肠内营养中应用谷氨酰胺对重症急性胰腺炎大鼠继发感染的影响

目的探讨早期肠内营养中应用谷氨酰胺（Gln）对重症急性胰腺炎（SAP）大鼠继发感染的影响。方法雄性SD大鼠95只，随机分成模型1天组、对照组、SAP＋肠外营养（PN）组、SAP＋肠内营养（EN）组、SAP＋EN＋Gln组，除模型1天组外，各组又分为4天喂养组和7天喂养组。分别在第4、7天检测各组各项指标，主要包括细菌移位、血浆内毒素、二胺氧化酶、肠转运功能。结果肠系膜淋巴结和肝培养出的细菌多为革兰氏阴性杆菌，以大肠杆菌为主，SAP＋EN、SAP＋EN＋Gln组显著低于SAP＋PN组（P〈0．01），SAP＋EN＋Gln组低于SAP＋EN组（P〈0．05）。各SAP组血浆内毒素水平显著高于对照组，以SAP＋PN组最高，SAP＋EN、SAP＋EN＋Gln组显著低于SAP＋PN组（P〈0．01），SAP＋EN组高于SAP＋EN＋Gln组（P〈0．05）。SAP＋PN7天组明显高于4天组（P〈0．05）。各SAP组发病4天后血浆DAO活性明显下降（P〈0．01）；7天后SAP＋EN＋Gln组回升和对照组无差异，其余各SAP组继续下降，以SAP＋PN组为甚（P〈0．01）。各SAP组的肠转运系数显著降低（P〈0．05，P〈0．01）；SAP＋EN、SAP＋EN＋Gln组显著高于SAP＋PN组（P〈0.05，P〈0．01）；SAP＋EN＋Gln和SAP＋EN组之间差异无显著性。结论肠内营养中应用谷氨酰胺能更有效地降低SAP大鼠肠道细菌移位，从而减少胰腺继发感染的发生。     

支链氨基酸强化的肠内肠外营养对肝硬化大鼠术后肝功能及血浆氨基酸谱的影响

目的探讨支链氨基酸（BCAA）强化的早期肠内肠外营养支持对肝硬化大鼠肝部分切除术后肝功能及血浆氨基酸谱的影响,为临床合理应用提供实验依据。方法将24只雄性SD肝硬化大鼠按编号法随机分为肠内营养组、肠内营养＋BCAA组和肠外营养＋BCAA组3组,3组大鼠行肝部分切除术后1d分别进行等热量等氮量营养支持,共5d。术后6d测定大鼠体重、肝功能、前白蛋白、转铁蛋白、肝组织白蛋白mRNA的表达、Ki67蛋白表达指数及进行血浆氨基酸谱分析。结果与术前比较,术后肠内营养、肠内营养＋BCAA、肠外营养＋BCAA组大鼠体重均减轻（P〈0．05）。术后肠外营养＋BCAA组较肠内营养组碱性磷酸酶水平升高（P〈0．05）;肠外营养＋BCAA组较肠内营养＋BCAA组血清天冬氨酸氨基转移酶、丙氨酸氨基转移酶、碱性磷酸酶水平升高（P〈0．05）。术后肠外营养＋BCAA组较肠内营养、肠内营养＋BCAA组血清前白蛋白水平降低（P〈0．05）,转铁蛋白差异无统计学意义（P〉0．05）。肠外营养＋BCAA、肠内营养＋BCAA组较肠内营养组血浆中亮氨酸、异亮氨酸明显升高,酪氨酸、苯丙氨酸、精氨酸、色氨酸明显降低（P〈0．05）;肠外营养＋BCAA组与肠内营养＋BCAA组比较,各氨基酸浓度差异均无统计学意义（P〉0．05）。肠外营养＋BCAA、肠内营养＋BCAA组较肠内营养组总氨基酸及芳香族氨基酸均降低,BCAA及BCAA／芳香族氨基酸比值升高（P〈0．05）。肠外营养＋BCAA组较肠内营养、肠内营养＋BCAA组肝组织白蛋白mRNA表达水平及Ki67蛋白表达指数均降低（P〈0．05）。结论BCAA强化的肠内肠外营养支持能改善肝硬化大鼠血浆氨基酸失衡,恢复血浆中BCAA／芳香族氨基酸比值;肠内营养在改善肝功能、促进肝脏蛋白质合成和肝硬化大鼠肝切除术后残肝肝再生方面优于肠外营养,但仍不能提高血浆白蛋白水平。     

不同营养途径对肝硬化大鼠肝切除术后肝脏功能的影响

目的探讨不同营养途径对肝硬化大鼠肝切除术后肝脏功能的影响。方法6只正常大鼠作为正常对照组,30只肝硬化大鼠随机分为术前组6只,肝切除＋颈内静脉插管术后1d组6只,肝切除＋颈内静脉插管术后行肠外营养（PN）5d组6只,肝切除＋胃造瘘术后1d组6只,肝切除＋胃造瘘术后行肠内营养（EN）5d组6只。测定大鼠肝功能、肝组织白蛋白（ALB）mRNA的表达和肝组织信号传导和转录激活因子-3（STAT-3）、细胞增殖核抗原Ki67的蛋白表达。结果与PN5d组比较,EN5d组血清谷草转氨酶、谷丙转氨酶、碱性磷酸酶显著下降（P〈0．05）,血清ALB、胰岛素样生长因子-1显著升高（P〈0．05）,肝组织ALBmRNA表达显著升高（P〈0．05）,肝组织STAT-3、Ki67蛋白表达显著升高（P〈0．05）。结论EN可以加快肝硬化大鼠肝切除术后肝功能的恢复,避免胆汁淤积,促进肝脏蛋白合成和残肝再生。     

生态免疫肠内营养对重症胰腺炎大鼠肠屏障功能和胰腺炎症的影响

目的评估生态免疫肠内营养对重症胰腺炎（SAP）大鼠肠屏障功能和胰腺的影响。方法64只SPF大鼠随机分为对照组、SAP组、SAP普通肠内营养组（EEN组）和SAP生态免疫肠内营养组（EIN组）,分别于造模后第4天和第7天检测各组大鼠的多脏器细菌易位情况、血浆内毒素水平、肠黏膜通透性,并进行胰腺病理评分和回肠末端病理检查等。结果SAP组、EEN组和EIN组大鼠的总细菌易位率明显高于对照组（P〈0．05）,EEN组和EIN组明显低于SAP组（P〈0．05）,EIN组明显低于EEN组（P〈0．05）。SAP组、EEN组和EIN组大鼠的血浆内毒素水平和肠系膜上静脉血FD．40浓度均明显高于对照组（P〈0．01）,EEN组和EIN组均明显低于SAP组（P〈0．01）,EIN组的血内毒素水平亦低于EEN组（P〈0．05）。SAP组、EEN组和EIN组大鼠病理各单项及总评分均明显高于对照组（P〈0．01）;EEN和EIN组则明显低于SAP组（P〈0．01）;EIN组的各单项评分与EEN组差异无统计学意义（P〉0．05）,但总评分明显低于EEN组（P〈0．05）。EIN组大鼠肠黏膜较其他组明显增厚,绒毛茂盛。结论早期肠内营养对SAP大鼠肠屏障和胰腺具有保护作用,其中生态免疫肠内营养较普通肠内营养效果更佳。     

ω-3多不饱和脂肪酸对肠道缺血再灌注损伤和淋巴干结扎的影响

目的研究肠道缺血再灌注损伤时肠淋巴干结扎和ω-3多不饱和脂肪酸（ω-PUFA）对肠道和远隔组织的影响。方法将40只SD大鼠胃造瘘后随机分为假手术组、肠内营养（EN）组、EN＋淋巴千结扎（EN＋L）组、ω-PUFA组和ω—PUFA＋L组5组,每组8只。营养干预7d后,用动脉夹夹闭肠系膜上动脉60min,结扎组同时进行肠淋巴干结扎,假手术组只开腹。术后继续原营养干预3d后,分别测定肠道通透性、肠黏膜形态、细菌培养和血中细胞因子。结果缺血再灌注3d后,EN组和EN＋L组大鼠体重较造瘘前明显下降（P〈0．05）,EN＋L组大鼠体重明显低于ω-PUFA组和ω-PUFA＋L组（P〈0．05）。缺血再灌注后第1天,各组大鼠的L／M值均显著增加（P〈0．05或P〈0．01）,缺血再灌注后第3天,EN＋L组、ω-PUFA组和ω-PUFA＋L组大鼠的L／M值均明显低于缺血再灌注后第1天（P〈0．05）,EN组和EN＋L组大鼠的L／M值明显高于ω-PUFA＋L组（P〈0．05）;ω—PUFA组和ω-PUFA＋L组大鼠的空肠黏膜厚度及绒毛高度均明显高于假手术组、EN组和EN＋L组（P〈0．05或P〈0．01）,其回肠黏膜厚度及绒毛高度也均明显高于EN组（P均〈0．05）;ω-PUFA＋L组大鼠的血清内毒素和肿瘤坏死因子-α水平明显低于EN组（P〈0．05）,白细胞介素（IL）-6水平明显低于ω-PUFA组（P〈0．05）,IL-1β水平明显低于其余各组（P〈0．05）;EN组大鼠的肺细胞凋亡指数明显高于其余4组（P〈0．05）,其诱导型一氧化氮合酶和髓过氧化物酶浓度明显高于ω-PUFA＋L组（P〈0．05）,EN＋L组大鼠的诱导型一氧化氮合酶浓度也明显高于ω-PUFA＋L组（P〈0．05）。结论缺血60min可引起大鼠肠道损伤、肠屏障功能障碍、通透性增加;再灌注72h后,肠道损伤部分恢复,通透性较缺血后降低,细菌内毒素仍存在移位,肺仍有凋亡细胞。肠淋巴管结扎可弱化肺组织损伤,促进肠黏膜修复,维护肠屏障功能,减少内毒素移位和减轻系统炎症反应。添加ω-3PUFA的EN显著优于普通EN。     

重症急性胰腺炎早期肠内营养支持

目的 探讨早期肠内营养在重症急性胰腺炎（SAP）治疗中的安全性和有效性。方法回顾性分析71例SAP患者临床资料，经综合治疗观察患者血清白蛋白、前白蛋白、TNF-α、CD4／CD8、IgG水平。随机分为肠内营养组（EN组，36例）和全肠外营养组（TPN组，35例）。结果 EN组治疗后14d患者血清CD4/CD8及IgG水平比TPN组明显升高（P〈0．05）；EN组治疗后7、14d时TNF-α水平与治疗前比较明显下降（P〈0．05），且与TPN组比较明显减少（P〈0．05）；EN组治疗后血清前白蛋白水平较治疗前明显增高（P〈0．05），且与TPN组比较亦有显著性增高（P〈0．05）。结论早期肠内营养对SAP治疗是安全、有效的，可提高血清前白蛋白水平，改善其营养状况，增强肠道黏膜屏障，抑制炎性因子释放，增强患者的免疫功能，改善SAP的预后。     

肠内和肠外联合阶段性营养对重症急性胰腺炎患者治疗效果的影响

目的观察肠内和肠外联合阶段性营养对重症急性胰腺炎患者治疗效果的影响。方法重症急性胰腺炎患者45例分为完全胃肠外营养组（TPN组，n=25）和肠内营养加肠外阶段性营养组（PN＋EN组，n=20）。观察两组治疗结果以及临床指标的变化。结果营养支持后PN＋EN组的APACHEⅡ评分和CT评分均显著低于TPN组（P〈0．01）。营养支持两周后两组患者的血糖、血清淀粉酶和血肌酐水平均较营养支持前显著下降（P〈0．01），血清白蛋白、总蛋白、血钙水平均较营养支持前显著升高（P〈0．01），但是两组的各项指标比较无显著性差异（P〉0．05）。PN＋EN组患者的感染并发症发生率显著低于TPN组（P〈0．01），平均住院天数也显著短于TPN组（P〈0．01）。结论肠内和肠外联合阶段性营养支持方式的疗效优于完全胃肠外营养，对重症急性胰腺炎的治疗起了积极作用。     

老年恶性消化道肿瘤患者术后应用肠内营养的安全性及疗效

目的 评价老年恶性消化道肿瘤患者术后应用肠内营养支持的安全性及临床疗效。方法 将108例老年恶性消化道肿瘤患者随机分为肠内营养（EN）组（45例）、肠外营养（PN）组（45例）及对照组（常规输液）组（18例），EN组术后24小时输注肠内营养液，PN组经外周静脉输注营养液。观察临床和测定营养支持前后患者的血糖、肝肾功能和电解质以及营养和免疫指标的变化。结果 EN组肠功能恢复时间明显短于PN组和对照组（P＜0．001）；血糖、肝肾功能、电解质、补体C3、C4和总补体溶血活性在各组问差异无显著性；EN、PN组治疗后体重、前白蛋白、白蛋白、IgG、IgM、IgA及C-反应蛋白均显著高于对照组（P＜0．05，P＜0．01），而EN组与PN组间差异无显著性；EN组、PN组治疗后CD3^＋、CD4^＋、CD4^＋／CD8^＋均显著高于对照组（P＜0．01），且EN组治疗后CD4^＋、CD4^＋／CD8^＋亦高于PN组（P＜0．05）。结论 肠内营养安全、实用有效，对体质差、年龄大的恶性消化道肿瘤患者更为适合。     

免疫增强型肠内营养对重症急性胰腺炎大鼠炎症反应和免疫功能的影响

目的 探讨免疫增强型肠内营养对重症急性胰腺炎大鼠炎症反应和免疫功能的影响。方法 采用牛磺胆酸钠逆行注射法建立重症急性胰腺炎模型，将60只建模成功的胰腺炎大鼠分为全肠外营养组（TPN组）、普通肠内营养组（EN组）和免疫增强型肠内营养组（SEN组），每组20只。肠外营养和肠内营养分别采用经右颈内静脉插管输液和经胃造口空肠上段给液的方式。各组自建模成功后均应用乙酸奥曲肽皮下注射做基础治疗。于第24小时，第4、7、14天分别收集血液标本，测定血浆细胞因子TNF-α、IL-10及外周血中的CD3＋、CD4＋、CD8＋淋巴细胞亚群。并于建模前取10只大鼠测定血清淀粉酶，作为正常对照，建模后24小时，第3、5、7、9、11、14天检测各组血清淀粉酶。结果 （1）建模24小时，3组动物血浆TNF-α水平均相对较高，而IL-10处于相对较低水平；第4天时，3组TNF-α均明显下降，而IL-10浓度升高并达到最高水平，此时3组间的TNF-α和IL-10水平差异均无显著性（P〉0．05）；第7天和第14天时，3组的TNF-α和IL-10均呈不同程度下降趋势，其中SEN组TNF-α和IL-10水平明显低于TPN组和EN组（P〈0．05），而后两组间差异无显著性（P〉0．05）。（2）建模24小时，3组的CD3＋、CD4＋和CD4＋／CD8＋均升高；第7天和第14天时SEN组的CD3＋、CD4＋和CD4＋／CD8＋均明显高于TPN组和EN组（P〈0．05），但CD8＋变化不大。（3）建模24小时，3组血淀粉酶明显升高，此后均逐渐下降；第7天时，SEN组的血淀粉酶达正常范围，而TPN组和EN组的血淀粉酶在第9天才接近正常水平。SEN组14天死亡率明显低于TPN组和EN组（P〈0．05）。结论 免疫增强型肠内营养能有效调节重症急性胰腺炎大鼠细胞因子水平，增强免疫功能，缩短病程，降低死亡率。     

早期肠内营养在急性重症胰腺炎肠衰竭中的研究

目的探讨早期经肠内营养管行肠内营养（EN）在急性重症胰腺炎（SAP）肠衰竭中应用的安全性和有效性。方法2007年2月至2009年3月收治SAP患者23例,随机分为全胃肠外营养（TPN）组11例,EN组12例。观察两组腹胀缓解时间、肠道运动恢复时间、腹腔细菌感染率、多器官功能障碍综合征（MODS）发生率及治愈率的差异。结果EN组腹胀缓解时间[（6．4±1．3）d]、肠道运动恢复时间[（3．7±1．6）d]、腹腔细菌感染率[16．7％（2／12）]、MODS发生率[8．3％（1／12）]均低于TPN组[分别为（9．3±2．1）d、（7．2±3．5）d、45．5％（5／11）、27．3％（3／11）]（P〈0．01）,治愈率也明显提高但差异无统计学意义。结论早期EN在SAP肠衰竭中的应用是安全有效的。     

鱼油脂肪乳对肠外营养大鼠小肠黏膜紧密连接形态结构的影响

目的:探讨鱼油脂肪乳剂(富含ω-3多不饱和脂肪酸)对肠外营养(PN)大鼠肠上皮细胞紧密连接(TJ)形态和紧密连接蛋白occludin表达的影响。方法:将大鼠随机分为四组,即对照组(正常喂食),PN(禁食5d行PN支持)组,小剂量鱼油治疗(1 ml/kg鱼油静脉注射+PN)组和大剂量鱼油治疗(2 ml/kg鱼油静脉注射+PN)组。观察大鼠术后小肠黏膜上皮细胞TJ形态,以及紧密连接蛋白occludin的表达分布。结果:PN组大鼠治疗5 d后肠上皮细胞TJ结构部分缺失、间隙增宽;大剂量鱼油治疗组TJ形态基本完整,小剂量鱼油治疗组TJ部分缺失。各组occludin蛋白总量无明显差异。提取脂筏组分,发现与对照组比,三个实验组脂筏区域中occludin蛋白表达均明显减少,但大剂量鱼油治疗组较PN组明显升高(P<0.01)。结论:长期PN可导致TJ结构的缺失,脂筏区域紧密连接蛋白occludin表达减少。大剂量鱼油脂肪乳剂可维持occludin蛋白在脂筏区域的表达,保护小肠黏膜细胞TJ的结构和功能。     

Effect of colonic bacterial metabolites on Caco-2 cell paracellular permeability in vitro

One common effect of tumor promoters is increased tight junction (TJ) permeability. TJs are responsible for paracellular permeability and integrity of the barrier function. Occludin is one of the main proteins responsible for TJ structure. This study tested the effects of physiological levels of phenol, ammonia, primary bile acids (cholic acid, CA, and chenodeoxycholic acid, CDCA), and secondary bile acids (lithocholic acid, LCA, and deoxycholic acid, DCA) on paracellular permeability using a Caco-2 cell model. Paracellular permeability of Caco-2 monolayers was assessed by transepithelial electrical resistance (TER) and the apical to basolateral flux of [14C]-mannitol. Secondary, but not primary, bile acids increased permeability as reflected by significantly decreased TER and increased mannitol flux. Both phenol and ammonia also increased permeability. The primary bile acid CA significantly increased occludin expression (P < 0.05), whereas CDCA had no significant effect on occludin expression as compared to the negative control. The secondary bile acids DCA and LCA significantly increased occludin expression (P < 0.05), whereas phenol had no significant effect on the protein expression as compared to the negative control. This suggests that the increased permeability observed with LCA, DCA, phenol, and ammonia was not related to an effect on occludin expression. In conclusion, phenol, ammonia, and secondary bile acids were shown to increase paracellular permeability and reduce epithelial barrier function at doses typical of levels found in fecal samples. The results contribute to the evidence these gut microflora-generated products have tumor-promoting activity.     

Implications of Indirect Biomarkers of Intestinal Permeability in the Stools of Newborns and Infants with Perinatal Risk Factors for Intestinal Colonization Disorders and Infant Feeding Patterns

Background: The intestinal microbiota of pregnant women and factors disturbing the microbial balance of their gastrointestinal tract during the perinatal period may be the cause of dysbiosis and thus intestinal permeability syndrome in their children. The purpose of this study was to analyze the implications of intestinal permeability parameters in the stools of newborns and infants with perinatal risk factors for intestinal colonization disorders (the route of delivery, antibiotic therapy in the neonatal period and the abandonment of breastfeeding). Methods: The study included 100 mother–child pairs. All children were born from uncomplicated and term pregnancies (between 37 and 42 weeks of gestation). In order to determine the parameters of dysbiosis and intestinal permeability, we determined the concentrations of zonulin and occludin in stool samples taken from all children at 0 (i.e., at birth), 3, 6 and 12 months of age. Elevated levels of lipopolysaccharide (LPS) are associated with metabolic diseases and its presence may be indicative of TJ injury and the onset of leaky gut syndrome. To indirectly determine the presence of endotoxemia, the concentrations of lipopolysaccharide were also measured in stool samples taken from all children at 0, 3, 6 and 12 months of age. We analyzed the relationship between the markers studied and perinatal risk factors for impaired intestinal colonization, including the mode of delivery, the method of feeding, and a family history of allergy. Results: During the first 3 months of infant life, higher concentrations of fecal occludin and zonulin were most often accompanied by higher values of fecal LPS. Similarly, higher concentrations of zonulin were accompanied by higher values of occludin. There were no significant differences in the stool concentrations of the studied markers during the first year of life between children born by caesarean section and those born naturally. In addition, the method of feeding had no significant effect on the changes in the concentrations of the determined fractions. Antibiotic therapy was associated only with an increase in the fecal occludin concentration after birth, without any effect on zonulin, occludin or LPS levels. The use of probiotic therapy in infants resulted in a decrease in only LPS concentrations at 3 months of age, with no effect on zonulin or occludin concentrations at 0, 6 and 12 months. Conclusions: Perinatal factors related to intestinal permeability are important during the first 3 months of infant life. However, we found that the mode of delivery had no influence on the parameters of infant intestinal leakage during the first year of life. In addition, the mode of infant feeding—breast or exclusively formula—did not significantly affect the changes in the concentrations of LPS, zonulin or occludin in the stools of children. A short-term increase in occludin concentrations after delivery in the stools of children from mothers undergoing antibiotic therapy indicates a negative but reversible influence of intrapartum antibiotics on the intestinal integrity of children in the perinatal period. Probiotic therapy seems to have a positive effect on reducing endotoxemia in children during the first 3 months of life. The presence of LPS at 3 months did not affect intestinal tightness at any of the later measured periods of the infants’ lives.     

Effect of Colonic Bacterial Metabolites on Caco-2 Cell Paracellular Permeability In Vitro

One common effect of tumor promoters is increased tight junction (TJ) permeability. TJs are responsible for paracellular permeability and integrity of the barrier function. Occludin is one of the main proteins responsible for TJ structure. This study tested the effects of physiological levels of phenol, ammonia, primary bile acids (cholic acid, CA, and chenodeoxycholic acid, CDCA), and secondary bile acids (lithocholic acid, LCA, and deoxycholic acid, DCA) on paracellular permeability using a Caco-2 cell model. Paracellular permeability of Caco-2 monolayers was assessed by transepithelial electrical resistance (TER) and the apical to basolateral flux of [ ¹⁴ C]-mannitol. Secondary, but not primary, bile acids increased permeability as reflected by significantly decreased TER and increased mannitol flux. Both phenol and ammonia also increased permeability. The primary bile acid CA significantly increased occludin expression (P < 0.05), whereas CDCA had no significant effect on occludin expression as compared to the negative control. The secondary bile acids DCA and LCA significantly increased occludin expression (P < 0.05), whereas phenol had no significant effect on the protein expression as compared to the negative control. This suggests that the increased permeability observed with LCA, DCA, phenol, and ammonia was not related to an effect on occludin expression. In conclusion, phenol, ammonia, and secondary bile acids were shown to increase paracellular permeability and reduce epithelial barrier function at doses typical of levels found in fecal samples. The results contribute to the evidence these gut microflora-generated products have tumor-promoting activity.     

Toll-like receptor 4 increases intestinal permeability through up-regulation of membrane PKC activity in alcoholic steatohepatitis

Intestinal hyperpermeability is a causal factor for the development of alcoholic endotoxemia and steatohepatitis. However, the mechanisms governing this link remain unknown. The purpose of this study was to determine whether toll-like receptor 4 (TLR4) is involved in ethanol's deleterious effects on the intestinal barrier. Caco-2 cells were incubated in vitro with 1–10% ethanol. The results indicated that ethanol had a dose-dependent effect in increasing TLR4 expression and intercellular permeability. Then the effects of TLR4 on protein kinase C (PKC) and the intercellular junction protein occludin were assessed with and without pretreatment with a TLR4 inhibitor. The results indicated that TLR4 increased nonspecific PKC activity and reduced the expression of phosphorylated occludin in the membrane, which increased intercellular permeability. These effects were prevented by pretreatment with TLR4 mAb.     

Glutamine Restores Tight Junction Protein Claudin‐1 Expression in Colonic Mucosa of Patients With Diarrhea‐Predominant Irritable Bowel Syndrome

Background:Recent studies showed that patients with diarrhea‐predominant irritable bowel syndrome (IBS‐D) had an increased intestinal permeability as well as a decreased expression of tight junctions. Glutamine, the major substrate of rapidly dividing cells, is able to modulate intestinal permeability and tight junction expression in other diseases. We aimed to evaluate, ex vivo, glutamine effects on tight junction proteins, claudin‐1 and occludin, in the colonic mucosa of patients with IBS‐D. Materials and Methods: Twelve patients with IBS‐D, diagnosed with the Rome III criteria, were included (8 women/4 men, aged 40.7 ± 6.9 years). Colonic biopsy specimens were collected and immediately incubated for 18 hours in culture media with increasing concentrations of glutamine from 0.6–10 mmol/L. Claudin‐1 and occludin expression was then measured by immunoblot, and concentrations of cytokines were assessed by multiplex technology. Claudin‐1 expression was affected by glutamine (P < .05, analysis of variance). In particularly, 10 mmol/L glutamine increased claudin‐1 expression compared with 0.6 mmol/L glutamine (0.47 ± 0.04 vs 0.33 ± 0.03, P < .05). In contrast, occludin expression was not significantly modified by glutamine. Interestingly, glutamine effect was negatively correlated to claudin‐1 (Pearson r = ‐0.83, P < .001) or occludin basal expression (Pearson r = ‐0.84, P < .001), suggesting that glutamine had more marked effects when tight junction protein expression was altered. Cytokine concentrations in culture media were not modified by glutamine treatment. Conclusion: Glutamine increased claudin‐1 expression in the colonic mucosa of patients with IBS‐D. In addition, glutamine effect seems to be dependent on basal expression of tight junction proteins.     

Computational identification of interplay between phosphorylation and O-β-glycosylation of human occludin as potential mechanism to impair hepatitis C virus entry

Hepatitis C virus (HCV) is one of the leading causes of liver diseases. Several host factors that facilitate the attachment and entry of HCV have been discovered, of which human occludin seems to be the most promising. Studies have shown that activity of occludin is dependent upon its phosphorylation status, and that during HCV infection deregulation of phosphorylated occludin collectively leads to a reduction in tight junction (TJ) integrity of hepatocytes and favors HCV entry. However, detailed information of the posttranslational modifications (PTMs) of occludin still remains largely unknown. In addition to phosphorylation, serine/threonine residues of several proteins are also regulated by a unique type of modification known as O-β-glycosylation and this crosstalk serves as a functional switch. To identify the O-β-glycosylation potential and how interplay between phosphorylation and O-β-glycosylation can be exploited for the inhibition of HCV entry, here we report a computational analysis of PTMs of human occludin. Several conserved phosphorylation residues and kinases that can alter the ability of occludin to regulate the integrity of TJs were identified. In addition to previously reported Tyr residues, two additional Tyr residues (Tyr29 and Tyr287) were identified as target sites of Src kinase. To our knowledge, this is the first study to report the O-β-GlcNAc potential of occludin and target sites of ERK (Ser8, Ser310, and Thr345), GSK-3 (Ser8, Ser341) and Cdk5 (Thr376). Furthermore, based on findings from this study, a potential novel interplay between phosphorylation and O-β-glycosylation at the two Yin Yang sites (Ser408 and Ser490) is also proposed.     

缺血性脑卒中对犬肠屏障功能的影响

目的 研究缺血性脑卒中对犬肠黏膜屏障功能的影响以及细胞凋亡和紧密连接蛋白在其中的可能机制.方法 杂犬20只,按随机数字表法分为梗死组和假手术组,每组10只.梗死组采用双硅柱置入法制作缺血性脑卒中模型,假手术组动物不置入栓子,其他同梗死组.光镜下观察肠黏膜形态;免疫组织化学法分析肠黏膜紧密连接蛋白闭锁蛋白和闭锁小带蛋白1的表达;采用TdT介导的dUTP缺口末端标记技术检测肠上皮细胞凋亡.结果 梗死组动物均形成脑梗死.和假手术组相比,梗死组紧密连接蛋白闭锁蛋白和闭锁小带蛋白1表达均明显降低(闭锁蛋白平均光密度值:0.20±0.01比0.22±0.01,P=0.007;闭锁小带蛋白1平均光密度值:0.20±0.01比0.22±0.02,P=0.008);梗死组凋亡指数明显增高(29.04 ±3.79比6.44±1.24,P=0.002);闭锁蛋白与闭锁小带蛋白1呈正相关(R=0.71,P=0.02);凋亡指数与闭锁蛋白和闭锁小带蛋白1均呈负相关(R=-0.91,P=0.00;R=-0.77,P=0.01).光镜下梗死组存在小肠病理损伤,假手术组小肠结构正常.结论 缺血性脑卒中时,肠道屏障功能发生损害.紧密连接蛋白闭锁蛋白和闭锁小带蛋白1表达降低,从而导致紧密连接破坏.肠黏膜细胞凋亡上调是肠屏障障碍的细胞学基础,凋亡是缺血性脑卒中时肠上皮细胞死亡的重要形式.     

Kaempferol enhances intestinal barrier function through the cytoskeletal association and expression of tight junction proteins in Caco-2 cells

Kaempferol, a natural flavonoid present in fruits, vegetables, and teas, provides beneficial effects for human health. We investigated the promotive effect of kaempferol on tight junction (TJ) barrier integrity in human intestinal Caco-2 cell monolayers. Transepithelial electrical resistance (TER; a TJ integrity marker) across the monolayers rapidly and markedly increased during the first 6 h after kaempferol administration and remained elevated until 48 h without any changes in the lucifer yellow or dextran fluxes. Immunoblot analysis demonstrated that kaempferol promoted the actin cytoskeletal association of the TJ proteins, zonula occludens (ZO)-1, ZO-2, occludin, claudin-1, claudin-3, and claudin-4, which was associated with the increase in TER. Kaempferol-mediated ZO-2 and claudin-4 expression was relatively smaller or occurred later than the kaempferol-promoted cytoskeletal association. Confocal microscopy showed that kaempferol-induced assembly of occludin and claudin-3 occurred at the TJ at 6 h postadministration. Extraction of cholesterol with methyl-β-cyclodextrin suppressed the kaempferol-mediated increase in TER. Sucrose density gradient centrifugation showed that the kaempferol treatment increased the TJ protein distributions in the cholesterol-rich lipid microdomain fraction. Taken together, these results indicate that the membrane lipid microdomain is involved in the kaempferol-mediated promotion of TJ protein assembly and intestinal TJ integrity.     

Glucagon-Like Peptide-2 Improve Intestinal Mucosal Barrier Function in Aged Rats

Glucagon-like peptide-2 (GLP-2) plays a major role in repairing impaired intestinal mucosa, but its mechanism in the improvement of intestinal barrier function during the aging process remains unclear. In this study, 26-month-old male Sprague-Dawley rats were randomized to control group and GLP-2 group treated with a dose of 250 μg?kg-1?d-1 by intraperitoneal injection. After 14 days of treatment, intestinal mucosal morphometric changes were observed by light microscopy and transmission electron microscopy (TEM). Small intestinal permeability was evaluated by fluorescein isothiocyanate (FITC)-labeled dextran. The mRNA and protein expression of Zonula Occludens-1 (ZO-1), occludin, claudin-1 and the GLP-2 receptor (GLP-2R) were detected by Real-time PCR and Western blot. Our results showed that GLP-2 administration significantly improved the age-related atrophy of intestinal mucosa and villi and increased small intestinal permeability. The mRNA and protein expression of ZO-1and occludin in ileum were up regulated in the GLP-2-treated old rats. In addition, the serum GLP-2 levels were negatively correlated with small intestinal permeability measured by FITC-dextran levels (r=-0.610, P<0.01). Taking all these data together, it is concluded that GLP-2 improved small intestinal epithelial barrier function in aged rats mainly by facilitating intestinal mucosa growth, alleviating the increased small intestinal permeability and increasing ZO-1 and occludin expression. Our observations provide evidence for the clinical significance of GLP-2 in preventing the intestinal epithelial barrier dysfunction during aging.