Small Intestinal Mucosal Barrier Research Articles

Arsenic exposure induces small intestinal toxicity in mice by barrier damage and inflammation response via activating RhoA/ROCK and TLR4/Myd88/NF-κB signaling pathways

Numerous studies have shown that arsenic (As) is an important hazardous metalloid that is commonly considered to have systemic toxicity. The main pathway of arsenic exposure is oral; however, many of the events that occur during its passage through the gastrointestinal tract are unclear, and there are few reports on the effect of arsenic on small intestinal mucosal barrier. This study aimed to investigate arsenic-induced mucosal barrier damage in the small intestine of mice induced by oral exposure and its potential mechanisms. In the present study, histomorphometric and immunohistochemical analyses showed that arsenic-treated mice exhibited signs of irregularly arranged and atrophied small intestinal villi, reduced villus lengths, inflammatory cells infiltration, along with up-regulated expression of inflammatory factors TNF-α, IL-6 and IL-1β in the small intestine of mice. The myeloperoxidase (MPO) activity was also increased in As-exposed mice. Transmission electron microscopy (TEM) analysis demonstrated that intestinal epithelial tight junctions (TJs) were impaired in the small intestines of mice in As group. In addition, arsenic down-regulated mRNA levels of TJ-related genes (ZO-1, ZO-2, occludin, claudin-1, and claudin-7) and protein levels of ZO-1, occludin and claudin-1 were significantly reduced in arsenic-treated groups, while arsenic also increased levels of TLR4, Myd88, NF-κB, RhoA, and ROCK mRNA and protein expression. In summary, these results indicate that the small intestine toxicity in mice evoked by arsenic was correlated with the activation of TLR4/Myd88/NF-κB and RhoA/ROCK pathways.

Estrogen deficiency aggravates fluoride-induced small intestinal mucosa damage and junctional complexes proteins expression disorder in rats

To investigate the effect of estrogen deficiency on the small intestinal mucosal barrier induced by fluoride (F), F exposure models of ovariectomy (OVX) rats (surgically removed ovaries) and non-OVX rats (normal condition) were established by adding sodium fluoride (NaF) (0, 25, 50, and 100 mg/L, calculated by F ion) in drinking water for 90 days. The intestinal mucosal histomorphology, mucosal barrier function, and protein expression levels of tight junctions (TJs), adhesion junctions (AJs), and desmosomes were evaluated in the duodenum, jejunum, and ileum. Hematoxylin-eosin (HE) staining and 5-Bromo-2-deoxyUridine (BrdU) measurement showed that excessive F-induced damage to intestinal epithelial cells and inhibited the proliferation of intestinal epithelial cells, eventually decreasing the number of goblet cells and decreasing glycoprotein secretion, as indicated by Alcian blue and periodic acid-Schiff (AB-PAS) and periodic acid-Schiff (PAS) staining. Further immunofluorescence analysis demonstrated that excessive F decreased the protein expression levels of occludin, zonula occludens-1 (ZO-1), E-cadherin, and desmoplakin (P < 0.05, P < 0.01) and enhanced the expression of claudin-2 (P < 0.01), suggesting that cell-to-cell junctions were disrupted. Collectively, F exposure impaired the small intestinal mucosal barrier by inducing damage to intestinal epithelial cells and inhibiting intestinal epithelial cell proliferation. Disorders in the junctional complex protein expression blocked the synergy between intercellular communication and aggravated mucosal injury. In particular, estrogen deficiency exacerbated F-induced enterotoxicity, which provides new explanations for the development and severity of intestinal disease in postmenopausal women with high-F areas.

Heat stress of gilts around farrowing causes oxygen insufficiency in the umbilical cord and reduces piglet survival

Late gestating sows are susceptible to high ambient temperatures, possibly causing farrowing complications and reducing piglet survival. This experiment aimed to quantify in the days leading up to farrowing the impact of sow heat stress (HS) on farrowing physiology and survival of the piglets. Pregnant primiparous sows (gilts) were allocated to either thermoneutral control (CON, n = 8; constant 20 °C) or cyclical HS conditions (n = 8; 0900 h to 1700 h, 30 °C; 1700 h to 0900 h, 28 °C) from d 110 of gestation until farrowing completion. Gilt respiration rate, skin temperature and rectal temperature were recorded daily, and farrowing duration was quantified by video analyses. Blood samples were collected from the piglet umbilical vein at birth. At 48 h of age, piglet growth was quantified by morphometric analyses. The thermal exposure model induced HS and respiratory alkalosis in the gilts, as indicated by increased respiration rate, rectal temperature, skin temperature (all P < 0.001), plasma cortisol (P = 0.01) and blood pH (P < 0.001). Heat-stressed gilts took longer to start expelling placentae (P = 0.003), although the active farrowing duration was not significantly different between treatments. Stillbirth rates were higher in the HS group (P < 0.001), with surviving piglets at birth having lower umbilical vein partial pressure of oxygen (P = 0.04), oxygen saturation rate (P = 0.03) and tending to have increased lactate concentrations (P = 0.07). At birth, piglet skin meconium staining scores were greater in the HS group (P = 0.022). At 48 h of age, piglets from the HS group had reduced small intestinal length (P = 0.02), reduced jejunal crypt depth (P = 0.02) and lighter absolute brain weight (P = 0.001). In contrast, piglet BW, growth rate, relative organ weight and small intestinal mucosal barrier function did not change between treatments. Collectively, these findings demonstrated gilt HS during late gestation caused farrowing complications and reduced the umbilical oxygen supply to the piglets at parturition, leading to increased risks of piglet stillbirth with implications on impaired neonatal survivability and development.

Baicalin regulates autophagy to interfere with small intestinal acute graft-versus-host disease

Acute graft-versus-host disease (aGVHD) is the main complication of and cause of death after allogeneic hematopoietic stem cell transplantation. Baicalin can protect the small intestinal epithelial cells of rats against TNF-α-induced injury and alleviate enteritis-related diarrhea. To verify whether baicalin can protect the small intestinal mucosal barrier by regulating abnormal autophagy and interfering with intestinal aGVHD, a mouse model of aGVHD was established. CB6F1 micewere intravenously injected with a suspension of mononuclear cells derived from BALB/c donor mouse bone marrow and splenic tissue after treatment with 60Co X-rays. After treatment with different doses of baicalin for 15 days, the survival time, serum TNF-α and IL-10 levels, and autophagy markers levels in the intestine were assessed. A cell model of intestinal barrier dysfunction was also used to verify the effect of baicalin. The results showed that baicalin significantly prolonged the survival time, significantly reduced the aGVHD pathology score and clinical score by decreasing the TNF-α level with increasing the IL-10 level compared with the control. Transmission electron microscopy examination showed that baicalin treatment increased the number of autophagic vacuoles and led to the recovery of mitochondrial structures in the intestinal mucosal epithelial cells of mice and in Caco-2 cells. Western blotting results showed that baicalin treatment enhanced autophagy in vivo by regulating the AMPK/mTOR autophagy pathway. Similar results were observed in vitro in Caco-2 cells. Furthermore, the effect of baicalin was reduced after combination treatment with the autophagy inhibitor 3-methyladenine(3-MA). Baicalin can decrease the severity of small intestinal aGVHD by regulating autophagy by influencing imbalances in inflammatory cytokine levels and mucosal barrier damage, thus baicalin may have potential as a new treatment for aGVHD.

Effects of dietary protein level on small intestinal morphology, occludin protein, and bacterial diversity in weaned piglets.

Due to the physiological characteristics of piglets, the morphological structure and function of the small intestinal mucosa change after weaning, which easily leads to diarrhea in piglets. The aim of this study was to investigate effects of crude protein (CP) levels on small intestinal morphology, occludin protein expression, and intestinal bacteria diversity in weaned piglets. Ninety‐six weaned piglets (25 days of age) were randomly divided into four groups and fed diets containing 18%, 20%, 22%, and 24% protein. At 6, 24, 48, 72, and 96 h, changes in mucosal morphological structure, occludin mRNA, and protein expression and in the localization of occludin in jejunal and ileal tissues were evaluated. At 6, 24, and 72 h, changes in bacterial diversity and number of the ileal and colonic contents were analyzed. Results showed that structures of the jejunum and the ileum of piglets in the 20% CP group were intact. The expression of occludin mRNA and protein in the small intestine of piglets in the 20% CP group were significantly higher than those in the other groups. As the CP level increased, the number of pathogens, such as Clostridium difficile and Escherichia coli, in the intestine increased, while the number of beneficial bacteria, such as Lactobacillus, Bifidobacterium, and Roseburia, decreased. It is concluded that maintaining the CP level at 20% is beneficial to maintaining the small intestinal mucosal barrier and its absorption function, reducing the occurrence of diarrhea, and facilitating the growth and development of piglets.

Ulinastatin protects intestinal mucosal barrier by inhibiting the activation of intestinal NLRP3 inflammasomes in septic rats

To explore the damage of the intestinal mucosal barrier of septic rats by the activation of NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasomes and the role of Ulinastatin (UTI) on the expression of intestinal nuclear factor-κB (NF-κB)/NLRP3 inflammasome signaling pathway in septic rats. According to the random number table method, 64 male Wistar rats were divided into sham operation group (Sham group), cecal ligation and puncture (CLP) group, UTI treatment group (100 kU/kg UTI was intraperitoneally injected 1, 6, 12 and 18 hours after CLP), and UTI pretreatment group (100 kU/kg UTI was given 1 hour before CLP), with 16 rats in each group. The survival of rats was observed after 24 hours, and the blood was collected from abdominal aorta at 24 hours after modeling, then rats were killed and their ileum tissues were taken. Hematoxylin-eosin (HE) staining was used to observe histopathological changes and Chiu score. The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and intestinal fatty acid binding protein (I-FABP) in serum were detected by enzyme linked immunosorbent assay (ELISA). The protein expression of NF-κB p65 in intestinal tissue was detected by Western blotting. The expression of intestinal tight junction proteins Claudin-1, Occludin and the inflammasome NLRP3, apoptosis-associated speck-like protein containing CARD (ASC) and caspase-1 were detected by immunohistochemistry. Compared with Sham group, the 24-hour survival rate of CLP group was significantly reduced. Histopathological results showed that the CLP group had severe edema of mucosa and submucosal stroma with obvious infiltration of inflammatory cells and disordered villi arrangement. Some glands were incomplete, and the villus structure was severely damaged. The Chiu score was significantly increased. The levels of TNF-α, IL-1β, I-FABP in serum and the protein expression of NF-κB p65 in intestinal tissue were significantly increased. The positive expressions of NLRP3, caspase-1 and ASC were also significantly increased. However, the positive expression of tight junction protein in small intestine tissue such as Occludin and Claudin-1 were significantly reduced. It suggested that when sepsis occurs, small intestinal mucosal barrier dysfunction happens, and mucosal permeability increases, while tight junction protein expression decreases, NLRP3 inflammasome and its upstream molecule NF-κB p65 were activated. After UTI treatment and UTI pretreatment intervention, although there was no significant difference in 24-hour survival compared with CLP group (62.5%, 68.8% vs. 43.8%, both P > 0.05), the intestinal tissue damage of septic rats was significantly improved. Specifically: Chiu score and the levels of TNF-α, IL-1β, I-FABP in serum were significantly decreased [Chiu score: 3.37±0.25, 3.23±0.16 vs. 4.08±0.13, TNF-α (ng/L): 147.62±20.74, 140.71±24.81 vs. 222.82±16.84, IL-1β (ng/L): 80.64±5.68, 78.11±4.75 vs. 133.73±3.92, I-FABP (μg/L): 38.29±3.60, 35.88±4.52 vs. 59.81±4.66, all P < 0.05]; the protein expression of NF-κB p65 was significantly decreased (NF-κB p65/β-actin: 0.65±0.10, 0.69±0.11 vs. 0.99±0.10, both P < 0.05), the positive expressions of Claudin-1 and Occludin in the small intestine tissue were increased [Claudin-1 positive expression area: (19.43±3.08)%, (23.99±6.27)% vs. (7.77±2.03)%; Occludin positive expression area: (19.58±4.75)%, (23.28±3.68)% vs. (11.69±4.30)%, all P < 0.05], while the positive expressions of NLRP3, caspase-1, ASC were decreased [NLRP3 positive expression area: (7.80±3.14)%, (6.86±2.63)% vs. (14.44±3.68)%; caspase-1 positive expression area: (10.62±3.52)%, (9.49±3.09)% vs. (26.69±8.05)%; ASC positive expression area: (9.95±2.81)%, (10.53±3.61)% vs. (24.16±5.48)%, all P < 0.05]. However, there was no significant difference in the improvement effect between UTI treatment group and UTI pretreatment group. Intestinal barrier dysfunction in sepsis may be related to the activation of NLRP3 inflammasomes in the intestinal mucosa. The protective effect of UTI in the intestinal mucosa may be related to inhibiting the activation of NLRP3 inflammasomes in the intestinal mucosa, but UTI pretreatment has no obvious advantage compared with UTI treatment.

The Mechanism of Mica on Protecting NSAIDs Induced Small Intestinal Mucosal Barrier Injury via ncRuPAR-PAR2 Pathway

Objective: The study was to detect the role of ncRuPAR and Protease activated receptor (PAR)2 in the mechanism of Non-steroidal anti-inflammatory drugs(NSAIDs) induced small intestinal mucosal barrier injury. And to clarify the possible mechanism of mica in treatment NSAIDs induced small intestinal disease. Methods: Caco-2 cells were cultured and divided into 6 groups: control group, diclofenac sodium group, control RNAa group, ncRuPAR RNA activation (RNAa) group, diclofenac sodium + mica group, diclofenac sodium + FSLLRY-NH2 group. 40 μmol/L diclofenac sodium treatment for 24 hours to establish NSAIDs induced small intestinal epithelial cell injury model, 10% mica-containing serum treatment for 24h, 100uM PAR2 blocker FSLLRY-NH2 treatment for 24h. Blank vector or ncRuPAR overexpression vector were transfected into cells for 72h. Then Transwell assay were performed to detect cell permeability, qPCR were used to detect RNA expression of PAR2 and ncRuPAR, and western blot were performed to detect PAR2, occludin and claudin-1 expression. Result: NSAIDs-induced small intestinal epithelial cell injury model was successfully built. Compared with the control group, ncRuPAR and PAR2 were up-regulated, occludin and claudin-1 were down-regulated, cell permeability were increased in diclofenac sodium group (P 0.05). Compared with the control RNAa group, overexpression of ncRuPAR significantly up-regulated the expression of ncRuPAR and PAR2, down-regulated the expression of occludin and claudin-1, and increased cell permeability (P<0.01). Conclusion: When NSAIDs induced small intestinal injury occurs, ncRuPAR activation could up-regulate PAR2, causing the destruction of intestinal epithelial cell tight junction and the increase of intestinal epithelial cells permeability. Mica-containing serum has a similar effect as PAR2 blocker, which could antagonize the above-mentioned damage process and play a role in protecting the mechanical barrier function of the intestinal mucosa. Funding Statement: This research was supported by funding from Zhejiang Provincial Natural Science Foundation of China under Grant No.LY18H030001； the Medicine and Health Science and Technology Plan Projects in Zhejiang province(2017KY413), Traditional Chinese Medicine Science and Technology Plan of Zhejiang Province(2017ZA089, 2016ZB071, 2015ZZ012, 2014ZA030); National Natural Science Foundation of China (81573760);Medical Health Platform Plan Projects of Zhejiang Province (2015RCA020); Zhejiang Provincial Natural Science Foundation of China (LY16H030010). Declaration of Interests: The authors state: There is no conflict of interest. Ethics Approval Statement: Not required.

Dietary l-Tryptophan Supplementation Enhances the Intestinal Mucosal Barrier Function in Weaned Piglets: Implication of Tryptophan-Metabolizing Microbiota.

l-Tryptophan (Trp) is known to play an important role in the health of the large intestine. However, a role of dietary Trp in the small-intestinal mucosal barrier and microbiota remains poorly understood. The present study was conducted with weaned piglets to address this issue. Postweaning piglets were fed for 4 weeks a corn- and soybean meal-based diet supplemented with 0 (Control), 0.1, 0.2, or 0.4% Trp. The small-intestinal microbiota and serum amino acids were analyzed by bacterial 16S rRNA gene-based high-throughput sequencing methods and high-performance liquid chromatography, respectively. The mRNA levels for genes involved in host defense and the abundances of tight-junction proteins in jejunum and duodenum were measured by real time-PCR and Western blot techniques, respectively. The concentrations of Trp in the serum of Trp-supplemented piglets increased in a dose-dependent manner. Compared with the control group, dietary supplementation with 0.2–0.4% Trp reduced the abundances of Clostridium sensu stricto and Streptococcus in the jejunum, increased the abundances of Lactobacillus and Clostridium XI (two species of bacteria that can metabolize Trp) in the jejunum, and augmented the concentrations of secretory immunoglobulin A (sIgA) as well as mRNA levels for porcine β-defensins 2 and 3 in jejunal tissues. Moreover, dietary Trp supplementation activated the mammalian target of rapamycin signaling and increased the abundances of tight-junction proteins (zonula occludens (ZO)-1, ZO-3, and claudin-1) in jejunum and duodenum. We suggested that Trp-metabolizing bacteria in the small intestine of weaned pigs primarily mediated the beneficial effects of dietary Trp on its mucosal integrity, health, and function.

Effect of absorption-modifying excipients, hypotonicity, and enteric neural activity in an in vivo model for small intestinal transport

The small intestine mucosal barrier is physiologically regulated by the luminal conditions, where intestinal factors, such as diet and luminal tonicity, can affect mucosal permeability. The intestinal barrier may also be affected by absorption-modifying excipients (AME) in oral drug delivery systems. Currently, there is a gap in the understanding of how AMEs interact with the physiological regulation of intestinal electrolyte transport and fluid flux, and epithelial permeability. Therefore, the objective of this single-pass perfusion study in rat was to investigate the effect of three AMEs on the intestinal mucosal permeability at different luminal tonicities (100, 170, and 290 mOsm). The effect was also evaluated following luminal administration of a nicotinic receptor antagonist, mecamylamine, and after intravenous administration of a COX-2 inhibitor, parecoxib, both of which affect the enteric neural activity involved in physiological regulation of intestinal functions. The effect was evaluated by changes in intestinal lumen-to-blood transport of six model compounds, and blood-to-lumen clearance of 51Cr-EDTA (a mucosal barrier marker). Luminal hypotonicity alone increased the intestinal epithelial transport of 51Cr-EDTA. This effect was potentiated by two AMEs (SDS and caprate) and by parecoxib, while it was reduced by mecamylamine. Consequently, the impact of enteric neural activity and luminal conditions may affect nonclinical determinations of intestinal permeability. In vivo predictions based on animal intestinal perfusion models can be improved by considering these effects. The in vivo relevance can be increased by treating rats with a COX-2 inhibitor prior to surgery. This decreases the risk of surgery-induced ileus, which may affect the physiological regulation of mucosal permeability.

Short-chain fatty acids augment rat duodenal mucosal barrier function.

What is the central question of this study? Small intestinal epithelium is exposed to high concentrations of short-chain fatty acids (SCFAs), but their role in regulating intestinal mucosal barrier function and motility is not fully understood. What is the main finding and its importance? By perfusing the duodenal segment in anaesthetized rats, we show that acetate and propionate significantly decrease mucosal paracellular permeability and transepithelial net fluid flux and increase mucosal bicarbonate secretion. Likewise, SCFAs administered i.v. decrease mucosal permeability but decrease bicarbonate secretion. Altered luminal chemosensing or aberrant signalling in response to SCFAs might contribute to symptoms observed in patients with suppressed mucosal barrier function. Short-chain fatty acids (SCFAs) are produced by bacterial fermentation in the large intestine, particularly from diets containing fibres and carbohydrates. The small intestinal epithelium is exposed to SCFAs derived mainly from oral bacteria or food supplementation. Although luminal nutrients are important in regulation of intestinal functions, the role of SCFAs in regulation of small intestinal mucosal barrier function and motility has not been fully described. The aim of the present study was to elucidate the effects of acetate and propionate on duodenal mucosal barrier function and motility. Rats were anaesthetized with thiobarbiturate, and a 30mm segment of proximal duodenum with an intact blood supply was perfused. The effects on duodenal bicarbonate secretion, blood-to-lumen clearance of 51 Cr-EDTA, motility and transepithelial net fluid flux were investigated. Perfusion of the duodenum with acetate or propionate significantly decreased mucosal paracellular permeability and transepithelial net fluid flux and significantly increased bicarbonate secretion. Acetate or propionate administered as an i.v. infusion decreased the mucosal paracellular permeability, but significantly decreased bicarbonate secretion. Luminal SCFAs changed the duodenal motility pattern from migrating motor complexes to fed patterns. Systemic administration of glucagon-like peptide-2 induced increases in both bicarbonate secretion and net fluid absorption, but did not change motility. Glucagon-like peptide-2 infusion during luminal perfusion of SCFAs significantly reduced the motility. In conclusion, SCFAs decreased duodenal paracellular permeability and net fluid flux. Short-chain fatty acids induced opposite effects on bicarbonate secretion after luminal and i.v. administration. Presence of SCFAs in the lumen induces fed motility patterns. Altered luminal chemosensing and aberrant signalling in response to SCFAs might contribute to symptoms observed in patients with suppressed barrier function.

Small intestinal permeability in older adults.

It is not yet clear whether intestinal mucosal permeability changes with advancing age in humans. This question is of high importance for drug and nutrition approaches for older adults. Our main objective was to answer the question if small intestinal barrier integrity deteriorates with healthy aging. We conducted a cross‐sectional study including the pooled data of 215 nonsmoking healthy adults (93 female/122 male), 84 of whom were aged between 60 and 82 years. After a 12‐h fast, all participants ingested 10 g of lactulose and 5 g of mannitol. Urine was collected for 5 h afterwards and analyzed for test sugars. The permeability index (PI = lactulose/mannitol) was used to assess small intestinal permeability. Low‐grade inflammation defined by high‐sensitivity C‐reactive protein ≥1 mL/L and kidney function (estimated glomerular filtration rate) were determined in the older age group. The PI was similar in older compared to younger adults (P =0.887). However, the urinary recovery of lactulose and mannitol was lower in the older adults and this change was neither associated with urinary volume nor glomerular filtration rate. The PI was not significantly correlated with low‐grade inflammation or presence of noninsulin‐dependent type 2 diabetes. However, it significantly deteriorated in the copresence of both conditions compared to low‐grade inflammation alone (P =0.043) or type 2 diabetes alone (P =0.015). Small intestinal mucosal barrier does not deteriorate with age per se. But low‐grade inflammation coupled with minor disease challenges, such as type 2 diabetes, can compromise the small intestinal barrier.

Small intestinal permeability in older adults

AbstractIt is not yet clear whether intestinal mucosal permeability changes with advancing age in humans. This question is of high importance for drug and nutrition approaches for older adults. Our main objective was to answer the question if small intestinal barrier integrity deteriorates with healthy aging. We conducted a cross‐sectional study including the pooled data of 215 nonsmoking healthy adults (93 female/122 male), 84 of whom were aged between 60 and 82 years. After a 12‐h fast, all participants ingested 10 g of lactulose and 5 g of mannitol. Urine was collected for 5 h afterwards and analyzed for test sugars. The permeability index (PI = lactulose/mannitol) was used to assess small intestinal permeability. Low‐grade inflammation defined by high‐sensitivity C‐reactive protein ≥1 mL/L and kidney function (estimated glomerular filtration rate) were determined in the older age group. The PI was similar in older compared to younger adults (P = 0.887). However, the urinary recovery of lactulose and mannitol was lower in the older adults and this change was neither associated with urinary volume nor glomerular filtration rate. The PI was not significantly correlated with low‐grade inflammation or presence of noninsulin‐dependent type 2 diabetes. However, it significantly deteriorated in the copresence of both conditions compared to low‐grade inflammation alone (P = 0.043) or type 2 diabetes alone (P = 0.015). Small intestinal mucosal barrier does not deteriorate with age per se. But low‐grade inflammation coupled with minor disease challenges, such as type 2 diabetes, can compromise the small intestinal barrier.

Protective effect of bone marrow mesenchymal stem cells in intestinal barrier permeability after heterotopic intestinal transplantation.

To explore the protective effect of bone marrow mesenchymal stem cells (BM MSCs) in the small intestinal mucosal barrier following heterotopic intestinal transplantation (HIT) in a rat model. BM MSCs were isolated from male Lewis rats by density gradient centrifugation, cultured, and analyzed by flow cytometry. The HIT models were divided into a non-rejection group, saline-treated rejection group (via penile vein), and BM MSC-treated group (via penile vein). Intestinal mucosal barrier injury was estimated by diamine oxidase (DAO) and D-lactic acid (D-LA) expression levels. Tumor necrosis factor-α (TNF-α), interferon-γ (INF-γ), interleukin-10 (IL-10), and transforming growth factor-β (TGF-β) were detected by enzyme-linked immunosorbent assay. Ultrastructural change of tight junctions (TJs) was observed under transmission electron microscope. Expression levels of the TJ proteins occludin and zona occludens (ZO)-1, affected by the inflammatory factors, were measured using real-time polymerase chain reaction and Western blotting. The pathological score at each time point after surgery indicated significantly less serious injury in the BM MSCs-treated group than in the rejection group (P < 0.05). In the former, graft levels of DAO and D-LA were reduced, and TNF-α and INF-γ production was inhibited (at day 7: 10.6473 ± 0.0710 vs 17.2128 ± 0.4991, P < 0.05; 545.1506 ± 31.9416 vs 810.2637 ± 25.1175, P < 0.05). IL-10 and TGF-β production was increased greatly (at day 7: 125.7773 ± 4.7719 vs 80.3756 ± 2.5866, P < 0.05; 234.5273 ± 9.3980 vs 545.1506 ± 31.9416, P < 0.05). There was increased expression of occludin and ZO-1 protein (at day 7: 0.2674 ± 0.0128 vs 0.1352 ± 0.0142, P < 0.05; at day 5: 0.7189 ± 0.0289 vs 0.4556 ± 0.0242, P < 0.05) and mRNA (at day 7: 0.3860 ± 0.0254 vs 0.1673 ± 0.0369, P < 0.05; at day 5: 0.5727 ± 0.0419 vs 0.3598 ± 0.0242, P < 0.05). BM MSCs can improve intestinal barrier permeability, repair TJs, and increase occludin and ZO-1 protein expression. With altered cytokine levels, they can protect the intestinal mucosa after transplantation.

Role of serotonin in fatty acid-induced non-alcoholic fatty liver disease in mice

BackgroundSaturated fatty acids are thought to be of relevance for the development of non-alcoholic fatty liver disease and obesity. However, the underlying mechanisms are poorly understood. In previous studies we found that food-derived carbohydrates such as fructose alter the intestinal serotonergic system while inducing fatty liver disease in mice. Here, we examined the effect of fatty acid quantity (11% versus 15%) and quality (saturated, monounsaturated, or polyunsaturated fatty acids) on hepatic fat accumulation, intestinal barrier and the intestinal serotonergic system.MethodsC57BL/6 mice had free access to diets enriched with one of the three fatty acids or standard diet, for 8 weeks. In an additional experiment mice were fed diets enriched with saturated, monounsaturated fatty acids or standard diet supplemented with tryptophan (0.4 g/(kg.d), 8 weeks) or not. Hepatic fat accumulation, small intestinal barrier impairment and components of the serotonergic system were measured with RT-PCR, western blot or immunoassays. For statistical analysis t-test and one-way ANOVA with Tukey’s post hoc test and Bartlett’s test for equal variances was used.ResultsHepatic triglycerides, liver weight and liver to body weight ratio were significantly changed depending on the fat quality but not fat quantity. In contrast, fat quantity but not quality decreased the expression of the tight junction proteins occludin and claudin-1 in the small intestine. These changes seemed to result in enhanced portal vein endotoxin concentrations and fatty liver disease after feeding diet enriched with saturated and monounsaturated fatty acids but not polyunsaturated fatty acids. Neither fatty acid quantity nor quality significantly influenced the intestinal serotonergic system. Similarly, tryptophan supplementation had no impact on small intestinal barrier or fatty liver disease.ConclusionIn conclusion, diets rich in saturated or monounsaturated fatty acids promote the development of fatty liver disease in mice, likely by a dysfunction of the small intestinal mucosal barrier.

Enzymatically Structured Emulsions in Simulated Gastrointestinal Environment: Impact on Interfacial Proteolysis and Diffusion in Intestinal Mucus

Fundamental knowledge of physicochemical interactions in the gastrointestinal environment is required in order to support rational designing of protein-stabilized colloidal food and pharmaceutical delivery systems with controlled behavior. In this paper, we report on the colloidal behavior of emulsions stabilized with the milk protein sodium caseinate (Na-Cas), and exposed to conditions simulating the human upper gastrointestinal tract. In particular, we looked at how the kinetics of proteolysis was affected by adsorption to an oil-water interface in emulsion and whether the proteolysis and the emulsion stability could be manipulated by enzymatic structuring of the interface. After cross-linking with the enzyme transglutaminase, the protein was digested with use of an in vitro model of gastro-duodenal proteolysis in the presence or absence of physiologically relevant surfactants (phosphatidylcholine, PC; bile salts, BS). Significant differences were found between the rates of digestion of Na-Cas cross-linked in emulsion (adsorbed protein) and in solution. In emulsion, the digestion of a population of polypeptides of M(r) ca. 50-100 kDa was significantly retarded through the gastric digestion. The persistent interfacial polypeptides maintained the original emulsion droplet size and prevented the system from phase separating. Rapid pepsinolysis of adsorbed, non-cross-linked Na-Cas and its displacement by PC led to emulsion destabilization. These results suggest that structuring of emulsions by enzymatic cross-linking of the interfacial protein may affect the phase behavior of emulsion in the stomach and the gastric digestion rate in vivo. Measurements of ζ-potential revealed that BS displaced the remaining protein from the oil droplets during the simulated duodenal phase of digestion. Diffusion of the postdigestion emulsion droplets through ex vivo porcine intestinal mucus was only significant in the presence of BS due to the high negative charge these biosurfactants imparted to the droplets. This implies that the electrostatic repulsion produced can prevent the droplets from being trapped by the mucus matrix and facilitate their transport across the small intestine mucosal barrier.

The role of mesenteric lymph in pathogenesis of systemic inflammatory response syndrome and systemic complications following severe acute pancreatitis in rats

To observe the effect of mesenteric lymph duct ligation (MLDL) on systemic inflammatory response syndrome (SIRS) and systemic complications in severe acute pancreatitis (SAP) in rats. Twenty-four Sprague-Dawley (SD) male rats were randomly divided into sham operation group, SAP group, and SAP+MLDL group, with 8 rats in each group. Sham operation group underwent laparotomy only. SAP complicated with multiple organ injury model was reproduced by retrograde injection of sodium taurocholate into the pancreatic duct in SD rats. In SAP+MLDL group MLDL was performed before reproduction of SAP. Rats were sacrificed at 24 hours after operation. The terminal ileum were collected for examination under light microscope. The samples of pancreas, lung and liver were collected for measuring the level of myeloperoxidase (MPO). The mesenteric lymph nodes were harvested for standard bacteriologic culture. The blood levels of diamine oxidase (DAO), D-lactate, endotoxin, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) were also measured. The blood levels of DAO [(0.64 + or - 0.17) kU/L vs. (0.37 + or - 0.07) kU/L], D-lactate [(8.16 + or - 1.79) ng/L vs. (3.24 + or - 1.00) ng/L], endotoxin [(0.068 + or - 0.019) kEU/L vs. (0.033 + or - 0.009) kEU/L], TNF-alpha [(65.21 + or - 13.38) ng/L vs. (22.16 + or - 5.04) ng/L] and IL-6 [(7.95 + or - 1.83) ng/L vs. (4.26 + or - 1.23) ng/L] in SAP group were increased significantly comparing with those in sham operation group (all P<0.01). The contents of pulmonary tissue [(9.07 + or - 2.52) U/g vs. (4.38 + or - 1.29) U/g], pancreatic [(5.14 + or - 1.24) U/g vs. (2.88 + or - 0.75) U/g] and liver [(6.36 + or - 1.63) U/g vs. (3.19 + or - 0.96) U/g] MPO were increased significantly in SAP group comparing with those in sham operation group (all P<0.01). The rate of bacterial translocation to mesenteric lymph nodes in SAP group was significantly higher than that of the sham operation group (75.0% vs. 0, P< 0.01). Compared to SAP group, the contents of DAO [(0.50 + or - 0.13) kU/L], D-lactate [(6.23 + or - 1.25) ng/L] , endotoxin [(0.048 + or - 0.014) kEU/L], TNF-alpha [(48.50 + or - 13.23) ng/L] and IL-6 [(6.06 + or - 1.64) ng/L] were significantly decreased in SAP+MLDL group (P<0.05 or P<0.01). The levels of pulmonary [(6.58 + or - 1.96) U/g], pancreatic [(4.01 + or - 1.05) U/g] and liver [(4.64 + or - 1.34) U/g] MPO, as well as the rate of bacterial translocation to mesenteric lymph nodes (12.5%) were significantly lower in SAP+MLDL group than those in SAP group (all P<0.05). MLDL decreases bacteria/ endotoxin translocation and reduces the systemic inflammatory response by interrupting the lymph flow from the injured gut into the bloodstream. This procedure may protect the small intestine mucosal barrier function and other organs by reducing the neutrophil aggregation and the release of cytokines.

N-acetylcysteine prevents intestine barrier functional disturbance and"second attack"following sodiuln taurocholate-induced severe acute pancreatitis in rats

Objective To evaluate the protective effect of N-acetylcysteine(NAC)on the small intestinal mucosal barrier and other organs in severe acute pancreatitis(SAP)rats.Methods Fifty-four Wistar male rats were divided into sham operation group(n=6),SAP group(n=24),and SAP+NAC group(300 mg/kg body wt)(n=24).Sham operation group underwent laparotomy only.SAP model was established by retrograde injection of 5%sodium taurocholate into the bile-pancreatic duct in Wistar rats.The rats in SAP+NAC group were administered with NAC 300 mg/kg body wt intaperitoneally 2 h before SAP model establishment.Rats were killed at 3,6,12 and 24 h after operation.The terminal ileum was collected for examination under light microscopy.The samples of the pancreas,the lung and the liver were collected for measuring the levels of myeloperoxidase(MPO).The blood levels of diamine oxidase(DAO).tumor necrosis factor Mpha(TNF-α)and interleukin-6(IL-6)were also measured.Results The contents of TNF-α,IL-6 and DAO in SAP group were increased significantly at all the 4 time points as compared with those in sham operation group(P＜0.01).The levels of MPO in the pancreas.the lung and liver of SAP group were increased significantly at 6,12 and 24 h as compared with those in sham operation group(P＜0.05 or P＜0.01).As compared with SAP group,the contents of TNF-α and IL-6 were signifieanfly decreased in SAP+NAC group at all the 4 time points(P＜0.05 or P＜0.01),while the level of DAO significantly decreased in SAP+NAC group except for the 3-h time point(P＜0.05 or P＜0.01).The contents of MPO in the pancreas,the lung and the liver were significantly decreased in SAP+NAC group at 12 and 24 h after operation(P＜0.05 or P＜0.01).Conclusion NAC may protect the small intestine mucosal barrier and other organs by reducing the polymorphonuclear leucocyte detaining,which might be relate to the reduction of neutrophil aggregation by decreasing the release of TNF-α and IL-6. Key words: N-acetyleysteine; Acute pancreatitis; Intestine barrier functional disturbance:Cytokines

Effect of reproductive status on uptake of latex microparticles in rat small intestine

This study investigates whether pregnancy or lactation affects microparticle uptake across the small intestinal mucosal barrier, since aspects of gastrointestinal physiology such as motility may be altered in these conditions. It also reports on validation of the model by several methods and discusses the findings in relation to possible mechanisms.Anaesthetised, pregnant, lactating, virgin female or male adult rats were gavaged with fluorescent latex microparticles. The small intestine was removed and fixed either 5 or 30 min later and successive segments of equal length were examined with fluorescence microscopy. Minor adjustments were made to experimental methods to explore details of the uptake mechanism.Control sections contained no particles. All experimental samples showed luminal and surface particles and also contained particles within the tissue, most associated with villous absorptive enterocytes. Particle uptake was greatest at the 30-min time-point, when maximum uptake was usually in the proximal jejunum; although in the early lactating group, this was shifted distally. Total tissue uptake was increased in pregnant and early lactating groups, mainly at villous absorptive and mucus-secreting cells. Accumulation and progression of particles was reflected in increased numbers in the lamina propria. These data were validated by several methods, including particle detection in the blood and mesenteric lymph nodes in some groups.At both time-points, uptake profiles for pregnancy and early lactation differed from those of other groups, implying possible links between particle uptake and hormone levels, surface mucus and tight junction patency.

Disruption of the Small-Intestine Mucosal Barrier After Intestinal Occlusion: A Study with Light and Electron Microscopy

Disruption of the Small-Intestine Mucosal Barrier After Intestinal Occlusion: A Study with Light and Electron Microscopy

Disruption of the Small-Intestine Mucosal Barrier After Intestinal Occlusion: A Study with Light and Electron Microscopy

It is known that the gut may serve as a reservoir for various microorganisms, which under specific circumstances may intrude into the systemic circulation, causing systemic infections. The aim of the present study was to estimate the "critical time" of disruption of the small-intestine mucosal barrier in conditions of experimentally induced intestinal occlusion, based on the histopathological alterations observed under light and electron microscopy. Forty rabbits underwent small-intestine obstruction through ligation with a nonabsorbable suture. Blood cultures from portal vein and inferior vena cava, as well as cultures from the peritoneal fluid, a hepatic fragment, and a mesenteric lymph node, were obtained before the ligation (0 h). The same cultures were repeated at 4 and 8 h (group A, 20 rabbits) and at 6 and 12 h after the ligation (group B, 20 rabbits). Small-intestine specimens proximal to the occlusion were taken for examination under the optic and electronic microscope in the same time intervals. Five of 20 rabbits of group A died within 4 h and 6 of 20 rabbits of group B died within 6 h after the operation. All anaerobic cultures were negative. All aerobic cultures that became positive developed Escherichia coli colonies. Intestinal epithelium of dead animals was transformed to cuboid with destruction of goblet cells and alteration in secretion of acid polysaccharides. The mucosal appearance of all rabbits that survived 12 hours after ligation was the same. The disruption of the mucosal barrier begins 4 h after complete intestinal occlusion. At 12 h after complete intestinal occlusion, the disruption is total with different degrees of severity.