于小肠上皮细胞腔面膜上的氯离子跨膜转运是肠道水、钠、钾、碳酸氢根等物质分泌的原动力,很多腹泻的发生机制都离不开肠道上皮细胞膜上氯离子通道的参与,如CFTR和CaCC两种氯离子通道与霍乱毒素和旅行者腹泻的发生有直接关系。因此,对肠上皮细胞上氯离子通道的研究以及开发通道特异性的药物为临床治疗腹泻提供重要的理论基础。我们发现GABA能信号系统在小肠上皮细胞表达,外源性GABAA受体激动剂引起小肠液的大量分泌。因此我们推测,GABAA受体作为氯离子通道受体,有可能参与腹泻的发生过程。本部分我们对GABA能信号系统在食物过敏原诱导的腹泻过程中的作用及其机制进行了详细探讨。
方法
免疫组织化学同第一部分
食物致敏原诱导的腹泻动物模型的建立
健康成年雄性BALB/c小鼠(20-25g),随机分为对照组、OVA (50mg, Ovalbumin,卵白蛋白)诱导模型组、OVA诱导和Gabazine (100μM)处理组、OVA诱导和Picrotoxin(100μM)处理组(非特异性GABAA受体阻断剂)。实验前禁食,自由饮水。所有动物在处理第一天均用硫酸铝佐剂稀释的OVA (1mg)腹腔内注射,致敏全部小鼠。从第七天开始,对照组给予生理盐水250μl灌胃,模型组和处理组给予OVA和相应药物溶于生理盐水中250μl灌胃,1小时后观察并记录动物粪便性状和排便反应。隔天灌胃一次,共灌胃10次后牺牲动物,收集动物回肠进行免疫组织化学染色和Western blot检测。
Western Blot
动物组织蛋白获取和检测步骤同第一部分。
IL-13处理后的IEC-18细胞,预冷的PBS冲洗3次,用裂解液破碎和收集细胞,4℃离心,取离心上清液蛋白定量,加入上样缓冲液后变性蛋白,进行SDS-PAGE电泳。湿转方法将蛋白转移至PVDF膜,5%脱脂牛奶室温封闭1小时,TTBS冲洗3次,每次10分钟,加入一抗,4℃环境下孵育过夜。TTBS冲洗3次,每次10分钟,加入辣根过氧化物酶标记的二抗,室温条件下孵育1小时,TTBS洗3次后,ECL显影。
统计分析
动物模型指标观察：灌胃后1小时观察动物粪便稀薄、透明、不成型视为腹泻模型成功,计数每次灌胃后模型成功动物的个数,依次累计,直至牺牲动物。以每次灌胃后各组动物腹泻发生率作为统计指标作图分析。
结果
1, OVA诱导的过敏性动物腹泻模型在第7次灌胃时达到100%成功；对照组动物无动物发生腹泻；用Gabazine和Picrotoxin处理的两组动物,明显降低小鼠腹泻的发生率并延缓腹泻的进程。
2,OVA诱导的腹泻动物回肠表达GAD67和GABAA受体β2/β3、π亚单位比对照组显著增高。GABAA受体阻断剂则降低动物肠道β2/β3蛋白的表达,而小肠上皮细胞和腔面膜上的谷氨酸脱羧酶65/67、π亚单位表达则增加。
3,IL-13作用4分钟和4.5分钟时最高。而GABAA受体β2/β3亚单位蛋白表
达量亦在IL-13处理后4分钟开始增高。
结论
1,γ-氨基丁酸能信号系统在过敏性动物腹泻发病过程中表达明显增加,特异性或非特异性阻断该系统后,动物经OVA诱导后腹泻的发病率明显降低,且发病进程减缓。
2,Y-氨基丁酸能信号系统在腹泻发病过程中的上调很可能是由PI3K-AKT-GABAA-R信号通路介导完成。
Part1GABAergic signal system is expressed in ileum epithelium and is involved in the regulation of fluid secretion
Objective
y-aminobutyric acid (GABA for short) is one of the most important inhibitory neurotransmitter in mammalian CNS which mediates the fast inhibition of neurons. Physiologically, GABA is synthesized by glutamate decarboxylase (GAD) from glutamate in neurons which are called GABAergic neurons. And there are two isoforms of GAD:GAD65and67identified by the different molecular weight. GAD65is found to sit in the GABA-contained vesicles near the axon terminals, and the GABA is released to the synaptic cleft acting on the GABAA receptors in postsynaptic membrane mediating fast inhibition in adult brain. While GAD67is distributed randomly in the cytosol, and GABA synthesized by GAD67is thought to act on GABA receptors located in extrasynaptic membrane. As for the release mechanism of cytosolic GABA remains obscure. GABAA receptors are chloride ion channels. GABA or its analogous activates GABAA receptors whose opening leads to chloride influx with membrane hyperpolarization as a consequent. In immature neurons or some types of neurons, GABAA receptor hyperpolarized cell membrane to excite cells. GABA, GABA receptors and GAD are termed as GABAergic signal system. Besides CNS, GABAergic signal system is found in many peripheral tissues and organs like lungs, reproductive system, kidney as well as gastrointestinal tract. GABA and GABAA receptors are reported to play important roles in regulating pulmonary epithelial fluid and mucus secretion. The system is found to be upregulated in gastric and colon carcinoma, but whether it is there in normal gastrointestinal tract is still unknown despite its physiological functions. We hypothesized that GABAergic signal system is expressed in human and animal intestine, which might contributes to the fluid and mucus secretion when activated.
Methods
RT-PCR
RNAs were extracted from IEC-18cell line and cortex of healthy male Wistar rat by Trizol. RT-PCR KIT was used to reproduce cDNA(complementary DNA). And GABAA receptor subunits and GAD DNA were amplified using their primers. The DNA bands were got by running DNA agar gel.
Immunohistochemistry
Paraffin embedded slice staining:fresh tissues were soaked in4%PFA (Paraformal dehyde) for24hours before paraffin embedding. Slice the tissue with4-5μm thickness. Followed by deparaffination, hydration and antigen repair. Then block the slice with5%serum for1hour at room temperature. Incubate the slices at4℃overnight with primary antibody after the serum blocking. Rinse slices3times with PBS the next day,5minutes per rinse. Incubate with fluorescence labeled secondary antibodies for1hour at room temperature. Rinse slices3times and5minutes per time. Seal the slice with75%glycerol buffer and take pictures with fluorescence microscope.
IEC-18cell staining:cells were planted on coverslips coated with10%Poly-L-Lysine. Fix cells with4%PFA for10minutes at room temperature, followed by PBS rinses for3times,5minutes per time. The rest procedures are the same as the paraffin embedded slice staining.
Western Blot Analysis
Fresh tissues and cells were homogenized, centrifuged at12000g for10min at4℃.Total proteins were fractionated on a5%to10%gradient SDS-PAGE (sodium d odecylsulfate polyacrylamide gel electrophoresis). They were transferred to0.45μm P olyvinylidene Fluoride (PVDF) membranes. Membranes were blocked in blocking bu ffer (5%non-fat dry milk, TTBS) for60min at room temperature, incubated with pri mary antibody at4℃overnight. After washing10minutes for three times, the PVDF membranes were incubated for1h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies followed by3times TTBS washing. Finally, immunoreactive proteins were detected by ECL (Electrochemiluminescence) plus.
Electrophysiology
IEC-18cells were incubated on PDL (Poly-D-Lysine) coated coverslips for24hours. Record cells using Axon700B amplifier. The cells were bathed in extracellular solution containing (in mmol/1):155NaCl,1.3CaC12,5.4KC1,25HEPES, and33glucose (pH7.4, osmolarity315mosmol/kgH2O) while the recording pipette was filled with intracellular solution containing (in mmol/1)155KC1,15KOH,10HEPES,2MgC12,1CaC12,10EGTA and2Tetraethylammonium (pH7.35, osmolarity:315mosmol/kgH2O).
In vivo intestinal fluid secretion experiment
Healthy male BALB/c mice (weight between25-30g) were used in this experiment. Animals were fast overnight while free drinking. Animals were anesthetized with2%pentobarbital sodium (45-50mg/kg intraperitoneal injection). Body temperature of mice was maintained between36and38℃during surgery using a heating pad. An abdominal incision (～1.5cm) was made to expose the small intestine, and a closed loop of empty ileum (20mm of length) proximal to the cecum was isolated by sutures. Ileum loops were injected with100μl normal saline (NS) alone or NS containing drugs. The abdominal incision was closed with sutures, and the animal was allowed to recover from anesthesia. Five hours later, sacrifice the mice, and ileum loops were isolated. The ileum loops were weighted after removal of mesentery and connective tissues. After the lumen fluid was released through a longitudinal incision, the weight and length of ileum loops were measured. The ratio of intestinal fluid weight to intestinal length was calculated.
Statistical Analysis
In the intestinal fluid secretion experiment, the relative values of intestinal fluid weight to intestinal length were used to analyze the difference between groups.
All the values in these experiments were presented as mean±SEM. One way analysis of variance (ANOVA) and the student's t-test was used to analyze. P<0.05was considered to be a significant difference.
Results
1, RT-PCR results showed GAD65/67and GABAA receptor subunits mRNA expression in intestinal epithelial cells. And GAD65/67,β2/β3and%subunits of GABAA receptor were found to be expressed in protein level using Western Blot technique.
2, Immunohistochemistry staining located GAD65/67to be in cytosol of epithelial cells, while β2/β3and π subunits of GABAA receptor sit in the apical side of intestinal villi.
3, GAD65/67and π subunits of GABAA receptor are also found in epithelial cells of human ileum.
4, Whole cell patch clamp of IEC-18cells showed an inward current triggered by1mM GAB A when holding cell membrane potential at-60mV. Muscimol, the selective GABAA receptor agonist, works the same as GAB A on IEC-18cells.5, In the intestinal fluid secretion experiment, GAB A (100μm) and Muscimol (10μm) showed similar increasing fluid accumulation in intestine, while the increased fluid secretion can only be partly blocked by TTX1μm, Tetrodotoxin). Gabazine, the selective GABAA receptor blocker, decreased intestinal fluid accumulation caused by GABA.
Conclusion
1, GABAergic signal system is functionally expressed in mouse, rat and human intestinal epithelial cells.
2, Exogenous GABAA receptor agonist and antagonist activate/deactivate GABAA receptor to regulate intestinal fluid secretion.
Part Ⅱ The role of GABAergic signal system in diarrhea and the underlying mechanisms
Objective
Diarrhea happens commonly in patients with digestive diseases, and there are kinds of mechanisms involved in diarrhea. Viruses and bacteria toxins invade intestinal mucous leading to increased permeability of the intestinal epithelium, and disturb secretion and absorption of the epithelia, thus leading to diarrhea. Chloride secretion from the apical membrane to lumen is the driving force for other ions and water efflux. The malfunction of chloride channel activity is always the direct reason for diarrhea. For example, Cholera and Travelers'diarrhea need the involvement of CFTR and CaCC channels. Both of CFTR and CaCC are chloride channels whose activating leads to water, electrolytes and mucus accumulation in intestinal lumen. It's important to discuss the roles that chloride channels. play in physiological and pathophysiological progress of intestine. Targeting chloride channels in intestinal epithelial membrane might be an alternative way to treat diarrhea.
GABAA receptors are found to be functionally expressed in intestinal epithelial cells according to our previous results. Since GABAA receptors are chloride channels too, it's necessary to explore the functional significance of GABAergic signal system. We aim to research the roles that GABAergic signal system plays in food allergen induced diarrhea and the underlying mechnisms.
Methods
Immunohistochemistry procedure is the same as part1.
Food allergen induced diarrhea animal model experiment protocol
Healthy male adult BALB/c mice (body weight between20-25g) were divided into four groups randomly:control group, OVA (50mg, Ovalbumin) induced diarrhea group, OVA mixed with Gabazine (selective GABAA receptor antagonist), OVA mixed with Picrotoxin (nonselective GABAA receptor blocker). All the animal were injected with OVA (1mg) diluted in aluminum potassium sulfate adjuvant to sensitize animals. At the7th day after sensitization, mice were fasted for4h. Mice in different groups were given NS (control), OVA (50mg), OVA mixed with gabazine (1.2mg/kg), OVA mixed with picrotoxin (1.2mg/kg) via intragastric administration. The properties of mouse stool were observed1h after intragastric administration of NS and drugs. The occurrence of diarrhea in mice was determined by comparing the property of stools before and after OVA or drug administration. Mice were sacrificed by cervical dislocation after10times of intragastric drug administration every other day. The ileum (0.5cm from cecum,2-3cm length) was excised from each mouse and was prepared for immunohistochemistry assay or Western blot.
Western Blot Analysis
Tissue protein samples obtaining and testing procedure is the same as Part1.
Cell protein analysis:IEC-18cells were rinsed with cold PBS3times after being treated with IL-13. Cell lysis was used to break cells followed by centrifuging at4℃. Protein is in the supernatant. Proteins were denatured at100℃for5minutes. Then they were allowed to go through the SDS-PAGE. Proteins were transferred to PVDF membrane. After blocking PVDF membrane with5%non-fat dry milk in TTBS for60min at room temperature, incubate membrane with primary antibody at4℃overnight. After washing10minutes for three times, the PVDF membranes were incubated for1h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies followed by3times TTBS washing. Finally, immunoreactive proteins were detected by ECL (Electrochemiluminescence) plus.
Statistical Analysis
The properties of mouse stool were observed1h after intragastric administration of NS and drugs. Animals with loose or watery stool were considered to be diarrhea. The diarrhea incidence rate of each intragastric administration (diarrhea animal number to the total number of each group) was taken as the statistical index.
Results
1, All the animals showed diarrhea symptom after the7th intragastric administration with OVA, while the NS group animals were of no diarrhea. Diarrhea incidence was significantly reduced when treating animals with OVA and GABAA receptor antagonists (Gabazine and Picrotoxin).
2, OVA treatment increased expression of GAD67, π and β2/β3subunits of GABAA receptor in mouse ileum. The increased expression of β2/β3subunits was reversed by GABAA receptor antagonists, Gabazine and Picrotoxin, while GAD65/67and π subunit were still in higher expression.
3, Phosphorylated-AKT473and phosphorylated-AKT308reached the highest level at
4and4.5minute after IL-13treatment on IEC-18cells. And GABAA receptor β2/β3subunits expression began to increase at4minute.
Conclusion
1, GABAergic signal system expression was significantly increased during the progress of allergic diarrhea, while GABAA receptors antagonists alleviated diarrhea symptoms and slowed down diarrhea incidence.
2, PI3K-AKT-GABAA receptor signal pathway might account for the upregulation of GABAergic signal system.
引文
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