Intestinal Epithelial Cell Migration Research Articles

MiR-329b-5p Affects Sheep Intestinal Epithelial Cells against Escherichia coli F17 Infection.

Diarrhea is the most common issue in sheep farms, typically due to pathogenic Escherichia coli (E. coli) infections, such as E. coli F17. microRNA, a primary type of non-coding RNA, has been shown to be involved in diarrhea caused by pathogenic E. coli. To elucidate the profound mechanisms of miRNA in E. coli F17 infections, methods such as E. coli F17 adhesion assay, colony counting assay, relative quantification of bacterial E. coli fimbriae gene expression, indirect immune fluorescence (IF), Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), Western blotting (WB), and scratch assay were conducted to investigate the effect of miR-329b-5p overexpression/knock-down on E. coli F17 susceptibility of sheep intestinal epithelial cells (IECs). The findings indicated that miR-329b-5p enhances the E. coli F17 resistance of sheep IECs to E.coli F17 by promoting adhesion between E. coli F17 and IEC, as well as IEC proliferation and migration. In summary, miR-329b-5p plays a crucial role in the defense of sheep IECs against E. coli F17 infection, providing valuable insights into its mechanism of action.

Fatty Acid Oxidation Promotes Apoptotic Resistance and Proinflammatory Phenotype of CD4+ Tissue-resident Memory T cells in Crohn’s Disease

Background and aimsAs the most abundant memory T cells and major source of tumor necrosis factor α (TNF-α) in the intestinal mucosa of Crohn’s disease (CD) patients, CD4+ tissue-resident memory T (TRM) cells play a critical role in CD pathogenesis. We investigated the role of metabolic reprogramming in the regulation of proinflammatory and apoptosis-resistant phenotype for CD4+ TRM cells. MethodsCD4+ TRM cells were collected from intestinal resection tissues from control and CD patients. Transcriptomic and metabolomic analysis were performed to identify metabolic characteristics of CD4+ TRM cells. ELISA and qPCR experiments were utilized to assess cytokines level in CD4+ TRM cells, activation-induced cell apoptosis (AICD) rate was evaluated by flow cytometry. Transwell assay and wound healing assay were performed to detect the effect of CD4+ TRM cells on the migration of normal intestinal epithelial cells. ResultsTranscriptomic data combined with unbiased metabolomic analysis revealed an increased fatty acid oxidation (FAO) phenotype existed in CD4+ TRM cells from CD patients. The lipidomic data and stable isotope tracer experiments demonstrated that CD4+ TRM cells upregulated their lipid lipolysis and fatty acid uptake to fuel FAO in CD patients. Mechanistically, the activated NF-κB signaling increased transcription of genes involved in lipid lipolysis, fatty acid uptake and oxidation in CD4+ TRM cells from CD patients. Targeting FAO of CD4+ TRM cells reversed their apoptosis-resistant and proinflammatory phenotype in CD Patients. ConclusionCD4+ TRM cells process an accelerated FAO mediated by activated NF-κB signaling in CD patients, targeting FAO could reverse their apoptosis-resistant and proinflammatory phenotype. These findings shed a new light on the pathogenic mechanism investigation and novel therapy development in CD patients.

Luminol-conjugated cyclodextrin biological nanoparticles for the treatment of severe burn-induced intestinal barrier disruption.

The breakdown of intestinal barrier integrity occurs after severe burn injury and is responsible for the subsequent reactions of inflammation and oxidative stress. A new protective strategy for the intestinal barrier is urgently needed due to the limitations of the traditional methods. Recently, the application of nanoparticles has become one of the promising therapies for many inflammation-related diseases or oxidative damage. Herein, we developed a new anti-inflammatory and antioxidant nanoparticle named luminol-conjugated cyclodextrin (LCD) and aimed to evaluate its protective effects in severe burn-induced intestinal injury. First, LCD nanoparticles, engineered with covalent conjugation between luminol and β-cyclodextrin (β-CD), were synthesized and examined. Then a mouse burn model was successfully established before the mouse body weight, intestinal histopathological manifestation, permeability, tight junction (TJ) expression and pro-inflammatory cytokines were determined in different groups. The proliferation, apoptosis, migration and reactive oxygen species (ROS) of intestinal epithelial cells (IECs) were assessed. Intraepithelial lymphocytes (IELs) were isolated and cultured for analysis by flow cytometry. LCD nanoparticle treatment significantly relieved the symptoms of burn-induced intestinal injury in the mouse model, including body weight loss and intestinal permeability abnormalities. Moreover, LCD nanoparticles remarkably recovered the mechanical barrier of the intestine after severe burn, renewed TJ structures, promoted IEC proliferation and migration, and inhibited IEC apoptosis. Mechanistically, LCD nanoparticles dramatically alleviated pro-inflammation factors (tumor necrosis factor-α, IL-17A) and ROS accumulation, which could be highly involved in intestinal barrier disruption. Furthermore, an increase in IL-17A and the proportion of IL-17A+Vγ4+ γδ T subtype cells was also observed in vitro in LPS-treated Vγ4+ γδ T cells, but the use of LCD nanoparticles suppressed this increase. Taken together, these findings demonstrate that LCD nanoparticles have the protective ability to ameliorate intestinal barrier disruption and provide a therapeutic intervention for burn-induced intestinal injury.

An Engineered Butyrate-Derived Polymer Nanoplatform as a Mucosa-Healing Enhancer Potentiates the Therapeutic Effect of Magnolol in Inflammatory Bowel Disease.

Colonic epithelial damage and dysregulated immune response are crucial factors in the progression and exacerbation of inflammatory bowel disease (IBD). Nanoenabled targeted drug delivery to the inflamed intestinal mucosa has shown promise in inducing and maintaining colitis remission, while minimizing side effects. Inspired by the excellent antioxidative and anti-inflammatory efficacy of naturally derived magnolol (Mag) and gut homeostasis regulation of microbiota-derived butyrate, we developed a pH/redox dual-responsive butyrate-rich polymer nanoparticle (PSBA) as an oral Mag delivery system for combinational therapy of IBD. PSBA showed a high butyrate content of 22% and effectively encapsulated Mag. The Mag-loaded nanoparticles (PSBA@Mag) demonstrated colonic pH and reduction-responsive drug release, ensuring efficient retention and adhesion in the colon of colitis mice. PSBA@Mag not only normalized the level of reactive oxygen species and inflammatory effectors in inflamed colonic mucosa but also restored the epithelial barrier function in both ulcerative colitis and Crohn's disease mouse models. Importantly, PSBA promoted the migration and healing ability of intestinal epithelial cells in vitro and in vivo, sensitizing the therapeutic efficacy of Mag in animal models. Moreover, transcriptomics and metabolism analyses revealed that PSBA@Mag mitigated inflammation by suppressing the production of pro-inflammatory cytokines and chemokines and restoring the lipid metabolism. Additionally, this nanomedicine modulated the gut microbiota by inhibiting pathogenic Proteus and Escherichia-Shigella and promoting the proliferation of beneficial probiotics, including Lachnoclostridium, Lachnospiraceae_NK4A136_group and norank_f_Ruminococcaceae. Overall, our findings highlight the potential of butyrate-functionalized polymethacrylates as versatile and effective nanoplatforms for colonic drug delivery and mucosa repair in combating IBD and other gastrointestinal disorders.

The casein-derived peptide YFYPEL alleviates intestinal epithelial cell dysfunction associated with NEC by regulating the PI3K/AKT signaling pathway.

Necrotizing enterocolitis (NEC) is a life-threatening risk to the health of neonates, but thus far, there is no very effective treatment. Although many studies have confirmed the therapeutic role of peptides in diseases, the effect of peptides in NEC remains poorly understood. This study investigated the role of casein-derived peptide YFYPEL in NEC cells and animal models. We synthesized YFYPEL and analysed its protective effects on NEC both in vitro and in vivo. YFYPEL integration in the intestine increased rat survival and clinical conditions, lowered the incidence of NEC, alleviated bowel inflammation, and enhanced intestinal cell migration. Furthermore, YFYPEL significantly decreased interleukin 6 expression and increased intestinal epithelial cell migration. Moreover, YFYPEL alleviated intestinal epithelial cell dysfunction through the PI3K/AKT pathway, as demonstrated by western blotting and bioinformatics analysis. A selective PI3K activator reversed the protective effect of YFYPEL on lipopolysaccharide-stimulated intestinal epithelial cells. Our study showed that YFYPEL reduced inflammatory cytokine expression and enhanced migration by regulating the PI3K/AKT pathway. The use of YFYPEL may thus develop into a novel modality in NEC treatment.

Comparison and Investigation of Exosomes from Human Amniotic Fluid Stem Cells and Human Breast Milk in Alleviating Neonatal Necrotizing Enterocolitis

In view of the devastating impact of neonatal necrotizing enterocolitis (NEC) on newborns, the research on its intervention is particularly important. Although exosomes from human amniotic fluid stem cells (AFSC) and human breast milk (HBM) can protect against NEC, their mechanisms remain unclear. Here, we intend to compare the intervention effects of two types of exosomes on NEC mouse model and reveal their respective regulatory mechanisms. In general, both AFSC-derived exosomes (AFSC-exos) and HBM-derived exosomes (HBM- exos) can alleviate NEC- associated intestinal injury, significantly reduce NEC score, and reduce systemic and ileal inflammation and NEC related brain injury during experimental NEC. However, the mode and mechanism of action of the two sources of exosomes were not identical. In vivo, the number of ileal crypts was more significantly restored after HBM-exos intervention than AFSC-exos, and in vitro, HBM-exos preferentially inhibited the inflammatory response of intestinal epithelial cells (IECs), whereas AFSC-exos preferentially regulated the migration of IECs. Mechanistically, GO and KEGG analyses revealed the different therapeutic mechanisms of AFSC-exos and HBM-exos in NEC. Taken together, our results illustrate that AFSC-exos and HBM-exos reduce the severity of experimental NEC and intestinal damage through different mechanisms, supporting the potential of cell-free or breast milk free exosome therapy for NEC.Graphical

Epithelial JAM-A is fundamental for intestinal wound repair in vivo.

Junctional adhesion molecule-A (JAM-A) is expressed in several cell types, including epithelial and endothelial cells, as well as some leukocytes. In intestinal epithelial cells (IEC), JAM-A localizes to cell junctions and plays a role in regulating barrier function. In vitro studies with model cell lines have shown that JAM-A contributes to IEC migration; however, in vivo studies investigating the role of JAM-A in cell migration–dependent processes such as mucosal wound repair have not been performed. In this study, we developed an inducible intestinal epithelial–specific JAM-A–knockdown mouse model (Jam-aERΔIEC). While acute induction of IEC-specific loss of JAM-A did not result in spontaneous colitis, such mice had significantly impaired mucosal healing after chemically induced colitis and after biopsy colonic wounding. In vitro primary cultures of JAM-A–deficient IEC demonstrated impaired migration in wound healing assays. Mechanistic studies revealed that JAM-A stabilizes formation of protein signaling complexes containing Rap1A/Talin/β1 integrin at focal adhesions of migrating IECs. Loss of JAM-A in primary IEC led to decreased Rap1A activity and protein levels of Talin and β1 integrin, and it led to a reduction in focal adhesion structures. These findings suggest that epithelial JAM-A plays a critical role in controlling mucosal repair in vivo through dynamic regulation of focal adhesions.

ZINC40099027 promotes monolayer circular defect closure by a novel pathway involving cytosolic activation of focal adhesion kinase and downstream paxillin and ERK1/2.

ZINC40099027 (ZN27) is a specific focal adhesion kinase (FAK) activator that promotes murine mucosal wound closure after ischemic or NSAID-induced injury. Diverse motogenic pathways involve FAK, but the direct consequences of pure FAK activation have not been studied, and how ZN27-induced FAK activation stimulates wound closure remained unclear. We investigated signaling and focal adhesion (FA) turnover after FAK activation by ZN27 in Caco-2 cells, confirming key results in CCD841 cells. ZN27 increased Caco-2 FAK-Y-397, FAK-Y-576/7, paxillin-Y-118, and ERK 1/2 phosphorylation and decreased FAK-Y-925 phosphorylation, without altering FAK-Y-861, p38, Jnk, or Akt phosphorylation. ZN27 increased FAK-paxillin interaction while decreasing FAK-Grb2 association. ZN27 increased membrane-associated FAK-Y-397 and FAK-Y-576/7 phosphorylation and paxillin-Y-118 and ERK 1/2 phosphorylation but decreased FAK-Y-925 phosphorylation without altering Src or Grb2. Moreover, ZN27 increased the fluorescence intensity of GFP-FAK and pFAK-Y397 in FAs and increased the total number of FAs but reduced their size in GFP-FAK-transfected Caco-2 cells, consistent with increased FA turnover. In contrast, FAK-Y397F transfection prevented ZN27 effects on FAK size and number and FAK and pFAK fluorescent intensity in FAs. We confirmed the proposed FAK/paxillin/ERK pathway using PP2 and U0126 to block Src and MEK1/2 in Caco-2 and CCD841 cells. These results suggest that ZN27 promotes intestinal epithelial monolayer defect closure by stimulating autophosphorylation of FAK in the cytosol, distinct from classical models of FAK activation in the FA. Phosphorylated FAK translocates to the membrane, where its downstream substrates paxillin and ERK are phosphorylated, leading to FA turnover and human intestinal epithelial cell migration.

Effects of Spermidine on Cell Proliferation, Migration, and Inflammatory Response in Porcine Enterocytes.

Polyamines have been demonstrated to be beneficial to porcine intestinal development. Our previous study showed that putrescine mitigates intestinal atrophy in weanling piglets and suppresses inflammatory response in porcine intestinal epithelial cells, it is still unknown the role of spermidine in mediating putrescine function. The current study aimed to investigate the effect of spermidine on the proliferation, migration, and inflammatory response in porcine intestinal epithelial cells (IPEC-J2 cell line). The effects of spermidine on proliferation and migration of IPEC-J2 cells were measured. Difluoromethyl ornithine (DFMO) and diethylglyoxal bis (guanylhydrazone) (DEGBG) were used to block the production of putrescine and spermidine, respectively. A cell inflammation model was established with lipopolysaccharides (LPS) stimulation. Gene expression and protein abundance were determined by real-time quantitative PCR and western blotting, respectively. Spermidine significantly enhanced cell proliferation in DFMO (or/and) DEGBG treated IPEC-J2 cells (p < 0.05). Pretreatment with putrescine restored cell growth inhibited by DFMO but did not prevent the decrease in cell proliferation caused by DEGBG (p > 0.05). Similarly, spermidine but not putrescine significantly elevated the rate of migration in DEGBG treated IPEC-J2 cells (p < 0.05). Spermidine deprivation by DEGBG dramatically enhanced mRNA abundance of pro-inflammatory cytokines IL-8, IL-6, and TNF-α (p < 0.05), and the addition of spermidine attenuated excessive expression of those inflammatory pro-inflammatory cytokines, moreover, spermidine but not putrescine suppressed the phosphorylation of NF-κB induced by DEGBG. Spermidine supplementation also significantly suppressed LPS-induced the expression of TNF-α. The present study highlights a novel insight that putrescine may be converted into spermidine to modulate cell proliferation, migration, and inflammatory response on porcine enterocytes.

Polysaccharides from Panax ginseng promote intestinal epithelial cell migration through affecting the Ca2+ related regulators

Background and aimPanax ginseng, a key herbal medicine of replenishing Qi and tonifying Spleen, is widely used in the treatment of gastrointestinal diseases in East Asia. In this study, we aim to investigate the potential effects and mechanisms of polysaccharides from P. ginseng (PGP) on intestinal mucosal restitution which is one of the crucial repair modalities during the recovery of mucosal injury controlled by the Ca2+ signaling. MethodsRat model of intestinal mucosal injury was induced by indomethacin. The fractional cell migration was carried out by immunohistochemistry staining with BrdU. The morphological observations on intestinal mucosal injury were also performed. Intestinal epithelial cell (IEC-6) migration in vitro was conducted by scratch method. Western-blot was adopted to determine the expressions of PLC-γ1, Rac1, TRPC1, RhoA and Cav-1. Immunoprecipitation was used to evaluate the levels of Rac1/PLC-γ1, RhoA/TRPC1 and Cav-1/TRPC1. ResultsThe results showed that PGP effectively reduced the assessment of intestinal mucosal injury, reversed the inhibition of epithelial cell migration induced by Indomethacin, and increased the level of Ca2+ in intestinal mucosa in vivo. Moreover, PGP dramatically promoted IEC-6 cell migration, the expression of Ca2+ regulators (PLC-γ1, Rac1, TRPC1, Cav-1 and RhoA) as well as protein complexes (Rac1/PLC-γ1, Cav-1/TRPC1 and RhoA/TRPC1) in vitro. ConclusionPGP increases the Ca2+ content in intestinal mucosa partly through controlling the regulators of Ca2+ mobilization, subsequently promotes intestinal epithelial cell migration, and then prevents intestinal mucosal injury induced by indomethacin.

Myosin chaperone, UNC‐45A, is a novel regulator of intestinal epithelial barrier integrity and repair

The intestinal epithelium creates a protective barrier separating the luminal content and the immune system in the gut. Leakiness of the intestinal epithelial barrier and inefficient healing of mucosal wounds represent common manifestations of inflammatory bowel diseases (IBD). Integrity of the gut barrier is mediated by different adhesive structures, including adherens junctions (AJ), tight junctions (TJ) and focal adhesion (FA). Functions of these adhesive structures depend on their association with the cortical actin cytoskeleton and particularly, to a major actin motor protein, non‐muscle myosin II (NM‐II). While biochemical mechanisms that regulate activity of NM‐II in epithelial cells have been extensively investigated, little is known about assembly of the contractile myosin structures at the epithelial adhesion sites. In this study, we focused on UNC‐45A, a cytoskeletal chaperone that is essential for proper folding of NM‐II heavy chains and myofilament assembly. We found abundant expression of UNC‐45A in human intestinal epithelial cell (IEC) lines and in the epithelial layer of the normal human colon. Interestingly, protein levels of UNC‐45A were decreased in colonic epithelium of patients with ulcerative colitis in parallel to diminished expression of NM‐IIA and NM‐IIC isoforms. CRISPR/Cas9‐mediated knock‐out of UNC‐45A in HT‐29 and SK‐CO15 IEC disrupted epithelial barrier integrity, impaired AJ and TJ assembly, and cell migration. Consistently, decreased UNC‐45 expression increased permeability of the Drosophila gut in vivo. The mechanisms underlying barrier disruptive and anti‐migratory effects of UNC‐45A depletion involved disorganization of the actomyosin bundles at epithelial junctions and the migrating cell edge. Loss of UNC‐45A also decreased contractile forces at epithelial junctions and matrix adhesions. Expression of deletion mutants revealed roles for the myosin binding domain of UNC‐45A in controlling IEC barrier function, junctional assembly and migration. Our findings uncover a novel mechanism that regulates integrity and restitution of the intestinal epithelial barrier, which may be impaired during mucosal inflammation.

P-Cadherin Regulates Intestinal Epithelial Cell Migration and Mucosal Repair, but Is Dispensable for Colitis Associated Colon Cancer.

Recurrent chronic mucosal inflammation, a characteristic of inflammatory bowel diseases (IBD), perturbs the intestinal epithelial homeostasis resulting in formation of mucosal wounds and, in most severe cases, leads to colitis-associated colon cancer (CAC). The altered structure of epithelial cell-cell adhesions is a hallmark of intestinal inflammation contributing to epithelial injury, repair, and tumorigenesis. P-cadherin is an important adhesion protein, poorly expressed in normal intestinal epithelial cells (IEC) but upregulated in inflamed and injured mucosa. The goal of this study was to investigate the roles of P-cadherin in regulating intestinal inflammation and CAC. P-cadherin expression was markedly induced in the colonic epithelium of human IBD patients and CAC tissues. The roles of P-cadherin were investigated in P-cadherin null mice using dextran sulfate sodium (DSS)-induced colitis and an azoxymethane (AOM)/DSS induced CAC. Although P-cadherin knockout did not affect the severity of acute DSS colitis, P-cadherin null mice exhibited faster recovery after colitis. No significant differences in the number of colonic tumors were observed in P-cadherin null and control mice. Consistently, the CRISPR/Cas9-mediated knockout of P-cadherin in human IEC accelerated epithelial wound healing without affecting cell proliferation. The accelerated migration of P-cadherin depleted IEC was driven by activation of Src kinases, Rac1 GTPase and myosin II motors and was accompanied by transcriptional reprogramming of the cells. Our findings highlight P-cadherin as a negative regulator of IEC motility in vitro and mucosal repair in vivo. In contrast, this protein is dispensable for IEC proliferation and CAC development.

Loss of DRO1/CCDC80 in the tumor microenvironment promotes carcinogenesis.

Tumors are composed of the tumor cells and the surrounding microenvironment. Both are closely interwoven and interact by a complex and multifaceted cross-talk which plays an integral part in tumor initiation, growth, and progression. Dro1/Ccdc80 has been shown to be a potent suppressor of colorectal cancer and ubiquitous inactivation of Dro1/Ccdc80 strongly promoted colorectal carcinogenesis in ApcMin/+ mice and in a chemically-induced colorectal cancer model.The aim of the present study was to investigate whether Dro1/Ccdc80’s tumor suppressive function is tumor-cell-autonomous. Expression of Dro1/Ccdc80 in cancer cells had no effect on both colon tumor development in ApcMin/+ mice and formation of xenograft tumors. In contrast, DRO1/CCDC80 loss in the microenvironment strongly increased tumor growth in xenograft models, inhibited cancer cell apoptosis, and promoted intestinal epithelial cell migration. Moreover, stromal Dro1/Ccdc80 inactivation facilitated formation of intestinal epithelial organoids. Expression analyses showed Dro1/Ccdc80 to be significantly down-regulated in murine gastric cancer associated fibroblasts, in ApcMin/+ colon tumor primary stromal cells and in microdissected stroma from human colorectal cancer compared to normal, non-tumor stroma. Our results demonstrate epithelial derived DRO1/CCDC80 to be dispensable for intestinal tissue homeostasis and identify Dro1/Ccdc80 as tumor suppressor in the tumor microenvironment.

NIPEP-IBD; A SYNTHETIC PEPTIDE TARGETING NOVEL MOLECULE, INTEGRIN BETA 1, TO RESTITUTE INTESTINAL EPITHELIAL CELLS FOR INFLAMMATORY BOWEL DISEASE (IBD) TREATMENT

Abstract Under Inflammatory bowel disease (IBD), impaired intestinal barrier from lumen is highly associated with ongoing IBD symptoms and it also contributes as a precursor for IBD. Current IBD therapeutics lack on mucosal regeneration, which the unmet needs could be the next first in class therapeutics for IBD. Here, we propose NIPEP-IBD, a synthetically modified peptide that promotes adhesion, migration, and differentiation of intestinal epithelial cells via interaction of integrin beta 1. From NIBEC’s specialized peptide discovery platform screening, specifically sequenced peptide has been selected for regenerative functional peptide, termed NIPEP-IBD. NIPEP-IBD selectively targeted and bound to integrin beta 1 protein, and its interaction was also verified in vitro, using immunoprecipitation and immunofluorescence assays on epithelial cells. Consequently, NIPEP-IBD increased integrin beta 1 expression, followed by phosphorylated FAK and RhoA proteins. As FAK-RhoA signaling pathway is known to modulate cytoskeletal rearrangement and maturation of epithelial cells, tight junction markers and rapid wound closure of epithelial cells was accelerated by NIPEP-IBD induction. The effect of NIPEP-IBD was further analyzed in vivo IBD model. DSS-induced colitis mouse model was used to analyze therapeutic effect of NIPEP-IBD in each IP and PO administration, and impaired mucosa layer by DSS was recovered by NIPEP-IBD, verified with H&amp;E staining. Moreover, NIPEP-IBD treated mice showed reduced immune cell infiltration in colon tissue and reduced pro-inflammatory cytokines in plasma. In vivo fluorescence imaging system was further used to track distribution of NIPEP-IBD, and NIPEP-IBD specifically binds to impaired epithelium, co-localized with integrin beta 1 protein. The following GLP toxicity studies, ADME studies, and PK studies demonstrated that safety of NIPEP-IBD was verified as a therapeutic agent. Therefore, NIPEP-IBD is a safe and effective therapeutic peptide on IBD patients by restituting epithelial layer of colon and maintain its remission.

NIPEP-IBD; A SYNTHETIC PEPTIDE TARGETING NOVEL MOLECULE, INTEGRIN BETA 1, TO RESTITUTE INTESTINAL EPITHELIAL CELLS FOR INFLAMMATORY BOWEL DISEASE (IBD) TREATMENT

Under Inflammatory bowel disease (IBD), impaired intestinal barrier from lumen is highly associated with ongoing IBD symptoms and it also contributes as a precursor for IBD. Current IBD therapeutics lack on mucosal regeneration, which the unmet needs could be the next first in class therapeutics for IBD. Here, we propose NIPEP-IBD, a synthetically modified peptide that promotes adhesion, migration, and differentiation of intestinal epithelial cells via interaction of integrin beta 1. From NIBEC’s specialized peptide discovery platform screening, specifically sequenced peptide has been selected for regenerative functional peptide, termed NIPEP-IBD. NIPEP-IBD selectively targeted and bound to integrin beta 1 protein, and its interaction was also verified in vitro, using immunoprecipitation and immunofluorescence assays on epithelial cells. Consequently, NIPEP-IBD increased integrin beta 1 expression, followed by phosphorylated FAK and RhoA proteins. As FAK-RhoA signaling pathway is known to modulate cytoskeletal rearrangement and maturation of epithelial cells, tight junction markers and rapid wound closure of epithelial cells was accelerated by NIPEP-IBD induction. The effect of NIPEP-IBD was further analyzed in vivo IBD model. DSS-induced colitis mouse model was used to analyze therapeutic effect of NIPEP-IBD in each IP and PO administration, and impaired mucosa layer by DSS was recovered by NIPEP-IBD, verified with H&E staining. Moreover, NIPEP-IBD treated mice showed reduced immune cell infiltration in colon tissue and reduced pro-inflammatory cytokines in plasma. In vivo fluorescence imaging system was further used to track distribution of NIPEP-IBD, and NIPEP-IBD specifically binds to impaired epithelium, co-localized with integrin beta 1 protein. The following GLP toxicity studies, ADME studies, and PK studies demonstrated that safety of NIPEP-IBD was verified as a therapeutic agent. Therefore, NIPEP-IBD is a safe and effective therapeutic peptide on IBD patients by restituting epithelial layer of colon and maintain its remission.

CircRNA_Maml2 promotes the proliferation and migration of intestinal epithelial cells after severe burns by regulating the miR-93-3p/FZD7/Wnt/β-catenin pathway

BackgroundCircular RNA (circRNA) plays key regulatory roles in the development of many diseases. However the biological functions and potential molecular mechanisms of circRNA in the injury and repair of intestinal mucosa in mice after severe burns are yet to be elucidated.MethodsCell counting kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU), wound healing and transwell assays were used to detect cell proliferation and migration ability. Real-time quantitative PCR was used to identify the expression of circRNA, microRNA and messenger RNA. Nuclear and cytoplasmic separation experiments were employed to perceive the location of circRNA_Maml2. Finally, in vitro and in vivo experiments were conducted to study the repairing effect of circRNA_Maml2 on the intestinal mucosa of mice after severe burns.ResultsWhen compared with the control group, the expression of circRNA_Maml2 was significantly reduced in the severe burn group. Furthermore, overexpression of circRNA_Maml2 promoted the proliferation and migration of CT26.wt cells in vivo and the repair of damaged intestinal mucosa in vitro. CircRNA_Maml2 acted as a sponge adsorption molecule for miR-93-3p to enhance the expression of frizzled class receptor 7 and activate the downstream Wnt/β-catenin pathway, thereby promoting the repair of the intestinal mucosa.ConclusionsOur findings demonstrate that circRNA_Maml2 regulates the miR-93-3p/FZD7/Wnt/β-catenin pathway and promotes the repair of damaged intestinal mucosa. Hence, circRNA_Maml2 is a potential therapeutic target to promote intestinal mucosal repair.

Study on the effect and mechanism of recombinant human intestinal trefoil factor on intestinal mucosal injury and repair in burned mice

Study on the effect and mechanism of recombinant human intestinal trefoil factor on intestinal mucosal injury and repair in burned mice

Effect of calcium-sensing receptor on the migration and proliferation of porcine intestinal epithelial cells

The rapid healing of impaired intestinal surface plays a role in maintaining intestinal homeostasis. This study investigated the effect of calcium-sensing receptor (CaSR) on the migration and proliferation of intestinal porcine epithelial cells (IPEC-J2). Results showed that cell migration area and width were increased by R568 (CaSR activator) and decreased by NPS2143 (CaSR inhibitor). The protein level of GTP-rac1 and the phosphorylation of phospholipase C gamma 1 (PLCγ1) were increased by 2 µM R568. Furthermore, R568 + 120 µM NSC23766 (Rac1 inhibitor) and R568 + 1 µM U73122 (PLCγ1 inhibitor) decreased the protein level of GTP-rac1 and the phosphorylated PLCγ1, respectively, and both inhibited cell migration compared with R568. In addition, spermine increased the protein expression levels of CaSR and the levels of GTP-rac1 and the phosphorylated PLCγ1 and thereby promoted the migration of IPEC-J2 cells. Moreover, R568 improved the proliferation of the IPEC-J2 cells. Spermine increased cell proliferation, but NPS2143 incubated with spermine decreased cell proliferation compared with the spermine group. This study suggests that CaSR activation increased cell migration by activating Rac1 and PLCγ1 signaling and improved cell proliferation, and both effects were regulated by spermine by activating CaSR.

The Selective 5-HT1A Agonist SR57746A Protects Intestinal Epithelial Cells and Enteric Glia Cells and Promotes Mucosal Recovery in Experimental Colitis.

Neurotrophic growth factors can stabilize the intestinal barrier by preventing the apoptosis of enteric glial cells (EGCs) and enterocytes. We reasoned that a selective 5-HT1A receptor agonist may have neuroprotective properties in the gut and that topical application of SR57746A might be an effective treatment strategy in inflammatory bowel disease (IBD). The therapeutic potential of 5-HT1A receptor agonist SR57746A in IBD was evaluated in vitro (nontransformed NCM460 colonic epithelial cells, SW480 colorectal carcinoma cells) and in vivo (murine dextran sulfate sodium [DSS] colitis and CD4-T-cell transfer colitis). In vitro, we analyzed the effect of SR57746A on apoptosis in intestinal epithelial cells (IECs) and EGCs, and upon proliferation, migration, and intracellular signaling in IECs. In vivo, the effect of topical application of SR57746 on disease activity and on histological and endoscopic findings was compared with intraperitoneal infliximab and placebo, respectively. The SR57746A activates PI3-K/AKT- and ERK-signaling in IECs. Depending on ERK- and AKT activation, SR57746A potently prevents apoptosis of IECs without inducing proliferation or migration in these cells. Moreover, SR57746A prevented apoptosis in EGCs in vitro. Topical SR57746A treatment significantly reduced mucosal injury in 2 experimental murine colitis models and was as effective as intraperitoneal infliximab treatment. Treatment with SR57746A prevents inflammatory cell damage and apoptosis in IECs and EGCs, similar to the neurotrophic effects of EGCs on IECs. Topical treatment with SR57746A could be a candidate for clinical evaluation in the treatment of IBD.

Effects of spermine on the proliferation and migration of porcine intestinal epithelial cells

Whether spermine promotes the repair of porcine intestinal epithelium damage through Ras-related C3 botulinum toxin substrate 1 (Rac1)/phospholipase C-γ1 (PLC-γ1) signaling remains unclear. The current study investigated the effects of spermine addition on the proliferation and migration of IPEC-J2 cells and the effects of Rac1/PLC-γ1 signaling on cell migration. We showed that the inhibitors of Rac1 (NSC-23766) and PLC-γ1 (U73122) reduced cell migration and decreased the protein levels of Rac1 and PLC-γ1 in the cells. Moreover, spermine promoted the proliferation and migration of the IPEC-J2 cells, that is, 1 µM spermine exhibited the best effect, and spermine treatment increased the protein levels of Rac1 and PLC-γ1. Further experiments showed that spermine treatment increased cell migration and enhanced Rac1 and PLC-γ1 protein levels, compared with NSC-23766 and U73122 treatments with spermine. In conclusion, spermine treatment promoted the repair of damaged porcine intestinal epithelium by accelerating cell proliferation and migration mediated by Rac1/PLC-γ1 signaling.