Intestinal Mucosal Epithelium Research Articles

Effects of Methylmethionine Sulfonium Chloride on Activity and Tight Junction Protein Expression of Intestinal Porcine Jejunum Epithelial Cells (IPEC-J2)

Background: The intestinal mucosal epithelium acts as a physical and biochemical barrier and plays an important role in regulating of barrier function and immune homeostasis. Methylmethionine sulfonium chloride (MMSC) is a multifaceted amino acid that is critical to the normal physiology of the gastrointestinal tract. The present study investigated the effects of extracellular MMSC on intestinal epithelial cell line (IPEC-J2). Methods: IPEC-J2 cells were treated with 0.1, 0.5 and 1 mM MMSC, respectively for an additional 24 h. CCK-8 assay was used to evaluate cell proliferation. The cell Annexin V-FITC/PI apoptosis were analyzed by flow cytometry (FCM) method. The mRNA transcript and protein expression levels of tight junction proteins in IPEC-J2 cells were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting (WB). Result: The results showed that MMSC could stimulate IPEC-J2 cells proliferation and inhibit cell apoptosis. In addition, the RT-qPCR and WB results indicated that 0.5 mM MMSC significantly increased the mRNA and protein expression of tight junction proteins, including occludin, claudin-1 and zonula occludin-1 (Zo-1). These findings may provide valuable information to investigate further the possible mechanism and function of MMCS on the intestinal barrier function.

Effect of acupoint thread-embedding on tight junction of intestinal mucosa epithelium in rats with ulcerative colitis

To observe the effect of acupoint thread-embedding on tight junction of intestinal mucosal epithelial barrier in rats with ulcerative colitis (UC) under the state of "deficiency and stasis", and to explore its mechanism. Sixty male SD rats were randomly divided into a control group (n=12) and a UC model group (n=48). The rats were gavaged with adenine and folium sennae by stages to prepare the "deficiency and stasis" state, and then 5% 2,4,6-trinitrobenzene sulfonic acid (TNBS, 100 mg/kg) and 0.25 mL 50% ethanol were used to induce UC model. Thirty rats with successful model establishment were selected and randomly divided into a model group, a medication group and a thread-embedding group, 10 rats in each group. The rats in the thread-embedding group were intervened with thread-embedding at "Zusanli" (ST 36), "Tianshu" (ST 25), "Geshu" (BL 17), "Pishu" (BL 20), "Shenshu" (BL 23) and "Dachangshu" (BL 25), once every 14 days, totaling 3 times. The rats in the medication group were gavaged with sulfasalazine, once a day. The rats in the control group, model group and thread-embedding group were gavaged with the same amount of 0.9% sodium chloride solution at the same time for 42 days. The general condition and body weight of rats in each group were observed. The appearance and morphological changes of colonic tissue were observed by naked eye and HE staining. The serum levels of calmodulin-dependent protein kinaseⅡ(CaMKⅡ) and myosin light chain kinase (MLCK) were detected by ELISA. The mRNA levels of colonic tight junction (TJ) proteins including occludin, claudin1, claudin2 and ZO-1were detected by quantitative real-time PCR. The protein expressions of occludin, claudin1, claudin2, ZO-1 and CaMKⅡ, MLCK were detected by Western blot. Compared with the control group, in the model group the body weight was decreased (P<0.01), colon tissue was significantly swelled with bleeding and a large number of inflammatory cells infiltration, and the colon tissues damage index score was increased (P<0.01), serum CaMKⅡ and MLCK levels were increased (P<0.01), and the mRNA and protein expression levels of occludin, claudin1 and ZO-1 in colon tissue were decreased (P<0.01), the mRNA and protein expression levels of claudin2 were increased (P<0.01), and the protein expression levels of CaMKⅡ and MLCK were increased (P<0.01). Compared with the model group, in the medication group and thread-embedding group the weight was increased (P<0.01), the colonic lesions were significantly alleviated, the tissues damage index scores were decreased (P<0.01), the serum CaMKⅡ and MLCK contents were decreased (P<0.01), the mRNA and protein expression levels of occludin, claudin1 and ZO-1 in colonic tissue were increased (P<0.01, P<0.05), the mRNA and protein expression levels of claudin2 were decreased (P<0.01), and the protein expression levels of CaMKⅡ and MLCK were decreased (P<0.01, P<0.05). There was no significant difference of each index between the medication group and thread-embedding group (P>0.05). The thread-embedding could repair the tight junction of intestinal mucosa epithelium and reduce the permeability of intestinal mucosa epithelium, which may be related to the decrease of the expression of CaMKⅡ, MLCK and other protein kinases.

Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium.

Porcine aminopeptidase N (APN), a membrane-bound metallopeptidase abundantly present in small intestinal mucosa, can initiate a mucosal immune response without any interference such as low protein expression, enzyme inactivity, or structural changes. This makes APN an attractive candidate in the development of vaccines that selectively target the mucosal epithelium. Previous studies have shown that APN is a receptor protein for both enterotoxigenic Escherichia coli (E. coli) F4 and transmissible gastroenteritis virus. Thus, APN shows promise in the development of antibody-drug conjugates or novel vaccines based on APN-specific antibodies. In this study, we compared production of APN-specific monoclonal antibodies (mAbs) using traditional hybridoma technology and recombinant antibody expression method. We also established a stably transfected Chinese hamster ovary (CHO) cell line using pIRES2-ZSGreen1-rAbs-APN and an E. coli expression BL21(DE3) strain harboring the pET28a (+)-rAbs-APN vector. The results show that antibodies expressed in pIRES2-ZSGreen1-rAbs-APN-CHO cells and mAbs produced using hybridomas could recognize and bind to the APN protein. This provides the basis for further elucidation of the APN receptor function for the development of therapeutics targeting different APN-specific epitopes.

Anti-inflammatory effects of Radix Aucklandiae herbal preparation ameliorate intestinal mucositis induced by 5-fluorouracil in mice

Ethnopharmacological relevance5-Fluorouracil (5-FU) is a chemotherapy agent that is widely used in clinical oncologic practice. However, intestinal mucositis is the most frequently occurring side effect of cancer therapy with 5-FU. Based on a literature survey, Radix Aucklandiae herbal preparation potentially ameliorates intestinal mucositis in 5-FU-treated mice. Aim of the studyThe aim of this study was to investigate the inflammation and gastrointestinal regulation of intestinal mucositis induced by 5-FU, including the intestinal morphology, as well as the reduction in food intake, body weight loss, and diarrhea. Materials and methodsIntestinal mucositis was induced in mice by 5-FU (30 mg/kg, i.p., for 5 consecutive days). The dose-dependent Radix Aucklandiae herbal preparation (0.3, 1, and 3 g/kg/day, p.o.), loperamide (3 mg/kg/day, p.o.) or celecoxib (40 mg/kg/day, p.o.) was concurrently administered until the 7th day. Physical status observation, diarrhea assessment, serum proinflammatory cytokine levels, intestinal villus height and crypt depth, and total goblet cells from tissues were assessed. ResultsThe dosage regimen of 5-FU administration caused severe intestinal mucositis in mice, including damage to the intestinal morphology, accompanied by a reduction in food intake, body weight loss, and diarrhea. The high-dose Radix Aucklandiae herbal preparation significantly relieves 5-FU-induced intestinal mucositis by enhancing proliferative activity in epithelial crypts; improving anepithymia, body weight loss, and diarrhea; and displaying protective effects on goblet cells in intestinal mucosal epithelia. Activation of NF-κB in the intestinal mucositis model was also suppressed by the Radix Aucklandiae herbal preparation, suggesting that it is a potent inhibitor of NF-κB and proinflammatory cytokines, such as IL-1β, IL-6, TNF-α, and COX-2. ConclusionsOur data support the conclusion that the Radix Aucklandiae herbal preparation could effectively ameliorate 5-FU-induced gastrointestinal toxicity and be applied clinically for the prevention of intestinal mucositis during chemotherapy.

Changes of Kinesin Family Member 18 Pathway in Epithelial Mesenchymal Transformation and Effect of Butyrate on Migration and Invasion of Colon Cancer Cells

Colon cancer is a common digestive system disease with an increasing incidence. Severe migration and invasion aggravates the deterioration of colon cancer patients. Previous studies have found that epithelial mesenchymal transition (EMT) is closely associated with early transference of colon carcinoma and abnormal changes occur in KIF18 signaling pathway. Butyrate protects colonic mucosa with a considerable effect on the colon. This study predicts that butyrate may reverse EMT process of colon cancer cells through KIF18 signaling pathway, thereby inhibiting cell migration and invasion. In this experiment, EMT model of colon cancer was used to investigate migration and invasion. Human colon cancer cell line SW1116 was cultured and assigned into control group (0 mmol/L butyrate), low concentration group (2 mmol/L), medium concentration group (4 mmol/L), and high concentration group (10 mmol/L). After 72 hours, cell migration and invasion was analyzed by Transwell assays. E-cadherin, Vimentin, and KIF18 level was detected by Western blot and quantitative real-time PCR. After treatment, cell migration and invasion was significantly inhibited compared to control dose-dependently. In addition, Vimentin and KIF18 mRNA level was significantly lower and E-cadherin mRNA was higher in treatment groups than control group in a dose-dependent manner (P &lt; 0.05). Consistently, the profile of protein level of these molecules was similar to mRNA expression profile. Electron microscope showed that after treatment with butyrate, the surface protuberances of colon cancer cells were abnormally increased, especially the vesicular protuberances, which were the microvilli of intestinal mucosal epithelium. In conclusion, KIF18 is crucial in EMT of colon cancer cells. Butyrate may elevate E-cadherin and suppress Vimentin and KIF18 by activating KIF18 signaling, thus inhibiting invasion and migration.

Intestinal Stem Cells and Immune Cell Relationships: Potential Therapeutic Targets for Inflammatory Bowel Diseases.

The mammalian intestine is the largest immune organ that contains the intestinal stem cells (ISC), differentiated epithelial cells (enterocytes, Paneth cells, goblet cells, tuft cells, etc.), and gut resident-immune cells (T cells, B cells, dendritic cells, innate lymphoid cell, etc.). Inflammatory bowel disease (IBD), a chronic inflammatory disease characterized by mucosa damage and inflammation, threatens the integrity of the intestine. The continuous renewal and repair of intestinal mucosal epithelium after injury depend on ISCs. Inflamed mucosa healing could be a new target for the improvement of clinical symptoms, disease recurrence, and resection-free survival in IBD treated patients. The knowledge about the connections between ISC and immune cells is expanding with the development of in vitro intestinal organoid culture and single-cell RNA sequencing technology. Recent findings implicate that immune cells such as T cells, ILCs, dendritic cells, and macrophages and cytokines secreted by these cells are critical in the regeneration of ISCs and intestinal epithelium. Transplantation of ISC to the inflamed mucosa may be a new therapeutic approach to reconstruct the epithelial barrier in IBD. Considering the links between ISC and immune cells, we predict that the integration of biological agents and ISC transplantation will revolutionize the future therapy of IBD patients.

Синдром диареи: новые акценты курации пациентов в условиях пандемии COVID-19

The digestive tract, like the respiratory organs, is the entrance gate for SARS-CoV-2. At the same time, diarrhea is one of the most common gastrointestinal symptoms among infected people. As the debut of COVID-19 clinical manifestations, diarrhea can be associated both with the direct cytopathic effect of the virus on the intestinal mucosal epithelium and with the gut microbiota modulation. During the period of the expanded clinical picture of COVID-19, diarrhea syndrome is commonly associated with the adverse event of drug therapy, primarily antibiotics, nonsteroidal anti-inflammatory drugs, antiviral agents, antiplatelet agents and anticoagulants, as well as with possible involvement in the pathological process of the liver and pancreas with a violation of their functional activity. Finally, after the infection, the repeated occurrence of diarrhea requires the exclusion of antibiotic-associated diarrhea, including those caused by Clostridium difficile, as well as ischemic colitis and post-infectious irritable bowel syndrome. Besides, despite the pandemic, it should not be forgotten about the possible debut of gastrointestinal disorder or an exacerbation of already existing chronic pathology. The article presents a brief algorithm for the differential diagnosis of diarrhea in the conditions of the COVID-19 pandemic. KEYWORDS: diarrhea, COVID-19 pandemic, antibiotic-associated diarrhea, ischemic colitis, post-infectious irritable bowel syndrome. FOR CITATION: Gaus O.V., Livzan M.A. Diarrhea syndrome: new accents of patient curation during COVID-19 pandemic. Russian Medical Inquiry. 2021;5(6):446–452 (in Russ.). DOI: 10.32364/2587-6821-2021-5-6-446-452.

Pretreatment with intestinal trefoil factor alleviates stress-induced gastric mucosal damage via Akt signaling.

BACKGROUNDStress-related gastric mucosal damage or ulcer remains an unsolved issue for critically ill patients. Stress ulcer prophylaxis has been part of routine intensive care, but uncertainty and controversy still exist. Co-secreted with mucins, intestinal trefoil factor (ITF) is reported to promote restitution and regeneration of intestinal mucosal epithelium, although the mechanism remains unknown.AIMTo elucidate the protective effects of ITF on gastric mucosa and explore the possible mechanisms.METHODSWe used a rat model of gastric mucosal damage induced by water immersion restraint stress and lipopolysaccharide-treated human gastric epithelial cell line to investigate the potential effects of ITF on damaged gastric mucosa both in vivo and in vitro.RESULTSITF promoted the proliferation and migration and inhibited necrosis of gastric mucosal epithelia in vitro. It also preserved the integrity of gastric mucosa by upregulating expressions of occludin and zonula occludens-1. In the rat model, pretreatment with ITF ameliorated the gastric mucosal epithelial damage and facilitated mucosal repair. The protective effects of ITF were confirmed to be exerted via activation of Akt signaling, and the specific inhibitor of Akt signaling LY249002 reversed the protective effects.CONCLUSIONITF might be a promising candidate for prevention and treatment of stress-induced gastric mucosal damage, and further studies should be undertaken to verify its clinical feasibility.

ROS Plays a Role in the Neonatal Rat Intestinal Barrier Damages Induced by Hyperoxia.

Background Hyperoxia treats a subset of critical neonatal illnesses but induces intestinal damage in neonatal pups. In this process, the intestinal flora and mucosal epithelium might be altered by hyperoxia. So the changes of the intestinal flora and mucosal epithelium were studied. Methods Neonatal rats were randomized into the model group that was exposed to hyperoxia and the control group that was maintained under normoxic conditions; then, intestinal lavage fluid and intestinal tissues were harvested. ELISA was used to detect D-lactic acid (D-LA), endotoxin (ET), diamine oxidase (DAO), intestinal fatty acid binding protein (i-FABP), liver-type fatty acid binding protein (L-FABP) and cytokines in the intestinal lavage of neonatal rats during hyperoxia. The intestinal zonula occluden-1 (ZO-1), occlusion protein (Occludin), and closure protein-4 (Claudin-4) of neonatal pups were detected by immunohistochemistry, western blotting, and real-time Polymerase chain reaction (RT-PCR) during hyperoxia. NCM460 cell survival rates were assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) during hyperoxia and administration of N-acetyl-L-cysteine (NAC). The expression levels of ZO-1, Occludin, and Claudin-4 in NCM460 cells were detected by immunohistochemistry, western blotting, and RT-PCR during hyperoxia and NAC. Results D-LA, ET, L-FABP, i-FABP, DAO, TNF-α, IL-10, and IFN-γ were significantly increased by hyperoxia, while ZO-1, Occludin, and Claudin-4 were clearly decreased in the hyperoxia group compared with the control group. NAC promoted cell survival, which was inhibited by hyperoxia. The cellular expression levels of ZO-1, Occludin, and Claudin-4, which were lowered by hyperoxia, were increased by NAC. Conclusion Hyperoxia causes injury of the intestinal mucosa, and ROS plays a role in this intestinal damage during hyperoxia.

Comprehensive two-dimensional liquid chromatography as a biomimetic screening platform for pharmacokinetic profiling of compound libraries in early drug development

A comprehensive two-dimensional liquid chromatography-based biomimetic platform (LCxLC) has been developed and validated for drug diffusion studies. Human serum albumin (HSA) and immobilized artificial membrane (IAM) were thereby used in the first (1D) and second (2D) separation dimension, respectively. While the former was meant to emulate the blood, the latter was instead intended to mimic the intestinal mucosa epithelium. Therefore, the experimental conditions, i.e. pH, temperature and buffer composition, were modulated to reflect faithfully in vivo conditions. 30 compounds, whose effective intestinal permeability (Peff) assayed in situ on humans by a validated technique was known from the literature, were used as model drugs.A good and orthogonal separation was achieved for the whole dataset, although for a better distribution of the most polar compounds in the elution window a segmented gradient elution program had to be employed. Interestingly, the passively uptaken compounds having the most favourable Peff populated a specific area of the 2D plots, implying that the affinity for HSA and IAM has to lie in specific ranges in order for a compound to be satisfactorily absorbed from the intestinal lumen.Although these results should be regarded as preliminary, this work paves an entirely new and unprecedented way to profile pharmaceutically relevant compounds for their in vivo absorption and distribution potential.

Inhibition of miRNA-29a regulates intestinal barrier function in diarrhea-predominant irritable bowel syndrome by upregulating ZO-1 and CLDN1.

Diarrhea-predominant irritable bowel syndrome (IBS-D) is a common chronic functional gastrointestinal disorder. MicroRNAs (miRNAs) have been identified to be involved in different physiological and pathological processes. In this study, the role of miRNA-29a in the potential mechanism underlying the function of the intestinal mucosal barrier in IBS-D was analyzed. Human intestinal mucosal epithelia from patients with IBS-D (diagnosed as meeting the Rome IV criteria) and healthy volunteers were collected. An IBS-D mouse model was established via induction with trinitro-benzene-sulfonic acid (TNBS), and the mice were injected with miRNA-29a inhibitor. Using transmission electron microscopy (TEM), the epithelial ultrastructure of the human intestinal mucosa was examined. Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis, the expression level of miRNA-29a was assessed. ELISA was used to analyze the activity of D-lactate (D-LA) and diamine oxidase (DAO). Through immunohistochemistry, RT-qPCR and western blotting, the expression of tight junction protein ZO-1 (ZO-1) and claudin-1 (CLDN1) was examined. In the human intestinal mucosal epithelia from patients with IBS-D, miRNA-29a was upregulated, ZO-1 and CLDN1 were downregulated, and the junctional complex (JC) was faint and discontinuous. In the IBS-D mouse model, treatment with miRNA-29a inhibitor downregulated D-LA and DAO activity, and increased the expression of ZO-1 and CLDN1 in the intestinal mucosal epithelium. In conclusion, the present study revealed that miRNA-29a is involved in the pathogenesis of IBS-D, probably by downregulating ZO-1 and CLDN1 expression, suggesting that miRNA-29a is likely to be an important regulator of intestinal barrier function and could be a possible therapeutic target for IBS-D.

Molecular characterization of pathogenic Escherichia coli isolated from diarrheic and in-contact cattle and buffalo calves.

Escherichia coli field isolates from calves were characterized and categorized into the most significant diarrheagenic pathotypes using polymerase chain reaction (PCR) assays with different specific primers. The used PCR systems were designed to detect sequences representing the group-specific virulence genes encoding fimbriae f5 (K99), Shiga toxins (stx1 and stx2), heat-stable enterotoxins (st), heat-labile enterotoxins (lt), intimin (eae), hemolysin (hylA), and EAEC heat-stable enterotoxin (astA). In the present work, a total of 150 E. coli field isolates were recovered from 150 fecal swabs collected from 100 diarrheic and 50 apparently healthy in-contact cattle and buffalo calves under 3 months old. Out of these 150 isolated E. coli, 106 isolates from 77 diarrheic and 29 in-contact calves harbored one or more of the investigated virulence genes. The pathotyping of the isolates could classify them into shigatoxigenic E. coli (STEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), and enteroaggregative E. coli (EAEC) with a 30.7, 2.7, 12.7, and 7.3% distribution, respectively. Meanwhile, the detection rates of f5, stx1, stx2, st, lt, eae, hylA, and astA genes were 17.3, 27.3, 6.7, 10, 37.3, 17.7, 9.3, and 20.7%, respectively. These virulence genes were found either single or in different combinations, such as stx/eae, stx/st/f5, eae/st/f5, or st/lt/f5. Four attaching-effacing shigatoxigenic E. coli isolates (AE-STEC) harboring stx/eae were retrieved from diarrheic calves. Although none of the stx-or eae-positive isolates was verified as O157:H7, STEC isolates detected in apparently healthy calves have potential pathogenicity to humans highlighting their zoonotic importance as reservoirs. Atypical combinations of ETEC/STEC and ETEC/EPEC were also detected in percentages of 14.7 and 2.7%, respectively. Most of these atypical combinations were found more in buffalo calves than in cattle calves. While STEC and EPEC isolates were detected more in cattle calves than in buffalo calves, ETEC isolates were the same in the two species. The pathogenic E. coli infection in calves was recorded to be higher in the first weeks of life with the largest numbers of virulence factor-positive isolates detected at the age of 4 weeks. Histopathological examination of five intestinal samples collected from four dead buffalo calves revealed typical attaching and effacing (AE) lesion which was correlated with the presence of intimin encoding virulence gene (eae). Other lesions characterized by hemorrhagic enteritis, shortening and fusion of intestinal villi and desquamation of the lining epithelium of intestinal mucosa had also been detected.

Study of the Histopathologic Effects of Probiotic Lactobacillus acidophilus in Exposure to E. coli O157: H7 in Zebrafish Intestine

Background: Over the past three decades, the use of probiotics has increased as growth promoters and effective supplements to reduce the pathogenicity of pathogens. In this regard, Lactobacillus bacteria are among the most common probiotics used, as they can help strengthening the digestive system and therefore reducing intestinal hystopathological damage when encountering pathogens. Objectives: This study aimed to evaluate the histopathologic effects of Lactobacillus acidophilus as a dietary supplement in zebrafish ration, as an appropriate laboratory model, and in the exposure to Escherichia coli O157: H7. Methods: In this project, 48 fish were grouped in 4 aquariums and monitored for 30 days; control group (C1A1) received basic ration; B1A1 group received control group ration and were exposed to E. coli O157: H7; Treatment 1 (T1A1) received basic ration containing L. acidophilus with no exposure to E. coliO157: H7, and Treatment 2 (T2A2) received basic ration containing L. acidophilus with exposure to E. coli O157: H7. During 30 days of the experiment, the samples were taken from the intestinal tissue in the days 15, 27, and 30 for the histopathological examinations. Results: The results of the findings showed a significant increase in the length of the intestinal villi and the number of goblet cells in the studied tissue in the group treated with a ration containing probiotic supplements compared to the control group (P < 0.001, P < 0.01, P < 0.05). Also, in the group exposed to E. coli O157: H7, histopathological changes including mild edema, inflammatory cell accumulation in the intestinal mucosal tissue, severe necrosis and epithelium loss in the intestinal tissue were evident. These symptoms were much lower in the group fed with probiotic. Conclusions: According to the obtained data, it can be concluded that feeding fish using L. acidophilus supplement can produce very beneficial effects in reducing tissue damage caused by E. coli O157: H7 infection in zebrafish intestines as a laboratory model.

Reactive Changes of Endocrine Cells of Intestinal Mucosal Epithelium with Administration of Melatonin or Doxylamine Succinate (Electron Microscopic Study)

Using an electron microscopy method, the structure of endocrinocytes of mucosal epithelium of duodenum, colon, and rectum in Wistar line rats was studied after daily administration of melatonin or doxylamine succinate for 1 month. It was demonstrated that morphological traits of increased functional activity (which is often accompanied by intracellular regeneration phenomena) are observed 1 day after the last administration of the studied substances. Mitochondria of endocrinocytes are primarily subject to changes. In addition, a large number of poorly differentiated endocrine cells were registered in the intestinal epithelium of rats subjected to experimental exposure, which indicates the activation of cambial elements of the epithelium. In all studied cases, D1 cells were more frequently found in the epithelium than other types of endocrinocytes.

Multivesicular bodies containing exosomes in immune-related cells of the intestine in zebrafish (Danio rerio): Ultrastructural evidence

Exosomes are secreted from various cells by multivesicular bodies (MVBs) that fuse with the plasma membrane and are involved in the intestinal immune response to maintain intestinal homeostasis. Here, we demonstrate the ultrastructural characteristics of MVBs and their exosomes in immune-related cells of the zebrafish intestine, including goblet cells (GCs), mitochondria-rich cells (MRCs), high endothelial cells (HECs) and lymphocytes. In GCs, MVBs with a low electron density were present under the nucleus. MVBs with exosomes were observed among mucin granules. “Heterogeneous” MVBs were identified within the cytoplasm around mucin granules. MRCs were observed in the intestinal mucosa epithelium, including “open-type” MRCs and “close-type” MRCs. Typical MVBs were identified in these MRCs. MVBs with a variety of exosomes were observed in the HECs of the capillary located in the lamina propria (LP). The HEC basement membrane budded outward to LP cells to form a plurality of basal blebs, later containing a large number of exosomes. MVBs also existed in the LP lymphocytes. A schematic diagram of the ultrastructural distribution of MVBs and their exosomes in the intestinal mucosal immune-related cells was created. Our findings provide cytological evidence for the source and ultrastructural distribution of exosomes within the different intestine cells of zebrafish. Component analysis and immunological functions of exosomes require future study.

Comparative transcriptomics and histopathological analysis of crucian carp infection by atypical Aeromonas salmonicida

Aeromonas salmonicida is a ubiquitous fish pathogen known to cause furunculosis. With the emergence of new subtypes and the expansion of the host range, it has threatened the health of a variety of marine and freshwater fish, particularly the non-salmonids, manifesting differently from the classical furunculosis. Although there have been reports of infection by atypical strains on the crucian carp, the pathogenesis and tissue pathology remain unclear. In this study, transcriptomics and histopathology were used to analyze the immune response and lesions of crucian carp infected with A. salmonicida. Comparative analysis showed 6579 differentially expressed genes (DEGs) (3428 down-regulated and 3151 up-regulated) were identified on day 5 post-infection (5 dpi). Further annotation and analysis revealed that the DEGs were enriched in enzyme regulator activity, response to oxidative stress, iron ion homeostasis and other functions, and mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB), toll-like receptor (TLR), and nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) etc., and immune-related signaling pathways. Meanwhile, the four C-type lysozyme genes found in all DEGs were significantly up-regulated after infection. In addition, there was severe bleeding on the body of the infected fish. Also, the intestine, liver, spleen, and kidney showed varying degrees of inflammatory damage, especially the goblet cell hyperplasia of intestinal mucosa epithelium and degeneration and necrosis of renal tubular epithelium cells. Additionally, with the increase in pathogen concentration, the cumulative mortality increased, the severity of lesions in the hindgut and head-kidney tissues increased. The relative expression levels of four immune-related genes (TNF-α, IL-1β, IL-11, C-lysozyme) were also significantly upregulated, compared with the control (P < 0.05). In conclusion, this study provides a scientific basis for further study on the immune response, pathological diagnosis, and prevention of crucian carp infection caused by atypical A. salmonicida.

Interleukin-13 and its signaling pathway is associated with obesity-related colorectal tumorigenesis.

The incidence of colorectal cancer (CRC) has been on the rise, which is linked to the increasing prevalence of obesity, based on global epidemiological evidence. Although chronic inflammation is implicated in tumor development, the mechanisms underlying obesity‐associated CRC remain unknown. Here, we sought to identify the inflammatory cytokines and their roles in obesity‐related colorectal tumorigenesis using cytokine array analyses in a mouse model. Colorectal tumorigenesis was induced through i.p. injection of azoxymethane once a week for 6 weeks in 6‐week‐old female WT C57Black/6J mice and the obesity diabetes model mouse KK/TaJcl, KK‐Ay/TaJcl. The formation of aberrant crypt foci and colorectal tumors were more frequent in obese mice compared with WT mice, and both serum interleukin (IL)‐13 and IL‐13 receptor (R) expression in the normal intestinal mucosal epithelium were significantly increased in the obese mice. Furthermore, addition of IL‐13 to a human CRC cell line and a human colon organoid culture altered the phenotype of intestinal epithelial cells. Knockdown experiments further revealed that IL‐13Rα1 dominantly induced mucosal proliferation. Collectively, These results suggest an association between anti‐inflammatory cytokines and colorectal carcinogenesis, and provide new research directions for cancer prevention strategies. In particular, inflammation provoked by obesity, notably by increased expression of the cytokine IL‐13, could play an important role in the carcinogenesis of obesity‐related CRC.

Effects of vitamin D3 on intestinal mucosal barrier of mice with severe burns

Objective: To explore the effects of vitamin D3 on intestinal mucosal barrier of mice with severe burns. Methods: Forty-two C57BL/6C male mice aged eight to twelve weeks were divided into vitamin D3 vehicle+ sham injury group of seven mice, vitamin D3 vehicle+ burn injury group of fourteen mice, vitamin D3+ sham injury group of seven mice, and vitamin D3+ burn injury group of fourteen mice according to random number table. Mice in vitamin D3 vehicle+ sham injury group and vitamin D3 vehicle+ burn injury group were injected with vehicle of vitamin D3 at a dose of 0.1 mL intraperitoneally at 1, 24, and 48 h before burn experiment. Mice in vitamin D3+ sham injury group and vitamin D3+ burn injury group were injected with vitamin D3 at a dose of 100 ng/kg dissolved in 0.1 mL vehicle intraperitoneally at the same time points. Mice in vitamin D3 vehicle+ burn injury group and vitamin D3+ burn injury group were inflicted with 30% total body surface area full-thickness dermal scald (hereinafter referred to as burn) on the back by 98 ℃ hot water for 3 to 4 seconds. And mice in vitamin D3 vehicle+ sham injury group and vitamin D3+ sham injury were treated with 37 ℃ water on the back for 3 to 4 seconds to simulate injury. Seven mice in vitamin D3 vehicle+ sham injury group and seven mice in vitamin D3+ sham injury group at post injury hour (PIH) 24, and seven mice in vitamin D3 vehicle+ burn injury group and seven mice in vitamin D3+ burn injury group at PIH 6 and 24 were sacrificed respectively to collect mesentery lymph nodes, spleens, livers, and intestinal tissue. The mesentery lymph nodes, spleens, and livers of mice in each group were collected to observe growth of bacteria, and number of bacteria was counted. Intestinal tissue of mice in each group was collected to detect protein expressions of zonal occludin 1 (ZO-1) and occludin by immunohistochemistry staining method, distribution of ZO-1 by immunofluorescence staining method, and expression of occludin by Western blotting. Data were processed with Kruskal-Wallis H test, Nemenyi test, one-way analysis of variance, t test, and Bonferroni correction. Results: (1) At PIH 6 and 24, bacterial counts of mesentery lymph nodes, livers, and spleens of mice in vitamin D3 vehicle+ burn injury group were significantly higher than those of mice in vitamin D3 vehicle+ sham injury group (P<0.05). At PIH 6, bacterial counts of livers and spleens of mice in vitamin D3+ burn injury group were significantly lower than those of mice in vitamin D3 vehicle+ burn injury group (P<0.05). At PIH 24, bacterial counts of mesentery lymph nodes and livers of mice in vitamin D3+ burn injury group were significantly lower than those of mice in vitamin D3 vehicle+ burn injury group (P<0.05). (2) At PIH 6 and 24, expressions of ZO-1 and occludin of intestinal tissue of mice in vitamin D3 vehicle+ burn injury group were significantly lower than those of mice in vitamin D3 vehicle+ sham injury group, and expressions of ZO-1 and occludin of intestinal tissue of mice in vitamin D3+ burn injury group were close to those of mice in vitamin D3+ sham injury group. At PIH 6 and 24, expressions of ZO-1 and occludin of intestinal tissue of mice in vitamin D3+ burn injury group were significantly higher than those of mice in vitamin D3 vehicle+ burn injury group. (3) At PIH 6 and 24, compared with that of mice in vitamin D3 vehicle+ sham injury group, distribution of ZO-1 of intestinal mucosal epithelium of mice in vitamin D3 vehicle+ burn injury group was discontinuous. Distribution of ZO-1 of intestinal mucosal epithelium of mice in vitamin D3+ sham injury group was normal, and the distribution of ZO-1 of intestinal mucosal epithelium of mice in vitamin D3+ burn injury group was with good continuity. (4) At PIH 6 and 24, expressions of occludin of intestinal tissue of mice in vitamin D3 vehicle+ burn injury group were 0.720±0.003, 0.638±0.052 respectively, significantly lower than 0.918±0.003 of mice in vitamin D3 vehicle+ sham injury group (t=57.33, 5.36, P<0.05). At PIH 6 and 24, expressions of occludin of intestinal tissue of mice in vitamin D3+ burn injury group were 0.994±0.058, 1.064±0.060, close to 0.938±0.023 of mice in vitamin D3+ sham injury group (t=0.91, 1.96, P>0.05). At PIH 6 and 24, expressions of occludin of intestinal tissue of mice in vitamin D3 vehicle+ burn injury group were significantly lower than those of mice in vitamin D3+ burn injury group (t=4.75, 5.35, P<0.05). Conclusions: Intestinal bacterial translocation can occur in the early stage of severe burn. And vitamin D3 plays a protective role in the intestinal mucosal barrier post severe burn to reduce the bacterial translocation by protecting tight junction proteins of intestinal epithelium.

P117 PLATELET ACTIVATING FACTOR AND TNFα HAVE SYNERGISTIC EFFECTS IN PROMOTING INTESTINAL EPITHELIAL MIGRATION AND MUCOSAL WOUND REPAIR

Epithelial cells migrate and proliferate to cover denuded mucosal surfaces. The intestinal mucosal infiltrating immune cells and epithelium secrete an array of inflammatory mediators that have been proposed to modulate wound repair. Here, we report that Platelet Activating Factor (PAF), a molecule considered to be a pro-inflammatory mediator and its receptor (PAFR) are upregulated in healing colonic mucosal wounds. We show that in intestinal epithelial cells the PAFR up-regulation is downstream of TNF-α mediated signaling leading to enhanced wound repair after PAF treatment. In vitro PAF signaling promotes cell migration by activation of focal adhesion kinase that regulates dynamics of integrin containing cell-matrix contacts. On the converse, PAFR deficient mice display delayed wound closure. In addition, intraperitoneal administration of a neutralizing TNF-α antibody inhibited intestinal mucosal wound repair. Our findings highlight a cross-talk between PAFR and TNF-α in orchestrating intestinal epithelial repair.

P117 PLATELET ACTIVATING FACTOR AND TNFα HAVE SYNERGISTIC EFFECTS IN PROMOTING INTESTINAL EPITHELIAL MIGRATION AND MUCOSAL WOUND REPAIR

Epithelial cells migrate and proliferate to cover denuded mucosal surfaces. The intestinal mucosal infiltrating immune cells and epithelium secrete an array of inflammatory mediators that have been proposed to modulate wound repair. Here, we report that Platelet Activating Factor (PAF), a molecule considered to be a pro-inflammatory mediator and its receptor (PAFR) are upregulated in healing colonic mucosal wounds. We show that in intestinal epithelial cells the PAFR up-regulation is downstream of TNF-α mediated signaling leading to enhanced wound repair after PAF treatment. In vitro PAF signaling promotes cell migration by activation of focal adhesion kinase that regulates dynamics of integrin containing cell-matrix contacts. On the converse, PAFR deficient mice display delayed wound closure. In addition, intraperitoneal administration of a neutralizing TNF-α antibody inhibited intestinal mucosal wound repair. Our findings highlight a cross-talk between PAFR and TNF-α in orchestrating intestinal epithelial repair.