Peroxisome proliferator-activated receptor gamma activation promotes intestinal barrier function by improving mucus and tight junctions in a mouse colitis model

Background and aimsDefects in mucus and intestinal epithelia can lead to intestinal inflammation in colitis. Reduced peroxisome proliferator-activated receptor gamma (PPAR_γ) in the mucosa may contribute to inflammation. However, the roles of PPAR_γ in the intestinal barrier remain poorly understood.MethodsChronic colitis was induced in C57BL/6 mice by administration of dextran sulfate sodium (DSS) for 27 days. Three days before DSS treatment, mice were treated with the PPAR_γ agonist rosiglitazone (Ro) orally at 20 mg kg⁻¹ day⁻¹.ResultsThe colitis based on disease activity index and colonic histopathology was significantly ameliorated in the DSS + Ro group. Additionally, mice in the DSS + Ro group had a thicker mucous layer than those in DSS + NS group, and muc2 mRNA expression was elevated significantly along with the mouse atonal homolog, SAM-pointed domain-containing Ets-like factor, and anterior gradient 2 genes. Moreover, tight junctions were up-regulated, whereas long myosin light chain kinase and phosphorylation of the myosin II light chain were lower in DSS + Ro mice. Similarly, after HT-29 and Caco-2 cells were treated by LPS or LPS + Ro, PPAR_γ activation by Ro could effectively improve the intestinal barrier, including intestinal mucus and tight junctions.ConclusionsOur results demonstrate that activated PPAR_γ could effectively promote intestinal mucus integrity by increasing the number of goblet cells, the glycosylation of mucins, and tight junctions via an MLCK-dependent mechanism.     

肠黏膜细胞的紧密连接与肠壁通透性的研究进展

肠黏膜细胞的紧密连接是构成肠黏膜屏障的重要结构基础,在调节肠黏膜通透性中发挥着重要的作用.其结构的破坏,可导致肠壁通透性增高,引起细菌移位、全身炎症反应及多器官功能受损.本文就肠黏膜紧密连接的结构和功能、与通透性的影响因素及改善措施进行了综述.     

Structure of tight junctions in epithelia with different permeability.

Freeze-fracture studies have shown a network of intramembrane fibrils in the tight junctions of epithelia. A direct correlation between the number of fibrils and junctional permeability has been suggested by previous studies. However, we have made two groups of observations showing that junctional permeability is not univocally related to the complexity of the network revealed by freeze-fracture. (i) The tight junctions of the rabbit ileum mucosa are permeable to lanthanum, although they have a complex network of fibrils resembling the junctions of toad urinary bladder, which are impermeable to lanthanum. (ii) The tight junctions of the toad urinary bladder are normally of low permeability; however, when the luminal solution is made hypertonic with lysine, junctional permeability markedly increases and lanthanum permeates through the tight junctions. In freeze-fracture replicas, no differences between the fibrils of control and lysine-treated bladders were found. Our results indicate that junctional permeability is controlled not only by the complexity of the fibrilar network, but that some features of the junctions or the fibrils themselves, not yet revealed by electron microscopy, play a central role in regulating epithelial permeability.     

Role of gut microbiota on intestinal barrier function in acute pancreatitis

Acute pancreatitis(AP) is a common gastrointestinal disorder. Approximately15%-20% of patients develop severe AP. Systemic inflammatory response syndrome and multiple organ dysfunction syndrome may be caused by the massive release of inflammatory cytokines in the early stage of severe AP,followed by intestinal dysfunction and pancreatic necrosis in the later stage. A study showed that 59% of AP patients had associated intestinal barrier injury,with increased intestinal mucosal permeability, leading to intestinal bacterial translocation, pancreatic tissue necrosis and infection, and the occurrence of multiple organ dysfunction syndrome. However, the real effect of the gut microbiota and its metabolites on intestinal barrier function in AP remains unclear. This review summarizes the alterations in the intestinal flora and its metabolites during AP development and progression to unveil the mechanism of gut failure in AP.     

Vibrio cholerae produces a second enterotoxin, which affects intestinal tight junctions.

Attenuated Vibrio cholerae vaccine strains specifically mutated in genes encoding cholera toxin (CT) are still capable of causing mild to moderate diarrhea. Culture supernatants of V. cholerae strains, both CT-positive and CT-negative, were examined in Ussing chambers, and a toxin was found that increases the permeability of the small intestinal mucosa by affecting the structure of the intercellular tight junction, or zonula occludens. The activity of this toxin is reversible, heat-labile, sensitive to protease digestion, and found in culture supernatant fractions containing molecules between 10 and 30 kDa in size. Production of this factor (named ZOT for zonula occludens toxin) correlates with diarrheagenicity of V. cholerae strains in volunteers and may represent another virulence factor of infectious diarrhea that must be eliminated to achieve a safe and effective live oral vaccine against cholera.     

An oligopeptide permeates intestinal tight junctions at glucose-elicited dilatations. Implications for oligopeptide absorption

Turnover of the Na(+)-glucose cotransporter in the apical membrane of intestinal absorptive cells elicits alterations in tight-junction structure including the appearance of intrajunctional dilatations. Paralleling these structural responses, epithelial permeability to ions and nutrient-sized solutes increases. However, it is not known how these observed permeability changes specifically relate to the structural alterations elicited by glucose. Using a hemeconjugated peptide tracer (MP-11; mol wt, approximately 1900), the present study shows that the glucose-elicited tight-junction dilatations are specific anatomical sites of junctional permeation. This peptide tracer penetrates tight junctions selectively at sites of dilatations and is detected focally within the paracellular space. This same tracer does not penetrate junctions when glucose is not present. A heme-conjugated macromolecule (horseradish peroxidase; mol wt, approximately 40,000) is excluded by both glucose-exposed and glucose-unexposed tissues. The results of this study show a paracellular pathway for small peptides that is regulated during Na(+)-glucose-activated absorption. It is speculated that the paracellular pathway may contribute to the meal-related oligopeptide absorption that is known to occur and has previously been wholly attributed to the transcellular route.     

Heat stress modulated gastrointestinal barrier dysfunction: role of tight junctions and heat shock proteins

Increased environmental temperature exerts a visible impact on an individual’s physiology. At the onset of heat stress, there is an increase in core body temperature which triggers peripheral vasodilation and sweating in an effort to dissipate the elevated body heat. The increase in peripheral circulation however reduces blood flow to the internal organs which are thus adversely affected. In particular, the gastrointestinal (GI) tract gets adversely affected during hyperthermia resulting in loosening of the tight junctions (TJs) that finally leads to higher intestinal permeability. At the cellular level, elevated levels of heat shock proteins (HSPs) induced in response to heat stress mediated cytoprotection by maintaining proper protein folding, releasing survival signals and preserving cytoskeleton integrity. Recent studies have indicated that HSPs play a crucial role in maintaining the localization of TJ proteins. Dietary supplements have also shown to have a positive effect on the maintenance of intestinal TJs. Therefore, it becomes imperative to understand the cellular, molecular and physiological alterations in response to heat stress in GI tract. In the present report, the effect of thermal stress on GI tract has been summarized. Specific role of HSPs along with mitogen activated protein (MAP) kinase signaling pathway in response to hyperthermia has also been discussed.     

Establishment of tight junctions between epithelial cells.

Epithelia serve as barriers to the diffusion of solutes between body compartments, and must do so despite the frequent loss of cells. When single cells are experimentally removed from the Necturus gallbladder epithelium, contiguous cells migrate to fill the defect within 30 min. Electrophysiological measurements show that the local electrical resistance across the epithelium in the region of a wound returns to normal in the same period of time; electron microscopy demonstrates that tight junctions are formed concurrently. Physiologically functional and morphologically recognizable tight junctions can thus be established within 30 min, demonstrating a mechanism for the rapid restoration of epithelial integrity after cell loss.     

Hyperhomocysteinemia increases permeability of the blood-brain barrier by NMDA receptor-dependent regulation of adherens and tight junctions

Hyperhomocysteinemia (HHcy) increases permeability of the blood-brain barrier, but the mechanisms are undetermined. Homocysteine (Hcy) is an agonist of the neuronal N-methyl-D-aspartate receptor (NMDAr). We tested the hypothesis that HHcy disrupts the blood-brain barrier by an NMDAr-dependent mechanism in endothelium. In brain microvascular endothelial cells, there was no change in expression of the adherens junction protein VE-cadherin with Hcy treatment, but there was a significant decrease in the amount of β-catenin at the membrane. Moreover, Hcy caused nuclear translocation of β-catenin and attachment to the promoter for the tight junction protein claudin-5, with concomitant reduction in claudin-5 expression. Using a murine model of HHcy (cbs(+/-)), treatment for 2 weeks with an NMDAr antagonist (memantine) rescued cerebrovascular expression of claudin-5 and blood-brain barrier permeability to both exogenous sodium fluorescein and endogenous IgG. Memantine had no effect on these parameters in wild-type littermates. The same results were obtained using an in vitro model with brain microvascular endothelial cells. These data provide the first evidence that the NMDAr is required for Hcy-mediated increases in blood-brain barrier permeability. Modulating cerebral microvascular NMDAr activity may present a novel therapeutic target in diseases associated with opening of the blood-brain barrier in HHcy, such as stroke and dementia.     

早期肠内谷氨酰胺补给对烫伤大鼠肠黏膜细胞紧密连接的影响

目的:了解早期肠内谷氨酰胺补给对烫伤后肠黏膜屏障功能的保护作用, 并探讨其可能的作用机制.方法:选取健康SD大鼠随机分为标准肠内营养组(EN组)和谷氨酰胺补给组(EN+Gln组), 每组32只, 制成烫伤动物模型(30% TBSA Ⅲ度烫伤), 伤后早期分别给予标准肠内营养制剂(能全力)和标准肠内营养制剂+谷氨酰胺, 于第1、4、7、10天观察血清D-乳酸, Western blot半定量分析紧密连接结构蛋白Occludin和RTPCR分析ZO-1 mRNA表达的变化.结果:烫伤后血清D-乳酸水平升高, EN组在观察时间内血清D-乳酸水平没有恢复至伤前水平, EN+Gln组在伤后4 d恢复(4.5±0.8 mg/Lvs 3.8±0.6 mg/L); Western blot半定量分析显示EN+Gln组伤后Occludin表达先下降后上升,在4、7 d时明显高于EN组(1.18±0.14 vs 0.79±0.09, 1.59±0.16 vs 1.12±0.13, 均P<0.05);烧伤后1 d时, 2组ZO-1 mRNA表达与伤前比较均明显下降(0.71±0.19, 0.76±0.17 vs 1.00, 均P<0.05), 4 d时EN+Gln组与EN组比较差异有显著的统计学意义(1.17±0.16 vs 0.76±0.15,P<0.05), 其余时间点各组间差异均无统计学意义.结论:早期肠内谷氨酰胺补给支持能快速逆转烧伤后TJ结构蛋白Occludin、ZO-1表达的下降, 从而改善肠黏膜的屏障功能.     

Role of the intestinal barrier in inflammatory bowel disease

A critical function of the intestinal mucosa is to form a barrier that separates luminal contents from the interstitium. The single layer of intestinal epithelial cells （IECs） serves as a dynamic interface between the host and its environment. Cell polarity and structural properties of the epithelium is complex and is important in the development of epithelial barrier function. Epithelial cells associate with each other via a series of intercellular junctions. The apical most intercellular junctional complex referred to as the Apical Junction Complex （AJC） is important in not only cell-cell recognition, but also in the regulation of paracellular movement of fluid and solutes. Defects in the intestinal epithelial barrier function have been observed in a number of intestinal disorders such as inflammatory bowel disease （IBD）. It is now becoming evident that an aberrant epithelial barrier function plays a central role in the pathophysiology of IBD. Thus, a better understanding of the intestinal epithelial barrier structure and function in healthy and disease states such as IBD will foster new ideas for the development of therapies for such chronic disorders.     

肠道菌群失衡在急性胰腺炎肠黏膜屏障损伤中的作用

急性胰腺炎(acute pancreatitis,AP)是临床常见的急腹症,其发病率呈逐年增高趋势.AP病情进展迅速,15％～20％的患者发展为重症急性胰腺炎(severe acute pancreatitis,SAP),病死率高达20％～30％.SAP常可导致肠道功能障碍,包括肠黏膜屏障损伤和肠道动力障碍,引起肠道细...     

姜黄素对肠道屏障的作用及机制

姜黄素是一种从姜科植物的根茎中提取而得的橙黄色多酚类化学物质,是中药姜黄、郁金等的主要有效成分,被广泛用于食品添加、化妆品、染料等。近些年大量研究表明姜黄素还存在较高的医疗价值,具有抗炎、抗氧化、抗肿瘤、抗凋亡、抗纤维化、免疫调节等作用,可用于多种疾病[1],已逐渐成为国内外的一个研究热点。     

Ethanol-induced activation of myosin light chain kinase leads to dysfunction of tight junctions and blood-brain barrier compromise

BACKGROUND: Brain endothelial cells form the blood-brain barrier (BBB) that regulates solute and macromolecule flux in and out of the brain, leukocyte migration, and maintains the homeostasis of the central nervous system. BBB dysfunction is associated with disruption of tight junctions (TJ) in the brain endothelium. We propose that alcohol abuse may impair BBB permeability through TJ modification. METHODS: Primary cultured bovine brain microvascular endothelial cells (BBMEC) were treated with 50 mM ethanol (EtOH), and monolayer tightness was assessed by measurement of transendothelial electrical resistance (TEER). Changes in TEER were correlated with alterations in TJ protein distribution [occludin, zonula occludens-1 (ZO-1), claudin-5] using immunofluorescence (IF). Expression of myosin light chain (MLC) kinase (MLCK), ZO-1, claudin-5, and phosphorylated MLC, occludin and claudin-5 were determined by immunoprecipitation and Western blot. EtOH-induced changes in monocyte migration across in vitro BBB constructs were also examined. RESULTS: EtOH induced a decrease in TEER of BBMEC monolayers that was reversed by EtOH withdrawal. Treatment of BBMEC with EtOH or its metabolite, acetaldehyde, prior to monocyte application resulted in a 2-fold increase in monocyte migration across the BBB. IF demonstrated decrease in claudin-5 staining, occludin translocation from cell borders to cytoplasm and gap formation in EtOH-treated BBMEC monolayer. These changes paralleled significant increase in phosphorylation of MLC, occludin and claudin-5. EtOH-treated BBMEC showed reduction of total occludin and claudin-5 without changes in ZO-1 or MLC. TEER decrease, changes in occludin/claudin staining, increase in MLC, occludin and claudin-5 phosphorylation and enhanced monocyte migration across the BBB were all reversed by inhibition of MLCK. Inhibition of EtOH metabolism in BBMEC also reversed these events. CONCLUSION: These results suggest that EtOH activates MLCK leading to phosphorylation of MLC, occludin and claudin-5. Cytoskeletal alterations (MLC) and TJ changes (occludin and claudin-5 phosphorylation) result in BBB impairment (decrease in TEER). TJ compromise is associated with increased monocyte migration across the BBB.     

肠屏障在自身免疫性肝炎发病中的作用

目的观察并分析肠屏障在自身免疫性肝炎(AIH)发病中的作用,为阐释AIH的发病机制和探索基于肠道的治疗策略提供方向。方法纳入2017年1至12月在天津医科大学总医院就诊的14例AIH患者(AIH无肝硬化组6例,AIH肝硬化组8例)和10名健康对照(健康对照组),采用ELISA法检测各组血清D-乳酸、二胺氧化酶(DAO)含量,实时荧光定量PCR检测各组回肠末端组织紧密连接蛋白[闭锁小带蛋白-1(ZO-1)、闭合蛋白(occludin)]、细胞因子[IL-2、干扰素γ、IL-4和IL-10]和TLR4的相对表达量,蛋白质印迹法检测回肠末端组织分泌型免疫球蛋白A(sIgA)的相对表达量。选取30只BALB/c小鼠分为空白组、葡聚糖硫酸钠(DSS)组、刀豆蛋白A(ConA)组、DSS+ConA组、DSS+灌菌+ConA组,每组6只,检测各组小鼠结肠组织ZO-1和闭合蛋白的相对表达量,血清转氨酶水平(ALT、AST)和肝组织学炎症活动度Knodell评分。统计学方法采用独立样本t检验和单因素方差分析。结果AIH肝硬化组和AIH无肝硬化组的血清D-乳酸和DAO水平均高于健康对照组[(1768.2±147.1)μg/L、(436.2±197.0)μg/L比(100.2±10.9)μg/L和(11.5±2.5)U/L、(5.4±0.9)U/mL比(3.5±0.9)U/mL],且AIH肝硬化组血清D-乳酸和DAO水平最高,差异均有统计学意义(t=5.512、36.010、4.088和9.443,F=396.958、46.640,P均<0.01)。AIH肝硬化组的肠黏膜ZO-1、闭合蛋白的相对表达量均低于健康对照组(0.20±0.14比1.67±0.51,0.12±0.09比0.90±0.21),AIH无肝硬化组ZO-1的相对表达量低于健康对照组(0.99±0.37比1.67±0.51),差异均有统计学意义(t=8.641、7.407、2.295,P均<0.05)。AIH肝硬化组回肠末端组织中IL-2、干扰素γ的相对表达量均高于健康对照组(1.11±0.43比0.24±0.16和3.50±1.90比0.32±0.30),肠黏膜sIgA的相对表达量低于健康对照组(0.506±0.024比1.081±0.102),差异均有统计学意义(t=4.679、3.981、5.493,P均<0.05);AIH肝硬化组和AIH无肝硬化组IL-10的相对表达量均低于健康对照组(0.30±0.20、0.42±0.24比0.84±0.23),回肠末端组织TLR4的相对表达量均高于健康对照组(8.74±5.13、6.74±3.65比0.89±0.70),差异均有统计学意义(t=3.095、4.816、3.856、3.685,P均<0.05)。DSS+ConA组肠黏膜ZO-1和闭合蛋白的相对表达量均低于ConA组(0.14±0.08比0.98±0.13和0.09±0.02比0.98±0.16),血清ALT、AST水平和Knodell评分均高于ConA组[(5496.67±618.83)U/L比(3325.00±1030.06)U/L、(8825.00±1165.35)U/L比(5433.33±1691.14)U/L和(18.00±2.00)分比(9.33±3.01)分],差异均有统计学意义(t=13.480、13.520、4.427、4.045、-2.892,P均<0.01)。DSS+灌菌+ConA组肠黏膜ZO-1和闭合蛋白的相对表达量均高于DSS+ConA组(0.46±0.08比0.14±0.08和0.53±0.15比0.09±0.02),血清ALT、AST水平均低于DSS+ConA组[(4343.33±252.16)U/L比(5496.67±618.83)U/L和(6123.33±1086.60)U/L比(8825.00±1165.35)U/L],差异均有统计学意义(t=6.928、7.122、4.228、4.153,P均<0.01)。结论AIH患者的肠道通透性增高、肠屏障被破坏,且肝硬化患者较非肝硬化患者的程度更严重。肠屏障被破环会加重ConA诱导的免疫性肝损伤,而保护和修复肠屏障则可相对减轻ConA诱导的免疫性肝损伤。     

Rho kinase regulates tight junction function and is necessary for tight junction assembly in polarized intestinal epithelia

BACKGROUND & AIMS: Tight junctions are crucial determinants of barrier function in polarized intestinal epithelia and are regulated by Rho guanosine triphosphatase. Rho kinase (ROCK) is a downstream effector of Rho. METHODS: A specific inhibitor of ROCK, Y-27632, was used to examine the role of ROCK in the regulation of tight junctions in model intestinal (T84) cells by electrophysiologic, biochemical, morphologic, and molecular biologic approaches. RESULTS: ROCK inhibition induced reorganization of apical F-actin structures and enhanced paracellular permeability but did not alter the distribution or detergent solubility of tight junction proteins. Confocal microscopy showed colocalization of a subpool of ROCK with the tight junction protein zonula occludens 1. Inhibition of ROCK function by a dominant negative mutant of ROCK also produced reorganization of apical F-actin structures without disruption of tight junctions. ROCK inhibition in calcium switch assays showed that ROCK is necessary for the assembly of tight and adherens junctions. Upon calcium repletion, occludin, zonula occludens 1, and E-cadherin failed to redistribute to the intercellular junctions; assembly of the apical F-actin cytoskeleton was prevented; and barrier function failed to recover. CONCLUSIONS: We suggest that ROCK regulates intact tight junctions via its effects on the F-actin cytoskeleton. ROCK is also critical for assembly of the apical junctional proteins and the F-actin cytoskeleton organization during junctional formation.     

Intraepithelial gammadelta+ lymphocytes maintain the integrity of intestinal epithelial tight junctions in response to infection

BACKGROUND & AIMS: Intestinal epithelial integrity and permeability is dependent on intercellular tight junction (TJ) complexes. How TJ integrity is regulated remains unclear, although phosphorylation and dephosphorylation of the integral membrane protein occludin is an important determinant of TJ formation and epithelial permeability. We have investigated the role intestinal intraepithelial lymphocytes (iIELs) play in regulating epithelial permeability in response to infection. METHODS: Recombinant strains of Toxoplasma gondii were used to assess intestinal epithelial barrier function and TJ integrity in mice with intact or depleted populations of iIELs. Alterations in epithelial permeability were correlated with TJ structure and the state of phosphorylation of occludin. iIEL in vivo reconstitution experiments were used to identify the iIELs required to maintain epithelial permeability and TJ integrity. RESULTS: In the absence of gammadelta+ iIELs, intestinal epithelial barrier function and the ability to restrict epithelial transmigration of Toxoplasma and the unrelated intracellular bacterial pathogen Salmonella typhimurium was severely compromised. Leaky epithelium in gammadelta+ iIEL-deficient mice was associated with the absence of phosphorylation of serine residues of occludin and lack of claudin 3 and zona occludens-1 proteins in TJ complexes. These deficiencies were attributable to the absence of a single subset of gammadelta T-cell receptor (TCR-Vgamma7+) iIELs that, after reconstituting gammadelta iIEL-deficient mice, restored epithelial barrier function and TJ complexes, resulting in increased resistance to infection. CONCLUSIONS: These findings identify a novel role for gammadelta+ iIELs in maintaining TJ integrity and epithelial barrier function that have implications for understanding the pathogenesis of intestinal inflammatory diseases associated with disruption of TJ complexes.