Intestinal Epithelial Cell Proliferation Research Articles

DR3 Regulates Intestinal Epithelial Homeostasis and Regeneration After Intestinal Barrier Injury

Tumor necrosis factor (TNF) superfamily member tumor necrosis factor-like protein 1A (TL1A) has been associated with the susceptibility and severity of inflammatory bowel diseases. However, the function of the tumor necrosis factor-like protein 1A and its receptor death receptor 3 (DR3) in the development of intestinal inflammation is incompletely understood. We investigated the role of DR3 expressed by intestinal epithelial cells (IECs) during intestinal homeostasis, tissue injury, and regeneration. Clinical phenotype and histologic inflammation were assessed in C57BL/6 (wild-type), Tl1a-/- and Dr3-/- mice in dextran sulfate sodium (DSS)-induced colitis. We generated mice with an IEC-specific deletion of DR3 (Dr3ΔIEC) and assessed intestinal inflammation and epithelial barrier repair. Invivo intestinal permeability was assessed by fluorescein isothiocyanate dextran uptake. Proliferation of IECs was analyzed by bromodeoxyuridine incorporation. Expression of DR3 messenger RNA was assessed by fluorescent in situ hybridization. Small intestinal organoids were used to determine exvivo regenerative potential. Dr3-/- mice developed more severe colonic inflammation than wild-type mice in DSS-induced colitis with significantly impaired IEC regeneration. Homeostatic proliferation of IECs was increased in Dr3-/- mice, but blunted during regeneration. Cellular localization and expression of the tight junction proteins Claudin-1 and zonula occludens-1 were altered, leading to increased homeostatic intestinal permeability. Dr3ΔIEC mice recapitulated the phenotype observed in Dr3-/- mice with increased intestinal permeability and IEC proliferation under homeostatic conditions and impaired tissue repair and increased bacterial translocation during DSS-induced colitis. Impaired regenerative potential and altered zonula occludens-1 localization also were observed in Dr3ΔIEC enteroids. Our findings establish a novel function of DR3 in IEC homeostasis and postinjury regeneration independent of its established role in innate lymphoid cells and T-helper cells.

YAP regulates intestinal epithelial cell proliferation through activation of STAT3 in DSS-induced colitis and associated cancer.

Ulcerative colitis (UC) is a chronic, relapsing inflammation of the colon. Impaired epithelial repair is an important biological features of UC. Accelerating intestinal epithelial repair to achieve endoscopic mucosal healing has become a key goal in UC. Yes-associated protein (YAP) is a key transcriptional coactivator that regulates organ size, tissue growth and tumorigenesis. Growing studies have focused on the role of YAP in intestinal epithelial regeneration. This study explore the molecular mechanism for the role YAP in modulating colonic epithelial proliferation, repair, and the development of colitis associated cancer. We constructed the acute colitis mouse model through successive 5 days of 3% dextran sulfate sodium salt (DSS) induction. Then YAP-overexpressed mouse model was constructed by intraperitoneal injection the YAP overexpressed and negative control lentivirus into DSS mice. On the 5th day of DSS induction and the 5th day of normal drinking water after removing DSS (5+5 d), the mice were killed by spinal dislocation. The colon was taken to measure the length, and the bowel 1-2 cm near the anal canal was selected for immunohistochemical and Western blotting. We used YAP over-expressed colonic epithelial cells and small interfering signal transducer and activator of transcription 3 (STAT3) RNA to probe the regulation of YAP on STAT3, using cell counting kit-8 and scratch assays to explore the role of YAP on colonic epithelial cell proliferation. Finally, we conducted co-immunoprecipitation to test the relationship between YAP and STAT3. After DSS treatment, the expression of YAP was dramatically diminished in crypts. Compared with the empty control mice, overexpression of YAP drastically accelerated epithelial regeneration after DSS induced colitis, presenting with more intact of structural integrity in intestinal epithelium and a reduction in the number of inflammatory cells in the mucosa. Further Western blotting, functional experiment and co-immunoprecipitation analyses showed that the expression of YAP in nucleus was significantly increased by 2 h post DSS cessation, accompanied with up-regulated total protein levels of STAT3 and phosphorylated-STAT3 (p-STAT3). Overexpression of YAP enhanced the expression of STAT3, p-STAT3, and their transcriptional targets including c-Myc and Cyclin D1. In addition, it promoted the proliferation and the "wound healing" of colonic cells. However, these effects were reversed when silencing STAT3 on YAP-overexpressed FHC cells. Moreover, protein immunoprecipitation indicated that YAP could directly interact with STAT3 in the nucleus, up-regulatvng the expressvon of STAT3. Finally, during the process of CAC, overexpression of YAP mutant caused the down-regulated expression of STAT3 and inhibited the development and progress of CAC. YAP activates STAT3 signaling in regulation of epithelial cell proliferation and promotes mucosal regeneration after DSS induced colitis, which may serve as a potential therapeutic target in UC. However, persistent and excessive YAP activation may promote CAC development.

Drinking alkaline mineral water confers diarrhea resistance in maternally separated piglets by maintaining intestinal epithelial regeneration via the brain-microbe-gut axis

IntroductionDiarrhea has the fourth-highest mortality rate of all diseases and causes a large number of infant deaths each year. The maternally separated (MS) piglet (newly weaned piglet) is an excellent model to investigate the treatment of diarrhea in infants. Drinking alkaline mineral water has the potential to be therapeutic in gastrointestinal disorders, particularly diarrhea, but the supporting evidence from system studies and the mechanisms involved have yet to be reported. ObjectivesThis study aims to determine whether drinking alkaline mineral water confers diarrhea resistance in MS piglets under weaning stress and what the fundamental mechanisms involved are. MethodsMS piglets were used to create a stress-induced intestinal disorder-diarrhea susceptibility model. A total of 240 MS piglets were randomly divided into two groups (6 pens/group and 20 piglets/pen). IPEC-J2 cell line was used for in vitro evaluation. An alkaline mineral complex (AMC) water was employed, and its effect on the hypothalamus-pituitary-adrenocortical (HPA) axis, gut microbes, gut morphology, and intestinal epithelial cell (IEC) proliferation and differentiation were investigated using a variety of experimental methodology. ResultsAMC water reduced diarrhea rate in MS piglets by inhibiting the HPA axis, ameliorating gut microbiota structure, and stimulating IEC proliferation and differentiation. Apparently, the brain-microbe-gut axis is linked with AMC water conferring diarrhea resistance in piglets. Mechanistically, AMC water decreased stress hormones (COR and Hpt) secretion by suppressing HPA axis, which then increased the abundance of beneficial gut microbes; accordingly, maintained the proliferation of IEC and promoted the differentiation of intestinal stem cells (ISC) into goblet cell and Paneth cell by activating the Wnt/β-catenin signaling pathway. In the absence of gut microbiota (in vitro), AMC activated the LPS-induced Wnt/β-catenin signaling inhibition in IPEC-J2 cells and significantly increased the number of Lgr5 + cells, whereas had no effect on IPEC-J2 differentiation. ConclusionDrinking alkaline mineral water confers diarrhea resistance in MS piglets by maintaining intestinal epithelial regeneration via the brain-microbe-gut axis; thus, this study provides a potential prevention strategy for young mammals at risk of diarrhea.

Bacteroides fragilis strain ZY-312 promotes intestinal barrier integrity via upregulating the STAT3 pathway in a radiation-induced intestinal injury mouse model.

Radiation-induced intestinal injury is characterized by intestinal barrier impairment. However, the therapeutic effects of probiotics for intestinal epithelial barrier repair in a mouse model of radiation-induced intestinal injury remain unclear. Previously, we isolated a strain of Bacteroides fragilis from the feces of a healthy infant and named it as B. fragilis strain ZY-312 (B. fragilis). In this study, we showed that B. fragilis can ameliorate radiation-induced intestinal injury in mice, manifested by decreased weight loss, intestinal length shortening, and intestinal epithelial cell (IEC) shedding. Moreover, we found that B. fragilis promoted IEC proliferation, stem cell regeneration, mucus secretion, and tight junction integrity by upregulating the STAT3 signaling pathway, through an experimental verification in Stat3 △IEC mice (STAT3 defects in intestinal epithelial cells). Thus, the underlying protective mechanism of B. fragilis in radiation-induced intestinal injury is related to IEC proliferation, stem cell regeneration, goblet cell secretion, and tight junction repair via activation of the STAT3 signaling pathway. In addition, the therapeutic effects of B. fragilis were studied to provide new insights into its application as a functional and clinical drug for radiation-induced intestinal injury after radiotherapy.

Glycoprotein (GP)96 Is Essential for Maintaining Intestinal Epithelial Architecture by Supporting Its Self-Renewal Capacity

Glycoprotein (GP)96 is an endoplasmic reticulum-resident master chaperone for cell surface receptors including the Wnt co-receptors low-densitylipoprotein-receptor-related protein 5/6. Intestinal epithelial cell (IEC)-specific deletion of Gp96 is embryonically lethal. However, the role of GP96 in adult intestinal tissue and especially within the intestinal stem cell (ISC) niche is unknown. Here, we investigated how GP96 loss interferes with intestinal homeostasis by compromising viability, proliferation, and differentiation of IECs. Tamoxifen was used to induce Cre-mediated deletion of Gp96 in GP96-VillincreERT2 (Cre recombinase-Estrogen-Receptor Transgene 2) mice and intestinal organoids. With H&E and immunofluorescence staining we assessed alterations in intestinal morphology and the presence and localization of IEC types. Real-time polymerase chain reaction and Western blot analysis were performed to explore the molecular mechanisms underlying the severe phenotype of Gp96 KO mice and organoids. IEC-specific deletion of Gp96 in adult mice resulted in a rapid degeneration of the stem cell niche, followed by complete eradication of the epithelial layer and death within a few days. These effects were owing to severe defects in ISC renewal and premature ISC differentiation, which resulted from defective Wnt and Notch signaling. Furthermore, depletion of GP96 led to massive induction of endoplasmic reticulum stress. Although effects on ISC renewal and adequate differentiation were partly reversed upon activation of Wnt/Notch signaling, viability could not be restored, indicating that reduced viability was mediated by other mechanisms. Our work shows that GP96 plays a fundamental role in regulating ISC fate and epithelial regeneration and therefore is indispensable for maintaining intestinal epithelial homeostasis.

The potential mechanism of Neu5Gc inducing colorectal cancer based on network pharmacology and experimental validation.

Colorectal cancer has high morbidity and mortality worldwide, especially in western countries; the incidence of colorectal cancer has been high, which is closely related to the high intake of red meat; and the N-glycolylneuraminic acid (Neu5Gc) is responsible for red meat-induced colorectal cancer. A large number of previous studies have suggested that exogenous Neu5Gc-activated inflammation induced the occurrence of colorectal cancer. However, it has not been known whether the Neu5Gc has a direct inducing effect on colorectal cancer. In this study, we found that Neu5Gc promoted the proliferation of colorectal cancer cells and normal intestinal epithelial cells, and further screened out 98 Neu5Gc targets related to the occurrence and development of colorectal cancer by network pharmacology. Subsequently, GO and KEGG enrichment analyses of these targets revealed that mainly enriched in the PI3K-Akt signaling pathway. Then, we selected SRC, HRAS, CDK2, CCNA2, and AKT2 as core targets based on the phenomena of the previous experiments and the available literature reports, and then we used AutoDock for molecular docking with Neu5Gc; the results found that these five genes could bind to Neu5Gc stably. In vitro experiments showed that the mRNA levels of SRC, HRAS, AKT2, CDK2, and CCNA2 were upregulated and the protein levels of HRAS, AKT2, and CCNA2 were enhanced in FHC and SW620 cells after Neu5Gc (100ng/mL) treatment. In conclusion, this study revealed that Neu5Gc probably acted as a carcinogen that stimulates the expression of proto-oncogene HRAS and the PI3K-Akt pathway and accelerated cell cycle progression. These findings revealed a novel mechanism that Neu5Gc promoted the occurrence and development of colorectal cancer.

Pattern of expression of CDX2 in colorectal cancer and its role in prognosis.

CDX2, a nuclear protein, is essential for the proliferation and development of intestinal epithelial cells and is frequently down-regulated during tumorigenesis. We have evaluated the pattern of CDX2 expression in all stages of colorectal cancer (CRC) and its association with prognosis. We performed CDX2 staining by immunohistochemistry (IHC) on the available biopsy blocks of patients of CRC registered in our institute from January 2014 to January 2018. CDX2 scoring was done using the semi-quantitative method. A total of 286 patients were registered during the study period, of which only 110 biopsy blocks were available for staining. Of 110 patients, 77 (70%) had colon cancer and 33 (30%) had rectal cancer. The median age was 54.2 years, with 62 (56.4%) being male and 48 (43.6%) female with a male to female ratio of 1.3:1. In the study cohort, 33 (30%) patients had stage II disease, 30 (27.3%) had stage III, and 47 (42.7%) had stage IV. Seventy-three (66.4%) were positive for CDX2 and 37 (33.4%) were negative. Loss of CDX2 expression was significantly associated with advanced stage, rectal site, poor grade of differentiation, and presence of lymphovascular invasion. With a median follow-up of 16 months, progression-free survival (PFS) at 2 years was 30% for CDX2-negative patients compared to 67% for CDX2-positive patients (P = 0.009), whereas the overall survival (OS) at 2 years was 46% for CDX2-negative versus 77% for CDX2-positive patients (P = 0.01). Loss of CDX2 expression is associated with advanced stage, higher tumor grade, presence of LVSI, worse PFS, and OS and thereby functions as a poor prognostic factor in CRC.

Ketogenic Diet and Beta-Hydroxybutyrate in Colorectal Cancer.

Colorectal cancer (CRC) is one of the leading causes of cancer-related death in the United States. Although certain genetic predispositions may contribute to one's risk for developing CRC, dietary and lifestyle factors may play an important role as well. In a recent study in Nature, Dmitrieva-Posocco et al, reveal a potential protective role of the ketogenic diet in colorectal cancer growth and progression. Administration of a ketogenic diet to CRC-bearing mice demonstrated a tumor-suppressive effect. Specifically, the ketone body β-hydroxybutyrate (BHB) exhibited the ability to suppress epithelial cell proliferation and inhibit tumor growth. BHB acts on cancer cells through regulation of homeodomain-only protein Hopx, known regulator of CRC. Furthermore, BHB requires a surface receptor Hcar to induce Hopx expression and suppress proliferation of intestinal epithelial cells. Taken together, these results describe a new therapeutic approach of using dietary intervention for the prevention and treatment of colorectal cancer.

Pattern of Expression of CDX2 in Colorectal Cancer and its Role in Prognosis: An Ambispective Observational Study

Abstract Introduction Caudal-type homeobox 2 (CDX2), a nuclear protein, is essential for the proliferation and development of intestinal epithelial cells and is frequently downregulated during tumorigenesis. CDX2 inhibits cell growth as well as stimulates differentiation by activating intestinal specific genes, thus lack of CDX2 favors tumor growth and aggressiveness. Objectives We aimed to evaluate the pattern of CDX2 expression in all stages of colorectal cancer (CRC) and study its association with baseline characteristics and prognosis. Materials and Methods Study was conducted as an ambispective observational study, enrolling cases of CRC retrospectively from January 2014 to July 2016 (30 months), and prospectively during next 18-month period till January 2018. We performed CDX2 staining by immunohistochemistry on the available biopsy blocks of CRC patients during the study period. Total 286 patients were registered during the study period, of which only 110 biopsy blocks were available for staining. CDX2 scoring was done by a semiquantitative method on whole tissue section for the intensity and percentage of the cells showing positivity. Correlation of CDX2 expression was done with baseline clinical and histopathologic characteristics, and survival. Results Of 110 patients, 77 (70%) constituted colon cancer and 33 (30%) were rectal cancer. The median age was 54.2 years, 62 (56.4%) being male and 48 (43.6%) female with male-to-female ratio 1.3:1. In the study cohort, 33 (30%) patients had stage II disease, 30 (27.3%) stage III, and 47 (42.7%) were stage IV. Seventy-three (66.4%) were positive for CDX2 and 37 (33.4%) were negative. Loss of CDX2 expression was significantly associated with advanced stage, rectal site, poor grade of differentiation, and presence of lymphovascular invasion (LVSI). With median follow-up of 16 months, progression-free survival (PFS) at 2 years was 30% for CDX2 negative patients compared with 67% for CDX2 positive (p = 0.009), while overall survival (OS) at 2 years was 46% for CDX2 negative versus 77% for positive patients (p = 0.01). Conclusion Loss of CDX2 expression is associated with advanced stage, higher tumor grade, presence of LVSI, and worse PFS and OS and thereby functions as a poor prognostic factor in CRC.

Modulation of intestinal epithelial cell proliferation and apoptosis by Lactobacillus gasseri SF1183

The gut microbiota exerts a variety of positive effects on the intestinal homeostasis, including the production of beneficial molecules, control of the epithelial barrier integrity and the regulation of the balance between host’s cell death and proliferation. The interactions between commensal bacteria and intestinal cells are still under-investigated and is then of paramount importance to address such interactions at the molecular and cellular levels. We report an in vitro analysis of the effects of molecules secreted by Lactobacillus gasseri SF1183 on HCT116 cells, selected as a model of intestinal epithelial cells. SF1183 is a L. gasseri strain isolated from an ileal biopsy of a human healthy volunteer, able to prevent colitis symptoms in vivo. Expanding previous findings, we show that bioactive molecules secreted by SF1183 reduce the proliferation of HCT116 cells in a reversible manner determining a variation in cell cycle markers (p21WAF, p53, cyclin D1) and resulting in the protection of HCT116 cells from TNF-alfa induced apoptosis, an effect potentially relevant for the protection of the epithelial barrier integrity and reconstitution of tissue homeostasis. Consistently, SF1183 secreted molecules increase the recruitment of occludin, a major component of TJ, at the cell–cell contacts, suggesting a reinforcement of the barrier function.

Postbiotic muramyl dipeptide alleviates colitis via activating autophagy in intestinal epithelial cells.

The pathogenesis of IBD is complicated and still unclear. Nucleotide-binding oligomerization domain 2 (NOD2) plays a significant role in regulating gut inflammation under the activation of muramyl dipeptide (MDP), which is used as a postbiotic. The study aimed to investigate the effect of MDP on the intestinal barrier in colitis and the mechanism involved. In this study, C57BL/6 mice were challenged with dextran sodium sulfate (DSS) for establishing a colitis model with the pre-treatment of MDP in vivo. Intestinal permeability was reflected by detecting the serum concentration of 4kDa Fluorescein Isothiocyanate-Dextran. The expression of inflammation, barrier-related proteins, and autophagy was tested by Western Blotting. Proliferation and apoptosis in intestinal epithelial cells were detected by immunohistochemistry. Caco-2 cells were exposed to lipopolysaccharide for imitating inflammation in vitro. The findings showed that administration of MDP ameliorated losses of body weight loss, gross injury, and histology score of the colon in the DSS-induced colitis mice. MDP significantly ameliorated the condition of gut permeability, and promoted intestinal barrier repair by increasing the expression of Zonula occludens-1 and E-cadherin. Meanwhile, MDP promoted proliferation and reduced apoptosis of intestinal epithelial cells. In the experiment group treated with MDP, LC3 was upregulated, and p62 was downregulated, respectively. These results suggested that MDP stimulation attenuates intestinal inflammation both in vivo and in vitro. Potentially, MDP reduced the intestinal barrier damage by regulating autophagy in intestinal epithelial cells. Future trials investigating the effects of MDP-based postbiotics on IBD may be promising.

Milk-Derived miR-22-3p Promotes Proliferation of Human Intestinal Epithelial Cells (HIECs) by Regulating Gene Expression.

MicroRNA (miRNA) is small non-coding RNA involved in gene silencing and post-transcriptional regulation of gene expression. Milk exosomes are microvesicles containing microRNAs (miRNAs). miR-22-3p (miR-22) is plentiful in human milk exosomes and may contribute to intestinal development since milk exosomes and microRNAs are resistant to gastrointestinal digestion in infants. After miR-22 mimics were transfected to human intestinal crypt-like epithelial cells (HIECs) using Lipofectamine for 24 h, RNA was isolated for microarray assay. Microarray results show that miR-22 markedly regulates gene expression, and the roles of miR-22 include promotion of proliferation, regulation of immune functions, and inhibition of apoptosis. Based on the microarray results and miR-22 predicted target genes, CCAAT/enhancer-binding protein δ (C/EBPδ) may be an important direct target of miR-22. C/EBPδ is a transcription factor that regulates numerous biological processes including cell proliferation. In miR-22 transfected HIECs, expression of the C/EBPδ gene was significantly inhibited. Silencing of the C/EBPδ gene by siRNA resulted in increased proliferation of HIECs. A luciferase assay showed that miR-22 specifically binds to the 3'-untranslated region of C/EBPδ mRNA. In summary, milk-derived miR-22 promotes intestinal proliferation by modifying gene expression, and C/EBPδ may be an important target for miR-22 involved in this effect.

Chenodeoxycholic Acid (CDCA) Promoted Intestinal Epithelial Cell Proliferation by Regulating Cell Cycle Progression and Mitochondrial Biogenesis in IPEC-J2 Cells.

Chenodeoxycholic acid (CDCA), a primary bile acid (BA), has been demonstrated to play an important role as a signaling molecule in various physiological functions. However, the role of CDCA in regulating intestinal epithelial cell (IEC) function remains largely unknown. Herein, porcine intestinal epithelial cells (IPEC-J2) were used as an in vitro model to investigate the effects of CDCA on IEC proliferation and explore the underlying mechanisms. IPEC-J2 cells were treated with CDCA, and flow cytometry and transcriptome analysis were adopted to investigate the effects and potential molecular mechanisms of CDCA on the proliferation of IECs. Our results indicated that adding 50 μmol/L of CDCA in the media significantly increased the proliferation of IPEC-J2 cells. In addition, CDCA treatment also hindered cell apoptosis, increased the proportion of G0/G1 phase cells in the cell cycle progression, reduced intracellular ROS, and MDA levels, and increased mitochondrial membrane potential, antioxidation enzyme activity (T-AOC and CAT), and intracellular ATP level (p < 0.05). RNA-seq results showed that CDCA significantly upregulated the expression of genes related to cell cycle progression (Cyclin-dependent kinase 1 (CDK1), cyclin G2 (CCNG2), cell-cycle progression gene 1 (CCPG1), Bcl-2 interacting protein 5 (BNIP5), etc.) and downregulated the expression of genes related to mitochondrial biogenesis (ND1, ND2, COX3, ATP6, etc.). Further KEGG pathway enrichment analysis showed that CDCA significantly enriched the signaling pathways of DNA replication, cell cycle, and p53. Collectively, this study demonstrated that CDCA could promote IPEC-J2 proliferation by regulating cell cycle progression and mitochondrial function. These findings provide a new strategy for promoting the intestinal health of pigs by regulating intestinal BA metabolism.

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.

Intestinal Epithelial STAT6 Activation Rescues the Defective Anti-Helminth Responses Caused by Ogt Deletion.

Dynamic regulation of intestinal epithelial cell (IEC) proliferation and differentiation is crucial for maintaining mucosa homeostasis and the response to helminth infection. O-GlcNAc transferase (OGT), an enzyme catalyzing the transfer of GlcNAc from the donor substrate UDP-GlcNAc onto acceptor proteins, has been proposed to promote intestinal epithelial remodeling for helminth expulsion by modifying and activating epithelial STAT6, but whether the IEC intrinsic OGT-STAT6 axis is involved in anti-helminth responses has not been tested in vivo. Here, we show that the inducible deletion of Ogt in IECs of adult mice leads to reduced tuft and goblet cell differentiation, increased crypt cell proliferation, and aberrant Paneth cell localization. By using a mouse model with concurrent Ogt deletion and STAT6 overexpression in IECs, we provide direct in vivo evidence that STAT6 acts downstream of OGT to control tuft and goblet cell differentiation in IECs. However, epithelial OGT regulates crypt cell proliferation and Paneth cell differentiation in a STAT6-independent pathway. Our results verify that protein O-GlcNAcylation in IECs is crucial for maintaining epithelial homeostasis and anti-helminthic type 2 immune responses.

PSXIII-B-8 Effect of Supplemental myo-Inositol on Growth Performance and Apparent Total Tract Digestibility of Weanling Piglets fed Reduced Protein Diets and Intestinal Cell Epithelial Barrier Integrity

Abstract Myo-inositol is a breakdown product of phytate produced in the gut through the action of phytase. Although the effect of phytase-released phosphorus on growth performance of animals has been well characterized, there is still little understanding of myo-inositol effects. The objectives of this study were to determine the effects of added myo-inositol to a phytate-rich low-protein diet on growth performance and apparent total tract digestibility (ATTD) in growing piglets as well as to determine whether myo-inositol could directly affect intestinal epithelial cell proliferation and function in intestinal porcine epithelial cells (IPEC-J2). A total of 128 weanling piglets were allotted to four dietary treatments consisting of eight replicates per treatment and four piglets per replicate in a randomized complete block design for four weeks. The experimental diets comprised the positive control (PC; 20% crude protein (CP), negative control (NC; 17% CP), negative control plus 2.0g/kg myo-inositol (NC+INO; 17% CP) and negative control plus 3000FTU/kg phytase (NC+PHY; 17% CP). Weekly average daily feed intake (ADFI), average daily gain (ADG), gain-feed ratio (G: F) were recorded. Phytase supplementation in the protein-deficient NC diet increased the G:F ratio (P&amp;lt; 0.05) without myo-inositol effect on growth performance. Phosphorus digestibility in the phytase supplemented group increased compared to the PC, NC, and NC+INO groups whereas plasma myo-inositol concentration was significantly higher (P&amp;lt; 0.05) in the NC+INO group. Due to lack of myo-inositol effect on growth performance, an additional in vitro study was conducted to determine direct effect of myo-inositol on the intestinal epithelium that might not be reflected in growth performance. Myo-inositol increased mRNA abundance of selected nutrient transporters in a concentration-dependent manner (P&amp;lt; 0.05), enhanced barrier integrity in the IPEC-J2 monolayer and reduced paracellular permeability of FITC-dextran (P&amp;lt; 0.05). In conclusion, despite the lack of myo-inositol effect on animal performance, the in-vitro data indicates that myo-inositol may directly regulate gut barrier integrity.

MiR-146a-5p promotes epithelium regeneration against LPS-induced inflammatory injury via targeting TAB1/TAK1/NF-κB signaling pathway

Intestinal inflammation often restricts the health and production of animals. MiR-146a has been proved to be an anti-inflammatory molecule in inflammatory disorders, but its role in the intestinal injury and regeneration remains unclear. The study aimed to explore the inflammatory response of intestinal epithelial cells (IECs) in intestinal tissue-specific miR-146a-5p knockout mouse models. We identified the role of miR-146a-5p in inhibiting inflammatory response and promoting proliferation under lipopolysaccharide (LPS) stimulation in vitro and vivo. LPS stimulation significantly increased the expression of TNF-α, IL6 and inhibited IPEC-J2 cell proliferation. Overexpression of miR-146a-5p can reverse the effect of LPS stimulation, and promote the proliferation of intestinal epithelial cells. In the LPS challenge experiment in intestine-specific miR-146a knock-out mice (CKO) and Floxp+/+ mice (CON), CKO mice were more sensitive to LPS stimulation, with more weight loss and more severe intestinal morphological damage than CON mice. Also, miR-146a-5p regulated LPS-induced intestinal injury, inflammation by targeting TAB1. Taken together, miR-146a may function as an anti-inflammatory factor in IECs by targeting TAB1/TAK1-IKK-NF-κB signaling pathway. miR-146a-5p may represent a promising biomarker for inflammatory disorders, and may provide an effective therapeutic method to alleviate weaning stress in piglets and some experimental basis to improve the intestinal health of livestock.

The miR-181 family regulates colonic inflammation through its activity in the intestinal epithelium.

The intestinal epithelium is a key physical interface that integrates dietary and microbial signals to regulate nutrient uptake and mucosal immune cell function. The transcriptional programs that regulate intestinal epithelial cell (IEC) quiescence, proliferation, and differentiation have been well characterized. However, how gene expression networks critical for IECs are posttranscriptionally regulated during homeostasis or inflammatory disease remains poorly understood. Herein, we show that a conserved family of microRNAs, miR-181, is significantly downregulated in IECs from patients with inflammatory bowel disease and mice with chemical-induced colitis. Strikingly, we showed that miR-181 expression within IECs, but not the hematopoietic system, is required for protection against severe colonic inflammation in response to epithelial injury in mice. Mechanistically, we showed that miR-181 expression increases the proliferative capacity of IECs, likely through the regulation of Wnt signaling, independently of the gut microbiota composition. As epithelial reconstitution is crucial to restore intestinal homeostasis after injury, the miR-181 family represents a potential therapeutic target against severe intestinal inflammation.

Transcriptome Profile Analysis of Intestinal Upper Villus Epithelial Cells and Crypt Epithelial Cells of Suckling Piglets.

Simple SummaryTo investigate the genetic reprogramming that drives intestinal epithelial cells (IECs) maturation along the crypt-villus axis, entCerocytes were sequentially isolated from the villus tip to the crypts of porcine small intestine. The present study obtained the intestinal upper villus epithelial cells (F1) and crypt epithelial cells (F3) of 21-day suckling piglets using the divalent chelation and precipitation technique. By mRNA high-throughput sequencing analysis of F1 and F3, it was shown that a total of 672 unigenes were differentially expressed between F1 and F3, including 224 highly expressed and 448 minimally expressed unigenes. The greatest number of differentially expressed genes enriched in signal transduction, e.g., Wnt, Hippo, TGF-beta, mTOR, PI3K-Akt, and MAPK signaling pathways, were closely related to the differentiation, proliferation, maturation and apoptosis of IECs. Our results provide important information for identifying new regulators of IECs differentiation and maturation. Moreover, they can also provide insights into the regulatory mechanisms underlying intestinal epithelial cell renewal and the rapid repair of intestinal mucosal oxidative damage.It is well known that the small intestinal epithelial cells of mammals rapidly undergo differentiation, maturation, and apoptosis. However, few studies have defined the physiological state and gene expression changes of enterocytes along the crypt-villus axis in suckling piglets. In the present study, we obtained the intestinal upper villus epithelial cells (F1) and crypt epithelial cells (F3) of 21-day suckling piglets using the divalent chelation and precipitation technique. The activities of alkaline phosphatase, sucrase, and lactase of F1 were significantly higher (p < 0.05) than those of F3. To explore the differences at the gene transcription level, we compared the global transcriptional profiles of F1 and F3 using RNA-seq analysis technology. A total of 672 differentially expressed genes (DEGs) were identified between F1 and F3, including 224 highly expressed and 448 minimally expressed unigenes. Functional analyses indicated that some DEGs were involved in the transcriptional regulation of nutrient transportation (SLC15A1, SLC5A1, and SLC3A1), cell differentiation (LGR5, HOXA5 and KLF4), cell proliferation (PLK2 and TGFB3), transcriptional regulation (JUN, FOS and ATF3), and signaling transduction (WNT10B and BMP1), suggesting that these genes were related to intestinal epithelial cell maturation and cell renewal. Gene Ontology (GO) enrichment analysis showed that the DEGs were mainly associated with binding, catalytic activity, enzyme regulator activity, and molecular transducer activity. Furthermore, KEGG pathway analysis revealed that the DGEs were categorized into 284 significantly enriched pathways. The greatest number of DEGs enriched in signal transduction, some of which (Wnt, Hippo, TGF-beta, mTOR, PI3K-Akt, and MAPK signaling pathways) were closely related to the differentiation, proliferation, maturation and apoptosis of intestinal epithelial cells. We validated the expression levels of eight DEGs in F1 and F3 using qRT-PCR. The present study revealed temporal and regional changes in mRNA expression between F1 and F3 of suckling piglets, which provides insights into the regulatory mechanisms underlying intestinal epithelial cell renewal and the rapid repair of intestinal mucosal damage.

CPT1 Mediated Ionizing Radiation-Induced Intestinal Injury Proliferation via Shifting FAO Metabolism Pathway and Activating the ERK1/2 and JNK Pathway.

The intestinal compensatory proliferative potential is a key influencing factor for susceptibility to radiation-induced intestinal injury. Studies indicated that the carnitine palmitoyltransferase 1 (CPT1) mediated fatty acid β-oxidation (FAO) plays a crucial role in promoting the survival and proliferation of tumor cells. Here, we aimed to explore the effect of 60Co gamma rays on CPT1 mediated FAO in the radiation-induced intestinal injury models, and investigate the role of CPT1 mediated FAO in the survival and proliferation of intestinal cells after irradiation. We detected the changed of FAO in the plasma and small intestine of Sprague Dawley (SD) rats at 24 h after 60Co gamma irradiation (0, 5 and 10 Gy), using target metabolomics, qRT-PCR, immunohistochemistry (IHC), western blot (WB) and related enzymatic activity kits. We then analyzed the FAO changes in radiation-induced intestinal injury models regardless of ex vivo (mice enteroids), or in vitro (normal human intestinal epithelial cell lines, HIEC-6). HIEC-6 cells were transduced with lentivirus vector GV392 and treated with puromycin for obtaining CPT1 stable knockout cell lines, named CPT1 KO. CPT1 enzymatic activities of HIEC-6 cells and mice enteroids were also inhibited by pharmaceutical inhibitor ST1326 and Etomoxir (ETO), to study the function of CPT1 in the survival and proliferation of HIEC-6 cells after 60Co gamma irradiation. We found that CPT1 mediated FAO was altered in the small intestine of the SD rats after irradiation, especially, the expression level and enzymatic activity of CPT1 were significantly increased. Similarly, the expression levels of CPT1 were also remarkably enhanced in mice enteroids and HIEC-6 cells after irradiation. CPT1 inhibition decreased the proliferation of the HIEC-6 cells and mice enteroids after irradiation partially by reducing the extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK) pathways activation, CPT1 inhibition also reduced the proliferation of mice enteroids after irradiation partially by down-regulating the Wnt/β-catenin signaling activity. In conclusion, our study indicated that CPT1 plays a crucial role in promoting intestinal epithelial cell proliferation after irradiation.