Etomidate Alleviates Ischemia-Anoxia Reperfusion Injury in Intestinal Epithelial Cells by Inhibiting the Activation of traf6-Regulated NF-KB Signaling

Objective: The present study aimed to investigate the role of etomidate in intestinal cell ischemia and hypoxia-reperfusion injury and potential mechanisms. Method: In this study, we establish the intestinal epithelial cells ischemia-reperfusion model in vitro. CCK8 was used to detect cell viability and flow cytometry assay was used to detect apoptosis levels of treated OGD/R model cells. ELISA measured the expression level of oxidative stress factors and inflammatory factors. Furthermore, western blot assay was used to detect the expression the apoptosis-related factors and TNFR-associated factors in treated OGD/R model cells. Result: Etomidate does not affect the activity of intestinal epithelial cells, and can protect intestinal epithelial cells to reduce ischemiareperfusion injury, and the expression of inflammatory factors and oxidative stress in cells with mild intestinal epithelial ischemia-reperfusion injury. Etomidate alleviates apoptosis of intestinal epithelial ischemia-reperfusion injury cells. Etomidate inhibits the activation of traf6-mediated NF-κB signal during ischemia-anoxia reperfusion of intestinal epithelial cells. Conclusion: Taken together, our study demonstrated that etomidate attenuates inflammatory response and apoptosis in intestinal epithelial cells during ischemic hypoxia-reperfusion injury and inhibits activation of NF-κB signaling regulated by TRAF6.

Improving gut function in pigs to prevent pathogen colonization

Intestinal pathogens causing either clinical or sub-clinical infections increase pig morbidity and (or) mortality, resulting in economic losses and wider socio-economic impacts on pig production. An optimally functioning gastrointestinal tract (GIT) is fundamental to combatting intestinal pathogen colonisation at all the stages of life. This requires successful development and maintenance of key gut functions: digestive function; the gastro-intestinal cell line barrier; gut-associated lymphoid tissue (GALT); and gut-associated microbiota. This chapter first discusses research on genes associated with pathogen resistance and porcine immune response. It then reviews risk factors associated with gut mucosa impairment as well as dietary strategies to control risk factors and improve gut functionality in preventing intestinal pathogen colonisation.

Fatty acids produced by the gut microbiota dampen host inflammatory responses by modulating intestinal SUMOylation

The gut microbiota produces a wide variety of metabolites, which interact with intestinal cells and contribute to host physiology. These metabolites regulate intestinal cell activities by modulating either gene transcription or post-translational modifications of gut proteins. The effect of gut commensal bacteria on SUMOylation, an essential ubiquitin-like modification in intestinal physiology, remains however unknown. Here, we show that short chain fatty acids (SCFAs) and branched chain fatty acids (BCFAs) produced by the gut microbiota increase protein SUMOylation in different intestinal cell lines in a pH-dependent manner. We demonstrate that these metabolites induce an oxidative stress which inactivates intestinal deSUMOylases and promotes the hyperSUMOylation of chromatin-bound proteins. In order to determine the impact of these modifications on intestinal physiology, we focused on the NF-kappaB signaling pathway, a key player in inflammation known to be regulated by SUMOylation. We demonstrated that the hyperSUMOylation induced by SCFAs/BCFAs inhibits the activation of the NF-kappaB pathway in intestinal cells by blocking the degradation of the inhibitory factor IkappaBalpha in response to TNFalpha. This results in a decrease in pro-inflammatory cytokines expression, such as IL8 or CCL20, as well as a decrease in intestinal epithelial permeability in response to TNFalpha. Together, our results reveal that fatty acids produced by gut commensal bacteria regulate intestinal physiology by modulating SUMOylation and illustrate a new mechanism of dampening of host inflammatory responses by the gut microbiota.

Mycotoxin Interactions along the Gastrointestinal Tract: In Vitro Semi-Dynamic Digestion and Static Colonic Fermentation of a Contaminated Meal

Aflatoxin B1 (AFB1) and ochratoxin A (OTA) naturally co-occur in several foods, but no studies have followed the fate of mycotoxins’ interactions along the gastrointestinal tract using in vitro digestion models. This study used a novel semi-dynamic model that mimics gradual acidification and gastric emptying, coupled with a static colonic fermentation phase, in order to monitor mycotoxins’ bioaccessibility by the oral route. AFB1 and OTA bioaccessibility patterns differed in single or co-exposed scenarios. When co-exposed (MIX meal), AFB1 bioaccessibility at the intestinal level increased by ~16%, while OTA bioaccessibility decreased by ~20%. Additionally, a significant increase was observed in both intestinal cell viability and NO production. With regard to mycotoxin–probiotic interactions, the MIX meal showed a null effect on Lactobacillus and Bifidobacterium strain growth, while isolated AFB1 reduced bacterial growth parameters. These results were confirmed at phylum and family levels using a gut microbiota approach. After colonic fermentation, the fecal supernatant did not trigger the NF-kB activation pathway, indicating reduced toxicity of mycotoxins. In conclusion, if single exposed, AFB1 will have a significant impact on intestinal viability and probiotic growth, while OTA will mostly trigger NO production; in a co-exposure situation, both intestinal viability and inflammation will be affected, but the impact on probiotic growth will be neglected.

The Protective Effects of Lactoferrin on Aflatoxin M1-Induced Compromised Intestinal Integrity

Aflatoxin M1 (AFM1), the only toxin with maximum residue levels in milk, has adverse effects on the intestinal barrier, resulting in intestinal inflammatory disease. Lactoferrin (LF), one of the important bioactive proteins in milk, performs multiple biological functions, but knowledge of the protective effects of LF on the compromised intestinal barrier induced by AFM1 has not been investigated. In the present study, results using Balb/C mice and differentiated Caco-2 cells showed that LF intervention decreased AFM1-induced increased intestinal permeability, improved the protein expression of claudin-3, occludin and ZO-1, and repaired the injured intestinal barrier. The transcriptome and proteome were used to clarify the underlying mechanisms. It was found that LF reduced the intestinal barrier dysfunction caused by AFM1 and was associated with intestinal cell survival related pathways, such as cell cycle, apoptosis and MAPK signaling pathway and intestinal integrity related pathways including endocytosis, tight junction, adherens junction and gap junction. The cross-omics analysis suggested that insulin receptor (INSR), cytoplasmic FMR1 interacting protein 2 (CYFIP2), dedicator of cytokinesis 1 (DOCK1) and ribonucleotide reductase regulatory subunit M2 (RRM2) were the potential key regulators as LF repaired the compromised intestinal barrier. These findings indicated that LF may be an alternative treatment for the compromised intestinal barrier induced by AFM1.

Gastrointestinal motility and Intestinal structure following oral exposure to acrylamide in Wistar rats

Introduction: Acrylamide, a byproduct of the cooking process, has been reported to be a toxicant with likely carcinogenic properties. Its impairment of gastric function has been previously reported. In this study its effects on gastrointestinal motility and intestinal structure was investigated in male Wistar rats.Methods: Forty-five rats (120-180g) were divided into 3 equal groups (n=15) and treated p.o with either 0.2ml distilled-water, or acrylamide (7.5mg/kg and 15mg/kg respectively) for 28days. Thereafter, gastric emptying and intestinal motility was assessed. Intestinal structure (duodenum, jejunum and ileum), mucosal and intestinal cell counts were evaluated using histological techniques.Results: Gastric emptying and intestinal transit time increased (p<0.05) in the experimental (acrylamidetreated; 7.5mg/kg and 15mg/kg) groups compared to control. Mucosal cell counts (duodenum, jejunum and ileum) and ileum intestinal cell counts (p<0.05) were reduced in the experimental groups compared to control. Compared to control, duodenal samples of the experimental groups showed severe coagulative necrosis and sloughing off of the villi, luminal filling with necrotic debris, disruption and necrosis of the crypts of Lieberkühn, moderate polymorphonuclear cell infiltration and vascular congestion. These pathologies albeit with less severity were also observed in the jejunum and ileum of acrylamide treated groups.Conclusion: Increased oral exposure to acrylamide impairs gastric emptying, intestinal motility, mucus secretion and compromises digestive and absorptive functions of the small intestines, especially the duodenum. These observations may be ascribed to acrylamide-induced impaired neuronal signaling, autonomic neuropathy, oxidative stress, inflammation and cell necrosis. Keywords: Acrylamide, gastrointestinal tract, gastric emptying, intestinal motility, small intestines

Pro-Resolving Factors Released by Macrophages After Efferocytosis Promote Mucosal Wound Healing in Inflammatory Bowel Disease

Nonresolving inflammation is a critical driver of several chronic inflammatory diseases, including inflammatory bowel diseases (IBD). This unresolved inflammation may result from the persistence of an initiating stimulus or from the alteration of the resolution phase of inflammation. Elimination of apoptotic cells by macrophages (a process called efferocytosis) is a critical step in the resolution phase of inflammation. Efferocytosis participates in macrophage reprogramming and favors the release of numerous pro-resolving factors. These pro-resolving factors exert therapeutic effects in experimental autoimmune arthritis. Here, we propose to evaluate the efficacy of pro-resolving factors produced by macrophages after efferocytosis, a secretome called SuperMApo, in two IBD models, namely dextran sodium sulfate (DSS)-induced and T cell transfer-induced colitis. Reintroducing these pro-resolving factors was sufficient to decrease clinical, endoscopic and histological colitis scores in ongoing naive T cell-transfer-induced colitis and in DSS-induced colitis. Mouse primary fibroblasts isolated from the colon demonstrated enhanced healing properties in the presence of SuperMApo, as attested by their increased migratory, proliferative and contractive properties. This was confirmed by the use of human fibroblasts isolated from patients with IBD. Exposure of an intestinal epithelial cell (IEC) line to these pro-resolving factors increased their proliferative properties and IEC acquired the capacity to capture apoptotic cells. The improvement of wound healing properties induced by SuperMApo was confirmed in vivo in a biopsy forceps-wound colonic mucosa model. Further in vivo analysis in naive T cell transfer-induced colitis model demonstrated an improvement of intestinal barrier permeability after administration of SuperMApo, an intestinal cell proliferation and an increase of α-SMA expression by fibroblasts, as well as a reduction of the transcript coding for fibronectin (Fn1). Finally, we identified TGF-β, IGF-I and VEGF among SuperMApo as necessary to favor mucosal healing and confirmed their role both in vitro (using neutralizing antibodies) and in vivo by depleting these factors from efferocytic macrophage secretome using antibody-coated microbeads. These growth factors only explained some of the beneficial effects induced by factors released by efferocytic macrophages. Overall, the administration of pro-resolving factors released by efferocytic macrophages limits intestinal inflammation and enhance tissue repair, which represents an innovative treatment of IBD.

Characterization of increased mucus production of HT29-MTX-E12 cells grown under Semi-Wet interface with Mechanical Stimulation

The intestinal mucus layer plays a crucial role in human health. To study intestinal mucus function and structure in vitro, the mucus-producing intestinal cell line HT29-MTX-E12 has been commonly used. However, this cell line produces only low amounts of the intestine-specific MUC2. It has been shown previously that HT29-MTX-E12 cells cultured under Semi-Wet interface with Mechanical Stimulation (SWMS) produced higher amounts of MUC2, concomitant with a thicker mucus layer, compared to cells cultured conventionally. However, it remains unknown which underlying pathways are involved. Therefore, we aimed to further explore the cellular processes underlying the increased MUC2 production by HT29-MTX-E12 cells grown under SWMS conditions. Cells grown on Transwell membranes for 14 days under static and SWMS conditions (after cell seeding and attachment) were subjected to transcriptome analysis to investigate underlying molecular pathways at gene expression level. Caco-2 and LS174T cell lines were included as references. We characterized how SWMS conditions affected HT29-MTX-E12 cells in terms of epithelial barrier integrity, by measuring transepithelial electrical resistance, and cell metabolism, by monitoring pH and lactate production per molecule glucose of the conditioned medium. We confirmed higher MUC2 production under SWMS conditions at gene and protein level and demonstrated that this culturing method primarily stimulated cell growth. In addition, we also found evidence for a more aerobic cell metabolism under SWMS, as shown previously for similar models. In summary, we suggest different mechanisms by which MUC2 production is enhanced under SWMS and propose potential applications of this model in future studies.