Apoptosis Of Colonic Epithelial Cells Research Articles

Nitrate ameliorates dextran sodium sulfate-induced colitis by regulating the homeostasis of the intestinal microbiota

Inflammatory bowel disease (IBD) involves chronic inflammation, loss of epithelial integrity, and gastrointestinal microbiota dysbiosis. Effective therapies for IBD have not been established. Accordingly, in this study, we evaluated the effects of inorganic nitrate, a potent nitric oxide (NO) donor and microbiota regulator, in a mouse model of dextran sodium sulfate (DSS)-induced colitis. Mice were pretreated with NaNO3 (2 mM) in their drinking water for 5 days, and NaCl was used as a control. Feces were collected for microbiota analyses. The results showed that oral administration of dietary nitrate could maintained colon consistency, improved colon length, maintained body weight, decreased apoptosis in colon epithelial cells, and ameliorated inflammatory cell infiltration in both the colon and peripheral blood. Microbiota profiling revealed that nitrate regulated dysbiosis. Analysis of the top bacteria at the genus level showed that Bacteroidales_S24-7_group_unidentified, Lactobacillus, Bacteroides, and Prevotellaceae_UCG-001 decreased in the DSS group compared with that in the normal group, whereas Lactobacillus, Ruminococcaceae_UCG-014, and Prevotellaceae_UCG-001 were increased in the DSS + NaNO3 group compared with that in the DSS group. The enriched bacteria in the nitrate group included Gordonibacter, Ureaplasama, and Lachnospiraceae_UCG-006. Moreover, microbiota analysis revealed that nitrate could partially decrease the enriched metabolic pathways (p53 signaling pathway and colorectal cancer pathway) compared with that in the DSS and DSS + NaCl groups. Overall, these findings indicated that nitrate could ameliorate DSS-induced colitis by decreasing inflammation, reducing apoptosis, and regulating the microbiota by activation of the NO3−/NO2−/NO pathway. Nitrate might be a potential treatment for colitis patients in the future clinical application.

LncRNA Mirt2 Is Downregulated in Ulcerative Colitis and Regulates IL-22 Expression and Apoptosis in Colonic Epithelial Cells.

lncRNA Mirt2 is a lipopolysaccharide- (LPS-) inducible inflammation inhibitor. We found that Mirt2 was downregulated in plasma of ulcerative colitis (UC) patients and the downregulation of Mirt2 distinguished UC patients from healthy controls. IL-22 was also downregulated in UC patients and positively correlated with Mirt2. Mirt2 overexpression led to upregulated, while Mirt2 siRNA silencing led to inhibited secretion of IL-22 from colonic epithelial cells treated with LPS. In addition, under LPS treatment, Mirt2 overexpression led to decreased, while Mirt2 siRNA silencing led to increased apoptotic rate of colonic epithelial cells. Therefore, Mirt2 is downregulated in ulcerative colitis and regulates IL-22 expression in colonic epithelial cells.

Clostridium difficile Toxin A Upregulates Bak Expression through PGE2 Pathway in Human Colonocytes.

Clostridium difficile toxin A is known to cause colonic epithelial cell apoptosis, which is considered the main causative event that triggers inflammatory responses in the colon, reflecting the concept that the essential role of epithelial cells in the colon is to form a physical barrier in the gut. We previously showed that toxin A-induced colonocyte apoptosis and subsequent inflammation were dependent on prostaglandin E2 (PGE2) produced in response to toxin A stimulation. However, the molecular mechanism by which PGE2 mediates cell apoptosis in toxin A-exposed colonocytes has remained unclear. Here, we sought to identify the signaling pathway involved in toxin A-induced, PGE2-mediated colonocyte apoptosis. In non-transformed NCM460 human colonocytes, toxin A exposure strongly upregulated expression of Bak, which is known to form mitochondrial outer membrane pores, resulting in apoptosis. RT-PCR analyses revealed that this increase in Bak expression was attributable to toxin A-induced transcriptional upregulation. We also found that toxin A upregulation of Bak expression was dependent on PGE2 production, and further showed that this effect was recapitulated by an EP1 receptor agonist, but not by agonists of other EP receptors. Collectively, these results suggest that toxin A-induced cell apoptosis involves PGE2-upregulation of Bak through the EP1 receptor.

Berberine inhibits epithelial-mesenchymal transition and promotes apoptosis of tumour-associated fibroblast-induced colonic epithelial cells through regulation of TGF-β signalling

Tumour-associated fibroblasts (TAFs) mediate the differentiation of adjacent stromal cells. Berberine (BBR), a monomer of traditional Chinese herbs, exhibits a potent therapeutic effect against cancer. However, the effects of BBR on the differentiation of normal colonic epithelial cells induced by TAFs have not been determined. In the present study, we selected the TAF-like myofibroblast cell line CCD-18Co. CCD-18Co-derived conditioned medium (CM) and co-culture induced epithelial-mesenchymal transition (EMT) changes in colonic epithelial HCoEpiC cells with decreased E-cadherin and increased vimentin and α-SMA expression. In addition, CCD-18Co stimulated the expression of ZEB1 and Snail and promoted motility. We used LY364947, a TGF-β receptor kinase type I (TβRI) inhibitor, and BBR. Our results showed that LY364947 and BBR inhibited these phenomena. BBR decreased the expression of ZEB1 and Snail, and this effect was concentration dependent. BBR also downregulated the expression of TβRI, TβRII, Smad2/p-Smad2 and Smad3/p-Smad3. In addition, BBR induced apoptosis in EMT-like HCoEpiC cells in a concentration-dependent manner with upregulation of Bax and downregulation of Bcl-2. However, VX-702, an inhibitor of p38 MAPK, significantly suppressed the apoptosis rate. BBR promoted the expression of p38 MAPK and phosphorylated p38 MAPK. In conclusion, berberine inhibits EMT and promotes apoptosis in TAF-induced colonic epithelial cells through mediation of the Smad-dependent and SMAD-independent TGF-β signalling pathways.

Trichostatin a inhibits phenotypic transition and induces apoptosis of the TAF-treated normal colonic epithelial cells through regulation of TGF-β pathway.

Tumor-associated fibroblasts (TAFs) contribute to transdifferentiation of stromal cells in tumor microenvironment. Epithelial-mesenchymal transition (EMT) is a procedure of phenotypic remodeling of epithelial cells and extensively exists in local tumoral stroma. Histone deacetylase (HDAC) inhibitor Tricostatin A (TSA) and sodium butyrate (SB) are reported to play important roles in the regulation of biological behaviour of cancer cells. However, whether TSA or SB is involved in control of EMT in colon epithelial cells induced by TAFs remains unidentified. In present study, we used conditioned medium (CM) form TAF-like CCD-18Co cells to stimulate 2D- and 3D-cultured colon epithelial HCoEpiC cells for 24 h and 4 d. We found that the CCD-18Co CM triggered multiple morphological changes in HCoEpiCs including prolonged cell diameters, down-regulation of E-cadherin and up-regulation of vimentin and α-SMA. Besides, ZEB1 and Snail expression and migration were also promoted by the CM. These phenomena were abolised by 5 μg/ml LY364947, a TGF-β receptor inhibitor. CCD-18Co induced up-regulation of HDAC1 and HDAC2 in the 2D and 3D models, while no change of HDAC4 exprerssion was found. Treatment of 2 μg/ml TSA reversed the CCD-18Co-induced morphological changes and migration of the HCoEpiCs, and suppressed the downregulation of E-cadherin and upregulation of vimentin, α-SMA, ZEB1 and Snail. However, the suppressive effect of 4 mg/ml SB on the EMT was not observed. TSA down-regulated the expressions of Smad2/3, p-Smad2/3 amd HDAC4. Besides, TSA promoted the apoptosis rate (36.84 ± 6.52%) comparing with the CCD-18Co-treated HCoEpiCs (3.52 ± 0.85%, P < 0.05), with promotion of Bax (0.5893±0.0498 in 2D and 0.8867±0.0916 in 3D) and reduction of Bcl-2 (0.0476±0.0053 in 2D and 0.0294±0.0075 in 3D). TSA stimulated expression of phosphorylated-p38 MAPK in 2D (0.3472±0.0249) and 3D (0.3188±0.0248). After pre-treatment with p38 MAPK inhibitor VX-702 (0.5 mg/ml), the apoptosis rate of TSA was decreased in 2D (10.32%) and 3D (5.26%). Our observations demonstrate that epigenetic treatment with HDAC inhibitor TSA may be a useful therapeutic tool for the reversion of TAF-induced EMT in colon epithelium through mediating canonical Smads pathway and non-canonical p38 MAPK signalling.

SAT-138 ROLE OF GUT COMMENSAL BACTERIA IN ISCHEMIA/REPERFUSION INDUCED ACUTE KIDNEY INJURY

Emerging evidence suggests the critical role of gut commensal bacteria in health and diseases by modulating local and systemic immune response. However, the role of the microbiome and gut-derived immunity in the development of kidney ischemia/reperfusion injury (IRI) is largely unknown. The purpose of this study is to examine the role of the gut microbiome and mucosal immunity in the development of kidney IRI and distant organ damage. C57BL/6 mice were fed with a combination of oral antibiotics (neomycin, ampicillin, metronidazole and vancomycin) or vehicle for 2 weeks and then underwent bilateral renal pedicle clamping. Renal function, histological kidney injury, gut permeability, gut-derived local and systemic immune response as well as microbiome analyses were performed. IRI induced renal functional, histological deterioration was accompanied by alteration of the gut microbiome. Kidney IRI resulted in a significant decrease in the diversity index of the microbiome, as well as the number of operational taxonomic units (OTUs). Among them, the reduction of short-chain fatty acid producing species such as Ruminococcaceae, Bacteroides, Christensenellaae, Lachnospiraceae was prominent. Altered microbiome after IRI was associated with the development of “leaky gut” and colitis that were associated with increased number of colon epithelial cell apoptosis, neutrophil and M1 macrophage infiltration in the colon. Gut microbiota depletion by oral antibiotic treatment but not by systemic antibiotics significantly attenuated kidney injury, as well as distant organ damage. The renoprotective effect of oral antibiotic treatment was associated with partial restoration of “leaky gut” with preserved tight junction proteins, decreased number of colon epithelial cell apoptosis or neutrophil or M1 macrophage-mediated colitis. This protective effect was also associated with a reduction of systemic proinflammatory cytokines and increase of anti-inflammatory cytokine IL-10. More importantly, alteration of the gut microbiome in oral antibiotics fed mice was associated significantly increased number of CD4+ CD25+ Tregs and Foxp3 expression in cervical lymph node and spleen following IRI. Our data showed the critical role of the commensal gut microbiome in the development of kidney IRI injury as well as distant organ injury. Antibiotic modification of gut microbiome induced renoprotective effect is thought to be partially mediated by modulating peripheral and systemic immune response toward Treg mediated immune tolerance. Targeting the gut might offer a new paradigm in the prevention of treatment of AKI.

Long non-coding RNA CRNDE promotes cell apoptosis by suppressing miR-495 in inflammatory bowel disease

ObjectiveThis article aims to investigate the mechanism of microRNA-495 (miR-495) and long non-coding RNA CRNDE on the apoptosis of colonic epithelial cells in inflammatory bowel diseases (IBDs). MethodsThe mouse model of IBD was induced by dextran sulfate sodium (DSS), and human colonic epithelial cell lines (HT-29, LOVO, and Caco-2) were treated with DSS, and received cell transfection. RNA interference was used to down-regulate CRNDE expression. ResultsCRNDE and SOCS1 were highly expressed, but miR-495 was lowly expressed in the DSS-induced colitis tissues and colonic epithelial cell lines. Interference of CRNDE inhibited cell apoptosis of DSS-induced colonic epithelial cells. The interaction between CRNDE and miR-495 was confirmed by RNA immunoprecipitation and RNA pull-down assay. The target relationship between miR-495 and SOCS1 was confirmed by the luciferase reporter assay. CRNDE promoted DSS-induced colonic epithelial cell apoptosis via miR-495/SOCS1. CRNDE interference in DSS-induced colitis mouse model alleviated clinical manifestations of IBD. ConclusionsOur findings demonstrated that CRNDE promoted DSS-induced colonic epithelial cell apoptosis via suppressing miR-495 and increasing SOCS1, indicating CRNDE as a novel target of treating IBD.

Pharmacological mechanism of Sishen Wan® attenuated experimental chronic colitis by inhibiting wnt/β-catenin pathway

Ethnopharmacological relevanceSishen Wan (SSW) is a commercial and frequently used Chinese patent medicine listed in the Chinese Pharmacopeia, which is usually used to treat chronic colitis. Aim of the studyWe explored the pharmacological mechanism of Sishen Wan attenuated experimental chronic colitis by inhibiting Wnt/β-catenin pathway. Materials and methodsExperimental chronic colitis was induced by trinitrobenzene sulfonic acid (TNBS). The therapeutic effect of SSW were analyzed by index of colonic weight, colonic length, pathological score. Cytokines expression were analyzed by ELISA, while the apoptosis level was checked by TUNEL staining. These proteins of Wnt/β-catenin signaling pathway was analyzed by Western blot assay. ResultsRats with TNBS-induced chronic colitis were treated by SSW for 10 days. The efficacy of SSW was demonstrated by improved macroscopic and microscopic colonic damage. SSW increased the level of ATP in colonic mucosa, while SSW inhibited β-catenin, ubiquitination of Nemo-like-kinase-associated ring finger protein and T-cell factor, and expression of Wnt/β-catenin downstream proteins (including c-Myc, cyclo-oxygenase-2, cyclin D1, survivin, signal transducer and activator of transcription 3 and zipper-interacting protein kinase), and improved lymphoid enhancer factor ubiquitination and β-TrCP activity, followed by excessive apoptosis of colonic epithelial cells. ConclusionsSSW effectively attenuated experimental chronic colitis induced by TNBS, which was realized by inhibition of the Wnt/β-catenin signaling pathway.

Ameliorative effect of Alnus japonica ethanol extract on colitis through the inhibition of inflammatory responses and attenuation of intestinal barrier disruption in vivo and in vitro.

Inflammatory bowel disease (IBD) is chronic inflammation of the gastrointestinal tract caused by high levels of pro-inflammatory cytokines and epithelial barrier dysfunction. Alnus japonica Steud. (Betulaceae) has been used in traditional Asian medicine. However, the potential of A. japonica for the treatment of intestinal inflammation has not been investigated. This study investigated the effects of ethanol extract from A. japonica bark (AJE) on colonic mucosa injury in mice with dextran sodium sulfate (DSS)-induced colitis. Treatment with AJE ameliorated pathological damage and the histopathologic features of DSS-induced colitis. The administration of AJE also inhibits DSS-induced pro-inflammatory cytokines expression, including interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and cyclooxygenase (COX)-2. Notably, AJE administration attenuated the reduction of tight junction proteins, zonula occludens (ZO)-1 and occludin, in DSS-induced colitis. In addition, AJE increased heme oxygenase (HO)-1 expression and prevented DSS-induced apoptosis in colonic epithelial cells. Furthermore, in vitro studies demonstrated that AJE inhibits TNF-α-induced IL-8, IL-1β, and COX-2 expression in human intestinal epithelial HT-29 cells and tert-butyl hydroperoxide-induced reduction of ZO-1 and occludin expression in human intestinal epithelial Caco-2 cells. AJE-induced HO-1 protein expression was also found in both HT-29 and Caco-2 cells. Taken together, our findings demonstrated that AJE inhibits intestinal inflammation and protects against intestinal barrier disruption in mice with DSS-induced colitis in vivo and human intestinal epithelial cells in vitro. These results suggest that AJE might have beneficial effects for the treatment of IBD.

Bisphenol A suppresses proliferation and induces apoptosis in colonic epithelial cells through mitochondrial and MAPK/AKT pathways

Bisphenol A (BPA) can act as pathogenic agent on mammalian cells. However, whether BPA induces colonic epithelial cell damage remains unexplored. We used HCT116 cells as a model to investigate the negative effect of BPA on human colon cancer cells and explore the potential mechanism. Our results suggest that BPA decreased viability of HCT116 cells. BPA also caused serious oxidative damage to the colonic epithelium, as indicated by increased mitochondrial and intracellular reactive oxygen species (ROS) content and elevated malondialdehyde and H2O2 levels. Moreover, BPA depolarized the mitochondrial membrane potential and caused loss of mitochondrial integrity. Furthermore, BPA induced colonic epithelial cell apoptosis accompanied by upregulation of caspase3 and bax gene expression. Additionally, cell proliferation was inhibited significantly in HCT116 cells after the BPA treatment. We also studied the molecular mechanism involved in these effects and found that BPA inhibited proliferation of colonic epithelial cells through the mitogen activated protein kinase (MAPK) and AKT signaling pathways. Our data suggest that BPA triggers ROS generation as an initial step, followed by mediation of mitochondrial dysfunction, apoptosis, and proliferation of HCT116 cells. Moreover, MAPK/AKT signaling pathways were involved in BPA-induced toxicity of human colon cancer cells.

NoxO1 Controls Proliferation of Colon Epithelial Cells.

Reactive oxygen species (ROS) produced by enzymes of the NADPH oxidase family serve as second messengers for cellular signaling. Processes such as differentiation and proliferation are regulated by NADPH oxidases. In the intestine, due to the exceedingly fast and constant renewal of the epithelium both processes have to be highly controlled and balanced. Nox1 is the major NADPH oxidase expressed in the gut, and its function is regulated by cytosolic subunits such as NoxO1. We hypothesize that the NoxO1-controlled activity of Nox1 contributes to a proper epithelial homeostasis and renewal in the gut. NoxO1 is highly expressed in the colon. Knockout of NoxO1 reduces the production of superoxide in colon crypts and is not subsidized by an elevated expression of its homolog p47phox. Knockout of NoxO1 increases the proliferative capacity and prevents apoptosis of colon epithelial cells. In mouse models of dextran sulfate sodium (DSS)-induced colitis and azoxymethane/DSS induced colon cancer, NoxO1 has a protective role and may influence the population of natural killer cells. NoxO1 affects colon epithelium homeostasis and prevents inflammation.

Functional role of microRNA-135a in colitis

BackgroundInflammatory bowel disease (IBD) is one of the chronic gastrointestinal diseases with increasing risk of colon cancer development in the future. Apoptosis and inflammation play an important role in the etiology of this disease. MicroRNAs are associated with etiology of different diseases including IBD. In this study, we aimed to explore the role of miR-135a in the etiology of colitis in murine model of DSS-induced colitis.ResultsThe results showed that expression of miR-135a in colonic cells was suppressed and up-regulating miR-135a inhibited apoptosis and inflammation of colonic epithelial cells. Additionally, Hif1α was identified as the target gene of miR-135a which promoted apoptosis and inflammation as knockdown of Hif1α led to the suppression of both apoptosis and inflammation.ConclusionsOverexpression of miR-135a might be beneficial in IBD due to its anti-apoptosis and anti-inflammation effects in vitro.

A mitogenic lectin from Rhizoctonia bataticola arrests growth, inhibits metastasis, and induces apoptosis in human colon epithelial cancer cells.

The correlation between colorectal cancer (CRC) progression and altered expression of N-glycans can be considered in search for new biomarkers and anticancer agents to control CRC. Earlier N-glycan specific mitogenic lectin from Rhizoctonia bataticola (RBL) has been reported which has growth inhibitory and apoptotic effect on human ovarian and leukemic cells, but mitogenic effect on normal PBMCs revealing its clinical potential. Here, we report the effect of RBL on human colon cancer HT 29, SW480, and SW620 cell growth and its differential binding to human normal colon and cancer tissues. RBL has strong binding to both primary and metastatic colon cancer cells with MFI of 403, 404, and 192, respectively for HT 29, SW480, and SW620 cells. RBL shows dose and time dependent growth inhibitory effect with IC50 of 5, 6.4, and 6.8 µg/mL, respectively for HT 29, SW480, and SW620 cells. RBL inhibited the clonogenicity of colon carcinoma cells. RBL arrests metastatic SW620 cell growth at S phase, increased hypodiploid population by 6.1%, 14.3%, and 23.2%, respectively at 12, 24, and 36 h. Further, RBL induces SW620 cell apoptosis in time dependent manner, showed increased release of ROS and nuclear degradation compared to lectin untreated control. Adhesion, wound healing, invasion, and migration assays demonstrated anti-metastasis effect of RBL in SW620 cells apart from its growth inhibitory effect. Anti angiogenic effect of RBL was demonstrated by CAM assay. All these results show the promising potential of RBL both as diagnostic and therapeutic agent.

Costus root granules improve ulcerative colitis through regulation of TGF-β mediation of the PI3K/AKT signaling pathway.

Ulcerative colitis is a chronic nonspecific inflammatory disease that occurs in the colon and rectum. Costus root is a type of traditional Chinese medicine that exhibits antibacterial properties and serves an inhibitory role in the regeneration of gut bacteria. However, the molecular mechanisms underlying Costus root-mediated improvements in ulcerative colitis remain unclear. A complex formula of Costus root granules was created and investigated in the present study for its therapeutic effects in a rat model of ulcerative colitis. Ingredient dissolution into a traditional water decoction was used as a control. The potential mechanism mediated by Costus root granules was also analyzed in colonic epithelial cells isolated from the experimental rats. The results of the present study demonstrated that Costus root granule treatment inhibited inflammation in colonic tissue. Costus root granule treatment also suppressed the apoptosis of colonic epithelial cells isolated from the rat model of ulcerative colitis. Analyses of the underlying mechanisms of these effects indicated that the administration of Costus root granules increased transforming growth factor β expression, which activated the phosphoinositide 3-kinase/RAC-α serine/threonine-protein kinase signaling pathway in colonic epithelial cells. Notably, the administration of Costus root granules improved stomachache, diarrhea and hematochezia in and increased the body weight of, the ulcerative colitis rats. In conclusion, these results indicate that Costus root granules markedly ameliorate inflammation of the colonic epithelium, decrease the apoptosis of colonic epithelial cells and improve colonic function, which suggests that Costus root granules are an efficient agent for the treatment of ulcerative colitis.

Gut Epithelial Vitamin D Receptor Regulates Microbiota-Dependent Mucosal Inflammation by Suppressing Intestinal Epithelial Cell Apoptosis.

Recent studies show that colonic vitamin D receptor (VDR) signaling protects the mucosal epithelial barrier and suppresses colonic inflammation, but the underlying molecular mechanism remains to be fully understood. To investigate the implication of colonic VDR downregulation seen in patients with inflammatory bowel disease, we assessed the effect of gut epithelial VDR deletion on colonic inflammatory responses in an experimental colitis model. In a 2,4,6-trinitrobenzenesulfonic acid-induced colitis model, mice carrying VDR deletion in gut epithelial cells [VDRflox/flox (VDRf/f);Villin-Cre or VDRΔIEC] or in colonic epithelial cells (VDRf/f;CDX2-Cre or VDRΔCEC) developed more severe clinical colitis than VDRf/f control mice, characterized by more robust T-helper (TH)1 and TH17 responses, with greater increases in mucosal interferon (IFN)-γ+, interleukin (IL)-17+, and IFN-γ+IL-17+ T cells. Accompanying the severe mucosal inflammation was more profound colonic epithelial cell apoptosis in the mutant mice. Treatment with caspase inhibitor Q-VD-OPh dramatically reduced colitis severity and attenuated TH1 and TH17 responses in VDRΔCEC mice. The blockade of cell apoptosis also prevented the increase in mucosal CD11b+CD103+ dendritic cells (DCs), known to be critical for TH17-cell activation. Moreover, depletion of gut commensal bacteria with antibiotics eliminated the robust TH1 and TH17 responses and CD11b+CD103+ DC induction. Taken together, these observations demonstrate that gut epithelial VDR deletion aggravates epithelial cell apoptosis, resulting in increases in mucosal barrier permeability. Consequently, invading luminal bacteria activate CD11b+CD103+ DCs, which promote mucosal TH1 and TH17 responses. Therefore, gut epithelial VDR signaling controls mucosal inflammation by suppressing epithelial cell apoptosis.

Sulphate-reducing bacteria from ulcerative colitis patients induce apoptosis of gastrointestinal epithelial cells

The human microbiome consists of a multitude of bacterial genera and species which continuously interact with one another and their host establishing a metabolic equilibrium. The dysbiosis can lead to the development of pathology, such as inflammatory bowel diseases. Sulfide-producing prokaryotes, including sulphate-reducing bacteria (SRB) constituting different genera, including the Desulfovibrio, are commonly detected within the human microbiome recovered from fecal material or colonic biopsy samples. It has been proposed that SRB likely contribute to colonic pathology, for example ulcerative colitis (UC).The interaction of SRB with the human colon and intestinal epithelial cell lines has been investigated using Desulfovibrio indonesiensis as a model mono-culture and in a co-culture with E. coli isolate, and with SRB consortia from human biopsy samples.We find that D. indonesiensis, whether as a mono- or co-culture, was internalized and induced apoptosis in colon epithelial cells. This effect was enhanced in the presence of E. coli. The SRB combination obtained through enrichment of biopsies from UC patients induced apoptosis of a human intestinal epithelial cell line. This response was not observed in SRB enrichments from healthy (non-UC) controls. Importantly, apoptosis was detected in epithelial cells from UC patients and was not seen in epithelial cells of healthy donors. Furthermore, the antibody raised against exopolysaccharides (EPS) of D. indonesiensis cross reacted with the SRB population from UC patients but not with the SRB combination from non-UC controls. This study reinforces a correlation between the presence of sulphate-reducing bacteria and an inflammatory response in UC sufferers.

Cell cycle arrest and apoptosis induced by enteroaggregative Escherichia coli in cultured human intestinal epithelial cells.

Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen causing diarrhoeal diseases in multiple epidemiological and clinical settings. However, understanding of the pathogenesis of the disease caused by this organism is still suboptimal. Studies have indicated that enteric bacteria induced cell cycle arrest and apoptosis in host intestinal epithelial cells might play a vital role in the pathogenesis caused by these organisms. In this study an attempt was made to assess EAEC-induced apoptosis and cell cycle modulation in human intestinal epithelial cell lines. INT-407 and HCT-15 cells were infected with EAEC-T8 (clinical isolate) as well as plasmid cured variant of EAEC-T8 (EAEC-pT8). Propidium iodide staining was done to select the time of infection and the incubation period of the infected culture. Apoptosis was further assessed in EAEC infected both the cell lines by annexin-V-FLUOS & propidium iodide, cell death detection ELISA, DNA strand breaks and microscopic analysis. Further, the DNA content of the EAEC-infected cells at different phases of cell cycle was also monitored. We have found that EAEC could induce apoptosis in human small intestinal as well as colonic epithelial cell lines, which was assessed by the expression of phosphatidylserine on host cell surface, internucleosomal cleavage of host cell DNA and microscopic analysis of the characteristic apoptotic features of these cells. EAEC was also found to arrest cells at S phase and G2-M phase of the cell cycle. EAEC-T8 could induce maximum apoptosis and cell cycle modulation in both small intestinal and colonic epithelial cells. Further, we have observed that the plasmid of this organism had maximum contribution to these processes. The outcome of this study has undoubtedly led to a better understanding of the basic mechanism of pathogenesis caused by EAEC.

Abnormal apoptosis causes colitis and enteritis in patients with Hoyeraal–Hreidarsson syndrome due to DKC1 mutations

Background: Hoyeraal–Hreidarsson syndrome (HHS), the severe clinical variant of X-linked dyskeratosis congenita, is caused by germline mutations in telomere associated genes. HHS usually manifests within the first years of life and is characterized by progressive bone marrow failure, immunodeficiency, neurological features including microcephaly and developmental delay, as well as intrauterine growth retardation. The typical mucocutaneous manifestations are nail dysplasia, lacy skin pigmentation, and oral leukoplakia. Importantly, gastrointestinal involvement is reported in most patients with HHS, and may be the presenting feature. Given the spectrum of gastrointestinal diseases with a similar presentation, recognizing the unique gastrointestinal histopathology of HHS may facilitate earlier diagnosis and treatment. Methods: This case series highlights the gastrointestinal pathology findings of 2 patients with HHS caused by DKC1 gene mutations. Results: Gastrointestinal biopsies reveal loss of mucosal glands, regenerative glandular alterations and increased colonic epithelial cell apoptosis. Immunostaining of biopsies for cleaved caspase 3, a marker of cellular apoptosis, demonstrates abnormal nonapical and random locations of enterocyte exit which was further exacerbated by enteritis. Conclusion: Gastrointestinal involvement is usually the presenting feature of patients with HHS. This case series highlights the important role on gastrointestinal histopathology in facilitating a diagnosis of HHS. Statement of novelty: Detailed gastrointestinal biopsy images associated with HHS involving DKC1 mutations.

GYY4137 ameliorates intestinal barrier injury in a mouse model of endotoxemia

Intestinal barrier injury has been reported to play a vital role in the pathogenesis of endotoxemia. This study aimed to investigate the protective effect of GYY4137, a newly synthesized H2S donor, on the intestinal barrier function in the context of endotoxemia both in vitro and in vivo. Caco-2 (a widely used human colon cancer cell line in the study of intestinal epithelial barrier function) monolayers incubated with lipopolysaccharide (LPS) or TNF-α/IFN-γ and a mouse model of endotoxemia were used in this study.The results suggested that GYY4137 significantly attenuated LPS or TNF-α/IFN-γ induced increased Caco-2 monolayer permeability. The decreased expression of TJ (tight junction) proteins induced by LPS and the altered localization of TJs induced by TNF-α/IFN-γ was significantly inhibited by GYY4137; similar results were obtained in vivo. Besides, GYY4137 promoted the clinical score and histological score of mice with endotoxemia. Increased level of TNF-α/IFN-γ in the plasma and increased apoptosis in colon epithelial cells was also attenuated by GYY4137 in mice with endotoxemia. This study indicates that GYY4137 preserves the intestinal barrier function in the context of endotoxemia via multipathways and throws light on the development of potential therapeutic approaches for endotoxemia.

Effects of 5-aminosalicylic acid on proliferation and apoptosis of colonic epithelial cells in patients with ulcerative colitis

Objective To investigate proliferation and apoptosis of colonic epithelial cells from ulcerative colitis (UC) patients with active new-onset, remission and active recurrent stages, and to study the effects of 5-aminosalicylic acid (5-ASA) on proliferation and apoptosis of colonic epithelial cells during the treatment of inducing remission and maintanence remission. Methods From January 2002 to December 2012, twelve patients with mild-to-moderate UC who received treatment and long-term follow-up and achieved remission with 5-ASA and received long-term maintanence treatment with it were assigned to UC group. At the same period, 10 healthy individuals with negative endoscopy results or solitary colonic polyp were assigned to control group. The biopsy tissues from colonoscopy for pathological examination of patients in UC group at new-onset active stage, remission stage and recurrence stage were obtained. Levels of proliferation marker Ki-67 and apoptosis of colonic epithelial cells were determined by immunohistochemistry and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, respectively.Data were expressed by median(lower quartile, upper quartile). Kruskal-Wallis test and Mann-Whitney test were performed to compare the differences between groups and Bonferroni method was used for correction. Results The median proliferation indexes (PI) of colonic epithelial cells of UC patients at new-onset active period, remission period and recurrent active period were 31.65%(19.14%, 39.66%), 12.30%(11.11%, 14.10%) and 44.15%(33.65%, 52.45%), respectively, which were all higher than those of control group (7.89%(6.54%, 8.86%))there were statistically significant differentes among four groups (H=30.033, P<0.01); those of new onset active period and recurrent active period were both higher than remission period, and the differences were statistically significant (all P<0.05/6). The median apoptosis indexes (AI) of colonic epithelial cells in UC patients at new-onset active period, remission period and recurrent active period were 24.18%(17.81%, 27.16%), 44.19%(43.41%, 50.55%), 41.24%(33.78%, 46.24%), respectively, which were all higher than those of control group (2.65%(2.48%, 2.98%)), there were statistically significant differences among four groups (H=31.563, P<0.01); and those of remission and recurrent active period were both higher than new-onset active period (all P<0.05/6). The median apoptosis/proliferation ratios of control group, new onset active period, remission period and recurrent active period were 0.320 0(0.275 5, 0.425 0), 0.749 9(0.634 9, 1.115 8), 3.282 8(3.133 1, 4.406 8) and 1.008 2(0.801 9, 1.099 2), respectively, there were statically significant differences among four groups (H=29.441, P<0.01); those of new onset active period and recurrent active period were both lower than that of remission period and the differnces were statistically significant (both P<0.05/6). Conclusions Proliferation and apoptosis imbalance in colonic epithelial cells of UC patients is one of the important mechanisms for the pathgensis of UC. 5-ASA does not promote the proliferation of epithelial cells during UC remission period. Key words: Colitis, ulcerative; 5-aminosalicylic acid; Proliferation; Apoptosis