Colonic Epithelial Cell Proliferation Research Articles

NF-κB Inducing Kinase Attenuates Colorectal Cancer by Regulating Noncanonical NF-κB Mediated Colonic Epithelial Cell Regeneration

Dysregulated colonic epithelial cell (CEC) proliferation is a critical feature in the development of colorectal cancer. We show that NF-κB-inducing kinase (NIK) attenuates colorectal cancer through coordinating CEC regeneration/differentiation via noncanonical NF-κB signaling that is unique from canonical NF-κB signaling. Initial studies evaluated crypt morphology/functionality, organoid generation, transcriptome profiles, and the microbiome. Inflammation and inflammation-induced tumorigenesis was initiated in whole-body NIK knockout mice (Nik-/-)and conditional-knockout mice following administration of azoxymethane and dextran sulfate sodium (AOM/DSS). Human transcriptomic data revealed dysregulated noncanonical NF-κB signaling. In vitro studies evaluating Nik-/- crypts and organoids derived from mature, nondividing CECs and colonic stem cells (CSCs) exhibited increased accumulation and stunted growth, respectively. Transcriptomic analysis of Nik-/- cells revealed gene expression signatures associated with altered differentiation-regeneration. When assessed in vivo, Nik-/- mice exhibited more severe colitis with DSS administration and an altered microbiome characterized by increased colitogenic microbiota. In the inflammation-induced tumorigenesis model, we observed both increased tumor burdens and inflammation in mice where NIK is knocked out in CECs (NikΔCEC). Interestingly, this was not recapitulated when NIK was conditionally knocked out in myeloid cells (NikΔMYE). Surprisingly, conditional knockout of the canonical pathway in myeloid cells (RelAΔMYE) revealed decreased tumor burden and inflammation and no significant changes when conditionally knocked out in CECs (RelAΔCEC). Dysregulated noncanonical NF-κB signaling is associated with the development of colorectal cancer in a tissue dependent manner and defines a critical role for NIK in regulating gastrointestinal inflammation and regeneration associated with colorectal cancer.

Sec1 regulates intestinal mucosal immunity in a mouse model of inflammatory bowel disease

Inflammatory bowel disease (IBD) is a common immune-mediated condition with its molecular pathogenesis remaining to be fully elucidated. This study aimed to deepen our understanding of the role of FUT2 in human IBD, by studying a new surrogate gene Sec1, a neighboring gene of Fut2 and Fut1 that co-encodes the α 1,2 fucosyltransferase in mice. CRISPR/Cas9 was used to prepare Sec1 knockout (Sec1−/−) mice. IBD was induced in mice using 3% w/v dextran sulphate sodium. Small interfering RNA (siRNA) was employed to silence Sec1 in murine colon cancer cell lines CT26.WT and CMT93. IBD-related symptoms, colonic immune responses, proliferation and apoptosis of colon epithelial cells were assessed respectively to determine the role of Sec1 in mouse IBD. Impact of Sec1 on the expression of death receptor 5 (DR5) and other apoptosis-associated proteins were determined. Sec1 knockout was found to be associated with deterioration of IBD in mice and elevated immune responses in the colonic mucosa. Silencing Sec1 in CT26.WT and CMT93 cells led to greater secretion of inflammatory cytokines IL-1β, IL-6 and TNF-α. Cell counting kit 8 (CCK8) assay, flow cytometry and TUNEL detection suggested that Sec1 expression promoted the proliferation of colon epithelial cells, inhibited cell apoptosis, reduced cell arrest in G0/G1 phase and facilitated repair of inflammatory injury. Over-expression of DR5 and several apoptosis-related effector proteins was noticed in Sec1−/− mice and Sec1-silenced CT26.WT and CMT93 cells, supporting a suppressive role of Sec1 in cell apoptosis. Our results depicted important regulatory roles of Sec1 in mouse IBD, further reflecting the importance of FUT2 in the pathogenesis of human IBD.

Metabolic support by macrophages sustains colonic epithelial homeostasis

The intestinal epithelium has a high turnover rate and constantly renews itself through proliferation of intestinal crypt cells, which depends on insufficiently characterized signals from the microenvironment. Here, we showed that colonic macrophages were located directly adjacent to epithelial crypt cells in mice, where they metabolically supported epithelial cell proliferation in an mTORC1-dependent manner. Specifically, deletion of tuberous sclerosis complex 2 (Tsc2) in macrophages activated mTORC1 signaling that protected against colitis-induced intestinal damage and induced the synthesis of the polyamines spermidine and spermine. Epithelial cells ingested these polyamines and rewired their cellular metabolism to optimize proliferation and defense. Notably, spermine directly stimulated proliferation of colon epithelial cells and colon organoids. Genetic interference with polyamine production in macrophages altered global polyamine levels in the colon and modified epithelial cell proliferation. Our results suggest that macrophages act as "commensals" that provide metabolic support to promote efficient self-renewal of the colon epithelium.

FEATURES OF PROLIFERATION AND APOPTOSIS OF COLONIC EPITHELIOCYTES AFTER ELECTRON IRRADIATION AT A DOSE OF 8 GY

Intestinal epithelial cells are characterized by high mitotic activity and have high radiosensitivity to all types of ionizing radiation that cause their damage. In modern morphology and radiology, there is no data on the effect of active electrons on the intestinal mucosa, despite the promise and active implementation of this type of radiation for therapeutic purposes in oncology. Purpose of the study: immunohistochemical assessment of proliferation and apoptosis of colon epithelial cells in male Wistar rats after local irradiation with electrons at a dose of 8 Gy. Animals were irradiated using a linear accelerator NOVAC-11, at a dose rate of 1 Gy/min, energy of 10 MeV and frequency of 9 Hz, the diameter of the irradiation field of the pelvic-abdominal region was 100 mm. Animals from each group were removed from the experiment on the first and third days. Histological preparations of the colon (n=30) from two groups, the first control group (n=10) and the second experimental group (n=20), were examined by immunohistochemical method with antibodies to Ki-67 and Cas3. In preparations of rats bred one day after irradiation, a predominance of apoptotic processes was revealed. The number of positive cells stained with antibodies to caspase 3 was increased by 1.5 times in the experimental group compared to the control group, and the number of Ki-67-positive cells, on the contrary, was reduced by 1.5 times. On day 3 after electron irradiation in the epithelium of the colon, the number of immunopositive cells did not differ from control values. Thus, with local single irradiation of the pelvic-abdominal region of rats with electrons at a dose of 8 Gy, a decrease in the number of stem epithelial cells of the intestinal crypts of the colon is observed every other day, which is confirmed by a shift in the proliferative-apoptotic balance towards cell death. In the dynamics of observation on the third day of the experiment, an increase in the pool of proliferating cells is observed, followed by restoration of intestinal crypts.

Hungatella hathewayi impairs the sensitivity of colorectal cancer cells to 5-FU through decreasing CDX2 expression.

Hungatella hathewayi (H. hathewayi), also known as Clostridium hathewayi, has been reported to be accumulated in the colorectal cancer (CRC) samples. In addition, evidence has demonstrated that inoculation with H. hathewayi promotes the proliferation of colonic epithelial cells in mice. Herein, we explored H. hathewayi role in regulating the 5-fluorouracil (5-FU) resistance in CRC cells, and investigated the underlying mechanisms. H. hathewayi abundance in CRC tissues and the corresponding adjacent normal tissues was tested using qRT-PCR. Both parental and 5-FU resistance CRC cell lines were used to assess H. hathewayi role in regulating the 5-FU resistance of CRC cells using CCK-8, flow cytometry and animal experiments. H. hathewayi abundance was significantly increased in CRC tissues, and the high level of H. hathewayi was linked to lower overall survival rate. H. hathewayi treatment significantly weakened 5-FU effects on inhibiting cell growth and inducing cell apoptosis in CRC HCT116 and HT29 cells. In addition, H. hathewayi enhanced the 5-FU resistance of HCT116/5-FU and HT29/5-FU cells (the 5-FU resistance cell lines). In mechanism, H. hathewayi decreased the expression of CDX2, and increased the expression of nuclear accumulation of β-catenin. Overexpression of CDX2 abolished H. hathewayi-mediated enhancement in cell growth and inhibition in cell apoptosis in HCT116/5-FU and HT29/5-FU cells, as well as inhibited the expression and nuclear accumulation of β-catenin. In conclusion, H. hathewayi abundance was increased in CRC tissues, and the high level of H. hathewayi was linked to lower overall survival rate. In mechanisam, H. hathewayi treatment enhanced the 5-FU resistance of CRC cells through modulating CDX2/β-catenin signaling.

The beneficial effects of commensal E. coli for colon epithelial cell recovery are related with Formyl peptide receptor 2 (Fpr2) in epithelial cells

BackgroundFormyl peptide receptor 2 (Fpr2) plays a crucial role in colon homeostasis and microbiota balance. Commensal E. coli is known to promote the regeneration of damaged colon epithelial cells. The aim of the study was to investigate the connection between E. coli and Fpr2 in the recovery of colon epithelial cells.ResultsThe deficiency of Fpr2 was associated with impaired integrity of the colon mucosa and an imbalance of microbiota, characterized by the enrichment of Proteobacteria in the colon. Two serotypes of E. coli, O22:H8 and O91:H21, were identified in the mouse colon through complete genome sequencing. E. coli O22:H8 was found to be prevalent in the gut of mice and exhibited lower virulence compared to O91:H21. Germ-free (GF) mice that were pre-orally inoculated with E. coli O22:H8 showed reduced susceptibility to chemically induced colitis, increased proliferation of epithelial cells, and improved mouse survival. Following infection with E. coli O22:H8, the expression of Fpr2 in colon epithelial cells was upregulated, and the products derived from E. coli O22:H8 induced migration and proliferation of colon epithelial cells through Fpr2. Fpr2 deficiency increased susceptibility to chemically induced colitis, delayed the repair of damaged colon epithelial cells, and heightened inflammatory responses. Additionally, the population of E. coli was observed to increase in the colons of Fpr2−/− mice with colitis.ConclusionCommensal E. coli O22:H8 stimulated the upregulation of Fpr2 expression in colon epithelial cells, and the products from E. coli induced migration and proliferation of colon epithelial cells through Fpr2. Fpr2 deficiency led to an increased E. coli population in the colon and delayed recovery of damaged colon epithelial cells in mice with colitis. Therefore, Fpr2 is essential for the effects of commensal E. coli on colon epithelial cell recovery.

FGF21 Depletion Attenuates Colitis through Intestinal Epithelial IL-22-STAT3 Activation in Mice.

Fibroblast growth factor 21 (FGF21) is a glucose and lipid metabolic regulator. Recent research revealed that FGF21 was also induced by inflammatory stimuli. Its role in inflammatory bowel disease (IBD) has not been investigated. In this study, an experimental IBD model was established in FGF21 knockout (KO) and wild-type (WT) mice by adding 2.5% (wt/vol) dextran sodium sulfate (DSS) to their drinking water for 7 days. The severity of the colitis and the inflammation of the mouse colon tissues were analyzed. In WT mice, acute DSS treatment induced an elevation in plasma FGF21 and a significant loss of body weight in a time-dependent manner. Surprisingly, the loss of body weight and the severity of the colitis induced by DSS treatment in WT mice were significantly attenuated in FGF21 KO mice. Colon and circulating pro-inflammatory factors were significantly lower in the FGF21 KO mice compared to the WT mice. As shown by BrdU staining, the FGF21 KO mice demonstrated increased colonic epithelial cell proliferation. DSS treatment reduced intestinal Paneth cell and goblet cell numbers in the WT mice, and this effect was attenuated in the FGF21 KO mice. Mechanistically, FGF21 deficiency significantly increased the signal transducer and activator of transcription (STAT)-3 activation in intestinal epithelial cells and increased the expression of IL-22. Further study showed that the expression of suppressor of cytokine signaling-2/3 (SOCS 2/3), a known feedback inhibitor of STAT3, was significantly inhibited in the DSS-treated FGF2 KO mice compared to the WT mice. We conclude that FGF21 deficiency attenuated the severity of DSS-induced acute colitis, which is likely mediated by enhancing the activation of the IL-22-STAT3 signaling pathway in intestinal epithelial cells.

Diallyl Trisulfide, A Major Bioactive Constituent Of The Garlic, Promotes Colonic Mucosal Healing In Ulcerative Colitis Through Accelerating Focal Adhesion Assembly And Consequent Epithelial Cell Migration Via The Rab21-Integrin Β1-Fak Pathway.

Colonic mucosal healing is the terminal goal for the treatment of ulcerative colitis (UC), but there is currently no specific drug available. This study investigated the beneficial effect of diallyl trisulfide (DATS) on the colonic mucosal healing. Dextran sulfate sodium (DSS) was used to induce colitis in female C57BL/6 mice, and DATS was orally administered during the recovery period. DATS hardly impacted the inflammation of the colonic tissues, but significantly promoted the mucosal repair. DATS promoted the migration but not proliferation of colonic epithelial cells in the colitis mice. In addition, DATS accelerated the wound healing, cell migration, focal adhesion assembly and phosphorylation of focal adhesion kinase (FAK) of colonic epithelial cells in vitro, which were evidently reversed by combined use of FAK inhibitor PF-573228. Similar results were shown in colitis mice. Mechanically, DATS promoted the binding of Rab21 to integrin β1 and accelerated the endocytosis of integrin β1, which was significantly attenuated by the knockdown of Rab21. DATS promotes the binding of Rab21 to integrin β1 and the endocytosis of integrin β1, thereby increases FAK phosphorylation and focal adhesion assembly, finally accelerates the migration of colonic epithelial cells and mucosal healing. This article is protected by copyright. All rights reserved.

Rat tight junction proteins are disrupted after subchronic exposure to okadaic acid.

Okadaic acid (OA), a lipophilic phycotoxin distributed worldwide, causes diarrheic shellfish poisoning and even leads to tumor formation. Currently, the consumption of contaminated seafood is the most likely cause of chronic OA exposure, but there is a serious lack of relevant data. Here, the Sprague-Dawley rats were exposure to OA by oral administration at 100µg/kg body weight, and the tissues were collected and analyzed to assess the effect of subchronic OA exposure. The results showed that subchronic OA administration disturbed colonic mucosal integrity and induced colitis. The colonic tight junction proteins were disrupted and the cell cycle of colonic epithelial cells was accelerated. It is inferred that disruption of the colonic tight junction proteins might be related to the development of chronic diarrhea by affecting water and ion transport. Moreover, the accelerated proliferation of colonic epithelial cells indicated that subchronic OA exposure might promote the restitution process of gut barrier or induce tumor promoter activity in rat colon.

Dietary Fructooligosaccharide, Xylooligosaccharide and Gum Arabic Have Variable Effects on Cecal and Colonic Microbiota and Epithelial Cell Proliferation in Mice and Rats

Two experiments were conducted to determine if supplementing soluble fiber (fructooligosaccharide, xylooligosaccharide or gum arabic) to a semi-elemental diet would beneficially change cecal and colonic microbiota populations and enhance epithelial cell proliferation. Experiments 1 and 2 used identical dietary regimens; mice and rats were given free access to a powdered semi-elemental diet. Animals were assigned to one of the four following treatment groups: control, no supplemental dietary fiber, fructooligosaccharide, xylooligosaccharide and gum arabic. Dietary fiber was supplied via drinking water at 30 g/L. In Experiment 1 populations of Bifidobacteria and total anaerobic flora were enumerated from the contents of the cecum and colon of weanling mice. Consumption of fructooligosaccharide increased (P < 0.05) the concentrations of Bifidobacteria and the ratio of Bifidobacteria to total anaerobic flora. In Experiment 2 tissue from the cecum and distal colon of weanling rats was examined for morphological changes of the mucosa. Consumption of xylooligosaccharide increased (P < 0.05) cecal crypt depth and labeling index relative to the other three treatments. Consumption of gum arabic and the control diet increased (P < 0.01) cecal proliferation zone. Consumption of xylooligosaccharide and the control diet increased (P < 0.01) cecal cell density (number of cells in a vertical-half of the crypt). Distal colonic crypt depth was greatest (P < 0.05) in controls and rats fed fructooligosaccharide, intermediate in those fed gum arabic, and smallest in those fed xylooligosaccharide. These results suggest that fructooligosaccharide effectively stimulates growth of Bifidobacteria and xylooligosaccharide supports a modest enhancement of cecal epithelial cell proliferation.

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.

Insect peptide CopA3 promotes proliferation and PAX7 and MYOD expression in porcine muscle satellite cells

Insects are a valuable natural source that can produce a variety of bioactive compounds due to their increasing species diversity. CopA3 is an antimicrobial peptide derived from Copris tripartitus (i.e., the dung beetle). It is known to increase the proliferation of colonic epithelial and neuronal stem cells by regulating cell cycle. This research hypothesized that CopA3 can promote the proliferation of porcine muscle satellite cells (MSCs). The effects of CopA3 on porcine MSCs, which are important for muscle growth and regeneration, remain unclear. Here, we investigated the effects of CopA3 on porcine MSCs. According to viability results, we designed four groups: control (without CopA3) and three treatment groups (treated with 5,10, and 25 μg/mL of CopA3). At a CopA3 concentration of 5 μg/mL and 10 μg/mL, the proliferation of MSCs increased more than that observed in the control group. Furthermore, compared to that in the control, CopA3 treatment increased the S phase but decreased the G0/G1 phase ratio. Additionally, early and late apoptotic cells were found to be decreased in the 5 μg/mL group. The expressions of the myogenesis-related transcription factor PAX7 and MYOD proteins were significantly upregulated in the 5 μg/mL and 10 μg/mL groups, whereas the MYOG protein remained undetected in all group. This study suggested that CopA3 promotes muscle cell proliferation by regulating the cell cycle of MSCs and can regulate the activity of MSCs by increasing the expressions of PAX7 and MYOD.

Aspartate Alleviates Colonic Epithelial Damage by Regulating Intestinal Stem Cell Proliferation and Differentiation via Mitochondrial Dynamics.

Proliferation and differentiation of intestinal stem cells (ISCs) are crucial for functional restoration after injury, which can be regulated by nutritional molecules. Aspartate is implicated in maintaining intestinal barrier after injury, but underlying mechanisms remain elusive. Here, this study seeks to investigate if aspartate alleviates colonic epithelial damage by regulating ISC function, and to elucidate its mechanisms. Eight-week-old male C57BL/6 mice supplement with or without 1% L-aspartate are subjected to drinking water or 2.5% DSS to induce colitis. In this study, aspartate administration alleviates the severity of colitis, as indicated by reduced body weight loss, colon shortening, and inhibited pro-inflammatory cytokine expression in DSS-challenged mice. Additionally, aspartate promotes colonic epithelial cell proliferation and differentiation after DSS-induced damage in mice. Pretreatment with aspartate not only enhances ISC proliferation but also induces ISC differentiation toward enterocytes and goblet cells, which prevent TNF-α-induced colonoid damage. Mechanistically, aspartate ameliorates DSS/TNF-α-induced perturbation of mitochondrial metabolism and maintains mitochondrial dynamics in colonic epithelium and colonoids. Moreover, aspartate-mediated ISC proliferation and differentiation are primarily dependent on mitochondrial fusion rather than fission. The findings indicate that aspartate promotes ISC proliferation and differentiation to alleviate colonic epithelial damage by regulation of mitochondrial metabolism and dynamics.

Inhibition of GABAAR or Application of Lactobacillus casei Zhang Alleviates Ulcerative Colitis in Mice: GABAAR as a Potential Target for Intestinal Epithelial Renewal and Repair.

Emerging evidence indicates that the gamma−aminobutyric acid type A receptor (GABAAR) and Lactobacillus casei Zhang regulate colitis in a variety of ways, such as by participating in host immune and inflammatory responses, altering the gut microbiota, and influencing intestinal barrier function. However, not much is known about the mechanisms by which GABAAR and L. casei affect colon epithelial cell renewal and the interaction between GABAAR and L. casei during this process. To elucidate this, we established a dextran sulfate sodium (DSS)−induced model and measured the mouse body weights, colon length, the disease activity index (DAI), and histological scores. Our results indicated that inhibition of GABAAR alleviated the DSS−induced colitis symptoms, resulting in less weight loss and more intact colon tissue. Moreover, treatment with bicuculline (Bic, a GABAAR inhibitor) increased the levels of PCNA, β−catenin, and TCF4 in mice with colitis. Interestingly, open field test performances showed that inhibition of GABAAR also attenuated colitis−related anxiety−like behavior. By 16S RNA gene sequencing analysis, we showed that inhibition of GABAAR partially reversed the gut dysbacteriosis of DSS−induced mice and increased the abundance of beneficial bacteria. Additionally, L. casei Zhang supplementation inhibited the expression of GABAAR in mice with colitis, promoted the proliferation and renewal of colon epithelial cells, and alleviated anxiety−like behavior and intestinal microflora disorder in mice. Thus, GABAAR plays a key role in the beneficial effects of L. casei on DSS−induced colitis in mice.

YAP Activates STAT3 Signalling to Promote Colonic Epithelial Cell Proliferation in DSS-Induced Colitis and Colitis Associated Cancer.

Background and AimsYes-associated protein (YAP) is a key transcriptional coactivator of cell proliferation and differentiation. In this study, we sought to identify the roles of YAP in colonic epithelial regeneration and tumourigenesis.MethodsMurine DSS-induced colitis and YAP overexpression models were constructed via lentiviral intraperitoneal injection. Stable YAP-overexpressing cells, protein immunoprecipitation, and ChIP were used to deeply explore the molecular mechanism.ResultsWe found that the expression of YAP was dramatically diminished in the colonic crypts during the acute colitis phase, while YAP was strikingly enhanced to initiate tissue repair after DSS withdrawal. Overexpressing YAP in mice drastically accelerated epithelial regeneration, presenting with more intact structural integrity and reduced inflammatory cell infiltration in the mucosa. Further mechanistic studies showed that the expression of YAP in the nucleus was significantly increased by 2 h post-DSS removal, accompanied by upregulated protein levels of activated STAT3. Overexpression of YAP (YAPWT) elevated the expression of activated STAT3 and its transcriptional targets and strengthened the proliferation and “wound healing” ability of colonic cells. However, these effects were reversed when STAT3 was silenced in YAPWT cells. Moreover, YAP could directly interact with STAT3 in the nucleus, and c-Myc and CyclinD1 were the transcriptional targets. Finally, during colitis-associated cancer (CAC), YAPWT promoted the progression of CAC, while the phosphomimetic YAP downregulated the expression of STAT3 and inhibited the development and progression of CAC.ConclusionYAP activates STAT3 signalling to facilitate mucosal regeneration after DSS-induced colitis. However, excessive YAP activation in the colonic epithelium promotes CAC development.

Bioinformatics Profiling and Experimental Validation of 4 Differentially-Expressed LIM Genes in the Course of Colorectal-Adenoma-Carcinoma.

BackgroundLIM domain proteins play crucial roles in tumors by interacting with diverse proteins. However, their roles in the course of colorectal mucosa-adenoma-carcinoma remain unclear. This study aimed to depict their dynamic expression profiles and elucidate their potential functions in this transition course.Material/MethodsDifferentially-expressed LIM proteins (DELGs) in paired adenomas, carcinomas, and mucosae were identified using the GEO dataset (GSE 117606) and validated by immunohistochemistry using our tissue microarray. Kaplan-Meier survival analysis, WGCNA, module-trait analysis, and KEGG enrichment were conducted. The correlation of DELGs expression levels with immune infiltration was assessed using the ESTIMATE package and TISCH database. The role of DELGs of interest was validated using cell proliferation, migration, and invasion assays.ResultsFour DELGs were identified – LMO3, FHL1, NEBL, and TGFB1I1 – all of which were of significance in prognosis. Module-trait correlation and KEGG enrichment revealed their involvement in cancer-related signaling. Immunohistochemistry showed gradual downregulation of LMO3 but upregulation of NEBL in the mucosa-adenoma-carcinoma sequence. The opposite expression patterns were observed for FHL1 and TGFB1I1 in tumor epithelium and mesenchyme. High expression levels of the DELGs were correlated with increased infiltration of NK, NKT, and macrophages, except for NEBL. Importantly, LMO3 inhibited proliferation, migration, and invasion of colon epithelial cells.ConclusionsThis study identified 4 differentially-expressed LIM genes – LMO3, FHL1, TGFB1I1, and NEBL – and revealed they were involved in the mucosa-adenoma-carcinoma sequence via regulating cancer-related pathways, influencing epigenetic field, or affecting immune infiltration. Our findings provide new insights into the roles of LIM proteins in the course of mucosa-adenoma-carcinoma.

Interferon Regulatory Factor 4 (IRF4) Promotes Lipopolysaccharide-Induced Colonic Mucosal Epithelial Cell Proliferation by Regulating Macrophage Polarization

Introduction: Ulcerative colitis (UC) is a chronic disease characterized by diffuse inflammation of the mucosa of colon and rectum. Interferon regulatory factor 4 (IRF4) mediates macrophage anti-inflammatory phenotype (alternatively activated macrophages [M2]). This study aimed to investigate the mechanism of IRF4 in lipopolysaccharide (LPS)-induced colonic mucosal epithelial cell proliferation via the regulation of macrophage polarization. Methods: Human bone marrow-derived macrophages were subjected to interleukin 4 (IL-4) induction. M2 macrophages were identified using flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR). IRF4 expression in M2 macrophages was detected using Western blot and qRT-PCR. IRF4 expression was silenced in M2 macrophages. IL-10 mRNA expression and protein level were detected using qRT-PCR and Western blot. The binding relation between IRF4 and IL-10 was verified using dual-luciferase and chromatin immunoprecipitation assays. Macrophages under different treatments were cocultured with LPS-induced human colonic mucosal epithelial cells. The levels of inflammatory factors (TNF-α, IL-6, and IL-1β) were detected using enzyme-linked immunosorbent assay. The proliferation of inflammatory cells was measured using Cell Counting Kit-8 assay, and the healing of inflammatory cells was detected using wound healing assay. Results: M2 macrophages alleviated LPS-induced inflammatory responses. IRF4 bound to IL-10 and promoted IL-10 expression. Inhibition of IRF4 reduced IL-10 expression and attenuated the alleviating effect of M2 macrophages on inflammatory responses. Inhibition of IRF4 combined with IL-10 overexpression enhanced the promoting effect of M2 macrophages on inflammatory healing. Conclusion: IRF4 promoted colonic mucosal epithelial cell proliferation by increasing IL-10 expression and regulating macrophage polarization to M2 phenotype, which might be related to UC mucosal healing.

Hederacoside C ameliorates colitis via restoring impaired intestinal barrier through moderating S100A9/MAPK and neutrophil recruitment inactivation.

Hederacoside C (HSC) has attracted much attention as a novel modulator of inflammation, but its anti-inflammatory mechanism remains elusive. In the present study, we investigated how HSC attenuated intestinal inflammation in vivo and in vitro. HSC injection significantly alleviated TNBS-induced colitis by inhibiting pro-inflammatory cytokine production and colonic epithelial cell apoptosis, and partially restored colonic epithelial cell proliferation. The therapeutic effect of HSC injection was comparable to that of oral administration of mesalazine (200 mg·kg−1·d−1, i.g.). In LPS-stimulated human intestinal epithelial Caco-2 cells, pretreatment with HSC (0.1, 1, 10 μM) significantly inhibited activation of MAPK/NF-κB and its downstream signaling pathways. Pretreatment with HSC prevented LPS-induced TLR4 dimerization and MyD88 recruitment in vitro. Quantitative proteomic analysis revealed that HSC injection regulated 18 proteins in the colon samples, mainly clustered in neutrophil degranulation. Among them, S100A9 involved in the degranulation of neutrophils was one of the most significantly down-regulated proteins. HSC suppressed the expression of S100A9 and its downstream genes including TLR4, MAPK, and NF-κB axes in colon. In Caco-2 cells, recombinant S100A9 protein activated the MAPK/NF-κB signaling pathway and induced inflammation, which were ameliorated by pretreatment with HSC. Notably, HSC attenuated neutrophil recruitment and degranulation as well as S100A9 release in vitro and in vivo. In addition, HSC promoted the expression of tight junction proteins and repaired the epithelial barrier via inhibiting S100A9. Our results verify that HSC ameliorates colitis via restoring impaired intestinal barrier through moderating S100A9/MAPK and neutrophil recruitment inactivation, suggesting that HSC is a promising therapeutic candidate for colitis.

Functional Comparison of Clostridium butyricum and Sodium Butyrate Supplementation on Growth, Intestinal Health, and the Anti-inflammatory Response of Broilers.

Butyrate has been reported to promote proliferation of colonic epithelial cells and maintain intestinal barrier integrity in broilers. Although supplementation of Clostridium butyricum and sodium butyrate have been shown to confer benefits on broilers, their effects and mechanisms have not been compared. In this study, C. butyricum and sodium butyrate were added into the basal diet of broilers and their effects on growth performance, intestinal health, and anti-inflammatory response were analyzed. It was found that both C. butyricum and sodium butyrate showed good probiotic effects on broilers. Their effects on growth rate and expression of inflammation related genes were superior to that of the antibiotic oxytetracycline. Besides, the two dietary supplements improved intestinal structure integrity and secretion of inflammatory cytokines, whereas the antibiotic had negative effects. Comparison of the two supplements revealed that sodium butyrate more effectively improved the growth and intestinal structure of broilers than C. butyricum. On the contrary, C. butyricum was superior to sodium butyrate in promoting tight junction protein expression and anti-inflammatory response. In summary, this study demonstrates the positive effects of C. butyricum and sodium butyrate on broilers, and will serve as a reference for selection of appropriate butyrate supplementation for broilers in the breeding industry.

Endogenous NRG4 promotes ST3GAL4 expression, leukocyte infiltration, and inflammation in IL‐10R neutralization colitis

BackgroundThe ErbB family of receptor tyrosine kinases are essential for the regulation of colonic epithelial cell proliferation, differentiation, survival, and wound healing. We previously showed that ErbB4 is induced as a compensatory response by intestinal inflammation; treatment with exogenous NRG4 (an ErbB4 ligand) protects colonocytes against cytokine‐induced apoptosis and ameliorates murine experimental colitis. Exogenous NRG4 can further alleviate intestinal inflammation by suppressing the activity of pro‐inflammatory macrophages. However, the role of endogenous NRG4 in the intestine has not been described.HypothesisBased on our previous studies, we hypothesized that NRG4 deletion would exacerbate intestinal inflammation.MethodsColitis was induced in 5‐week‐old wild‐type (WT) and NRG4 knockout (KO) mice by i.p. injection with 1 mg/kg IL‐10R neutralizing antibody, weekly for four weeks. Inflammation was assessed by fecal lipocalin‐2 ELISA, plasma myeloperoxidase ELISA, and total spleen weight. Disease was assessed by histological scoring of paraffin embedded colon rolls. Cytokine (TNF, IL‐1b, IFNg, and IL‐6) expression was determined by qPCR on RNA from full thickness distal colon homogenates. ST3GAL4 expression was assessed by qPCR and immunohistochemistry. Intestinal immune cell populations were identified and analyzed by mass cytometry (CyTof) using FlowJo software.ResultsFecal lipocalin expression was elevated in all mice after IL‐10R antibody treatment. Surprisingly, WT mice displayed significantly more fecal lipocalin than NRG4 KO cage mates. Similarly, after 4 weeks, WT mice had significantly higher plasma myeloperoxidase (2‐fold, p&lt;0.001), increased spleen weight (57% more, p&lt;0.01), and more histological injury and inflammation (72% higher score, p&lt;0.05) versus their KO cage mates. Furthermore, mRNA levels for IFNg (3.7‐fold difference, p&lt;0.01) and IL‐6 (1.6‐fold difference, p&lt;0.05) were significantly higher in WT versus KO cage mates. CyTof analysis identified a reduction in CD8+ T cell populations, as well as alterations in overall T cell populations. Expression of ST3GAL4, a sialyltransferase that contributes to inflammatory leukocyte recruitment, was significantly reduced in NRG4‐null colons and epithelia (&gt;90% reduction, p&lt;0.01 and p&lt;0.001 respectively), suggesting a mechanism for altered T cell populations.ConclusionIn contrast to the protective effects of exogenous NRG4, endogenous NRG4 appears to participate in recruitment of CD8+ T cells and intestinal inflammation, possibly through regulation of ST3GAL4.