Cytoprotection and Cytothetic Effects of GLP-2 on Enterocytes from a Weaned Piglet Injured by β-conglycinin in Vitro

The experiment was conducted to study the effects and possible mechanism of GLP-2 on proliferation,metabolism and apoptosis of cultured enterocytes from a 28-d weaned piglet injured by exposure to β-conglycinin.A cell damage model was established to investigate cell proliferation, metabolism and apoptosis by exposing primary cell cultures of intestinal epithelial cells(IEC) to 1.2 and 2.4 mg/mL β-conglycinin.A 2×3 factorial experiment was then used to study the effect of different GLP-2 concentrations of(1×10-9,1×10-8 and 1×10-7mol/L),in combination with the two concentrations ofβ-conglycinin.Cells exposed to the allergenβ-conglycinin had decreased(P【0.05) MTT OD;decreased (P【0.01) protein retention and total protein content of cells;increased(P【0.01) LDH and caspase-3 activities and decreased(P【0.05) Na+,K+-ATPase activity.When GLP-2 was used in combination withβ-conglycinin,MTT OD,protein retention,total protein content and Na+,K+-ATPase activity significantly increased(P【0.05);LDH activity gradually decreased(P【0.05 or P【0.01) and Caspase-3 activity significantly decreased(P【0.01) with increasing concentrations of GLP-2.The results indicated thatβ-conglycinin had adverse effects on proliferation and integrity of IEC in vitro.GLP-2 relieved or prevented the adverse effects ofβ-conglycinin on proliferation and integrity of IEC by regulating Na+,K+- ATPase and Caspase-3 activities,and consequently affecting cell metabolism.

Tumor necrosis factor-alpha induces apoptosis of enterocytes in mice with fulminant hepatic failure

AIM: To explore the alterations of intestinal mucosa morphology, and the effects of tumor necrosis factor a (TNFα) on enterocyte apoptosis in mice with fulminant hepatic failure (FHF). METHODS: Liver damage was induced by lipopolysaccharide (LPS)/TNF-α in D-galactosamine (GaIN) sensitized BALB/c mice. There were 40 mice in normal saline (NS)-treated group, 40 mice in LPS-treated group, 40 mice in GaIN-treated group, 120 mice in GaIN/ LPS-treated group and 120 mice in GaIN/ TNFα-treated group. Each group was divided into five subgroups of eight mice each. Serum samples and liver, intestinal tissues were respectively obtained at 2, 6,9,12 and 24 h after administration. Anti-TNFa monoclonal antibody was injected intravenously into GaIN/LPS-treated mice. Serum TNFα levels were determined by enzyme linked immunosorbent assays (ELISA). Serum ALT levels were determined using an automatic analyzer. The intestinal tissues were studied under light microscope and electron microscope at 2, 6, 9,12 and 24 h in mice with fulminant hepatic failure, respectively. Enterocyte apoptosis was determined by terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) method. The expression of tumor necrosis factor receptor 1 (TNFR1) in intestinal tissue was tested by immunohistochemistry Envision Two Steps. RESULTS: Gut mucosa was morphologically normal at all time points in all groups, but typical apoptotic cells could be seen in all experimental groups under electron microscope. Apoptosis rate of gut mucosal epithelial cells were significantly increased at 6, 9 and 12 h, peaked at 12 h in mice with fulminant hepatic failure. TNFa induced apoptosis of enterocytes in mice with FHF. The integrated OD (IOD) levels of TNFa receptor 1 protein expressed in the intestine of mice with GaIN/LPS and GaIN/ TNFα induced FHF at 2, 6, 9, 12 and 24 h after GaIN/LPS and GaIN/TNFα administration were 169.54±52.62/905.79±111.84,11 350.67±2 133.26/28 160.37±4 601.67, 25 781.00±2 277.75/122 352.30±49 412.40, 5 241.53±3 007.24/ 49 157.93±9 804.88, 7 086.13±1 031.15/3 283.45±127.67, respectively, compared with those in control groups (with NS, LPS and GaIN administration, respectively). IOD level of TNFR1 changed significantly at 6, 9 and 12 h after GaIN/LPS and GalN/TNFa administration. The expression of TNFR1 protein was significantly higher at 9 h after GaIN/LPS and GaIN/TNFα administration than that in control groups. Protein expression of TNFR1 was positively correlated with enterocyte apoptosis. CONCLUSION: TNFα can induce apoptosis of enterocytes in mice with FHF. Anti-TNFα IgG can inhibit this role.

Epidermal growth factor improves intestinal morphology by stimulating proliferation and differentiation of enterocytes and mTOR signaling pathway in weaning piglets

Epidermal growth factor(EGF) has been shown to improve piglet intestinal morphology and epithelial recovery. In an attempt to further understand the mechanisms behind these improvements, this study tested the hypothesis that dietary EGF may affect intestinal morphology by stimulating the proliferation and differentiation of enterocytes in weaning piglets. In piglets receiving200 μg kg^-1 EGF, crypt depth and villus height increased(P<0.05). Adding 400 μg kg^-1 EGF increased villus height-to-crypt depth ratio(P<0.05), but reduced crypt depth(P<0.05). Dietary supplementation with 200 μg kg^-1 EGF significantly increased the number of Ki67-positive cells(P<0.01) and tended to increase the mRNA level of proliferating cell nuclear antigen(P<0.10).However, this supplementation decreased the expression level of intestinal fatty acid-binding protein(P<0.05). Piglets fed with400 μg kg^-1 EGF had an increased mRNA level of intestinal alkaline phosphatase(P<0.05). The phosphorylation of m TOR(mammalian target of rapamycin) was observed in the 200 μg kg^-1 EGF group. These results suggest that dietary supplementation with a low level of EGF improved piglet intestinal morphology through stimulating the proliferation and differentiation of enterocytes, and the mTOR signaling pathway may partly be involved in this process.

Single-cell analysis of cellular heterogeneity and interactions in the ischemia-reperfusion injured mouse intestine

Nine major cell populations among 46,716 cells were identified in mouse intestinal ischemia‒reperfusion(II/R)injury by single-cell RNA sequencing.For enterocyte cells,11 subclusters were found,in which enterocyte cluster 1(EC1),enterocyte cluster 3(EC3),and enterocyte cluster 8(EC8)were newly discovered cells in ischemia 45 min/reperfusion 720 min(I 45 min/R 720 min)group.EC1 and EC3 played roles in digestion and absorption,and EC8 played a role in cell junctions.For TA cells,after ischemia 45 min/reperfusion 90 min(I 45 min/R 90 min),many TA cells at the stage of proliferation were identified.For Paneth cells,Paneth cluster 3 was observed in the resting state of normal jejunum.After I 45 min/R 90 min,three new subsets were found,in which Paneth cluster 1 had good antigen presentation activity.The main functions of goblet cells were to synthesize and secrete mucus,and a novel subcluster(goblet cluster 5)with highly proliferative ability was discovered in I 45 min/R 90 min group.As a major part of immune system,the changes in T cells with important roles were clarified.Notably,enterocyte cells secreted Guca2b to interact with Gucy2c receptor on the membranes of stem cells,TA cells,Paneth cells,and goblet cells to elicit intercellular communication.One marker known as glutathione S-transferase mu 3(GSTM3)affected intestinal mucosal barrier function by adjusting mitogen-activated protein kinases(MAPK)signaling during II/R injury.The data on the heterogeneity of intestinal cells,cellular communication and the mechanism of GSTM3 provide a cellular basis for treating II/R injury.

Intestinal sugar transport

Carbohydrates are an important component of the diet. The carbohydrates that we ingest range from simple monosaccharides （glucose, fructose and galactose） to disaccharides （lactose, sucrose） to complex polysaccharides. Most carbohydrates are digested by salivary and pancreatic amylases, and are further broken down into monosaccharides by enzymes in the brush border membrane （BBM） of enterocytes. For example, lactase-phloridzin hydrolase and sucraseisomaltase are two disaccharidases involved in the hydrolysis of nutritionally important disaccharides. Once monosaccharides are presented to the BBM, mature enterocytes expressing nutrient transporters transport the sugars into the enterocytes. This paper reviews the early studies that contributed to the development of a working model of intestinal sugar transport, and details the recent advances made in understanding the process by which sugars are absorbed in the intestine.

Influence of starch sources and dietary protein levels on intestinal functionality and intestinal mucosal amino acids catabolism in broiler chickens

Background: There is growing interest in carbohydrate and protein nutrition to enhance the efficiency of animal production.Reduced-crude protein diets depress environmental pollution and feeding cost,but the challenge to their adoption is maintaining digestive function and growth performance of birds.The present study was conducted to evaluate the influence of different dietary starch sources and protein levels on intestinal functionality and mucosal amino acid catabolism.Methods: Six dietary treatments,based on maize and soybean meal,were offered to 360 AA+male chicks from 6 to 35 d post-hatch as a 3 × 2 factorial array.Either waxy rice or amylose was added to a conventional maize-soy diet to provide three sources of starch with different digestion rates and relatively high and low dietary protein levels.Growth performance,parameters of intestinal functionality and concentrations of free amino acid in the portal circulation were determined.Results: In the grower phase,starch source influenced(P < 0.02) weight gain as diets containing amylose supported significantly higher weight gains than waxy rice.Significant increase of ileal ATP concentrations and Na^+/K^+-ATPase activity were found in amylose treatment.Also,amylose decreased BrdU positive cell numbers and down-regulated m RNA expression for CASP-3.GOT activity in the ileum was higher(P < 0.01) in birds offered low protein diets and there was a trend(P = 0.057) for waxy rice as a starch source to increase ileal GOT activities.There was a significant influence on the concentration of seventeen amino acids in the portal circulation with tryptophan the one exception.Waxy rice as a starch source generated 13.6% and 22.4% numerically higher concentrations of non-essential amino acids than maize and amylose,respectively.Conclusions: Amino acid catabolism in the gut mucosa is subject to nutritional regulation.Given that amino acids can be spared from catabolism in the gut mucosa by supplementation of amylose,it follows their post-enteral availability would be improved and intestinal energy would be derived more efficiently from glucose.

Host-microbial interactions and regulation of intestinal epithelial barrier function: From physiology to pathology

The gastrointestinal tract is the largest reservoir of commensal bacteria in the human body, providing nutrients and space for the survival of microbes while concurrently operating mucosal barriers to confine the microbial population. The epithelial cells linked by tight junctions not only physically separate the microbiota from the lamina propria, but also secrete proinflammatory cytokines and reactive oxygen species in response to pathogen invasion and metabolic stress and serve as a sentinel to the underlying immune cells. Accumulating evidence indicates that commensal bacteria are involved in various physiological functions in the gut and microbial imbalances (dysbiosis) may cause pathology. Commensal bacteria are involved in the regulation of intestinal epithelial cell turnover, promotion of epithelial restitution and reorganization of tight junctions, all of which are pivotal for fortifying barrier function. Recent studies indicate that aberrant bacterial lipopolysaccharide-mediated signaling in gut mucosa may be involved in the pathogenesis of chronic inflammation and carcinogenesis. Our perception of enteric commensals has now changed from one of opportunistic pathogens to active participants in maintaining intestinal homeostasis. This review attempts to explain the dynamic interaction between the intestinal epithelium and commensal bacteria in disease and health status.

Intestinal mucosal adaptation

Intestinal failure is a condition characterized by malnutrition and/or dehydration as a result of the inadequate digestion and absorption of nutrients. The most common cause of intestinal failure is short bowel syndrome, which occurs when the functional gut mass is reduced below the level necessary for adequate nutrient and water absorption. This condition may be congenital, or may be acquired as a result of a massive resection of the small bowel. Following resection, the intestine is capable of adaptation in response to enteral nutrients as well as other trophic stimuli. Identifying factors that may enhance the process of intestinal adaptation is an exciting area of research with important potential clinical applications.

ALTERATION IN ENTEROCYTE GENE EXPRESSION MAY EXPLAIN STRUCTURAL AND FUNCTIONAL CHANGES FOLLOWING GLUTAMINE SUPPLEMENTED PARENTERAL NUTRITION

Following extensive bowel resection, the intestinal tract undergoes a variety of adaptive responses to enhance bowel function. The purpose of this study was to determine the effect of glutamine-supplemented parenteral nutrition on mucosal cellularity and gut function. In addition, enterocyte gene expression of two relevant systems was also characterized and related to the structural and functional changes that occurred. Male Wistar rats underwent a 60% small bowel resection and jugular vein catheterization and were randomized into two groups. The control group (n = 10) received a standard intravenous nutritional solution and the study group (n = 10) received a similar solution but enriched with alanylglutamine dipeptide. After 7 days blood was taken for amino acid analysis, and bowel was harvested to determine mucosal morphology and expression of mucosal cell glutaminase and IGF-I mRNA. Mesentery lymphnodes were cultured to determine the presence of bacteria and thus access bacteria translocation. Serum glutamine concentration and mucosal architecture were maintained in the study group compared to the controls. Seventy percent of lymphnodes were cultured positive in control vs. only 20% in the study group (P

Alterations in enterocyte mitochondrial respiratory function and enzyme activities in gastrointestinal dysfunction following brain injury

AIM: To determine the alterations in rat enterocyte mitochondrial respiratory function and enzyme activities following traumatic brain injury (TBI).

Growth hormone regulates intestinal ion transport through a modulation of the constitutive nitric oxide synthase-nitric oxide-cAMP pathway

AIM： Growth hormone （GH） directly interacts with the enterocyte stimulating ion absorption and reducing ion secretion induced by agonists of cAMR Since nitric oxide （NO） is involved in the regulation of transepithelial ion transport and acts as a second messenger for GH hemodynamic effects, we tested the hypothesis that NO may be involved in the resulting effects of GH on intestinal ion transport. METHODS： Electrical parameters reflecting transepithelial ion transport were measured in Caco-2 cell monolayers mounted in Ussing chambers and exposed to GH and cholera toxin （CT） alone or in combination, in the presence or absence of the NO synthase （NOS） inhibitor, Nω-nitro-L-arginine methyl ester （L-NAME）. Similar experiments were conducted to determine cAMP and nitrite/nitrate concentrations. NOS expression was assayed by Western blot analysis. RESULTS： L-NAME causes total abrogation of absorptive and antisecretory effects by GH on intestinal ion transport, In addition, L-NAME was able to inhibit the GH-effects on intracellular cAMP concentration under basal conditions and in response to CT, GH induced a Ca^2＋ -dependent increase of nitrites/nitrates production, indicating the involvement of the constitutive rather than the inducible NOS isoform, which was directly confirmed by Western blot analysis. CONCLUSION： These results suggest that the GH effects on intestinal ion transport, either under basal conditions or in the presence of cAMP-stimulated ion secretion, are mediated at an intracellular level by the activity of cNOS.

Macleaya cordata Extract Reduces Inflammatory Responses of Intestinal Epithelial Cells in Vitro

Background: The EU ban on antimicrobial growth promoters (AGP) has initiated a search for non-antibiotic alternatives. It has been demonstrated that certain antibiotics and non-antibiotic alternatives enhance growth by inhibiting inflammatory cells, i.e. neutrophils and macrophages in the intestine. There is very little information on the effect of anti-inflammatory compounds on intestinal epithelial cells, which are known to play an important role in intestinal inflammatory responses. In order to establish this, a porcine intestinal epithelial cell line (IPEC J2) was incubated with an adherent enterotoxigenic E. coli (ETEC) to stimulate inflammation, using a non-pathogenic non-adherent E. coli (EC) as a control. The influence of the presence of the anti-inflammatory compounds Macleaya cordata extract (MCE) and acetylsalicylic acid (ASA) on inflammatory transcriptional responses was studied. Results: ETEC induced a strong inflammatory response as was most evident from the expression of IL-1&beta;, IL-8 and TNF-&alpha;, whereas EC induced IL-1&beta;only. Co-incubation with MCE and ASA significantly reduced the responses of the pro-inflammatory cytokines, similarly for IL-1&beta;and TNF-&alpha;, but ASA was more effective than MCE in reducing the IL8 response. Conclusions: The present results suggest that the in vivo anti-inflammatory growth promoting effects of AGP and effective alternatives to AGP such as MCE and ASA are not restricted to inflammatory cells and also involve the more abundant enterocytes. This suggests a major role for epithelial cells in growth promotion livestock, and it further supports the notion that effective alternatives to AGP should have anti-inflammatory activity.

Overloading of differentiated Caco-2 cells during lipid transcytosis induces glycosylation mistakes in the Golgi complex

Overloading the intestine enterocytes with lipids induced alteration of the Golgi complex(GC;Sesorova et al.,2020)and could cause glycosylation errors.Here,using differentiated Caco-2 cells with the established 0[I]blood group phenotype(no expression of the blood antigens A and B[AgA,AgB]under normal conditions)as a model of human enterocytes we examined whether the overloading of these cells with lipids could cause errors in the Golgi-dependent glycosylation.We demonstrated that under these conditions,there were alterations of the GC and the appearance of lipid droplets in the cytoplasm.Rare cells produced AgA and AgB.This suggested that after overloading of enterocytes with lipids,AgA were mistakenly synthesized in individual enterocytes by the Golgi glycosyltransferases.These mistakes could explain why in the absence of AgA and AgB antibodies against them exist in the blood.

<i>Citrobacter rodentium</i>, a Gut Pathogen: The Yin and the Yang of Its Pathophysiology, Immunity and Clinical Manifestation in Mice

Pathogenic strains of E. coli including enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC) are principle causes for diarrhoea in many parts of the globe. Citrobacter rodentium (C. rodentium), a gram negative bacterium, is a murine pathogen that also utilizes type III secretion system and similar virulence factors to EPEC and EHEC and forms comparable attaching/effacing lesions in the intestines as EPEC and EHEC. The infection caused by C. rodentium in mice is usually self-limiting and results in only minor systemic effects with higher mortality in some susceptible mouse strains. All these characteristics have made the bacteria a commonly used model to study host immune responses to pathogenic E. coli infection. In this review, we focus on the impact of virulence factors of the pathogen;different immune components involved in the immune response and summarize their role during C. rodentium infection.

Endogenous mucin conveyed to the mucosa with microbes can assure lumen fermentation and large intestinal security-swine versus fowl

Endogenous protein leaving the ileum largely consists of accrued mucins from the upper gastrointestinal tract(GIT)that had resisted digestion.The amounts released rely on their mucosal generation during enteral feeding which vary with age as well as diet.These digestion resistant proteins of endogenous origin continue to be unavailable in the large intestine,whereas those of dietary origin provide amino acids that largely support the existing microbial population while denying limited amounts for absorption.Other mucins pre-exist within the large intestine as two layers at the lumen surface.A loose layer harboring a diverse microbial population is superimposed on the unstirred water layer(USWL)which simultaneously acts as an obstacle to microbes at the loose layer while performing as a molecular sieve for nutrients.The USWL is formed through interplay between enterocyte and goblet cells;however,the basis for presence of the loose layer is elusive.Large intestinal fermentation predominates within the colon of swine,whereas fowl employ their ceca.Motility within the colon of swine segregates fine materials into haustrae out-pocketings that parallel their placement within the ceca of fowl.Viscous mucins from small intestinal endogenous losses may envelop microbes within the large intestinal lumen to present successive adherents on the USWL that assemble its loose layer.The loose layer continually functions as a microbial reservoir in support of lumen fermentation.Microbial catabolism of mucin within the loose layer is known to be slow,but its proximity to the enterocyte is of advantage to enterocyte absorption with by-product amino acids fostering the USWL.

Calcium overload and reactive oxygen species accumulation induced by selenium deficiency promote autophagy in swine small intestine

Selenium(Se)deficiency can seriously affect the small intestine of swine,and cause diarrhea in swine.However,the specific mechanism of Se deficiency-induced swine diarrhea has rarely been reported.Here,to explore the damage of Se deficiency on the calcium homeostasis and autophagy mechanism of swine,in vivo and in vitro models of swine intestinal Se deficiency were established.Twenty-four pure line castrated male Yorkshire pigs(45 d old,12.50±1.32 kg,12 full-sibling pairs)were divided into 2 equal groups and fed Se-deficient diet(0.007 mg Se/kg)as the Se-deficiency group,or fed Se-adequate diet(0.3 mg Se/kg)as the control group for 16 weeks.The intestinal porcine enterocyte cell line(IPEC-J2)was divided into 2 groups,and cultured by Se-deficient medium as the Se-deficient group,or cultured by normal medium as the control group.Morphological observations showed that compared with the control group,intestinal cells in the Se-deficiency group were significantly damaged,and autophagosomes increased.Autophagy staining and cytoplasmic calcium staining results showed that in the Sedeficiency group,autophagy increased and calcium homeostasis was destroyed.According to the reactive oxygen species(ROS)staining results,the percentage of ROS in the Se-deficiency group was higher than that in the control group in the in vitro model.Compared with the control group,the protein and mRNA expressions of autophagy-calcium-related genes including Beclin 1,microtubule-associated proteins 1 A(LC3-1),microtubule-associated proteins 1 B(LC3-2),autophagy-related protein 5(ATG5),autophagy-related protein 12(ATG12),autophagy-related protein 16(ATG16),mammalian target of rapamycin(mTOR),calmodulin-dependent protein kinase kinaseβ(CAMKK-β),adenosine 5’-monophosphate-activated protein kinase(AMPK),sarco(endo)plasmic reticulum Ca2+-ATPase(SERCA),and calpain in the Se-deficiency group were significantly increased which was consistent in vivo and in vitro(P<0.05).Altogether,our results indicated that Se deficiency could destroy the calcium homeostasis of the swine small intestine to trigger cell autophagy and oxidative stress,which was helpful to explain the mechanism of Se deficiency-induced diarrhea in swine.

The profiles of mitochondrial respiration and glycolysis using extracellular flux analysis in porcine enterocyte IPEC-J2

The porcine intestinal mucosa require large amounts of energy for nutrient processing and cellular functions and is vulnerable to injury by weaning stress involving bioenergetics failure. The mitochondrial bioenergetic measurement in porcine enterocytes have not been defined. The present study was to establish a method to measure mitochondrial respiratory function and profile mitochondrial function of IPEC-J2 using cell mito stress test and glycolysis stress test assay by XF24 extracellular flux analyzer. The optimal seeding density and concentrations of the injection compounds were determined to be 40,000 cells/well as well as 0.5 μ M oligomycin, 1 μM carbonyl cyanide p-trifluoromethoxy-phenylhydrazone(FCCP) and 1 μM rotenone & antimycin A, respectively. The profiles of mitochondrial respiration and glycolysis confirmed that porcine enterocyte preferentially derived much more energy from glutamine than glucose. These results will provide a basis for further study of mitochondrial function and bioenergetics of the porcine small intestine.