Intestinal Epithelial Cell Apoptosis Research Articles

Synergetic responses of intestinal microbiota and epithelium to dietary inulin supplementation in pigs.

Inulin is a soluble dietary fiber that has been implicated in regulating the intestinal health. Here, we describe a synergetic response of intestinal microbiota and epithelial functions to increased intake of inulin in a porcine model. Twenty growing-pigs were randomly allocated to two groups (n = 10) and fed with a basal diet (BD) or BD containing 0.5% inulin (INU) for 21 days. We show that INU supplementation not only elevated villus height and the abundance of zonula occludens-1 (ZO-1), but also increased acetate and butyrate concentrations in cecum (P < 0.05). Moreover, INU decreased IL-6 and TNFα secretion, and reduced intestinal epithelial cell apoptosis in ileum and cecum (P < 0.05). Interestingly, we observed an elevated 16S rRNA gene copies in cecum after INU ingestion (P < 0.05). INU had no influence on overall diversity, but acutely altered the abundance of specific bacteria. INU decreased the abundance of phylum Proteobacteria in ileum, but increased the phylum Bacteroidetes in the ileum and cecum (P < 0.05). INU significantly elevated the Lactobacillus spp. and Bacteroides spp. in the ileum and cecum, respectively. Importantly, INU elevated the expression levels of GPR43, GLP-2, and ZO-1, but decreased the expression levels of histone deacetylase 1 (HDAC1) and TNFα in the ileum and cecum mucosa (P < 0.05). Moreover, INU also elevated the expression levels of GPR109A and angiopoietin-4 (ANG-4) in the cecum mucosa (P < 0.05). This study indicated how the intestinal microbiome and epithelium adapt to inulin ingestion, and furthered our understanding of the mechanisms behind the dietary fiber-modulated intestinal microbiota and health.

Macleaya cordata extracts exert antiviral effects in newborn mice with rotavirus-induced diarrhea via inhibiting the JAK2/STAT3 signaling pathway.

Accumulating evidence demonstrates that Macleaya cordata extract exerts antiviral and anti-inflammatory effects in various diseases. The present study aimed to investigate the potential effects of M. cordata on rotavirus SA11-induced diarrhea in mice. Diarrhea severity, levels of inflammatory cytokines, histological changes in the small intestine and the underlying mechanisms were evaluated in rotavirus-stimulated mice treated with 1, 2 and 4 mg/kg/day M. cordata or 4 mg/kg/day ribavirin (positive control). M. cordata treatment effectively ameliorated rotavirus-induced diarrhea in a dose-dependent manner by decreasing viral RNA levels. In addition, M. cordata reduced the release of pro-inflammatory cytokines including migration inhibitory factor, interleukin (IL)-8, IL-β, interferon-γ and tumor necrosis factor-α, and elevated the secretion of the anti-inflammatory cytokine IL-10 following rotavirus infection. M. cordata inhibited intestinal epithelial cell apoptosis and improved intestinal inflammation after rotavirus infection. The study also revealed that M. cordata exerted antiviral and anti-inflammatory effects on rotavirus-induced diarrhea by suppressing the Janus kinase 2 (JAK2)/STAT3 pathway, as reflected by decreased protein expression of phosphorylated (p)-JAK2 and p-STAT3. Overall, M. cordata effectively inhibited the inflammation caused by rotavirus, which was closely associated with the suppression of JAK2/STAT3 phosphorylation. These data suggested that M. cordata may be applied as a treatment for rotavirus-induced diarrhea.

GTS-21 Promotes α7 nAChR to Alleviate Intestinal Ischemia-Reperfusion-Induced Apoptosis and Inflammation of Enterocytes

BackgroundIntestinal ischemia-reperfusion injury is a serious intestinal disease, with main symptoms of inflammatory reaction and severe oxidative damage. In addition, GTS-21-induced α7 nAChR has been shown to exert anti-inflammatory effects and anti-oxidation effects in various organs. However, whether α7 nAChR can alleviate ischemia-reperfusion-induced intestinal injury is unclear.Material/MethodsWe used intestinal epithelial cells (IEC-6) to perform the experiments. Oxygen glucose deprivation/reoxygenation (OGD/R) was used to simulate the physiological environment of ischemia-reperfusion. First, the expression of α7 nAChR was determined in these cells which was cultured under OGD/R conditions. After that, the GTS-21 was used to treat these cells and the levels of inflammatory factors (TNF-α, IL-1β, IL-6, and IL-10) were assessed by ELISA. Next, the levels of ROS, SOD, and MDA were determined in IEC-6 cells. Finally, the apoptosis rates of IEC-6 cells were measured by flow cytometry.ResultsResults showed that the expression of TNF-α, IL-1β, and IL-6 was enhanced when the IEC-6 cells were cultured under OGD/R conditions. However, after treatment with GTS-21, the levels of these proinflammatory factors were suppressed. In addition, the levels of ROS and MDA were also inhibited and the expression of SOD was promoted after GTS-21 treatment. We also found that the ratios of apoptotic cells declined after GTS-21 treatment.ConclusionsGTS-21-induced α7 nAChR decreased the OGD/R-induced inflammatory response, oxidative damage, and apoptosis of intestinal epithelial cells.

Loganin attenuates intestinal injury in severely burned rats by regulating the toll-like receptor 4/NF-κB signaling pathway.

Severe burns may lead to intestinal inflammation and oxidative stress, resulting in intestinal barrier damage and gut dysfunction. Loganin, an iridoid glycoside compound, has been isolated from Cornus officinalis Sieb. et Zucc; however, its role in the treatment of burn injury is yet to be fully elucidated. Therefore, the present study examined the effect of loganin administration on burn-induced intestinal inflammation and oxidative stress after severe burns in male Sprague-Dawley rats. Histological injury was assessed by hematoxylin and eosin staining. Furthermore, cytokine expression in intestinal tissues was measured by ELISA and reverse transcription-quantitative PCR. Antioxidative activities were assessed by determining the levels of reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA). Apoptosis was detected by flow cytometry. Apoptosis-related proteins, toll-like receptor 4 (TLR4) protein and NF-κB translocation were examined by western blotting. Immunohistochemical staining was used to observe TLR4 and NF-κB p65 expression in intestinal tissues. The present study suggested that loganin administration significantly reduced burn injury-induced intestinal histological changes, tumor necrosis factor-α, interleukin (IL)-6 and IL-1β production and oxidative stress, evidenced by decreased ROS levels and MDA content (P<0.05). Furthermore, loganin increased SOD, CAT and GSH-Px levels and intestinal epithelial cell apoptosis. Loganin treatment also significantly inhibited activation of the TLR4/NF-κB signaling pathway in the intestine of severely burned rats (P<0.05). In conclusion, loganin reduced burns-induced intestinal inflammation and oxidative stress, potentially by regulating the TLR4/NF-κB signaling pathway.

Does Dysbiosis Drive CVID Enteropathy by Enhancing Bile Acid Cytotoxicity?

Abstract Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by defects in the humoral immune response. CVID patients commonly present with a gastrointestinal enteropathy characterized by intestinal malabsorption. CVID is linked with abnormal gut microbiota composition (i.e. dysbiosis), which may be a contributing factor to CVID enteropathy. Recently, we demonstrated that CD19−/− mice represent a spontaneous animal model of CVID enteropathy that develop an associated dysbiosis. We now expand upon this work to identify how microbial dysbiosis may contribute to CVID enteropathy. We first demonstrate a significant association between dysbiosis in CD19−/− mice with defects in bile acid (BA) homeostasis (increased luminal BA titers and decreased abundance of conjugated BA species). Enteropathy in CD19−/− mice is associated with enhanced apoptosis of intestinal epithelial cells (IECs). Adoptive transfer of WT B cells (but not CD19−/− B cells) reduces luminal BA concentrations, protects IECs from apoptosis, and resolves enteropathy, indicating that functional B cells are important regulators of BA homeostasis and gastrointestinal health. Additionally, dietary supplementation with a taurine conjugated BA also protects animals from disease demonstrating that enhanced BA deconjugation enhances disease severity. Finally, direct ablation of the ability of a model commensal microbe to deconjugate BAs also rescues animals from disease. Collectively, these data suggest that enhanced bacterial deconjugation of BAs in the gut contributes to CVID enteropathy by enhancing BA cytotoxicity to IECs. Thus, microbial BA deconjugation may represent a novel therapeutic target for the treat of CVID enteropathy.

SiRNA Interference with Notch1 Affects LPS-Induced Apoptosis of Rat Intestinal Epithelial Cells

Notch signaling is associated with the pathogenesis of ulcerative colitis (UC). This study investigated whether the Notch regulates intestinal epithelial cell apoptosis and reactive oxygen species (ROS) production. Rats were separated into UC model group and control group followed by analysis of the expression of Notch1, Hes1 and OLFM4 by real-time PCR and western blot, content of malondialdehyde (MDA), superoxide dismutase (SOD) and caspase-3 as well as cell apoptosis by flow cytometry. Under LPS stimulation conditions, IEC-6 cells were divided into LPS + siRNA-NC group and LPS + siRNA-Notch1 group and Notch1, Hes1 and OLFM4 level as well as ROS was measured. Compared with control group, the activity of caspase-3, MDA and apoptosis in colon tissue of UC model were significantly increased with decreased SOD activity and increased Notch1, Hes1 and OLFM4 expression. LPS treatment can significantly up-regulate the expression of Notch1, Hes1 and OLFM4 in IEC-6 cells, increase cell apoptosis and ROS production. Notch1 interference by siRNA significantly downregulated Notch1, Hes1, and OLFM4 in IEC cells, and increased cell apoptosis and ROS production. UC lesions can activate Notch signaling pathway in colon tissue, which may play a role in repair. Interference with Notch1 signaling may aggravate intestinal epithelial cell apoptosis and ROS production in an inflammatory environment.

Heat shock transcription factor 2 inhibits intestinal epithelial cell apoptosis through the mitochondrial pathway in ulcerative colitis

UC is a chronic inflammatory disease of the colonic mucosa and lacks effective treatments because of unclear pathogenesis. Excessive apoptosis of IECs damages the intestinal epithelial barrier and is involved in the progression of UC, but the mechanism is unknown. HSPs are important in maintaining homeostasis and regulate apoptosis through the mitochondrial pathway. In our previous studies, HSF2, an important regulator of HSPs, was highly expressed in UC patients and negatively correlated with inflammation in mice and IECs. Therefore, we hypothesized that HSF2 may protect against intestinal mucositis by regulating the apoptosis of IECs. In this study, a DSS-induced colitis model of hsf2−/− mice was used to explore the relationship between HSF2 and apoptosis in IECs for the first time. The expression of HSF2 increased in the WT + DSS group compared with that in the WT + H2O group. Moreover, the extent of apoptosis was more severe in the KO + DSS group than in the WT + DSS group. The results showed that HSF2 was negatively correlated with apoptosis in vivo. The expression of HSF2 in Caco-2 cells was changed by lentiviral transfection, and the expression of Bax, cytoplasmic Cyto-C, Cleaved Caspase-9 and Cleaved Caspase-3 were negatively correlated with the different levels of HSF2. These results suggest that HSF2 negatively regulates apoptosis of IECs through the mitochondrial pathway. This may be one of the potential mechanisms to explain the protective role of HSF2 in UC.

The influence of selected gastrointestinal parasites on apoptosis in intestinal epithelial cells.

Studies on the parasite–host interaction may provide valuable information concerning the modulation of molecular mechanisms as well as of the host immune system during infection. To date, it has been demonstrated that intestinal parasites may affect, among others, the processes of digestion in the gastrointestinal system of the host, thus limiting the elimination of the parasite, the immune response as well as inflammation. However, the most recent studies suggest that intestinal parasites may also affect modulation of the apoptosis pathway of the host. The present paper presents the latest scientific information on the influence of intestinal parasite species (Blastocystis sp., Giardia sp., Cryptosporidium sp., Trichuris sp., Entamoeba histolytica, Nippostrongylus brasiliensis, Heligmosomoides polygyrus) on the molecular mechanisms of apoptosis in intestinal epithelial cells. This paper stresses that the interdependency between the intestinal parasite and the host results from the direct effect of the parasite and the host’s defense reactions, which lead to modulation of the apoptosis pathways (intrinsic and extrinsic). Moreover, the present paper presents the role of proteins involved in the mechanisms of apoptosis as well as the physiological role of apoptosis in the host’s intestinal epithelial cells.

Astilbin ameliorates deoxynivalenol-induced oxidative stress and apoptosis in intestinal porcine epithelial cells (IPEC-J2).

Deoxynivalenol (DON) is a common mycotoxin, which often induces oxidative stress and cytotoxicity in humans and animals. Astilbin (AST), as a natural antioxidant, exhibits multiple pharmacological functions. The aim of this study was to investigate the effects of AST on alleviating DON-induced cytotoxicity in intestinal porcine epithelial cells (IPEC-J2). The results demonstrated that 0.5 μg/mL DON stimulation for 6 hours induced oxidative stress, inflammation and apoptosis in IPEC-J2 cells. AST enhanced the cell viability in a dose- and time-dependent manner. The addition of 20 μg/mL AST significantly increased cell viability, superoxide dismutase and catalase activities, Bcl-2 gene expression and the Bcl-2/Bax ratio (P < .05), and decreased lactate dehydrogenase release, malondialdehyde content and the relative expressions of genes associated with inflammation and apoptosis such as interleukin-6 and -8, tumor necrosis factor-alpha, cyclooxygenase-2, nuclear factor-kappaB, Bax and caspase-3 (P < .05). Simultaneously, zonula occludens-1, claudin-1 and PepT1 gene expressions were upregulated and occludin, ASCT2 and GLUT2 gene expressions were downregulated by the addition of AST, compared with the DON group (P < .05). These results indicated that 20 μg/mL AST could ameliorate oxidative stress, inflammation and apoptosis by enhancing antioxidant enzyme activities and intestinal barrier function, and reducing the expressions of inflammation and apoptosis genes, as well as improve the barrier function and nutrient transport and absorption in DON-induced IPEC-J2 cells.

Restoring mitochondrial function and normalizing ROS-JNK/MAPK pathway exert key roles in glutamine ameliorating bisphenol A-induced intestinal injury.

Bisphenol A (BPA) is toxic to the reproductive and nervous system, even carcinogenetic in humans and animals. However, few studies focused on effects of BPA on the intestinal tract. Here, we detected BPA-induced injuries on intestinal mucosa and explored a reliable approach to counteract BPA effects. C57BL/6J mice were gavage BPA or BPA accompanied with ingestion of 4% (w/w) of glutamine for 4-wks. In vitro, IEC-6 cells were treated with 0.4mmol/L BPA for 6hours mimicking acute injury and 0.2mmol/L BPA for 12hours followed with or without the inclusion of 4mmol/L glutamine for 12hours to determine cell renewal, mitochondrial function and ROS-JNK/MAPK pathway upon moderate BPA exposure. As results, BPA exposure caused severe intestinal injury, and disturbed intestinal epithelial cell proliferation and apoptosis, accompanied with mitochondrial malfunction and activated JNK/MAPK pathway as well. Notably, glutathione metabolism was implicated in BPA-induce injury. Glutamine could well rescue cell renewal and mitochondrial function from BPA exposure-induced injuries. In conclusion, we demonstrated impaired effect of BPA exposure on intestinal functions, which could be well counteracted by glutamine partly via restoring mitochondrial function and normalizing ROS-JNK/MAPK pathway. Thereby, we provided a novel application of glutamine to rescue intestinal injury.

N-Acetyl Serotonin Alleviates Oxidative Damage by Activating Nuclear Factor Erythroid 2-Related Factor 2 Signaling in Porcine Enterocytes.

Apoptosis of intestinal epithelial cells following oxidative stress is a major cause of mucosal barrier dysfunction and is associated with the pathogenesis of various gastrointestinal diseases. Although L-tryptophan (Trp) is known to improve intestinal integrity and function, a beneficial effect of N-acetyl serotonin (NAS), a metabolite of Trp, on the apoptosis of enterocytes and the underlying mechanisms remain largely unknown. In the present study, we showed that porcine enterocytes treated with 4-hydroxy-2-nonenal (4-HNE), a metabolite of lipid peroxidation, led to upregulation of apoptotic proteins, including Bax and cleaved caspase-3, and reduction of tight junction proteins. These effects of 4-HNE were significantly abrogated by NAS. In addition, NAS reduced ROS accumulation while increasing the intracellular concentration of glutathione (GSH), and the abundance of the Nrf2 protein in the nucleus and its downstream target proteins. Importantly, these protective effects of NAS were abrogated by Atra, an inhibitor of Nrf2, indicating a dependence on Nrf2 signaling. Taken together, we demonstrated that NAS attenuated oxidative stress-induced cellular injury in porcine enterocytes by regulating Nrf2 signaling. These findings provide new insights into a functional role of NAS in maintaining intestinal homeostasis.

Effects of probiotics on apoptosis and proliferation of intestinal epithelial cells in rats with necrotizing enterocolitis

Objective To study the effect of compound Lactobacillus acidophilus on the apoptosis of intestinal epithelial cells and expression of proliferating cell nuclear antigen (PCNA) in rats with necrotizing enterocolitis, so as to provide some theoretical reference for the application of compound Lactobacillus acidophilus in the prevention and treatment of necrotizing enterocolitis(NEC) for newborn infants. Methods Thirty-six 48-hour old newborn SD rats were randomly divided into normal group, model group and intervention group, with 12 rats in each group.The NEC model was established by hypoxia and cold stimulation for 3 days, and the intervention group was given the compound Lactobacillus acidophilus at the same time; after the model was established, all the rats were sacrificed after 12 hours of fasting and their intestinal tissues were excised by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) method to determine the condition of apoptosis and to detect expression of the PCNA gene in the intestinal tissues by fluorescent quantitative PCR. Results (1) The apoptosis rates of the intestinal tissues: in the model group, the normal group and the intervention group were (88.33±2.77)%, (4.75±0.75)% and (44.41±4.81)%, respectively, and the differences had statistical significance(all P<0.05). (2) The PCNA expression in the intestinal tissue by real-time PCR in the model group, normal group and intervention group were (9.28±3.26)×10-4, (15.35±1.91)×10-4 and (12.09±3.06)×10-4, respectively, and the differences had statistical significance(all P<0.05). Conclusions The apoptosis rate of intestinal epithelial cells increases and PCNA expression decreases when NEC occurs.Compound Lactobacillus acidophilus can reduce apoptosis, promote intestinal epithelial cell proliferation, and thus help to reduce the risk and severity of NEC in neonatal rats. Key words: Neonatal necrotizing enterocolitis; Apoptosis; Proliferating cell nuclear antigen; Compound Lactobacillus acidophilus

Clinically Approved Carbon Nanoparticles with Oral Administration for Intestinal Radioprotection via Protecting the Small Intestinal Crypt Stem Cells and Maintaining the Balance of Intestinal Flora.

The exploration of an old drug for new biomedical applications has an absolute predominance in shortening the clinical conversion time of drugs for clinical application. In this work, carbon nanoparticles suspension injection (CNSI), the first clinically approved carbon nanoparticles in China, is explored as a new nano-radioprotective agent for potent intestinal radioprotection. CNSI shows powerful radioprotective performance in the intestine under oral administration, including efficient free radical scavenging ability, good biosafety, high chemical stability, and relatively long retention time. For example, CNSI shows high reactive oxygen species (ROS) scavenging activities, which effectively alleviates the mitochondrial dysfunction and DNA double-strand breaks to protect the cells against radiation-induced damage. Most importantly, this efficient ROS scavenging ability greatly helps restrain the apoptosis of the small intestinal epithelial and crypt stem cells, which decreases the damage of the mechanical barrier and thus relieves radiation enteritis. Moreover, CNSI helps remove the free radicals in the intestinal microenvironment and thus maintain the balance of intestinal flora so as to mitigate the radiation enteritis. The finding suggests a new application of clinically approved carbon nanoparticles, which not only promotes the development of new intestinal radioprotector, but also has a great potential for clinical transformation.

MCL1 Is Required for Maintenance of Intestinal Homeostasis and Prevention of Carcinogenesis in Mice

Background & AimsIntestinal epithelial homeostasis depends on a tightly regulated balance between intestinal epithelial cell (IEC) death and proliferation. While the disruption of several IEC death regulating factors result in intestinal inflammation, the loss of the anti-apoptotic BCL2 family members BCL2 and BCL2L1 has no effect on intestinal homeostasis in mice. We investigated the functions of the antiapoptotic protein MCL1, another member of the BCL2 family, in intestinal homeostasis in mice.MethodsWe generated mice with IEC-specific disruption of Mcl1 (Mcl1ΔIEC mice) or tamoxifen-inducible IEC-specific disruption of Mcl1 (i-Mcl1ΔIEC mice); these mice and mice with full-length Mcl1 (controls) were raised under normal or germ-free conditions. Mice were analyzed by endoscopy and for intestinal epithelial barrier permeability. Intestinal tissues were analyzed by histology, in situ hybridization, proliferation assays, and immunoblots. Levels of calprotectin, a marker of intestinal inflammation, were measured in intestinal tissues and feces.ResultsMcl1ΔIEC mice spontaneously developed apoptotic enterocolopathy, characterized by increased IEC apoptosis, hyperproliferative crypts, epithelial barrier dysfunction, and chronic inflammation. Loss of MCL1 retained intestinal crypts in a hyperproliferated state and prevented the differentiation of intestinal stem cells. Proliferation of intestinal stem cells in MCL1-deficient mice required WNT signaling and was associated with DNA damage accumulation. By 1 year of age, Mcl1ΔIEC mice developed intestinal tumors with morphologic and genetic features of human adenomas and carcinomas. Germ-free housing of Mcl1ΔIEC mice reduced markers of microbiota-induced intestinal inflammation but not tumor development.ConclusionThe antiapoptotic protein MCL1, a member of the BCL2 family, is required for maintenance of intestinal homeostasis and prevention of carcinogenesis in mice. Loss of MCL1 results in development of intestinal carcinomas, even under germ-free conditions, and therefore does not involve microbe-induced chronic inflammation. Mcl1ΔIEC mice might be used to study apoptotic enterocolopathy and inflammatory bowel diseases.

Deficiency in the anti-apoptotic protein DJ-1 promotes intestinal epithelial cell apoptosis and aggravates inflammatory bowel disease via p53

Parkinson disease autosomal recessive, early onset 7 (PARK7 or DJ-1) is involved in multiple physiological processes and exerts anti-apoptotic effects on multiple cell types. Increased intestinal epithelial cell (IEC) apoptosis and excessive activation of the p53 signaling pathway is a hallmark of inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn's disease (CD). However, whether DJ-1 plays a role in colitis is unclear. To determine whether DJ-1 deficiency is involved in the p53 activation that results in IEC apoptosis in colitis, here we performed immunostaining, real-time PCR, and immunoblotting analyses to assess DJ-1 expression in human UC and CD samples. In the inflamed intestines of individuals with IBD, DJ-1 expression was decreased and negatively correlated with p53 expression. DJ-1 deficiency significantly aggravated colitis, evidenced by increased intestinal inflammation and exacerbated IEC apoptosis. Moreover, DJ-1 directly interacted with p53, and reduced DJ-1 levels increased p53 levels both in vivo and in vitro and were associated with decreased p53 degradation via the lysosomal pathway. We also induced experimental colitis with dextran sulfate sodium in mice and found that compared with DJ-1-/- mice, DJ-1-/-p53-/- mice have reduced apoptosis and inflammation and increased epithelial barrier integrity. Furthermore, pharmacological inhibition of p53 relieved inflammation in the DJ-1-/- mice. In conclusion, reduced DJ-1 expression promotes inflammation and IEC apoptosis via p53 in colitis, suggesting that the modulation of DJ-1 expression may be a potential therapeutic strategy for managing colitis.

LncRNACNN3-206 activates intestinal epithelial cell apoptosis and invasion by sponging miR-212, an implication for Crohn's disease

BACKGROUNDStatistics indicate that the incidence of Crohn’s disease (CD) is rising in many countries. The poor understanding on the pathological mechanism has limited the development of effective therapy against this disease. Previous studies showed that long noncoding RNAs (lncRNAs) could be involved in autoimmune diseases including CD, but the detailed molecular mechanisms remain unclear.AIMTo identify the differentially expressed lncRNAs in the intestinal mucosa associated with CD, and to characterize their pathogenic role(s) and related mechanisms.METHODSThe differential expression of lncRNAs was screened by high-throughput RNA sequencing, and the top candidate genes were validated in an expanded cohort by real-time PCR. The regulatory network was predicted by bioinformatic software and competitive endogenous RNA analysis, and was characterized in Caco-2 and HT-29 cell culture using methods of cell transfection, real-time PCR, Western blotting analysis, flow cytometry, and cell migration and invasion assays. Finally, these findings were confirmed in vivo using a CD animal model.RESULTSThe 3' end of lncRNACNN3-206 and the 3’ UTR of Caspase10 contain high-affinity miR212 binding sites. lncRNACNN3-206 expression was found to be significantly increased in intestinal lesions of CD patients. Activation of the lncRNACNN3-206-miR-212-Caspase10 regulatory network led to increased apoptosis, migration and invasion in intestinal epithelial cells. Knockdown of lncRNACNN3-206 expression alleviated intestinal mucosal inflammation and tissue damage in the CD mouse model.CONCLUSIONlncRNACNN3-206 may play a key role in CD pathogenesis. lncRNACNN3-206 could be a therapeutic target for CD treatment.

PNU-282987 Attenuates Intestinal Epithelial Barrier Dysfunction in LPS-Induced Endotoxemia.

PNU-282987, the α7 acetylcholine receptor(α7nAchR) agonist, has been repeatedly reported to play a key role in anti-inflammatory action of multiple disease. However, little is known about its effect on LPS-induced intestinal epithelial barrier dysfunction. This study investigated the protective effects and mechanisms of PNU-282987 on intestinal epithelial barrier dysfunction in lipopolysaccharide(LPS)-induced endotoxemic rats. Endotoxemia models were induced by intraperitoneal injection of 10mg/kg LPS. In the endotoxemic group, results showed increases in ileum mucosal permeability, ultrastructural damage of tight junction and redistribution of zonula occludens-1, apoptosis of intestinal epithelial cells and caspase-3 activation. These changes were significantly improved after PNU-282987 administration(P < 0.05). Pretreatment with α-bungarotoxin before PNU-282987 administration reversed the effects of PNU-282987(P < 0.05). These results indicate that PNU-282987 exerts protective effects on intestinal epithelial barrier dysfunction in LPS-induced endotoxemic rats, and its mechanism may involve the improvement of zonula occludens-1 and inhibition of enterocyte apoptosis in an α7nAchR-dependent manner.

Set up of an in vitro model to study early host-parasite interactions between newly excysted juveniles of Fasciola hepatica and host intestinal cells using a quantitative proteomics approach

Fasciola hepatica is the causative agent of fasciolosis, a parasitic zoonosis of global distribution causing significant economic losses in animal production and a human public health problem in low-income countries. Hosts are infected by ingestion of aquatic plants carrying metacercariae. Once ingested, the juvenile parasites excyst in the small intestine and, after crossing it, they follow a complex migratory route that lead the parasites to their definitive location in the bile ducts. Despite being a critical event in the progression of the infection, the available data on the cross-talk relationships between the parasite and the host at an early stage of the infection are scarce. The objective of the present work is to characterize the proteomic changes occurring in both the parasite and the host, through the development of a novel in vitro model, to shed light on the molecular pathways of communication between the newly excysted juveniles (NEJ) from F. hepatica and the host’s intestinal epithelium. For this, in vitro excystation of F. hepatica metacercariae was carried out and NEJ were obtained. Additionally, optimal conditions of growth and expansion of mouse primary small intestinal epithelial cells (MPSIEC) in culture were fine-tuned. Tegumentary and somatic parasite antigens (NEJ-Teg and NEJ-Som), as well as host cell protein lysate (MPSIEC-Lys) were obtained before and after 24 h co-culture of NEJ with MPSIEC. We used an isobaric tags for relative and absolute quantitation (iTRAQ)-based strategy to detect 191 and 62 up-regulated, and 112 and 57 down-regulated proteins in the NEJ-Teg and NEJ-Som extracts, respectively. Similarly, 87 up-regulated and 73 down-regulated proteins in the MPSIEC-Lys extract were identified. Taking into account the biological processes in which these proteins were involved, interesting mechanisms related to parasite development, invasion and evasion, as well as manipulation of the host intestinal epithelial cell adhesion, immunity and apoptosis pathways, among others, could be inferred, taking place at the host-parasite interface. The further understanding of these processes could constitute promising therapeutic targets in the future against fasciolosis.

P033 Combination of vedolizumab with tacrolimus is more efficient in reducing intestinal inflammation than vedolizumab alone in the DSS-induced acute colitis mouse model

Abstract Background The humanised monoclonal antibody vedolizumab is used in the treatment of ulcerative colitis (UC) and Crohn’s disease (CD). Vedolizumab reduces intestinal inflammation through inhibition of the integrin heterodimer α 4β 7, responsible for the homing of T cells to the intestinal mucosa. Recent studies have also shown a possible involvement of vedolizumab in the regulation of the innate immune system. Particularly in CD, only a fraction of patients respond to vedolizumab treatment, and combination therapy with immunosuppressant drugs, such as the calcineurin-inhibitor tacrolimus, might prove beneficial. The aim of this study was to assess if co-treatment of vedolizumab and tacrolimus is more efficient in reducing intestinal inflammation in an acute colitis mouse model and to unravel the underlying molecular mechanisms. Methods NOD-SCID-SGM3 mice were reconstituted with human CD34+ cells and treated with 3% dextrane sodium sulphate (DSS) in drinking water to induce acute colitis. Mice were treated with vedolizumab alone (30mg/kg, inject 3 days prior to DSS-start and 50mg/kg at day 0 and at day 4 of DSS-treatment), tacrolimus alone (1mg/kg/day intraperitoneally), or a combination of tacrolimus and vedolizumab during colitis induction. Results As expected, DSS-treatment induced colitis in mice as observed by weight loss, diarrhoea, colon shortening, and endoscopic signs of inflammation categorised by the MEICS score. This translated histologically to an increased immune cell infiltration and epithelial erosion. Vedolizumab and tacrolimus treatment alone did not significantly reduce colitis severity, although endoscopy showed slightly less severe inflammation in vedolizumab-treated mice. Combination of vedolizumab and tacrolimus, however, clearly reduced colonoscopy and histology scores. DSS-treatment increased the number of CD3 T cells and CD68 macrophages in the intestine, an effect counteracted by vedolizumab or tacrolimus alone and further pronounced by combination treatment. Particularly vedolizumab treatment, either alone or in combination, caused clear reduction of pro-inflammatory M1 macrophages. Additionally, vedolizumab alone or combined decreased the levels of intestinal epithelial cell apoptosis as indicated by staining for cleaved caspase-3. Conclusion Our data demonstrate that the anti-inflammatory effect of vedolizumab is potentiated by co-treatment with tacrolimus. Notably, the combination of both drugs was more efficient in reducing T-cell and macrophage infiltration into the intestine. This indicates that the combination of vedolizumab with immunosuppressant drugs might prove beneficial for patients that do not respond to vedolizumab-only therapy.

Effects of Stearic Acid on Proliferation, Differentiation, Apoptosis, and Autophagy in Porcine Intestinal Epithelial Cells.

Stearic acid (SA), a saturated long-chain fatty acid consisting of 18 carbon atoms, is widely found in feed ingredients, such as corn, soybeans, and wheat. However, the roles of SA in the renewal of intestinal epithelial cells remain unclear. In the present study, we found that 0.01-0.1 mM SA promoted IPEC-J2 cell differentiation and did not affect IPEC-J2 cell viability. In addition, the results showed that the viability of IPEC-J2 cells was inhibited by SA in a time- and dose-dependent manner at high concentrations. Flow cytometry and western blot analysis suggested that SA induced apoptosis, autophagy and ER stress in cells. In addition, the amounts of triglyceride were significantly increased upon challenge with SA. Moreover, the decrease in the viability of cells induced by SA could be attenuated by 4-PBA, an inhibitor of ER stress. In summary, SA accelerated IPEC-J2 cell differentiation at 0.01-0.1 mM. Furthermore, SA induced IPEC-J2 cell apoptosis and autophagy by causing ER stress.