HT-29 Colonic Epithelial Cells Research Articles

고당으로 유도된 산화적 스트레스에 대한 두충 잎 추출물의 장 상피 세포 보호 효과

This study investigated the protective effect of the aqueous extract of Eucommia ulmoides leaves (AEEL) against high glucose-induced human colon epithelial HT-29 cells. The 2,2'-azino-bis (3-ethyl benzothiazoline-6-sulfonic acid) (ABTS), 1,1-diphenyl-2-picrylhydrazy (DPPH) radical scavenging activities, ferric reducing/antioxidant power (FRAP), and malondialdehyde (MDA) analyses indicated that AEEL had significant antioxidant activities. The 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay showed that AEEL increased cell viability against high glucose-, H2O2-, and lipopolysaccharide (LPS)-induced cytotoxicity in HT-29 cells. Also, the 2'-7'-dichlorodihydrofluorescein diacetate (DCF-DA) assay indicated that AEEL decreased intracellular reactive oxygen species (ROS) against high glucose-, H2O2-, and lipopolysaccharide (LPS)-induced cytotoxicity in HT-29 cells. AEEL showed inhibitory activities against α-glucosidase and inhibited the formation of advanced glycation end products (AGEs). AEEL showed significant positive effects on the viability and titratable acidity of L. brevis. The high-performance liquid chromatogram (HPLC) analysis identified chlorogenic acid and rutin as the major compounds of AEEL. These results suggested that AEEL has the potential to be used as a functional food source to suppress blood glucose levels and protect the gut from high glucose-induced oxidative stress.

Anthrax lethal toxin and tumor necrosis factor-α synergize on intestinal epithelia to induce mouse death.

Bacillus anthracis lethal toxin (LT) is a determinant of lethal anthrax. Its function in myeloid cells is required for bacterial dissemination, and LT itself can directly trigger dysfunction of the cardiovascular system. The interplay between LT and the host responses is important in the pathogenesis, but our knowledge on this interplay remains limited. Tumor necrosis factor-α (TNF-α) is a pleiotropic pro-inflammatory cytokine induced by bacterial infections. Since LT accumulates and cytokines, predominantly TNF, amass during B. anthracis infection, co-treatment of TNF + LT in mice was used to mimic in vivo conditions for LT to function in inflamed hosts. Bone marrow transplantation and genetically engineered mice showed unexpectedly that the death of intestinal epithelial cells (IECs) rather than that of hematopoietic cells led to LT + TNF-induced lethality. Inhibition of p38α mitogen-activated protein kinase (MAPK) signaling by LT in IECs promoted TNF-induced apoptosis and necroptosis of IECs, leading to intestinal damage and mouse death. Consistently, p38α inhibition by LT enhanced TNF-mediated cell death in human colon epithelial HT-29 cells. As intestinal damage is one of the leading causes of lethality in anthrax patients, the IEC damage caused by LT + TNF would most likely be a mechanism underneath this clinical manifestation and could be a target for interventions.

High-Throughput Screening for Epigenetic Compounds That Induce Human β-Defensin 1 Synthesis

Antimicrobial host defense peptides (HDPs) are critically important for innate immunity. Small-molecule compounds with the ability to induce HDP synthesis are being actively explored for antimicrobial therapy. To facilitate the discovery of the compounds that specifically activate human β-defensin 1 (DEFB1) gene transcription, we established a cell-based high-throughput screening assay that employs HT-29/DEFB1-luc, a stable reporter cell line expressing the luciferase gene driven by a 3-Kb DEFB1 gene promoter. A screening of a library of 148 small-molecule epigenetic compounds led to the identification of 28 hits, with a minimum strictly standardized mean difference of 3.0. Fourteen compounds were further selected and confirmed to be capable of inducing DEFB1 mRNA expression in human HT-29 colonic epithelial cells. Desirably, the human cathelicidin antimicrobial peptide (CAMP) gene was also induced by these epigenetic compounds. Benzamide-containing histone deacetylase inhibitors (HDACi) were among the most potent HDP inducers identified in this study. Additionally, several major genes involved in intestinal barrier function, such as claudin-1, claudin-2, tight junction protein 1, and mucin 2, were differentially regulated by HDP inducers. These findings suggest the potential for the development of benzamide-based HDACi as host-directed antimicrobials for infectious disease control and prevention.

Hydrangenol, an active constituent of Hydrangea serrata (Thunb.) Ser., ameliorates colitis through suppression of macrophage-mediated inflammation in dextran sulfate sodium-treated mice.

Ulcerative colitis (UC) is a chronic disease of the colon characterized by mucosal damage and relapsing gastrointestinal inflammation. Hydrangea serrata (Thunb.) Ser. and its bioactive compound, hydrangenol, are reported to have anti-inflammatory effects, but few studies have investigated the effects of hydrangenol in colitis. In the present study, we evaluated for the first time the anti-colitic effects and molecular mechanisms of hydrangenol in a dextran sodium sulfate (DSS)-induced mouse colitis model. To investigate the anti-colitic effects of hydrangenol, DSS-induced colitis mice, HT-29 colonic epithelial cells treated with supernatant from LPS-inflamed THP-1 macrophages, and LPS-induced RAW264.7 macrophages were used. In addition, to clarify the molecular mechanisms of this study, quantitative real time-PCR, western blot analysis, TUNEL assay, and annexin V-FITC/PI double staining analysis were conducted. Oral administration of hydrangenol (15 or 30 mg kg-1) significantly alleviated DSS-induced colitis by preventing DAI scores, shortening colon length, and colonic structural damage. F4/80+ macrophage numbers in mesenteric lymph nodes and macrophage infiltration in colonic tissues were significantly suppressed following hydrangenol treatment in DSS-exposed mice. Hydrangenol significantly attenuated DSS-induced destruction of the colonic epithelial cell layer through regulation of pro-caspase-3, occludin, and claudin-1 protein expression. Moreover, hydrangenol ameliorated abnormal tight junction protein expression and apoptosis in HT-29 colonic epithelial cells treated with supernatant from LPS-inflamed THP-1 macrophages. Hydrangenol suppressed the expression of pro-inflammatory mediators, such as iNOS, COX-2, TNF-α, IL-6, and IL-1β through NF-κB, AP-1, and STAT1/3 inactivation in DSS-induced colon tissue and LPS-induced RAW264.7 macrophages. Taken together, our findings suggest that hydrangenol recovers the tight junction proteins and down-regulates the expression of the pro-inflammatory mediators by interfering with the macrophage infiltration in DSS-induced colitis. Our study provides compelling evidence that hydrangenol may be a candidate for inflammatory bowel disease therapy.

In Vitro Anti-Inflammatory Activity of Peptides Obtained by Tryptic Shaving of Surface Proteins of Streptococcus thermophilus LMD-9

Streptococcus thermophilus, a lactic acid bacterium widely used in the dairy industry, is consumed regularly by a significant proportion of the population. Some strains show in vitro anti-inflammatory activity which is not fully understood. We hypothesized that peptides released from the surface proteins of this bacterium during digestion could be implied in this activity. Consequently, we prepared a peptide hydrolysate by shaving and hydrolysis of surface proteins using trypsin, and the origin of peptides was checked by liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. Most of the identified peptides originated from bacterial cell surface proteins. The anti-inflammatory activity of peptide hydrolysate was investigated under inflammatory conditions in two cell models. Peptide hydrolysate significantly decreased secretion of pro-inflammatory cytokine IL-8 in lipopolysaccharide (LPS)-stimulated human colon epithelial HT-29 cells. It also reduced the production of pro-inflammatory cytokines IL-8, IL-1β and the protein expression levels of Pro-IL-1β and COX-2 in LPS-stimulated THP-1 macrophages. The results showed that peptides released from bacterial surface proteins by a pancreatic protease could therefore participate in an anti-inflammatory activity of S. thermophilus LMD-9 and could prevent low-grade inflammation.

Immunomodulatory role of Parkinson\u2019s disease 7 in inflammatory bowel disease

Recently the role of Parkinson’s disease 7 (PARK7) was studied in gastrointestinal diseases, however, the complex role of PARK7 in the intestinal inflammation is still not completely clear. Expression and localization of PARK7 were determined in the colon biopsies of children with inflammatory bowel disease (IBD), in the colon of dextran sodium sulphate (DSS) treated mice and in HT-29 colonic epithelial cells treated with interleukin (IL)-17, hydrogen peroxide (H2O2), tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β or lipopolysaccharide (LPS). Effect of PARK7 on the synthesis of IBD related cytokines was determined using PARK7 gene silenced HT-29 cells and 3,4,5-trimethoxy-N-(4-(8-methylimidazo(1,2-a)pyridine-2-yl)phenyl)benzamide (Comp23)—compound increasing PARK7 activity—treated mice with DSS-colitis. PARK7 expression was higher in the mucosa of children with Crohn’s disease compared to that of controls. While H2O2 and IL-17 treatment increased, LPS, TNF-α or TGF-β treatment decreased the PARK7 synthesis of HT-29 cells. PARK7 gene silencing influenced the synthesis of IL1B, IL6, TNFA and TGFB1 in vitro. Comp23 treatment attenuated the ex vivo permeability of colonic sacs, the clinical symptoms, and mucosal expression of Tgfb1, Il1b, Il6 and Il10 of DSS-treated mice. Our study revealed the role of PARK7 in the regulation of IBD-related inflammation in vitro and in vivo, suggesting its importance as a future therapeutic target.

UNC‐45A is a novel regulator of the intestinal epithelial cell migration

Intestinal epithelial cell migration is a fundamental homeostatic process that regulates establishment of the epithelial architecture in healthy gut and drives mucosal wound healing during intestinal inflammation. Epithelial cell migration depends on remodeling of the cytoskeleton, most notably actin filament turnover and activity of nonmuscle myosin II (NM II) motors. The mechanisms that regulate remodeling of the actomyosin cytoskeleton in migrating intestinal epithelial cells remain poorly understood. UNC-45A is a highly conserved member of the UNC45/CRO1/She4p family proteins and it acts as a chaperone facilitating NM II folding and self-assembly. While previous studies implicated this molecular chaperon in controlling motility of fibroblastic and some tumor cells, its roles in regulating intestinal epithelial cell migration remain unexplored. In the present study, CRISPR/Cas9 mediated gene editing was used to downregulate UNC-45A expression in HT29 and SK-CO15 human colonic epithelial cells. Loss of UNC-45A significantly decreased cell contractility in the collagen contraction assay, thereby indicating impaired activity of NM II motors. Importantly, knockout of UNC-45A significantly decreased both collective epithelial cell migration in a wound heling assay and individual cell migration in the Boyden chamber. The attenuated migration of UNC-45A-depleted epithelial cells was accompanied by their increased adhesion to the collagen matrix, disassembly of basal actin stress fibers and inhibited membrane protrusion at the migrating cell edge. Interestingly, UNC-45A expression was significantly downregulated in isolated intestinal epithelial cells and tissue sections obtained from the colonic mucosa of ulcerative colitis patients. Our results signify UNC-45A as a novel cytoskeletal regulator of intestinal epithelial cell migration, which functions could be inhibited in chronic inflammatory disorders.

A 75 kDa glycoprotein isolated from Cudrania tricuspidata Bureau induces colonic epithelial proliferation and ameliorates mouse colitis induced by dextran sulfate sodium.

Cudrania tricuspidata Bureau (CTB), a species of the Moraceae plant, has been used as a bruise recovery treatment. This study aimed to determine whether the 75 kDa phytoglycoprotein extracted from CTB has a regulatory effect on the proliferation of human colon epithelial cells and the pathological process of inflammatory bowel disease (IBD). We found that CTB glycoprotein significantly induces the proliferation of human colon epithelial HT-29 cells by activating protein kinase C. CTB glycoprotein stimulated the phosphorylation of c-Jun N-terminal kinase and transcription factor nuclear factor-κB, which are responsible for the expression of cell-cycle-related proteins (CDK2, CDK4, cyclin D1 and cyclin E) during its promotion of cell proliferation. Experimental colitis was induced in mice by adding dextran sulfate sodium to their drinking water at a concentration of 4% (W/V) for seven days. We found that CTB glycoprotein ameliorates the pathological process of IBD and lowers the disease activity index score, which was composed of body weight change, diarrhea, and hematochezia in ICR mice treated with dextran sulfate sodium. Hence, we suggest that CTB glycoprotein has the ability to prevent IBD by promoting cell proliferation signaling events via the activation of PKC, JNK and NF-κB in colon epithelial cells.

Pharmacological Inhibition and Activation of the Ca2+ Activated Cl- Channel TMEM16A.

TMEM16A is a Ca2+ activated Cl− channel with important functions in airways, intestine, and other epithelial organs. Activation of TMEM16A is proposed as a therapy in cystic fibrosis (CF) to reinstall airway Cl− secretion and to enhance airway surface liquid (ASL). This CFTR-agnostic approach is thought to improve mucociliary clearance and lung function in CF. This could indeed improve ASL, however, mucus release and airway contraction may also be induced by activators of TMEM16A, particularly in inflamed airways of patients with asthma, COPD, or CF. Currently, both activators and inhibitors of TMEM16A are developed and examined in different types of tissues. Here we compare activation and inhibition of endogenous and overexpressed TMEM16A and analyze potential off-target effects. The three well-known blockers benzbromarone, niclosamide, and Ani9 inhibited both TMEM16A and ATP-induced Ca2+ increase by variable degrees, depending on the cell type. Niclosamide, while blocking Ca2+ activated TMEM16A, also induced a subtle but significant Ca2+ store release and inhibited store-operated Ca2+ influx. Niclosamide, benzbromarone and Ani9 also affected TMEM16F whole cell currents, indicating limited specificity for these inhibitors. The compounds Eact, cinnamaldehyde, and melittin, as well as the phosphatidylinositol diC8-PIP2 are the reported activators of TMEM16A. However, the compounds were unable to activate endogenous TMEM16A in HT29 colonic epithelial cells. In contrast, TMEM16A overexpressed in HEK293 cells was potently stimulated by these activators. We speculate that overexpressed TMEM16A might have a better accessibility to intracellular Ca2+, which causes spontaneous activity even at basal intracellular Ca2+ concentrations. Small molecules may therefore potentiate pre-stimulated TMEM16A currents, but may otherwise fail to activate silent endogenous TMEM16A.

Beneficial effect of immunobiotic strains on attenuation of Salmonella induced inflammatory response in human intestinal epithelial cells.

Probiotic bacteria have the ability to modulate host immune responses and have potent therapeutic functional effects against several diseases, including inflammatory diseases. However, beneficial effects of probiotics are strain specific and their interactions with host immune cells to modulate inflammatory response are largely unknown. Intestinal epithelial cells (IECs), which are the first line of defense against invading pathogens, and connects between commensals/probiotics and immune system; therefore, in this study, we used human IECs to assess the probiotic effects of three selected Lactobacillus strains in vitro. An HT-29 colonic epithelial cell and HT-29/blood mononuclear cells co-culture system were stimulated with Lactobacillus followed by Salmonella for different hours, after which the mRNA level of cytokines, β-defensin-2 and negative regulators for TLR signaling and protein levels of ZO-1 and IκB-α were analyzed by real-time polymerase chain reaction and western blot analysis. L. brevis decreased Salmonella induced IL-6, IL-8, MCP-1 and IL-1β levels, whereas L. pentosus suppressed IL-6 and MCP-1 in HT-29 cells. Moreover, L. brevis was able to increase the mRNA levels of A20, Tollip, SIGIRR and IRAKM, while L. pentosus reduced the levels of A20, and IRAKM in response to Salmonella. In addition, decrease in protein level of TNF-α and increase in mRNA level of IL-10 was observed in L. brevis and L. pentosus treated HT-29 cells. Lactobacillus strains were differentially modulated ZO-1 and p-IκB-α in HT-29 cells treated with Salmonella. Overall, the results of this study indicate that Lactobacillus strains attenuate Salmonella induced inflammatory responses through beneficial modulation of TLR negative regulators and the NF-κB pathway.

Intestinal cytotoxicity induced by Escherichia coli is fully prevented by red wine polyphenol extract: Mechanistic insights in epithelial cells

Gastrointestinal infections are considered a serious public health problem in view of their high incidence and the increasing antibiotic resistance of the microorganisms involved in their pathogenesis, namely Escherichia coli. Consequently, finding new ways to prevent and/or threat these infections is urgent.In this study we investigated whether a well-characterised polyphenolic red wine extract is able to inhibit the cytotoxic effects induced by a clinical pathogenic Escherichia coli strain (E. coli 270) against HT-29 colon epithelial cells. Firstly, we provide evidences showing that the E. coli strain triggered the death of the intestinal epithelial cells through the production and release of a toxin. Then we support that, in a concentration dependent way, RWE through both, a direct interaction with bacterial exotoxin and the epithelial cells, prevented the action of the toxin on the cells, significantly reducing cell death.This intends to constitute a position paper as detailed mechanisms for the inhibition of E. coli-induced toxicity by polyphenols are yet to be completely unraveled. However, considering that the amount of red wine polyphenols used can be easily achieved in a normal diet, this study suggests that RWE may provide a readily available dietary product with potential benefit for the prevention and/or treatment of intestinal infections induced by intestinal pathogenic bacteria and may open new therapeutic avenues for the development of potential natural antimicrobial agents.

The Effect of IL-26 on TNF-α-Induced CXCL8 Responses by Colonic Epithelial Cell Lines

ABSTRACTTh17 cells of the intestine and colon can produce several important cytokines during mucosal inflammation. However, few studies have focused on the role of IL-26 in intestinal inflammations. Colonic epithelial cells express receptors for IL-26, and this cytokine has been shown to induce the HT-29 colonic epithelial cell line to produce the chemokine CXCL8. However, epithelial cells would function in a cytokine network environment during mucosal inflammation and any effect of IL-26 on colonic epithelial cell chemokine responses could be affected by the presence of other potent pro-inflammatory cytokines like TNF-α and IL-1. Therefore, we investigated the effect of IL-26 with TNF-α or IL-1 on colonic epithelial cell line secretion of CXCL8. IL-26 alone had no effect on HT-29 or DLD1 cell line CXCL8 secretion. Yet, IL-26 was found to significantly enhance TNF-α-induced, but not IL-1-induced, CXCL8 secretion, but only at high levels of TNF-α. Similar results were seen with DLD1 cells. IL-26 did not enhance TNF-α-induced CXCL8 mRNA levels and did not affect TNF-α-induced IκBα phosphorylation or degradation. However, signaling through ERK and p38 MAPK were determined to be involved in the enhancing effect of IL-26 on the TNF-α-induced CXCL8 secretion, perhaps through known post-translational effects. These results suggest that the role of IL-26 in intestinal inflammation may be limited to enhancing CXCL8 secretion in the presence high levels of TNF-α, such as may occur in inflammatory bowel disease.Abbreviations: DMEM, Dulbecco’s Modified Eagle’s Medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IBD, inflammatory bowel disease; IL, interleukin; ITS, insulin, transferrin, selenium; TBS, Tris buffered saline; TNF, tumor necrosis factor.

Antitumor potential of new low molecular weight antioxidative preparations from the white rot fungus Cerrena unicolor against human colon cancer cells

The aim of this study was to investigate the anticancer and antioxidant activities of low molecular weight subfractions isolated from secondary metabolites produced by the wood degrading fungus Cerrena unicolor. Human colon cancer cells (stage I) HT-29 and human normal colon epithelial cells CCD 841 CoTr were used in the research. The present study demonstrated that the low molecular weight subfractions exhibited inhibitory activity towards human colon cancer cells HT-29 at a concentration range of 25–200 μg/mL. All 6 subfractions inhibited proliferation of cells down to 47.5–9.2% at the highest concentrations in a dose-dependent manner. The most desired activity was exhibited by subfractions S, 3, 4, and 5, as the proliferation of HT-29 cells was inhibited to the greatest extent (16.5, 47.5, 42.7, and 26.1% of the control, respectively), while the effect on CCD 841 CoTr cells was the mildest (inhibition to 54.4, 71.4, 79.4, and 53.4%, compared to the control, respectively). The microscopic observation revealed that all extracts induced programmed cell death, i.e. apoptosis (up to 44.4% (subfraction 6) towards HT-29 and less than 20% (most fractions) towards CCD 841 CoTr), with no or a significantly low level of necrosis in both cell lines at the same time.

Modulation of Gut Microbiota Composition by Serotonin Signaling Influences Intestinal Immune Response and Susceptibility to Colitis.

Background & AimsSerotonin (5-hydroxytryptamine [5-HT]) is synthesized mainly within enterochromaffin (EC) cells in the gut, and tryptophan hydroxylase 1 (Tph1) is the rate-limiting enzyme for 5-HT synthesis in EC cells. Accumulating evidence suggests the importance of gut microbiota in intestinal inflammation. Considering the close proximity of EC cells and the microbes, we investigated the influence of gut-derived 5-HT on the microbiota and the susceptibility to colitis.MethodsGut microbiota of Tph1-/- and Tph1+/- mice were investigated by deep sequencing. Direct influence of 5-HT on bacteria was assessed by using in vitro system of isolated commensals. The indirect influence of 5-HT on microbiota was assessed by measuring antimicrobial peptides, specifically β-defensins, in the colon of mice and HT-29 colonic epithelial cells. The impact of gut microbiota on the development of dextran sulfate sodium–induced colitis was assessed by transferring gut microbiota from Tph1-/- mice to Tph1+/- littermates and vice versa, as well as in germ-free mice.ResultsA significant difference in microbial composition between Tph1-/- and Tph1+/- littermates was observed. 5-HT directly stimulated and inhibited the growth of commensal bacteria in vitro, exhibiting a concentration-dependent and species-specific effect. 5-HT also inhibited β-defensin production by HT-29 cells. Microbial transfer from Tph1-/- to Tph1+/- littermates and vice versa altered colitis severity, with microbiota from Tph1-/- mice mediating the protective effects. Furthermore, germ-free mice colonized with microbiota from Tph1-/- mice exhibited less severe dextran sulfate sodium–induced colitis.ConclusionsThese findings demonstrate a novel role of gut-derived 5-HT in shaping gut microbiota composition in relation to susceptibility to colitis, identifying 5-HT–microbiota axis as a potential new therapeutic target in intestinal inflammatory disorders.

Differences in the fecal microbiota of neonates born at home or in the hospital

Research on the neonatal microbiome has been performed mostly on hospital-born infants, who often undergo multiple birth-related interventions. Both the hospital environment and interventions around the time of birth may affect the neonate microbiome. In this study, we determine the structure of the microbiota in feces from babies born in the hospital or at home, and from vaginal samples of their mothers. We included 35 vaginally-born, breast-fed neonates, 14 of whom delivered at home (4 in water), and 21 who delivered in the hospital. Feces from babies and mothers and maternal vaginal swab samples were collected at enrollment, the day of birth, followed by days 1, 2, 7, 14, 21, and 28. At the time of birth, the diversity of the vaginal microbiota of mothers delivering in the hospital was lower than in mothers delivering at home, and showed higher proportion of Lactobacillus. Among 20 infants not exposed to perinatal maternal antibiotics or water birth, fecal beta diversity differed significantly by birth site, with hospital-born infants having lower Bacteroides, Bifidobacterium, Streptococcus, and Lactobacillus, and higher Clostridium and Enterobacteriaceae family (LDA > 3.0), than babies born at home. At 1 month of age, feces from infants born in the hospital also induced greater pro-inflammatory gene expression (TLR4, IL-8, occludin and TGFβ) in human colon epithelial HT-29 cells. The results of this work suggest that hospitalization (perinatal interventions or the hospital environment) may affect the microbiota of the vaginal source and the initial colonization during labor and birth, with effects that could persist in the intestinal microbiota of infants 1 month after birth. More research is needed to determine specific factors that alter bacterial transmission between mother and baby and the long-term health implications of these differences for the developing infant.

Role of microRNA-223 in the regulation of poly(ADP-ribose) polymerase in pediatric patients with Crohn’s disease

Objectives: Crohn’s disease (CD) is a multifactorial disease, characterized by oxidant-induced tissue injury with a possible activation of poly(ADP-ribose) polymerase (PARP)-1. MicroRNAs (miRs) can offer a potential link between the genetic susceptibility, environmental and immunologic factors in the pathogenesis of CD. Previously, PARP-1 was identified as a direct target gene of miR-223 in an epithelial cell line. Our aim was to examine PARP activation and miR-223 expression in colonic biopsies of pediatric CD. To support our in vivo findings, the effect of lipopolysaccharide (LPS) on same parameters was examined in HT-29 colonic epithelial cell line.Methods: Colonic biopsies were taken from patients with macroscopically inflamed and intact mucosa with CD and controls. LPS treated HT-29 cells served as our in vitro model. To analyze the PARP-1 expression real-time PCR, Western blot and immunohistochemical analyses were used. PARP-1 enzymatic activity was assessed on the basis of poly(ADP-ribosyl)ated proteins. Expression of miR-223 was examined by real-time PCR.Results: PARP-1 mRNA and miR-223 expression was significantly elevated, however, the amount of PARP-1 protein and poly(ADP-ribose) was reduced in pediatric CD compared to controls. LPS incubation did not affect the expression of PARP-1 mRNA, however, decreased miR-223 expression, and enhanced PARP-1 activity.Conclusions: In our study, we showed that the expression of miR-223 is up-regulated and poly(ADP-ribosyl)ation is reduced in pediatric patients with CD. Moreover, we confirmed their opposite change in LPS treated epithelial cells, too. These data suggest that the hypofunctionality of PARP-1 may play a potential role in the pathomechanism of CD.

CF₃-Substituted Mollugin 2-(4-Morpholinyl)-ethyl ester as a Potential Anti-inflammatory Agent with Improved Aqueous Solubility and Metabolic Stability.

Although mollugin, the main ingredient of the oriental medicinal herb Rubia cordifolia, has considerable anti-inflammatory effects, it has poor aqueous solubility as well as poor metabolic and plasma stability. To overcome these shortfalls, various mollugin derivatives have been synthesized and evaluated for their ability to inhibit U937 monocyte cell adhesion to HT-29 colonic epithelial cells in TNF-α- or IL-6-induced models of colon inflammation. The 2-(4-morpholinyl)-ethyl ester of CF3-substituted mollugin (compound 15c) showed good water solubility, improved metabolic and plasma stability, and greater inhibitory activity than mesalazine in both the TNF-α- and IL-6-induced colonic epithelial cell adhesion assays, suggesting that 15c is a potential anti-inflammatory agent.

P-344 - Modulation of tight junctions in intestinal epithelial cells by red wine polyphenols

The etiology of Inflammatory bowel diseases (IBD) is not yet completely understood but a complex interplay among genetic, immunological, and environmental factors is known to induce a deregulated synthesis of a variety of proinflammatory mediators, including cytokines, reactive oxygen species (ROS) and nitric oxide (•NO), resulting in a disruption of the epithelial barrier and excessive tissue injury. Several studies showed that the barrier dysfunction in IBD is mainly due to a specific downregulation of key tight junction (TJ) -associated proteins. Here, using a cellular model of intestinal inflammation consisting of cytokines-stimulated HT-29 colon epithelial cells, we show that a polyphenolic red wine extract (RWE) significantly increased the mRNA of key TJ proteins as occludin, claudin-5 and ZO-1, not only preventing their decrease under inflammatory conditions but also enhancing their expression levels above those observed in control cells. Also, RWE prevented the cytokines-induced increase of claudin-2 mRNA. Taken together, these results suggest that, under inflammatory stimuli, RWE is able to promote the barrier function by affecting TJ protein expression and could potentially be useful in the treatment/prevention of IBD.

Inhibition of Protein Translation Initiation and Disruption of the Intestinal Epithelial Barrier in Mucosal Inflammation

Integrity of the intestinal epithelial barrier is regulated by two types of adhesive structures: intercellular junctions and focal adhesions (FA) that control epithelial cell attachment to each other and to the extracellular matrix (ECM), respectively. Disassembly of adherens junctions (AJ) and tight junctions (TJ) plays key roles in the breakdown of the intestinal epithelial barrier during mucosal inflammation. Abnormal architecture and functions of FA significantly contribute to attenuated restitution of the inflamed intestinal mucosa. Proinflammatory cytokines such as interferon (IFN)‐γ and tumor necrosis factor (TNF)‐α, are known to trigger AJ/TJ disassembly and attenuate wound healing in model intestinal epithelial cell monolayers. We hypothesized that inhibition of protein translation factors eIF4G1 and eIF4G2 could mediate cytokine‐induced disruption of the gut barrier and attenuation of epithelial restitution.Immunoblotting analysis of IFNγ and IFNγ/TNFα‐treated HT29 and T84 human colonic epithelial cells demonstrated significant downregulation of expression AJ proteins, E‐cadherin, p120 catenin, β‐catenin, and the TJ protein, occludin. By contrast quantitative RT‐PCR analysis did not reveal changes in mRNA levels of selected junctional proteins. Furthermore, expression of translation initiation factors eIF4G1 and eIF4G2 was downregulated by these proinflammatory cytokines in vitro, and eIF4G2 level was decreased in colonic mucosa of IBD patients. In order to examine functional roles of these translation initiation factors, eIF4G1 or eIF4G2 were selectively downregulated in well‐differentiated SK‐CO15 and HT29cF8 human colonic epithelial cell lines, as well as in stem‐cell like human intestinal epithelial cells (HIEC) by using either siRNA‐mediated or shRNA‐mediated knock‐down. Downregulation of either eIF4G1, or eIF4G2 increased permeability of SK‐CO15 epithelial cell monolayers and decreased expression of different adherens junction and tight junction proteins. Furthermore, depletion of these translation initiating factors inhibited collective wound healing and individual transfilter migration of both stem‐cell like and well‐differentiated intestinal epithelial cells. Interestingly, immunolabeling of newly‐translated proteins identified the epithelial leading edge as the hot‐spot site of active translation during wound healing. Finally, we observed a robust epithelial to myofibroblast transition in HIEC cells upon depletion of eIF4G1. The described results reveal a novel role for protein translation initiation in regulating intestinal epithelial barrier and epithelial restitution and suggest that this important homeostatic mechanism could be impaired during mucosal inflammation and IBD.Support or Funding InformationSupported by the Crohn's and Colitis Foundation of America grant 380973 to NGN and NIH grant DK108278 to AII.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In Vitro and Ex Vivo Effects of Silymarin Derivatives on Proinflammatory Cytokine Secretion Using a Murine Colonic Strip Model

AbstractSilymarin has anti-inflammatory properties and documented anti-colitis activity. Our prior study determined that in vitro treatment with certain extracted fractions of silymarin inhibited stimulated proinflammatory cytokine secretion from cell lines relevant to colitis. In this study, colitis was induced in mice by giving dextran sulfate sodium drinking water for 6 days. The ex vivo effects of crude silymarin extract, two different silymarin fractions, as well as commercially derived silibinin and isosilibinin were examined by determining the secretion of MIP-2, TNF-α, and IL-17 in cell culture media from colonic strips. Further, the effects of silymarin-derived treatments on IL-8 and TNF-α secretion induced by the colitis supernatant was characterized with HT-29 colonic epithelial and RAW 264.7 macrophage cell lines. Prominent inhibition of MIP-2 and TNF-α secretion from colonic strips of mice with/without dextran sulfate sodium-induced colitis was observed with various silymarin treatments. Further, inhibition of dual (IL-23+IL-1β) cytokine-stimulated secretion of IL-17 from colonic strips of mice was found with certain silymarin treatments. Significant attenuation of TNF-α secretion from colitis supernatant-stimulated RAW 264.7 cells was observed for crude silymarin extract and isosilibinin treatments. Finally, inhibition of IL-8 secretion from the colitis supernatant-stimulated HT29 colonic epithelial cell line was found with isosilibinin. These results contribute to the identification of silymarin-derived flavonoligans with optimal anti-inflammatory properties for further testing in colitis models.