γ-氨基丁酸改善2,4,6-三硝基苯磺酸-乙醇诱导的结肠炎肠黏膜屏障损伤作用

蒋廷媛¹^,, 岳源², 李芳华¹^,

1.湖北省宜昌市第二人民医院药剂科,宜昌 443000

2.武汉大学药学院,武汉 430071

JIANG Tingyuan¹^,, YUE Yuan², LI Fanghua¹^,

1.Department of Pharmacy,the Second People's Hospital of Yichang, Yichang 443000, China

2.School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China

作者简介蒋廷媛(1970-),女,湖北宜昌人,主管药师,研究方向:医院药学。电话:0717-6445092,E-mail:3218489554@qq.com。

通信作者李芳华(1970-),女,湖北人,博士,研究方向:医院药学。电话:0717-6211012,E-mail:1353554846@qq.com。

基金资助: 国家自然科学基金资助项目(81273523)

中图分类号: R965;R574 文献标识码: A 文章编号: 1004-0781(2018)08-0931-08

摘要:

目的探讨γ-氨基丁酸(GABA)改善2,4,6-三硝基苯磺酸(TNBS) -乙醇诱导的结肠炎肠黏膜屏障损伤作用。方法 SD大鼠随机分为正常对照组,TNBS-乙醇溶液模型对照组,造模后GABA 200,100,50 mg·kg^-1治疗组。造模后连续14 d,观察记录体质量变化、疾病活动指数(DAI);造模第15天,每组取5只,进行伊文思蓝染色。大鼠取结肠,组织学损伤评分,同时量取结肠长度、称重。通过WB法观察实验性结肠炎各组大鼠肠黏膜屏障连接蛋白及LC3表达情况。结果 GABA增加Caco-2细胞单层模型细胞电阻,减少FD4的渗透,降低单层模型通透性。GABA改善实验性结肠炎引起的体质量下降,增加DAI,降低结肠指数。另外,GABA降低实验性结肠炎大鼠伊文思蓝渗透性及结肠病理变化,显著提高大鼠Occludin、Claudin-4、ZO-1蛋白表达。实验性结肠炎大鼠肠细胞出现过度自噬,GABA抑制过度自噬。结论 GABA通过改善肠黏膜屏障损伤,缓解TNBS-乙醇溶液诱导的实验性结肠炎,故改善肠黏膜屏障损伤可能是治疗炎症性肠病(IBD)的新方法。

关键词: γ-氨基丁酸; ; 2,4,6-三硝基苯磺酸 ; 炎症性肠病 ; 肠黏膜屏障

Abstract:

Objective To investigate the protective effects of GABA against experimental inflammatory bowel disease (IBD) by enhanced intestinal barrier function and explore its underlying mechanisms. Methods Forty SD rats were randomly divided into normal control group, TNBS-alcohol model control group, 200, 100, 50 mg·kg^-1 GABA treatment group.After modeling for 14 days, the changes of body weight and disease activity indexes (DAI) were observed.On the 15th day after modeling, 5 rats in each group were subjected to Evans blue staining.For the remaining rats, colon was collected and evaluated with histological injury score; the length and weight of the colon was determined.The intestinal mucosal barrier connexin and LC3 expression in rats with experimental colitis were observed by WB method. Results The cell resistance of Caco-2 monolayer models was increased and the infiltration of FD4 and the monolayer permeability was reduced by GABA.GABA also improved body mass, increased DAI, and lowered colon index.In addition, GABA decreased the Evans blue permeability and pathological changes in rats with experimental colitis, and significantly increased the Occludin, Claudin-4 and ZO-1 protein expression in the rats.Intestinal autophagy appeared in experimental colitis rats, GABA inhibited excessive autophagy. Conclusion GABA could relieve TNBS-ethanol-induced experimental colitis, and the mechanism might be related to the improvement of intestinal mucosal barrier injury.

Key words: Gamma-aminobutyric acid ; 2,4,6-trinitrobenzene sulfonic acid ; Inflammatory bowel disease ; Intestinal mucosal barrier

炎症性肠病(inflammatory bowel disease,IBD)是一种病因和发病机制尚不十分确定的慢性肠道非特异性炎症疾病^[1]。目前采用的治疗药物有效率低,治疗现状不理想。因此,突破原有治疗限制,寻找新的治疗策略值得探索。研究表明,IBD患者一般有肠黏膜通透性增高^[2]。肠上皮细胞的黏膜屏障的破坏使肠内潜在免疫抗原被大量摄取,从而导致无节制的免疫反应^[3]。肠黏膜通透性增高可能是IBD 的原发因素^[4,5]。因此,增加肠黏膜屏障可能为防治IBD提供新的治疗手段。γ-氨基丁酸(gamma-aminobutyric acid,GABA)在中枢神经系统中是最主要的抑制性神经递质。GABA在实验性结肠炎致黏膜屏障损伤中的研究尚未见报道。本研究建立Caco-2黏膜屏障单层模型,进行GABA对体外肠黏膜屏障模型的作用及机制研究。同时探讨 GABA 对于 2,4,6-三硝基苯磺酸(2,4,6,-trinitrobenzene sulfonic acid,TNBS) -乙醇诱导的结肠炎肠黏膜屏障损伤的干预作用。

1 材料与方法

1.1 实验动物

SD 大鼠,SPF 级,体质量 200~250 g,40只,雄性,由武汉大学动物实验中心提供,动物生产许可证号:SCXK(鄂)2014-0004;动物合格证编号:00017226。在武汉大学动物实验中心饲养,动物房通风良好,室温(22±2) ℃,光照时间12 h、自由进食及饮水,实验前适应5 d。动物实验依据ARRIVE指南(Animal Research:Reporting In Vivo Experiments)进行,经武汉大学动物实验中心动物保健和使用委员会 (Institutional Animal Care and Use Committee,IACUC)批准。

1.2 试剂

TNBS (美国Sigma公司,批号:111M5001V);脂多糖 (lipopolysaccharides,LPS,美国Sigma公司,批号:L-2880,规格:50 μg);磷酸缓冲盐溶液(phosphate buffer saline,PBS,杭州吉诺生物医药技术有限公司);γ-氨基丁酸(γ-aminobutyrie acid,GABA,国药集团化学试剂有限公司,规格:500 g);RIPA 裂解液(碧云天生物技术研究所,批号:P0013B);增强化学发光法(enhanced chemiluminescence,ECL)试剂盒(碧云天生物技术研究所,批号:P0018);二喹啉甲酸法(bicinchoninic acid,BCA)蛋白浓度试剂盒(南京建成生物制品研究所,批号:2017110337)。

1.3 主要仪器

Milicell-ERS型电阻测定仪(美国Millipore公司)。DF-540 型荧光分光光度计(日本岛津)。倒置显微镜(重庆广电仪器有限公司)。蛋白凝胶电泳(上海天能科技有限公司)。DU800型紫外-可见分光光度计(Beckman公司)。

1.4 Caco-2细胞的培养

Caco-2细胞(购自中国典型培养物保藏中心,CCTCC 编号:GDC153)以含10%FBS,1%非必需氨基酸,1%L-谷氨酰胺以及100 U·L^-1青霉素和100 μg·L^-1链霉素双抗溶液的MEM完全培养基,于37 ℃,5% CO₂条件下培养。当细胞长至70%~80%时,以0.05%胰蛋白酶进行消化,传代。

1.5 Caco-2黏膜屏障单层模型的构建

当细胞达到80%融合后,以0.5×10⁶·(cm²)^-1细胞接种于24孔板的Transwell聚碳酸酯膜(直径12 mm,孔径0.4 μm)小室中。细胞培养期间用Milicell-ERS电阻测定仪测定单层Caco-2细胞的跨上皮电阻(transepithelial electrical resistance,TER)。细胞培养至10~11 d,TER在1 000~1 200 Ω·(cm²)^-1时,进行下一步实验^[6]。在Caco-2细胞培养液中加入LPS(50 μg·mL^-1),同时加入PBS,不同浓度GABA(用PBS溶解配置 400,80,16 μg·mL^-1 浓度梯度的 GABA 溶液,加入至培养液中使 GABA 的终浓度为40,8,1.6 μg·mL^-1),平行设置空白对照组(不加LPS及药物,加入等体积PBS),共同孵育培养24 h,测定单层Caco-2细胞的跨上皮电阻。同时,给药后21 h时向细胞培养液中加入荧光素标记葡聚糖(FITC-dextran,平均分子量4 000),孵育培养3 h。收集底室培养液,用DF-540 型荧光分光光度计(激发波长 495 nm,发射波长 520 nm)测定荧光强度,计算FITC-dextran含量。

1.6 TNBS-乙醇溶液造模

造模前所有实验大鼠禁食24 h,自由饮水。TNBS 溶解于体积分数为50%的乙醇溶液中,使TNBS 的终浓度为25 mg·mL^-1。实验大鼠以乙醚轻度麻醉后,将大鼠置于倒悬位,用一直径为2 mm、长约12 cm的橡胶输液管,将其表面涂以石蜡油润滑后,缓慢插入距大鼠肛门约8 cm处的结肠腔内,用无菌注射器将100 mg·kg^-1 TNBS-乙醇溶液注入结肠腔内,为防止药液流出肛门,将实验大鼠保持头朝下倒垂姿势约1 min后缓慢拔出橡胶输液管。根据疾病活动指数(disease activity index,DAI)判断模型是否成功。造模后大鼠体质量下降,大便松散,腹泻,并且出现大便隐血,即造模成功^[7]。

1.7 动物分组及给药

根据大鼠体质量,采用系统随机化法随机分为5组,除正常对照组外其他4组按上述方法造模,造模后自由饮食饲养3 d,第3天根据DAI判断模型全部成功。4组造模大鼠按照 DAI 评分重新采用系统随机化法随机分成4组,每组8只。开始给药,各组处理如下:GABA 大剂量(200 mg·kg^-1)、中剂量(100 mg·kg^-1)、小剂量(50 mg·kg^-1) 组分别灌胃给予 40,20,10 mg·mL^-1的 GABA 0.5 mL·(100 g)^-1。各组给药每天1次,持续11 d。实验期间,大鼠自由饮食、饮水,进行DAI评分观察。第14天,处死动物。所有大鼠进行结肠组织大体形态评分、结肠炎症组织病理学观察等指标测定。其中正常对照组、模型对照组、GABA大剂量(200 mg·kg^-1)行组织蛋白含量检测。

1.8 检测方法

1.8.1 DAI评分评分实验过程中每天观察大鼠体质量变化和大便性状,并且每天观察或用粪便隐血试纸检测大便带血情况,按文献方法行DAI 评分^[8]。

1.8.2 大鼠结肠黏膜渗透性检测按Lange法使用伊文思蓝(Evan’blue,EB)检测结肠通透性^[9]。大鼠麻醉,剖腹,剪断盲肠末端,温PBS由肛门开始冲洗整段结肠,结扎剪断的盲肠末端以及直肠与降结肠交汇处以形成闭合袢,并注入1.5% EB 0.5 mL,关腹腔。2 h后处死大鼠,取出结肠并剪开,乙酰半胱氨酸冲洗3次,滤纸吸干后称质量,然后加入甲酰胺2 mL,50 ℃水浴箱中提取48 h,660 nm下测定吸光度。

1.8.3 结肠组织大体形态评分两个盲态实验设计的独立观察者对结肠组织进行大体形态评分。解剖结肠,测量结肠长度和质量计算结肠单位长度质量。肛门至盲肠段结肠沿肠系膜纵轴剪开,20 ℃的0.9%氯化钠溶液冲洗干净,进行大体形态评分。参照文献^[10]:0=无损伤;1=轻度充血、水肿,无糜烂或溃疡;2=中度充血、水肿,糜烂或浅溃疡;3=重度充血、水肿,黏膜表面有坏死及溃疡形成,溃疡直径<1 cm;4=重度充血、水肿,坏死及溃疡形成,溃疡直径>1 cm。

1.8.4 大鼠结肠炎症组织病理学观察大鼠处死,取距离肛门8 cm处结肠,4%多聚甲醛固定,常规脱水,石蜡包埋,切片。进行苏木精-伊红(HE)染色和阿利新蓝(AB-PAS)染色。100倍光学显微镜下观察病理变化。

1.8.5 结肠组织蛋白含量检测取上述另一份结肠组织加入5倍量RIPA裂解液,匀浆。或者Caco-2细胞加入RIPA裂解液,超声破碎细胞。低温高速离心[4 ℃,12 000 r·min^-1(r=30 cm),20 min],取上清液后,测定并调节蛋白浓度。取预染蛋白Marker 5 μL,蛋白上样量30 μL,10%SDS-PAGE凝胶电泳,转印至PCDF膜,5%BSA封闭过夜(4 ℃),加入多克隆兔抗大鼠occludin一抗(Santa Cruz Biotechnology,1:200)、多克隆兔抗大鼠claudin-4一抗(Santa Cruz Biotechnology,1:200)、多克隆兔抗大鼠ZO-1(Santa Cruz Biotechnology,1:100),多克隆兔抗大鼠LC3一抗(Proteintech,1:400),兔抗大鼠β-actin一抗(Proteintech,1:2 000)室温孵育1.5 h,二抗(1:5 000)室温孵育1 h。化学发光法(ECL)显色,显影、定影,曝光后,拍照用Quantity One 测定灰度值。

1.9 统计学方法

采用SPSS13.0版统计软件,实验结果数据采用均数±标准差( $\bar{x}$ ±s)表示,多组间比较采用单因素方差分析;两组组间比较、组内比较用t检验,以P<0.05为差异有统计学意义。

2 结果

2.1 GABA对Caco-2细胞单层形成的体外黏膜屏障的影响

通过建立Caco-2细胞单层体外黏膜屏障模型,观察GABA对体外黏膜屏障完整性的影响。结果可见,LPS刺激后,单层细胞模型TER显著降低(P<0.05),GABA(40,8,1.6 μg·mL^-1)干预可以显著改善LPS刺激引起的TER降低,与模型对照组比较差异有统计学意义(P<0.05)。同时,LPS刺激后单层细胞模型对FITC-Dextran的通透量显著升高(P<0.01),而给予GABA(40,8,1.6 μg·mL^-1)干预后,FITC-Dextran通透量较模型对照组比较,显著降低(P<0.05或P<0.01)。即LPS会使Caco-2细胞单层模型通透性显著提高,而GABA可以显著降低LPS诱导的通透性的增加。见图1。

图1 5组Caco-2细胞中TER(A)及FD-4(B)的比较(x¯±s,n=3) 与正常对照组比较,^*1P<0.05;与模型对照组比较,^*2P<0.05

Fig.1 Comparison of TER(A) and FD-4(B) among five groups of Caco-2 cells(x¯±s,n=3) Compared with normal control group,^*1P<0.05;compared with model control group,^*2P<0.05

2.2 GABA对大鼠DAI评分的影响

实验期间,模型对照组于第3天死亡大鼠2只,第5天死亡大鼠1只,其余4组未出现大鼠死亡情况。给药期间,正常对照组大鼠毛发光泽、饮食饮水正常,体质量逐渐增加,活动敏捷,未出现稀便情况,大鼠肛周洁净;模型对照组大鼠自造模后第2天出现饮食下降,第4天出现血便,实验期间大鼠稀便,体质量降低,出现懒动,精神萎靡等症状。GABA给药组造模后初期出现腹泻、体质量下降,饮食减少现象,但程度较轻,且大鼠精神、食欲随给药时间延长逐渐好转,体质量逐步增加,腹泻在给药1周后逐渐消失。结果见图2。实验期间,与正常对照组比较,模型对照组大鼠体质量显著下降(F=4.5,P<0.01),GABA(200,100,50 mg·kg^-1)能显著改善实验性结肠炎引起的体质量下降(F分别为4.8,4.3,4.9,P<0.05或P<0.01)。如图2B所示,造模后各组大鼠DAI评分均出现增高趋势,与正常对照组比较,模型对照组大鼠DAI评分显著升高(F=3.6,P<0.01),而GABA(200,100,50 mg·kg^-1)能显著降低实验性结肠炎大鼠的DAI评分(F分别为4.0,3.3,3.1,P<0.05)。

图2 5组大鼠体质量变化(A)及DAI评分(B)的比较(x¯±s,n=8) 与正常对照组比较,^*1P<0.05;与模型对照组比较,^*2P<0.01,^*3P<0.05

Fig.2 Comparison of body weight(A) and DAI(B) among five groups of rats(x¯±s,n=8) Compared with normal control group,^*1P<0.05;compared with model control group,^*2P<0.01,^*3P<0.05

2.3 GABA对大鼠结肠组织大体形态损伤的影响

正常对照组大鼠的结肠均匀细长,颜色呈嫩粉色且色泽均一。TNBS造模形成的实验性结肠炎大鼠结肠段不同程度充血,肠壁增厚,伴有溃疡出现,且形成巨结肠,黏膜表面坏死,病变处黏膜呈黑褐色,其结肠重量和组织大体形态损伤评分显著高于正常对照组(F=8.5,P<0.01),结肠长度显著低于正常对照组(F=7.4,P<0.01)。而GABA组(200,100,50 mg·kg^-1)结肠恢复较好,充血减轻,未见明显巨结肠,仅个别出现小溃疡。可见GABA显著改善实验性结肠炎引起的大鼠肠道组织学损伤,显著改善了结肠质量和长度的改变,降低结肠组织大体形态损伤评分(P<0.05或P<0.01),且随给药剂量增加,评分呈递减趋势,说明GABA对实验性结肠炎的治疗呈浓度相关性。见图3。

图3 5组大鼠结肠长度(A)、质量(B)、形态(C)及组织学评分(D)比较(x¯±s,n=8) 与正常对照组比较,^*1P<0.05;与模型对照组比较,^*2P<0.01,^*3P<0.05

Fig.3 Comparison of colon length(A),weight(B),appearance(C) and histologic scores(D) among five groups of rats(x¯±s,n=8) Compared with normal control group,^*1P<0.05;compared with model control group,^*2P<0.01,^*3P<0.05

2.4 GABA对实验性结肠炎大鼠结肠病理变化的影响

大鼠结肠病理学观察可见,正常对照组大鼠结肠黏膜上皮完整连续,结构清晰,排列紧密有规则,未见炎症细胞浸润,无糜烂及溃疡形成。模型对照组大鼠结肠腺体结构破坏异常,黏膜、黏膜下层出现中性粒细胞、淋巴细胞等大量炎症细胞浸润,黏膜下层结构紊乱、水肿,结肠组织病理学评分显著高于正常对照组(P<0.01)。GABA(100,50 mg·kg^-1)给药组均可不同程度的减轻实验性结肠炎引起的大鼠结肠组织病变,结构清晰,腺体排列整齐,可见少量炎性细胞浸润,其组织病理学评分显著低于模型对照组(F=2.8或2.2,P<0.05或P<0.01)。且随给药剂量增加,组织病理学评分呈递减趋势,再次说明GABA对实验性结肠炎的治疗呈浓度相关性。见图4。

图4 5组大鼠结肠组织病理学切片及组织损伤评分的比较(×100,x¯±s,n=4) 与正常对照组比较,^*1P<0.05;与模型对照组比较,^*2P<0.01,^*3P<0.05

Fig.4 Comparison of histopathological image and tissue injury scores among five groups of rats(×100,x¯±s,n=4) Compared with normal control group,^*1P<0.05;compared with model control group,^*2P<0.01,^*3P<0.05

2.5 GABA对实验性结肠炎大鼠结肠AB-PAS染色的影响

大鼠结肠AB-PAS染色结果见图5。正常对照组大鼠的结肠杯状细胞呈蓝色均匀分布于肠黏膜上皮内,杯状细胞饱满;TNBS诱导的实验性结肠炎大鼠结肠杯状细胞数明显减少,模型对照组杯状细胞黏液滴染色积分光密度显著低于正常对照组(F=5.2,P<0.01)。GABA干预后,可明显改善TNBS-乙醇溶液诱导的实验性结肠炎大鼠结肠杯状细胞的减少,GABA给药组杯状细胞黏液滴染色积分吸光度显著高于模型对照组(F=3.3,P<0.05)。

图5 5组大鼠AB-PAS染色及IOD百分率的比较(×100,x¯±s,n=4) 与正常对照组比较,^*1P<0.05;与模型对照组比较,^*2P<0.05

Fig.5 Comparison of AB-PAS staining and IOD percentage among five groups of rats(×100,x¯±s,n=4) Compared with normal control group,^*1P<0.05;compared with model control group,^*2P<0.05

2.6 GABA对实验性结肠炎大鼠肠黏膜渗透性的影响

为观察GABA对于实验性结肠炎引起的肠黏膜渗透性的影响,进行大鼠伊文思蓝肠黏膜渗透性实验。实验结果见图6。实验性结肠炎的模型对照组大鼠结肠道组织伊文思蓝渗透量显著高于正常对照组(F=4.2,P<0.01)。给予GABA(200 mg·kg^-1)干预后,可以显著降低大鼠肠道伊文思蓝含量。由此可见,GABA能降低肠黏膜渗透,保护肠道黏膜屏障。

图6 GABA对实验性结肠炎大鼠肠黏膜渗透性的影响(x¯±s,n=6) 与正常对照组比较,^*1P<0.01;与模型对照组比较,^*2P<0.05

Fig.6 Effects of GABA on colonic mucosal permeability of rats with experimental colitis(x¯±s,n=6) Compared with normal control group,^*1P<0.05;compared with model control group,^*2P<0.05

2.7 GABA对实验性结肠炎大鼠肠黏膜屏障的影响

通过WB法观察实验性结肠炎各组大鼠肠黏膜屏障连接蛋白表达情况。结果见图7。模型对照组Occludin、Claudin-4、ZO-1蛋白表达显著低于正常对照组(F分别为3.6,8.5,7.2,P<0.05或P<0.01);给予GABA(80 mg·kg^-1)干预后,大鼠Occludin、Claudin-4、ZO-1阳性染色较模型对照组显著增加,蛋白表达显著高于模型对照组(F分别为2.6,3.5,4.2,P<0.05)。

图7 GABA对实验性结肠炎大鼠肠黏膜紧密连接蛋白的影响(x¯±s,n=4) 与正常对照组比较,^*1P<0.01;与模型对照组比较,^*2P<0.05

Fig.7 Effects of GABA on the expression of tight junction proteins in rats with experimental colitis(x¯±s,n=4) Compared with normal control group,^*1P<0.05;compared with model control group,^*2P<0.05

2.8 GABA对于LPS刺激下Caco-2自噬的影响

为观察GABA对于LPS刺激下Caco-2自噬的影响,免疫印迹法测定LPS刺激下Caco-2细胞中LC3蛋白表达情况,LC3蛋白表达情况以LC3Ⅱ与LC3Ⅰ的比值表示。实验结果见图8。LPS刺激下Caco-2后LC3Ⅱ与LC3Ⅰ的比值显著高于正常对照组,表明LPS刺激后Caco-2细胞内自噬显著增强。而GABA 200 mg·kg^-1给药后的LC3Ⅱ与LC3Ⅰ的比值显著降低,显著低于模型对照组。

图8 GABA对LPS刺激下Caco-2自噬的影响(x¯±s,n=4) 与正常对照组比较,^*1P<0.01;与模型对照组比较,^*2P<0.05

Fig.8 Effects of GABA on Caco-2 autophagy induced by LPS(x¯±s,n=4) Compared with normal control group,^*1P<0.01;compared with model control group,^*2P<0.05

2.9 GABA对于TNBS-乙醇溶液诱导的实验性结肠炎结肠组织自噬的影响

为观察GABA对于TNBS-乙醇溶液诱导的实验性结肠炎结肠组织自噬的影响,免疫印迹法测定TNBS-乙醇溶液诱导的实验性结肠炎结肠组织中LC3蛋白表达情况,LC3蛋白表达情况以LC3Ⅱ与LC3Ⅰ的比值表示。实验结果见图9。TNBS-乙醇溶液诱导的实验性结肠炎结肠组织LC3Ⅱ与LC3Ⅰ的比值均显著高于正常对照组,表明LPS刺激后TNBS-乙醇溶液诱导的实验性结肠炎结肠组织自噬显著增强。而GABA给药后的LC3Ⅱ与LC3Ⅰ的比值显著降低,显著低于模型对照组。

图9 GABA对TNBS诱导的结肠炎大鼠结肠组织自噬的影响(x¯±s,n=4) 与正常对照组比较,^*1P<0.01;与模型对照组比较,^*2P<0.05

Fig.9 Effects of GABA on colon autophagy of rats with TNBS-induced colitis(x¯±s,n=4) Compared with normal control group,^*1P<0.01;compared with model control group,^*2P<0.05

3 讨论

肠黏膜屏障功能异常是IBD发病的分子基础,IBD发病时肠黏膜屏障功能异常,肠腔内抗原物质向肠黏膜固有层移位。而抗原引起的肠道促炎因子的释放,又会进一步损伤肠道上皮细胞,破坏肠黏膜屏障^[11,12]。肠黏膜通透性增高可能是IBD 的原发因素。因此,增加肠黏膜屏障可能为防治IBD提供新的治疗手段。

GABA药物治疗已经被用于焦虑、戒酒、癫痫,以及诱导镇静、麻醉。GABA能信号系统不仅在中枢系统发挥着重要的作用,在外周系统中也起重要的调控作用^[13]。其在脾脏、胃、肝脏、肺以及血液单核细胞中都有功能性表达^[14]。在免疫系统中,GABA能系统可以调控炎症反应。GABA受体已被证实在免疫细胞上存在,可降低外周巨噬细胞炎症因子的产生^[15]。笔者尚未见国内有GABA与炎症性肠病的相关报道。而国外已有研究报道,GABA能用于炎症性肠病的防治^[16]。但是GABA是否能保护黏膜屏障,及其作用机制均需进一步研究。TNBS-乙醇溶液诱导的大鼠结肠炎组织学改变的许多特点与人类IBD相似。GABA能够改善大鼠体质量变化、结肠指数、DAI评分、肉眼组织学损伤评分等。这些指标能较好反映结肠炎症状和组织学宏观改变的特点和程度^[17]。

Caco-2单层模型为公认的肠黏膜屏障体外模型^[18]。使用结肠炎的重要诱导因子LPS诱导体外黏膜屏障损伤。GABA能够改善LPS刺激引起的细胞跨膜电阻降低、大分子物质的渗透性增加。为探讨GABA对于实验性结肠炎引起的肠黏膜的影响,进行实验结肠炎大鼠伊文思蓝肠黏膜渗透性实验。证明GABA能够显著改善TNBS-乙醇溶液引起的肠黏膜渗透性改变。

紧密连接蛋白被认为是维持肠黏膜屏障结构完整性的重要组成部分,并在肠黏膜屏障功能的维持中起到决定性作用。构成肠上皮细胞间的紧密连接蛋白主要有Occludin、Claudin、ZO-1等。GABA能够增加紧密连接蛋白Occludin,Claudin-4和ZO-1的表达,通过调节紧密连接蛋白的表达与装配维持肠黏膜屏障功能。

组织细胞通过自噬作用来降解细胞内的受损细胞器、蛋白质和核酸等内容物,并重新利用氨基酸、核糖等代谢物,从而维持细胞稳态。但过度的自噬能够诱导细胞发生程序性死亡^[19]。在TNBS-乙醇溶液诱导的实验性结肠炎动物模型,肠道组织出现过度自噬,表明TNBS-乙醇溶液诱导的实验性结肠炎结肠组织自噬显著增强,LC3Ⅱ与LC3Ⅰ的比值是正常对照组的5.4倍。而在LPS刺激的人上皮细胞Caco-2中,LC3Ⅱ与LC3Ⅰ的比值是正常对照组的5.5倍。由此可见,肠黏膜在强烈应激反应过程中出现过度自噬,导致Ⅱ型程序性细胞死亡。GABA能够显著抑制这种过度自噬。GABA对于肠黏膜屏障的保护作用可能与其抑制过度自噬有关。

由此可见,GABA能够抑制过度自噬,有效增强肠道黏膜屏障功能,对实验性结肠炎具有治疗作用。

The authors have declared that no competing interests exist.

参考文献

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Gene and cell therapy based treatment strategies for inflammatory bowel diseases

2011

... 炎症性肠病(inflammatory bowel disease,IBD)是一种病因和发病机制尚不十分确定的慢性肠道非特异性炎症疾病^[1].目前采用的治疗药物有效率低,治疗现状不理想.因此,突破原有治疗限制,寻找新的治疗策略值得探索.研究表明,IBD患者一般有肠黏膜通透性增高^[2].肠上皮细胞的黏膜屏障的破坏使肠内潜在免疫抗原被大量摄取,从而导致无节制的免疫反应^[3].肠黏膜通透性增高可能是IBD 的原发因素^[4,5].因此,增加肠黏膜屏障可能为防治IBD提供新的治疗手段.γ-氨基丁酸(gamma-aminobutyric acid,GABA)在中枢神经系统中是最主要的抑制性神经递质.GABA在实验性结肠炎致黏膜屏障损伤中的研究尚未见报道.本研究建立Caco-2黏膜屏障单层模型,进行GABA对体外肠黏膜屏障模型的作用及机制研究. 同时探讨 GABA 对于 2,4,6-三硝基苯磺酸(2,4,6,-trinitrobenzene sulfonic acid,TNBS) -乙醇诱导的结肠炎肠黏膜屏障损伤的干预作用. ...

Intestinal barrier functi-on:molecular regulation and disease pathogenesis

2009

Naringenin enhan-ces intestinal barrier function through the expression and cytoskeletal association of tight junction proteins in Caco-2 cells

2013

Importance of disrupted intestinal barrier in inflammatory bowel diseases

2011

Interleukin-13 is the key effector Th2 cytokine in ulcerative colitis that affects epithelial tight junctions,apoptosis,and cell restitution

2005

Wild jujube polysaccharides pro-tect against experimental inflammatory bowel disease by enabling enhanced intestinal barrier function

2015

... 当细胞达到80%融合后,以0.5×10⁶·(cm²)^-1细胞接种于24孔板的Transwell聚碳酸酯膜(直径12 mm,孔径0.4 μm)小室中.细胞培养期间用Milicell-ERS电阻测定仪测定单层Caco-2细胞的跨上皮电阻(transepithelial electrical resistance,TER).细胞培养至10~11 d,TER在1 000~1 200 Ω·(cm²)^-1时,进行下一步实验^[6].在Caco-2细胞培养液中加入LPS(50 μg·mL^-1),同时加入PBS,不同浓度GABA(用PBS溶解配置 400,80,16 μg·mL^-1 浓度梯度的 GABA 溶液,加入至培养液中使 GABA 的终浓度为40,8,1.6 μg·mL^-1),平行设置空白对照组(不加LPS及药物,加入等体积PBS),共同孵育培养24 h,测定单层Caco-2细胞的跨上皮电阻.同时,给药后21 h时向细胞培养液中加入荧光素标记葡聚糖(FITC-dextran,平均分子量4 000),孵育培养3 h.收集底室培养液,用DF-540 型荧光分光光度计(激发波长 495 nm,发射波长 520 nm)测定荧光强度,计算FITC-dextran含量. ...

Role of moxibustion in infl-ammatory responses during treatment of rat ulcerative colitis

2014

... 造模前所有实验大鼠禁食24 h,自由饮水.TNBS 溶解于体积分数为50%的乙醇溶液中,使TNBS 的终浓度为25 mg·mL^-1.实验大鼠以乙醚轻度麻醉后,将大鼠置于倒悬位,用一直径为2 mm、长约12 cm的橡胶输液管,将其表面涂以石蜡油润滑后,缓慢插入距大鼠肛门约8 cm处的结肠腔内,用无菌注射器将100 mg·kg^-1 TNBS-乙醇溶液注入结肠腔内,为防止药液流出肛门,将实验大鼠保持头朝下倒垂姿势约1 min后缓慢拔出橡胶输液管.根据疾病活动指数(disease activity index,DAI)判断模型是否成功. 造模后大鼠体质量下降,大便松散,腹泻,并且出现大便隐血,即造模成功^[7]. ...

Important role of Rho-kinase in the pathogenesis of cardiovascular inflammation and remodeling induced by long-term blockade of nitric oxide synthesis in rats

2002

... 1.8.1 DAI评分评分实验过程中每天观察大鼠体质量变化和大便性状,并且每天观察或用粪便隐血试纸检测大便带血情况,按文献方法行DAI 评分^[8]. ...

Glial-derived neurotro-phic factor regulates intestinal epithelial barrier function and inflammation and is therapeutic for murine colitis

2010

... 1.8.2 大鼠结肠黏膜渗透性检测按Lange法使用伊文思蓝(Evan’blue,EB)检测结肠通透性^[9].大鼠麻醉,剖腹,剪断盲肠末端,温PBS由肛门开始冲洗整段结肠,结扎剪断的盲肠末端以及直肠与降结肠交汇处以形成闭合袢,并注入1.5% EB 0.5 mL,关腹腔.2 h后处死大鼠,取出结肠并剪开,乙酰半胱氨酸冲洗3次,滤纸吸干后称质量,然后加入甲酰胺2 mL,50 ℃水浴箱中提取48 h,660 nm下测定吸光度. ...

Vanill-oid receptor-1 containing primary sensory neurones mediate dextran sulphate sodium induced colitis in rats

2003

... 1.8.3 结肠组织大体形态评分两个盲态实验设计的独立观察者对结肠组织进行大体形态评分.解剖结肠,测量结肠长度和质量计算结肠单位长度质量.肛门至盲肠段结肠沿肠系膜纵轴剪开,20 ℃的0.9%氯化钠溶液冲洗干净,进行大体形态评分.参照文献^[10]:0=无损伤;1=轻度充血、水肿,无糜烂或溃疡;2=中度充血、水肿,糜烂或浅溃疡;3=重度充血、水肿,黏膜表面有坏死及溃疡形成,溃疡直径<1 cm;4=重度充血、水肿,坏死及溃疡形成,溃疡直径>1 cm. ...

Cytokine regulation of tight junctions

2009

... 肠黏膜屏障功能异常是IBD发病的分子基础,IBD发病时肠黏膜屏障功能异常,肠腔内抗原物质向肠黏膜固有层移位.而抗原引起的肠道促炎因子的释放,又会进一步损伤肠道上皮细胞,破坏肠黏膜屏障^[11,12].肠黏膜通透性增高可能是IBD 的原发因素.因此,增加肠黏膜屏障可能为防治IBD提供新的治疗手段. ...

Defective epithelial barrier in chronic rhinosinusitis:the regulation of tight junctions by IFN-γ and IL-4

2012

Oppo-site role played by GABAA and GABAB receptors in the modulation of peristaltic activity in mouse distal colon

2014

... GABA药物治疗已经被用于焦虑、戒酒、癫痫,以及诱导镇静、麻醉.GABA能信号系统不仅在中枢系统发挥着重要的作用,在外周系统中也起重要的调控作用^[13].其在脾脏、胃、肝脏、肺以及血液单核细胞中都有功能性表达^[14].在免疫系统中,GABA能系统可以调控炎症反应.GABA受体已被证实在免疫细胞上存在,可降低外周巨噬细胞炎症因子的产生^[15].笔者尚未见国内有GABA与炎症性肠病的相关报道.而国外已有研究报道,GABA能用于炎症性肠病的防治^[16].但是GABA是否能保护黏膜屏障,及其作用机制均需进一步研究.TNBS-乙醇溶液诱导的大鼠结肠炎组织学改变的许多特点与人类IBD相似.GABA能够改善大鼠体质量变化、结肠指数、DAI评分、肉眼组织学损伤评分等.这些指标能较好反映结肠炎症状和组织学宏观改变的特点和程度^[17]. ...

GABA (A) receptor subunits RNA expression in mice peritoneal macrophages modulate their IL-6/IL-12 production

2007

Inhibitory role for GABA in autoimmune inflammation

2010

GABA and GABA receptors in the gastrointestinal tract:from motility to inflammation

2015

Glial-derived neurotro-phic factor regulates intestinal epithelial barrier function and inflammation and is therapeutic for murine colitis

2010

Activation of AMPK attenua-tes lipopolysaccharide-impaired integrity and function of blood-brain barrier in human brain microvascular endothelial cells

2014

... Caco-2单层模型为公认的肠黏膜屏障体外模型^[18].使用结肠炎的重要诱导因子LPS诱导体外黏膜屏障损伤.GABA能够改善LPS刺激引起的细胞跨膜电阻降低、大分子物质的渗透性增加.为探讨GABA对于实验性结肠炎引起的肠黏膜的影响,进行实验结肠炎大鼠伊文思蓝肠黏膜渗透性实验.证明GABA能够显著改善TNBS-乙醇溶液引起的肠黏膜渗透性改变. ...

mTOR依赖的自噬通路与神经变性疾病

2014

... 组织细胞通过自噬作用来降解细胞内的受损细胞器、蛋白质和核酸等内容物,并重新利用氨基酸、核糖等代谢物,从而维持细胞稳态.但过度的自噬能够诱导细胞发生程序性死亡^[19].在TNBS-乙醇溶液诱导的实验性结肠炎动物模型,肠道组织出现过度自噬,表明TNBS-乙醇溶液诱导的实验性结肠炎结肠组织自噬显著增强,LC3Ⅱ与LC3Ⅰ的比值是正常对照组的5.4倍.而在LPS刺激的人上皮细胞Caco-2中,LC3Ⅱ与LC3Ⅰ的比值是正常对照组的5.5倍.由此可见,肠黏膜在强烈应激反应过程中出现过度自噬,导致Ⅱ型程序性细胞死亡.GABA能够显著抑制这种过度自噬.GABA对于肠黏膜屏障的保护作用可能与其抑制过度自噬有关. ...