梗阻性黄疸对大鼠肠黏膜上皮细胞紧密连接蛋白ZO-1和肠黏膜通透性的影响

目的研究梗阻性黄疸对大鼠肠黏膜上皮细胞紧密连接蛋白ZO－1和肠黏膜通透性的影响。方法30只健康雄性Wistar大鼠随机分为3组：正常对照组、假手术组、梗阻性黄疸组（OJ组）。OJ组制成梗阻性黄疸动物模型,7d后各组同一时间点取材。检测门静脉血D-乳酸含量,免疫组化检测肠黏膜紧密连接蛋白ZO-I的分布。结果OJ组大鼠门静脉血D晋L酸水平显著高于正常对照组和假手术组（P〈0．05）,ZO-1染色变淡,分布不均匀,边缘粗糙,强阳性表达率下降。结论梗阻性黄疸时大鼠肠黏膜上皮紧密连接蛋白ZO－1分布紊乱、表达下降,肠黏膜通透性增加,肠黏膜屏障受损。     

大鼠弥漫性脑损伤后小肠黏膜超微结构与黏膜上皮细胞凋亡的动态变化

目的:观察弥漫性脑损伤后大鼠肠黏膜超微结构和上皮细胞凋亡的动态变化,分析二者在肠黏膜屏障功能障碍中的作用。方法:实验于2003-12/2005-12在华北煤炭医学院附属医院动物实验室进行。将雄性Wistar大鼠以随机数字表法分成对照组和伤后1,2,4,8,12,24,48,72,168h共9个时相组,每组15只。采用Marmarou脑损伤动物模型法对损伤组致大鼠重型弥漫性脑损伤,对照组只切开头皮,不行落体致伤,而后缝合切口。严格依照1,2,4,8,12,24,48,72,168h时间点,依次对各组大鼠回盲部近端2cm处切取直径为1mm小肠组织圈。对照组术后立即处死,处理同实验组。透射电镜下比较对照组和致伤组大鼠肠黏膜超微结构的改变,观察伤后小肠黏膜超微结构的动态变化。原位末端标记(TUNEL)法计数各组凋亡细胞的发生率。结果:实验大鼠全部进入结果分析,无脱失。①对照组大鼠肠黏膜上皮细胞微绒毛高而密,排列整齐,细胞间紧密连接完整,线粒体等细胞器结构正常。致伤组微绒毛短而稀,不规则,可见紧密连接开放,线粒体肿胀,内嵴断裂。②TUNEL法显示凋亡细胞胞核为棕褐色,固缩呈断片状,主要位于绒毛顶部,且随致伤时间的延长有由上向下发展的趋势。伤后1h细胞凋亡计数较对照组差异无显著性意义[(2.47±1.26),(2.16±0.83)个/100个细胞,P>0.05],伤后2h直至168h凋亡细胞数较对照组均显著增加[(6.17±2.23),(13.79±2.99),(16.68±2.91),(21.74±3.75),(16.16±2.63),(14.05±3.06),(16.26±2.90),(7.32±2.50)个/100个细胞,P<0.01]。结论:弥漫性脑损伤后,肠黏膜超微结构的改变包括紧密连接开放等是肠黏膜屏障功能障碍的形态学基础,黏膜上皮细胞凋亡过度在肠黏膜屏障功能障碍发生中可能起重要作用。     

大鼠肠屏障功能变化与不同强度游泳运动的关系

背景:研究在运动应激状态下肠道屏障损伤发生机制及规律,将为运动应激肠道屏障保护剂的研制提供理论依据。目的:观察不同强度游泳运动后大鼠肠屏障的变化。设计:随机对照动物实验。单位:湖北大学运动人体科学实验室和武汉大学医学院基础实验室。材料:选取3周龄健康雄性SD大鼠36只,随机分为对照组10只、适度运动组12只和过度性运动组14只,3组大鼠饲养条件一致。方法:①对照组平时不运动。②适度运动组:进行无负重游泳,前3d适应性游30min,并在1周内逐渐延长至60min,然后每天游泳1次,每周6次,训练6周。③过度运动组:前3d适应性游30min,并在1周内延长时间至120min,训练1周后,进行过度游泳训练。然后每天游泳1次,每周6次,持续4周。最后2周,每天早、晚1次,每周6次。主要观察指标:①大鼠肠屏障参数:血浆内毒素,肠黏膜通透性,细菌移位率。②大鼠肠黏膜组织结构。结果:①大鼠肠屏障参数结果:大鼠过度运动后血浆内毒素增加2倍,肠黏膜通透性提高2.5倍,细菌移位率增加230%。②适度运动对大鼠肠黏膜组织结构无明显影响,过度运动使大鼠肠上皮细胞内高尔基复合体和粗面内质网扩张,上皮细胞高度水肿,炎细胞浸润。结论:适度运动可以改善机体肠功能;过度运动造成机体肠屏障损伤,导致消化系统出现病理性征候群。     

细胞凋亡在腹部开放伤合并海水浸泡大鼠肠屏障功能障碍中的作用

目的：探讨肠黏膜上皮细胞的凋亡在大鼠腹部开放伤合并海水浸泡后肠屏障功能障碍中的作用。方法：观察腹腔海水浸泡后血浆二胺氧化酶（DAO）、内毒素（LPS）含量变化，肠组织丙二醛（MDA）、超氧化物歧化酶（SOD）含量，肠黏膜组织病理损伤程度；利用原位末端标记法计数肠黏膜上皮细胞凋亡指数。结果：腹部开放伤合并海水浸泡后，大鼠血浆DAO、LPS水平显著升高：肠组织MDA含量显著升高．SOD活性显著降低（P〈0．05或P〈0．01）；小肠病理组织学检查显示肠黏膜屏障受损：小肠黏膜上皮细胞凋亡指数显著增加（P〈0．01）。结论：腹部开放伤合并海水浸泡可导致大鼠肠屏障功能障碍，出现肠源性内毒素血症，肠组织氧自由基损伤，肠黏膜上皮细胞凋亡明显增加，上皮细胞凋亡增加在肠屏障功能障碍发生中可能起重要作用。[著者文摘]     

弥漫性脑损伤后大鼠肠黏膜上皮细胞的凋亡和核因子-κB的表达

目的 观察弥漫性脑损伤后大鼠肠黏膜上皮细胞凋亡以及核因子-κB（NF-κB）表达的动态变化，探讨两者在肠黏膜屏障功能障碍中的作用。方法 采用Marmarou模型致大鼠重型弥漫性脑损伤，150只雄性Wistar大鼠随机分成对照组和伤后1、2、4、8、12、24、48、72、168h组，共10组，利用免疫组化技术检测NF-κB在不同时相组的表达，采用原位末端标记（TUNEL）法计数不同时相组凋亡细胞。结果 致伤组NF-κB表达的积分光密度均显著大于对照组（P〈0．01）；伤后1h凋亡细胞数量即开始增加，于伤后12h达到峰值后逐渐下降，但直到168h均较对照组明显升高（P〈0.01）。结论 弥漫性脑损伤后激活NF-κB。肠黏膜上皮细胞凋亡增加，两者在肠黏膜屏障功能障碍发生中可能起重要作用。     

大鼠弥漫性脑损伤后小肠黏膜超微结构与黏膜上皮细胞凋亡的动态变化

目的：观察弥漫性脑损伤后大鼠肠黏膜超微结构和上皮细胞凋亡的动态变化，分析二者在肠黏膜屏障功能障碍中的作用。方法：实验于2003—12／2005-12在华北煤炭医学院附属医院动物实验室进行。将雄性Wistar大鼠以随机数字表法分成对照组和伤后1，2，4，8，12，24，48，72，168h共9个时相组，每组15只。采用Marmarou脑损伤动物模型法对损伤组致大鼠重型弥漫性脑损伤，对照组只切开头皮，不行落体致伤，而后缝合切口。严格依照1，2，4，8，12，24，48，72，168h时间点，依次对各组大鼠回盲部近端2cm处切取直径为1mm小肠组织圈。对照组术后立即处死，处理同实验组。透射电镜下比较对照组和致伤组大鼠肠黏膜超微结构的改变，观察伤后小肠黏膜超微结构的动态变化。原位末端标记（TUNEL）法计数各组凋亡细胞的发生率。结果：实验大鼠全部进入结果分析，无脱失。①对照组大鼠肠黏膜上皮细胞微绒毛高而密，排列整齐，细胞间紧密连接完整，线粒体等细胞器结构正常。致伤组微绒毛短而稀，不规则，可见紧密连接开放，线粒体肿胀，内嵴断裂。②TUNEL法显示凋亡细胞胞核为棕褐色，固缩呈断片状，主要位于绒毛顶部，且随致伤时间的延长有由上向下发展的趋势。伤后1h细胞凋亡计数较对照组差异无显著性意义[（2．47&;#177;1．26），（2．16&;#177;0．83）个／100个细胞，P〉0．051，伤后2h直至168h凋亡细胞数较对照组均显著增加[（6．17&;#177;2．23），（13．79&;#177;2．99），（16．68&;#177;2．91），（21．74&;#177;3．75），（16．16＋2．63），（14．05＋3．06），（16．26&;#177;2．90），（7．32＋2．50）个／100个细胞，P〈0．011。结论 弥漫性脑损伤后，肠黏膜超微结构的改变包括紧密连接开放等是肠黏膜屏障功能障碍的形态学基础，黏膜上皮细胞凋亡过度在肠黏膜屏障功能障碍发生中可能起重要作用。     

重症急性胰腺炎大鼠HMGB1对肠上皮细胞occludin表达的影响

目的 观察重症急性胰腺炎(SAP)大鼠肠组织中高迁移率族蛋白B1 (HMGB1)表达对肠黏膜上皮细胞紧密连接occludin蛋白表达的影响.方法 逆行胰胆管注射5％牛磺胆酸钠制作SAP模型.健康Wistar大鼠随机(随机数字法)分为对照组、SAP组、二硫代氨基甲酸吡咯烷(PDTC)处理组.测定血淀粉酶(AMY)、内毒素(LPS)及D-乳酸水平;光镜下观察胰腺和肠组织的病理变化;免疫组织化学法观察occludin分布和表达的变化;RT-PCR法检测大鼠肠组织中HMGB1的表达水平;Western blot法检测大鼠肠组织中HMGB1及occludin蛋白的表达水平.采用SPSS 13.0统计分析软件进行处理,P＜ 0.05为差异具有统计学意义.结果 在建模后24 h,SAP组大鼠血浆LPS及D-乳酸水平明显高于对照组,提示肠屏障通透性明显增加;PDTC处理组大鼠血浆LPS及D-乳酸水平明显低于SAP组(P＜0.05).SAP组大鼠肠组织HMGB1表达水平较对照组明显升高,而occludin蛋白的表达较对照组下降;PDTC组大鼠肠组织HMGB1表达水平明显低于SAP组,occludin水平较SAP组升高(P＜0.05).结论 SAP时,大鼠肠组织内HMGB1表达升高,通过降低occludin蛋白表达,来增加肠黏膜屏障通透性;PDTC可抑制HMGB1表达,上调occludin蛋白表达,改善肠黏膜屏障通透性.     

膳食纤维肠内营养对全肠道缺血再灌注大鼠肠屏障功能的影响

目的：通过建立大鼠全肠道缺血再灌注模型,探讨膳食纤维肠内营养对肠屏障功能的影响。方法：将实验大鼠随机分成4组,每组10只。第1组为对照组,第2组为缺血再灌注组（I/R组）,该两组为术后24h取材。第3组为普通肠内营养组（EN组）,第4组为膳食纤维肠内营养组（FEN组）,营养支持7d后取材。上述4组均行回、结肠形态学检查,细菌及内毒素移位、结肠固有层淋巴细胞分布检测,用循环D-乳酸法测定肠道通透性。结果：I/R组大鼠回、结肠黏膜萎缩,腺体稀疏,黏膜下层水肿、出血。FEN组肠黏膜形态学变化好于EN组。I/R组细菌移位率、血浆D-乳酸、血浆内毒素显著高于对照组;FEN组D-乳酸、内毒素水平低于EN组。I/R组结肠固有层CD4＋、CD8＋T淋巴细胞显著低于其余各组。FEN组CD4＋、CD8＋T淋巴细胞较EN组增多。结论：添加膳食纤维的肠内营养在维护肠黏膜屏障、改善肠道免疫功能、防止细菌及内毒素移位方面作用优于单纯肠内营养。膳食纤维能提高结肠局部免疫功能。     

紧密连接蛋白occludin在三硝基苯磺酸诱导大鼠结肠炎肠黏膜屏障中的作用研究

目的探讨紧密连接蛋白occludin在三硝基苯磺酸（trinitro-benzen-sulfonic acid,TNBS）诱导大鼠结肠炎肠黏膜屏障中的作用。方法建立大鼠结肠炎模型。SD大鼠18只,按随机数字表法分为TNBS诱导大鼠结肠炎模型组（n=10）和正常对照组（n=8）,进行疾病活动指数（DAI）和组织学损伤评分,用ELISA法测定结肠组织肿瘤坏死因子-α（TNF-α）、白介素-10（IL-10）和血清内毒素,采用免疫组织学染色检测紧密连接（tight junction,TJ）相关蛋白occludin的分布。结果TNBS诱导大鼠结肠炎后,和正常组比较,TJ结构遭到破坏,TJ相关蛋白的表达亦减少,结肠组织TNF-α水平升高、IL-10水平降低和血清内毒素水平升高（P〈0.05）。结论TNBS诱导大鼠结肠炎肠黏膜上皮细胞TJ相关蛋白occludin表达下降,肠黏膜上皮屏障的完整性被破坏,导致肠黏膜屏障功能低下,促发炎症反应。     

重型胰腺炎肠黏膜屏障紊乱141例临床研究

急性胰腺炎临床常见,轻型胰腺炎病死率低于3％,重型感染性胰腺炎病死率可高达30％,重型胰腺炎除了肠道消化吸收功能破坏及营养障碍外,还可以引起肠道屏障功能破坏,肠道通透性增加,免疫功能下降[1],使肠道细菌和内毒素得以通过肠屏障而转移至肠道外组织,导致全身脓毒症及多器官功能障碍综合征.因此,对胰腺炎早期肠黏膜屏障破坏的诊断和保护具有重要的意义[2].本研究通过监测重型胰腺炎患者尿中聚乙二醇4000 (polyethylenegly-col4000,PEG4000)吸收率,肠脂肪酸结合蛋白(IFABP)浓度,为早期重型胰腺炎肠屏障功能障碍引起细菌移位提供理论依据.     

细胞凋亡在急性重症胰腺炎肠道粘膜屏障功能障碍中的意义

目的：探讨细胞在凋亡在大鼠急性重症胰腺炎（ＡＳＰ）肠道粘膜屏障功能障碍中的意义。方法：用胰胆管内注射甘脱氧胆酸,结合静脉输注蛙皮素方法复制大鼠ＡＳＰ模型。分别对模型大鼠（ＡＳＰ）及假手术组大鼠（ＳＯ）于术后６、１２、２４小时取回肠粘膜组织进行光镜、电镜观察,并用分子生物学标记（ＴＵＮＥＬ）方法定量肠粘膜上皮细胞的凋亡指数。结果：ＡＳＰ组大鼠除制模２４小时光镜下可见肠粘膜上皮脱落及溃疡形成外,其它两     

急性重症胰腺炎大鼠肠粘膜内pH及氧代谢的改变

目的探讨肠粘膜内pH值及氧代谢改变在急性重症胰腺炎(ASP)肠粘膜屏障功能障碍中的作用。方法用胰胆管内注射甘脱氧胆酸结合静脉输注蛙皮素方法复制大鼠ASP模型，分别对ASP及假手术组(SO组)大鼠于术后6、12、24小时取门静脉,腹主动脉血行血气分析,据此计算回肠粘膜内pH值及其氧摄取率，同时取门静脉血测定门静脉血乳酸会计师。结果ASP组大鼠12及24小时肠道氧摄取率[分别为（45.88±6.05）%及(41.31±4.18)%]均较SO组[分别为(24.82±4.28)%和(23.08±3.44)%]显著增加(P均<0.001)以12小时最为显著,而ASP组制模后12及24小时肠粘膜内pH(分别为7.301±0.036及7.165±0.066)显著低于SO组(分别为7.410±0.047及7.390±0.044),P均<0.001,以24小时为最显著以24小时为最显著。门静脉血乳酸含量在12及24小时分别为(3.58±1.06)mmol/L及(7.97±1.05)mmol/L，均较SO组〔分别为(1.16±0.98)mmol/L及(1.24±0.29)mmol/L〕显著增加，门静脉血乳酸含量与粘膜内pH值呈负相关（r=0.868,P<0.001）。结论ASP时肠粘膜氧供减少，氧代谢障碍，肠粘膜内pH值降低，是肠粘膜屏障功能障碍的主要机制之一。     

Hydrophobicity of mucosal surface and its relationship to gut barrier function

Loss of the gut barrier has been implicated in the pathogenesis of the multiple organ dysfunction syndrome, and, thus, understanding the intestinal barrier is of potential clinical importance. An important, but relatively neglected, component of the gut barrier is the unstirred mucus layer, which through its hydrophobic and other properties serves as an important barrier to bacterial and other factors within the gut lumen. Thus, the goal of this study was to establish a reproducible method of measuring mucosal hydrophobicity and test the hypothesis that conditions that decrease mucosal hydrophobicity are associated with increased gut permeability. Hydrophobicity was measured in various segments of normal gut by measuring the contact angle of an aqueous droplet placed on the mucosal surface using a commercial goniometer. Second, the effect of the mucolytic agent N-acetyl cysteine on mucosal hydrophobicity and gut permeability was measured, as was the effects of increasing periods of in vivo gut ischemia on these parameters. Gut ischemia was induced by superior mesenteric artery occlusion, and gut permeability was measured by the mucosal-to-serosal passage of fluoresceine isothiocyanate-dextran (4.3 kDa) (FD4) across the everted sacs of ileum. Intestinal mucosal hydrophobicity showed a gradual increase from the duodenum to the end of the ileum and remained at high level in the cecum, colon, and rectum. Both N-acetyl cysteine treatment and ischemia caused a dose-dependent decrease in mucosal hydrophobicity, which significantly correlated increased gut permeability. Mucosal hydrophobicity of the intestine can be reproducibly measured, and decreases in mucosal hydrophobicity closely correlate with increased gut permeability. These results suggest that mucosal hydrophobicity can be a reliable method of measuring the barrier function of the unstirred mucus layer and a useful parameter in evaluating the pathogenesis of gut barrier dysfunction.     

Increased iNOS activity is essential for intestinal epithelial tight junction dysfunction in endotoxemic mice

We tested the hypothesis that increased production of nitric oxide (NO.) associated with lipopolysaccharide (LPS)-induced systemic inflammation leads to functionally significant alterations in the expression and/or targeting of key tight junction (TJ) proteins in ileal and colonic epithelium. Wild-type or inducible NO. synthase (iNOS) knockout male C57B1/6J mice were injected intraperitoneally with 2 mg/kg Escherichia coli O111:B4 LPS. iNOS was inhibited using intraperitoneal L-N(6)-(1-iminoethyl)lysine (L-NIL; 5 mg/kg). Immunoblotting of total protein and NP-40 insoluble proteins revealed decreased expression and decreased TJ localization, respectively, of the TJ proteins, zonula occludens (ZO)-1, ZO-2, ZO-3, and/or occludin in ileal mucosa and colonic mucosa (total protein only) after injection of C57B1/6J mice with LPS. Immunohistochemistry showed deranged distribution of ZO-1 and occludin in both tissues from endotoxemic mice. Endotoxemia was associated with evidence of gut epithelial barrier dysfunction evidenced by increased ileal mucosal permeability to fluorescein isothiocyanate-dextran (Mr=4 kDa) and increased bacterial translocation to mesenteric lymph nodes. Pharmacologic inhibition of iNOS activity using L-NIL or genetic ablation of the iNOS gene ameliorated LPS-induced changes in TJ protein expression and gut mucosal barrier function. These results support the view that at least one mechanism contributing to the pathogenesis of gastrointestinal epithelial dysfunction secondary to systemic inflammation is increased iNOS-dependent NO. production leading to altered expression and localization of key TJ proteins.     

Changes in gut mucosal nitric oxide synthase (NOS) activity after thermal injury and its relation with barrier failure

This study was designed to investigate changes in mucosal NOS activity after burns and its relation to barrier failure. In Experiment 1, female specific pathogen free (SPF) Sprague-Dawley rats underwent 35% total body surface area (TBSA) burn. One to six days after burn, intestinal permeability was determined from the plasma leakage of fluorescein isothiocyanate (FITC)-dextran 4400, intestinal mucosal cNOS and iNOS activity were assayed using Griess' reagent, and the cellular localization of iNOS was examined using immunostaining. In Experiment 2, S-methylisothiourea (SMT) was given (5 mg/kg, i.p. every 12 h) for 2 days to suppress inducible NOS (iNOS) activity after thermal injury. On postburn Day 2, the effect of SMT on gut mucosal NOS activity, intestinal permeability, and barrier function were evaluated. The activity of iNOS increased 24 h after the injury and up to a maximum of twofold on postburn Day 2, and decreased thereafter. The increase in iNOS activity in gut mucosa correlated well with the increase in intestinal permeability, an index for barrier failure (r = .776, p = .0002). Results from iNOS immunostaining showed that changes in mucosal iNOS activity after the burn occurred mainly in the enterocytes rather than in the macrophages. Administration of SMT decreased mucosal iNOS activity, intestinal permeability, and bacterial translocation incidence to mesenteric lymph node concurrently. In conclusion, thermal injury induces intestinal mucosal iNOS, which is principally in the enterocytes. The increased intestinal iNOS activity was closely related to barrier failure. SMT inhibited intestinal mucosal iNOS activity and prevented barrier failure as demonstrated by a decrease in BT occurrence and intestinal permeability.     

胆碱酯酶抑制剂中毒大鼠肠屏障功能和形态结构变化及宾赛克嗪治疗作用的研究

目的 观察胆碱酯酶抑制剂类神经性毒剂中毒后大鼠肠屏障功能和组织形态结构的变化以及宾赛克嚷的治疗作用.方法 40只雄性Wistar大鼠按随机数字表法均分为对照组、维埃克斯染毒模型组及宾赛克嗪1、3、9 mg/kg治疗组.皮下注射维埃克斯13μg/kg染毒;宾赛克嗪组在染毒后5 min腹腔注射相应剂量药物.各组于染毒后3 h取血,检测血浆D-乳酸浓度及二胺氧化酶(DAO)活性;同时取小肠组织,观察肠黏膜形态和超微结构变化.结果 与对照组比较.模型组血浆D-乳酸浓度和DAO活性均明显升高[D-乳酸:(87.752±22.906)mg/L比(29.072±6.546)mg/L,DAO:(6.72±0.93)U/L比(2.99±0.43)U/L,均P<0.01];宾赛克嗪1、3、9 mg/kg组呈现剂量依赖性降低血浆D-乳酸浓度和DAO活性,其中宾赛克嗪9 mg/kg组可逆转染毒后血浆D-乳酸浓度[(45.290±11.141)mg/L]和DAO活性C(3.17±0.68)U/L]增高(均P<0.01).光镜下观察模型组大鼠空肠和回肠肠壁变薄,皱壁变短,结构紊乱,固有层毛细血管扩张充血,黏膜间质水肿等病理变化;电镜下观察模型组大鼠回肠上皮细胞发生坏死,细胞器损伤,紧密连接破坏等变化.宾赛克嗪1、3、9 mg/kg组呈现剂量依赖性抑制小肠的病理变化.结论 胆碱酯酶抑制剂中毒时出现肠黏膜上皮细胞损伤,肠黏膜屏障功能破坏,通透性增加;宾赛克嗪能抑制中毒肠黏膜屏障结构和功能的损伤.     

Bile modulates intestinal epithelial barrier function via an extracellular signal related kinase 1/2 dependent mechanism

Objective Obstructive jaundice is frequently complicated by infections and has been associated with increased bacterial translocation and gut mucosal hyperpermeability in animal models. Proper expression of the tight junction (TJ) proteins ZO-1 and occludin is important for normal gut barrier function. We tested whether bile modulates intestinal epithelial ZO-1 and occludin expression.Animals (a) Male C57BL/6 mice; (b) male Sprague-Dawley rats.Interventions (a) Mice were subjected to common bile duct ligation (CBDL) or a sham procedure, and 96 h later all surviving animals were killed for measurement of ileal mucosal permeability to FITC-labeled dextran (everted gut sac technique), bacterial translocation to mesenteric lymph nodes, and ileal epithelial ZO-1 and occludin expression (western blots). (b) Rat IEC-6 enterocytic monolayers were incubated in the presence or absence of graded concentrations of rat bile and/or U0126, an inhibitor of extracellular signal related kinase (ERK) 1/2 activation.Results (a) Compared to sham-treated controls, CBDL significantly increased gut mucosal permeability and bacterial translocation and markedly decreased ileal epithelial expression of ZO-1 and occludin. In a follow-up in vivo experiment, gavaging mice with fresh rat bile twice daily significantly ameliorated the deleterious effects of CBDL on gut barrier function. (b) Addition of 1% (v/v) bile to media enhanced phosphorylation of ERK1/2, increased the expression of ZO-1 and occludin and decreased permeability to FITC-dextran. All of these bile-mediated effects were blocked by 10 µM U0126.Conclusions These data support the view that the presence of bile in the intestinal lumen is essential for normal gut barrier function, possibly because compounds present in bile initiate ERK1/2-dependent signaling that is essential for normal expression of key TJ proteins.This revised version was published online in April 2005 with a corrected section title.Grant support: 5R01 GM 37631-18 from the National Institutes of Health     

肠内肠外营养对大鼠肠道损伤后屏障功能恢复的实验研究

目的 比较肠内、肠外两种营养支持途径及不同营养物质对胃肠手术后大鼠肠屏障功能的恢复情况.方法 将40只雄性SD大鼠随机分为4组:空白对照组(C组)、肠外营养组(PN组)、普通肠内营养组(EN组)、富含谷氨酰胺的肠内营养组(G-EN组).PN组、EN组、G-EN组行盲肠切除+胃造口置管手术并联合使用阿莫西林50 mg+甲硝唑20 g两次进行干预.术后第1天起各组等氮等热卡连续给予营养支持7 d,于末段回肠5 cm处取肠段1 cm,在光镜下观察肠黏膜形态;采用酶联免疫吸附实验(ELISA)比较血浆D-乳酸含量,检测肠道黏膜通透性;双抗体夹心ABC-ELISA法检测肿瘤坏死因子水平(TNF-α);免疫组化法观察肠黏膜occludin蛋白表达.结果 PN组肠黏膜明显萎缩,其绒毛高度、黏膜厚度、隐窝深度、绒毛表面积及肠黏膜occludin蛋白表达均显著低于C组、EN组、G-EN组(P<0.05),D-乳酸及TNF-α水平显著高于C组、EN组、G-EN组(P<0.05);EN组肠黏膜萎缩较明显,其肠黏膜形态及肠黏膜occludin蛋白表达均低 于C组、G-EN组(P<0.05),D-乳酸及TNF-α水平显著高于C组及G-EN组(P<0.05);G-EN组肠道形态、肠黏膜occludin蛋白表达、D-乳酸及TNF-α水平与C组无统计学差异(P>0.05).结论 谷氨酰胺强化的肠内营养在维护肠黏膜屏障功能、减轻炎症反应,增加肠上皮occludin蛋白表达等方面优于单独使用肠内营养液或肠外营养液,更有助于胃肠手术后大鼠肠屏障功能的恢复.     

Endotoxin-induced bacterial translocation and mucosal permeability: role of xanthine oxidase, complement activation, and macrophage products

BACKGROUND AND METHODS: Previously, we documented that nonlethal doses of endotoxin injure the intestinal mucosal barrier and promote bacterial translocation from the gut to systemic organs. The current study was performed to determine the role of cytokines and complement activation in the pathogenesis of endotoxin-induced mucosal injury and bacterial translocation, as well as to quantify the magnitude of endotoxin-induced intestinal mucosal permeability. RESULTS: The frequency of endotoxin-induced bacterial translocation was similar between normal outbred (88%), complement deficient (67%), and macrophage-hyporesponsive (55%) mice, indicating that neither complement nor macrophage activation is necessary for endotoxin-induced bacterial translocation to occur. As early as 2 hrs after endotoxin challenge, there was evidence of a greater than two-fold increase in ileal (p = .008) but not jejunal (p = .11) permeability as measured by the clearance of 51Cr EDTA. Both the increase in endotoxin-induced ileal permeability and the occurrence of bacterial translocation were largely prevented by pretreatment with allopurinol, a competitive inhibitor of xanthine oxidase. CONCLUSIONS: These results suggest that endotoxin-induced bacterial translocation, mucosal injury, and ileal permeability are mediated via activation of xanthine oxidase, and not through complement activation or the liberation of macrophage products.     

Increased gut permeability and bacterial translocation in Pseudomonas pneumonia-induced sepsis

OBJECTIVE: Gut injury and barrier dysfunction may contribute to the pathogenesis of sepsis and multiple organ dysfunction syndrome. The objective of this study was to determine whether gut injury could be demonstrated in hyperdynamic, normotensive sepsis induced by Pseudomonas pneumonia. DESIGN: Randomized animal study. SETTING: University laboratory. SUBJECTS: Adult male Sprague-Dawley rats. INTERVENTIONS: Sepsis was induced by intratracheal instillation of Pseudomonas aeruginosa. MEASUREMENTS AND MAIN RESULTS: We measured gut mucosal and microvascular injury. In the first experiment, gut mucosal permeability was measured by 51Cr-EDTA uptake in control (n = 6), pneumonia 20-hr (n = 4), and pneumonia 40-hr (n = 4) groups. In the second experiment, microvascular permeability was measured by albumin extravasation, and morphologic abnormalities were scored in control (n = 6), pneumonia 20-hr (n = 9), and pneumonia 40-hr (n = 11) groups. Bacterial translocation to mesenteric lymph nodes was determined in both experiments. Cardiac index increased significantly in the pneumonia compared with control rats (64+/-2.1, 68+/-1.3, vs. 46+/-2 mL/min/100 g, p < .05; all results are listed in the order of pneumonia 20-hr, pneumonia 40-hr, and control groups as mean +/- SEM). Mean blood pressure was normal and was not different between groups (112+/-3, 111+/-2, vs. 118+/-2 mm Hg). 51Cr-EDTA recovery in urine 6 hrs after gavage increased significantly in both pneumonia groups vs. controls (17.5+/-2.2%, 17.9+/-7%, vs. 4+/-0.7%; p < .05). Albumin leak (tissue/plasma ratio) increased significantly in the middle and distal small intestine in the pneumonia 40-hr group vs. controls (0.68+/-0.05, 0.76+/-0.07, vs. 0.45+/-0.04, p < .05 in the middle small gut; 0.75+/-0.09, 0.85+/-0.07, vs. 0.51+/-0.05, p < .05 in the distal small gut). Bacterial translocation to mesenteric lymph nodes increased significantly in pneumonia 40-hr rats vs. controls (positive culture 67% vs. 8%; p < .05). CONCLUSIONS: This study demonstrates gut mucosal and microvascular injury and gut barrier dysfunction in normotensive sepsis secondary to bacterial pneumonia. The mechanism and significance of the injury need to be determined.