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Agaro-oligosaccharides (AGO), hydrolysis products of agarose, is known to have antioxidant and anti-inflammatory properties. Speculating that AGO is effective for preventing aging, we investigated the longevity-supporting effects of AGO and their mechanisms using Caenorhabditis elegans.Caenorhabditis elegans were fed AGO from young adulthood. The lifespan, locomotory activity, lipofuscin accumulation, and heat stress resistance of the worms were examined. To elucidate mechanisms of AGO-mediated longevity, we conducted comprehensive expression analysis using microarrays. Moreover, we used quantitative real-time PCR (qRT-PCR) to verify the genes showing differential expression levels. Furthermore, we measured the lifespan of loss-of-function mutants to determine the genes related to AGO-mediated longevity.AGO extended the lifespan of C. elegans, reduced lipofuscin accumulation, and maintained vigorous locomotion. The microarray analysis revealed that the endoplasmic reticulum-unfolded protein response (ER-UPR) and insulin/insulin-like growth factor-1-mediated signaling (IIS) pathway were activated in AGO-fed worms. The qRT-PCR analysis showed that AGO treatment suppressed sir-2.1 expression, which is a negative regulator of ER-UPR. In loss-of-function mutant of sir-2.1, AGO-induced longevity and heat stress resistance were decreased or cancelled completely. Furthermore, the pro-longevity effect of AGO was decreased in loss-of-function mutants of abnormal Dauer formation (daf) -2 and daf-16, which are IIS pathway-related genes.AGO delays the C. elegans aging process and extends their lifespan through the activations of ER-UPR and the IIS pathway.

Cancer cachexia and the associated skeletal muscle wasting are considered poor prognostic factors, although effective treatment has not yet been established. Recent studies have indicated that the pathogenesis of skeletal muscle loss may involve dysbiosis of the gut microbiota and the accompanying chronic inflammation or altered metabolism. In this study, we evaluated the possible effects of modifying the gut microenvironment with partially hydrolyzed guar gum (PHGG), a soluble dietary fiber, on cancer-related muscle wasting and its mechanism using a colon-26 murine cachexia model. Compared with a fiber-free (FF) diet, PHGG contained fiber-rich (FR) diet-attenuated skeletal muscle loss in cachectic mice by suppressing the elevation of the major muscle-specific ubiquitin ligases Atrogin-1 and MuRF1, as well as the autophagy markers LC3 and Bnip3. Although tight-junction markers were partially reduced in both FR and FF diet-fed cachectic mice, the abundance of Bifidobacterium, Akkermansia, and unclassified S24-7 family increased by FR diet, contributing to the retention of the colonic mucus layer. The reinforcement of the gut barrier function resulted in the controlled entry of pathogens into the host system and reduced circulating levels of lipopolysaccharide-binding protein (LBP) and IL-6, which in turn led to the suppression of proteolysis by downregulating the ubiquitin-proteasome system and autophagy pathway. These results suggest that dietary fiber may have the potential to alleviate skeletal muscle loss in cancer cachexia, providing new insights for developing effective strategies in the future.

Background The gut-liver axis has attracted much interest in the context of chronic liver disease pathogenesis. Prebiotics such as dietary fibers were shown to attenuate non-alcoholic fatty liver disease (NAFLD) by modulating gut microbiota. Partially hydrolyzed guar gum (PHGG), a water-soluble dietary fiber, has been reported to alleviate the symptoms of various intestinal diseases and metabolic syndromes. However, its effects on NAFLD remain to be fully elucidated. Aim To determine whether treatment with PHGG attenuates NAFLD development in mice through the gut-liver axis. Methods Seven-week-old male C57BL/6J mice with increased intestinal permeability were fed a control or atherogenic (Ath) diet (a mouse model of NAFLD) for 8 wk, with or without 5% PHGG. Increased intestinal permeability was induced through chronic intermittent administration of low-dose dextran sulfate sodium. Body weight, liver weight, macroscopic findings in the liver, blood biochemistry [aspartate aminotransferase (AST) and alanine aminotransferase (ALT), total cholesterol, triglyceride, free fatty acids, and glucose levels], liver histology, myeloperoxidase activity in liver tissue, mRNA expression in the liver and intestine, serum endotoxin levels in the portal vein, intestinal permeability, and microbiota and short-chain fatty acid (SCFA) profiles in the cecal samples were investigated. Results Mice with increased intestinal permeability subjected to the Ath diet showed significantly increased serum AST and ALT levels, liver fat accumulation, liver inflammatory (tumor necrosis factor-α and monocyte chemotactic protein-1) and fibrogenic (collagen 1a1 and α smooth muscle actin) marker levels, and liver myeloperoxidase activity, which were significantly attenuated by PHGG treatment. Furthermore, the Ath diet combined with increased intestinal permeability resulted in elevated portal endotoxin levels and activated toll-like receptor (TLR) 4 and TLR9 expression, confirming that intestinal permeability was significantly elevated, as observed by evaluating the lumen-to-blood clearance of fluorescein isothiocyanate-conjugated dextran. PHGG treatment did not affect fatty acid metabolism in the liver. However, it decreased lipopolysaccharide signaling through the gut-liver axis. In addition, it significantly increased the abundance of cecal Bacteroides and Clostridium subcluster XIVa. Treatment with PHGG markedly increased the levels of SCFAs, particularly, butyric acid, acetic acid, propionic acid, and formic acid, in the cecal samples. Conclusion PHGG partially prevented NAFLD development in mice through the gut-liver axis by modulating microbiota and downstream SCFA profiles.

COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which spread worldwide in 2020, is an urgent problem to be overcome. The ORF8 of SARS-CoV-2 has been suggested to be associated with the symptoms of COVID-19, according to reports of clinical studies. However, little is known about the function of ORF8. As one of the ways to advance the functional analysis of ORF8, mass production of ORF8 with the correct three-dimensional structure is necessary. In this study, we attempted to produce ORF8 protein by chemically inducible protein production system using tobacco BY-2 cells. An ORF8-producing line was generated by the Agrobacterium method. As a result, the production of ORF8 of 8.8 ± 1.4 mg/L of culture medium was confirmed. SDS-PAGE and nuclear magnetic resonance (NMR) analysis confirmed that the ORF8 produced by this system is a dimeric form with a single structure, unlike that produced in Escherichia coli. Furthermore, it was suggested that the ORF8 produced by this system was glycosylated. Through this study, we succeeded in producing ORF8 folded into a single structure in a chemically inducible protein production system using tobacco BY-2 cells. It is expected that the functional analysis of ORF8 will be advanced using the ORF8 produced by this system and that it will greatly contribute to the development of antibodies and therapeutic agents targeting ORF8.

Consumption of yacon (Smallanthus sonchifolius) is associated with beneficial effects such as prevention of metabolic diseases. Yacon root is known to contain various bioactive components including indigestible carbohydrates, but the alteration of intestinal environment after treatment with yacon has not been fully investigated. This study investigated yacon-containing diet effects on the intestinal environment in mice, including microbial composition, short-chain fatty acid levels, and mucus content. After mice were administered yacon-containing diet for 4 weeks, 16S rRNA gene sequencing analyses revealed their fecal microbiota profiles. Organic acid concentrations in cecal contents were measured using an HPLC system. Compared to the control group, yacon-containing diet-received mice had significantly higher the concentrations of succinic acid, lactic acid, acetic acid, and propionic acid. The fecal mucin content was also higher in yacon-containing diet-received mice. Results of 16S rRNA gene sequencing analyses showed that the relative abundances of 27 taxa differed significantly in yacon-containing diet-received mice. Furthermore, results show effects of yacon administration on intestinal inflammation using 2,4,6-trinitrobenzene sulfonic acid induced colitis model in mice. Increased colonic damage and myeloperoxidase activity after 2,4,6-trinitrobenzene sulfonic acid treatment were suppressed in yacon-containing diet-received mice. Results suggest that oral intake of yacon root modulates the intestinal environment, thereby inhibiting intestinal inflammation.

A probiotic is considered a live microbial feed supplement that has beneficial effects on the host. In this study, the probiotic property by which Enterococcus faecium HS-08 strengthens the immune system was investigated. Using a murine model, we evaluated the abilities of this strain to increase intestinal short-chain fatty acid contents and to induce the production of mucosal immunoglobulin A (IgA), which are crucial for mucosal immune systems. Various amounts (0%, 0.0038%, 0.038%, or 0.38%) of strain HS-08 cells were administered to BALB/cAJcl mice, which resulted in a dose-dependent increase of fecal IgA levels. A qRT-PCR analysis of Peyer’s patch cells revealed that the gene expression of retinal-dehydrogenase, interleukin 6, B-cell-activating factor, and a proliferation-inducing ligand were increased, which leads to IgA secretion via a T-cell-independent mechanism. The administration of 0.038% and 0.38% of strain HS-08 cells also increased fecal acetate levels, which plays an important role for maintaining immune functions. This cecal floral analysis and the stability of strain HS-08 against gastrointestinal digestion suggest that this strain can inhabit the host intestine. In conclusion, the administration of E. faecium HS-08 increased intestinal acetate levels and enhanced IgA secretion, which may result in strengthening of the mucosal immune system.

The components of lactic acid bacteria that exert immunostimulatory effects are of increasing interest for therapeutic and prophylactic options, such as alternatives to antibiotics, cognitive enhancements, and vaccine adjuvants. LTAs act as immunostimulatory molecules in the host innate immune system by interacting with pattern recognition receptors.

Betalain pigments are mainly produced by plants belonging to the order of Caryophyllales. Betalains exhibit strong antioxidant activity and responds to environmental stimuli and stress in plants. Recent reports of antioxidant, anti-inflammatory and anti-cancer properties of betalain pigments have piqued interest in understanding their biological functions. We investigated the effects of betalain pigments (betanin and isobetanin) derived from red-beet on amyloid-β (Aβ) aggregation, which causes Alzheimer's disease. Non-specific inhibition of Aβ aggregation against Aβ40 and Aβ42 by red-beet betalain pigments, in vitro was demonstrated using the thioflavin t fluorescence assay, circular dichroism spectroscopy analysis, transmission electron microscopy and nuclear magnetic resonance (NMR) analysis. Furthermore, we examined the ability of red-beet betalain pigments to interfere with Aβ toxicity by using the transgenic Caenorhabditis elegans model, which expresses the human Aβ42 protein intracellularly within the body wall muscle. It responds to Aβ-toxicity with paralysis and treatment with 50 μM red-beet betalain pigments significantly delayed the paralysis of C. elegans. These results suggest that betalain pigments reduce Aβ-induced toxicity.

Background Food allergy (FA) is a common disease in children; thus, a high level of safety is required for its prevention and treatment. Colonic regulatory T cells (Tregs) have been suggested to attenuate FA. We investigated the Treg-inducing ability and anti-FA effects of carotenoids, a pigment contained in vegetables and fruits. Methods C57BL/6N mice were fed a diet containing 0.01% (w/w) of lycopene, β-carotene, astaxanthin or lutein for 4 weeks, and the population of colonic Tregs was assessed. Subsequently, to evaluate the Treg-inducing ability of lycopene, splenic naive CD4+ T cells from BALB/c mice were cultured with anti-CD3/CD28 antibody, TGF-β and lycopene, and the frequencies of Tregs were examined. The effect of 0.1% (w/w) lycopene containing diet on FA was investigated in OVA-induced FA model BALB/c mice. Results In screening, only lycopene significantly increased the frequency and number of colonic Tregs. Lycopene also increased Treg differentiation in splenic naive CD4+ T cells. In FA mice, lycopene feeding significantly increased the number of colonic Tregs and attenuated allergic symptoms. The expression levels of IL-4, IL-9 and IL-13 mRNA in colonic mucosa were also significantly reduced by lycopene. IL-9 is known to induce proliferation of mast cells, and we found that lycopene feeding significantly reduced the number of mast cells in the colonic mucosa of FA mice. Conclusion Our results suggest that lycopene, a carotenoid present in many common foods on the market, may have the potential to induce colonic Tregs and suppress FA symptoms.

Bile acid sequestrants are used as medicinal drugs to treat dyslipidemia and type 2 diabetes. We found that cholestyramine, a bile acid sequestrant, increases cecal short-chain fatty acid (SCFA) production and intestinal immunoglobulin A (IgA) in C57BL/6J mice. In a 12-week high-fat diet study, feeding cholestyramine (2% (w/w)) significantly promoted C2-C4 SCFAs in the cecum by approximately 1.6-fold and fecal IgA by 1.8-fold. In an 8-week normal-fat diet study, feeding cholestyramine (1 and 2%) increased the cecal propionic acid content by approx. 2.0-fold. Fecal IgA was also significantly increased at 4 weeks (1%: 1.7-fold; 2%: 2.1-fold) and 8 weeks (1%: 1.8-fold; 2%: 2.0-fold) in the normal-fat diet study. These results indicate that bile acid sequestrants may exert their physiological functions, such as intestinal IgA production, through SCFA-dependent signaling pathways.

NEW FINDINGS What is the central question of this study? Exercise for type 2 diabetes patients treated with insulin therapy involves the risk of hypoglycaemia. Dipeptidyl peptidase-4 (DPP-4) inhibitors can be effective in combination with exercise because they reduce the incidence of hypoglycaemia. We evaluated the effect of this combination of treatments on hepatic lipid metabolism in diabetic KK/Ta mice. What is the main finding and its importance? The combination of a DPP-4 inhibitor and exercise, which lowers the risk of hypoglycaemia, is useful for improving insulin resistance by inhibiting excess insulin secretion and decreasing hepatic lipid accumulation, validated by downregulated CD36. ABSTRACT The role of exercise training in prevention of diabetes and/or dyslipidaemia has been firmly established. Dipeptidyl peptidase-4 (DPP-4) inhibitors improve insulin sensitivity and have attracted attention as therapeutics for hepatic lipid accumulation. The effect of a combination of DPP-4 inhibitor and exercise training on the prevention and treatment of hepatic lipid accumulation is unclear. Here, we investigated whether alogliptin, a DPP-4 inhibitor, enhances the preventive effect of exercise-induced hepatic lipid accumulation in diabetic mice. Balb/c and KK/Ta mice were fed a high-fat diet. Mice were divided into the following five groups: B, Balb/c mice; K, KK/Ta mice; K-A, KK/Ta mice with alogliptin (0.01%); K-Ex, KK/Ta mice with exercise training (3 days week-1 , 15-20 m min-1 for 30 min); and K-Ex+A, KK/Ta mice with alogliptin and exercise training (n = 8 or 9 mice per group). After 8 weeks, glucose, insulin and triglyceride concentrations in the blood and triglyceride levels in the liver were significantly lower in the K-Ex+A group than in the K group. The liver expression level of PPAR-γ in the K group was significantly higher than that in the other groups. Additionally, the liver CD36 expression level was significantly lower in the K-Ex+A and B groups than in the K group. Thus, combined therapy of a DPP-4 inhibitor with exercise training was effective against high-fat diet-induced hepatic lipid accumulation in KK/Ta mice. The results of this study provide useful support for the practice of safe exercise therapy even in diabetic patients who require treatment with a DPP-4 inhibitor.

Background and Aims Peroxiredoxin 4 (PRDX4), a secretory protein that is preferentially retained in the endoplasmic reticulum (ER), is encoded by a gene located on the X chromosome and highly expressed in colonic tissue. In this study, we investigated the role of PRDX4 by means of male PRDX4-knockout (PRDX4-/y) mice in the development of intestinal inflammation using a dextran sulfate sodium (DSS)-induced colitis model. Materials and methods Acute colitis was induced with DSS (2.5% in drinking water) in wild-type (WT) and PRDX4-/y male C57BL/6 mice. Histological and biochemical analyses were performed on the colonic tissues. Results PRDX4 was mainly localized in the colonic epithelial cells in WT mice. The disease activity index (DAI) scores of PRDX4-/y mice were significantly higher compared to those of WT mice. Specifically, PRDX4-/y mice showed marked body weight loss and shortening of colon length compared to WT mice, whereas the myeloperoxidase levels were increased in PRDX4-/y compared to WT mice. In addition, the mRNA expression levels of TNF-α and IFN-γ were significantly higher in the colonic mucosa of PRDX4-/y compared to WT mice. Moreover, the levels of CHOP and activated caspase 3 were higher in the colonic tissues of PRDX4-/y compared to WT mice following treatment with DSS. The ER also showed greater expansion in PRDX4-/y than WT mice, which was consistent with severe ER stress under PRDX4 deficiency. Conclusion Our results demonstrated that the lack of PRDX4 aggravated the colonic mucosal damage induced by DSS. Because PRDX4 functions as an ER thiol oxidase as well as an antioxidant, DSS induced oxidative damage and ER stress to a greater degree in PRDX4-/y than WT mice. These findings suggest that PRDX4 may represent a novel therapeutic molecule in intestinal inflammation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has the characteristic accessory protein ORF8. Although clinical reports indicate that ORF8 variant strains (Δ382 and L84S variants) are less likely to cause severe illness, functional differences between wild-type and variant ORF8 are unknown. Furthermore, the physicochemical properties of the ORF8 protein have not been analyzed. In this study, the physicochemical properties of the wild-type ORF8 and its L84S variant were analyzed and compared. Using the tobacco BY-2 cell production system, which has been successfully used to produce the wild-type ORF8 protein with a single conformation, was used to successfully produce the ORF8 L84S variant protein at the same level as wild-type ORF8. The produced proteins were purified, and their temperature and pH dependencies were examined using nuclear magnetic resonance spectra. Our data suggested that the wild-type and L84S variant ORF8 structures are highly stable over a wide temperature range. Both proteins displayed an aggregated conformation at higher temperature that reverted when the temperature was decreased to room temperature. Moreover, ORF8 precipitated at acidic pH and this precipitation was reversed when the solution pH was shifted to neutral. Interestingly, the L84S variant exhibited greater solubility than wild-type ORF8 under acidic conditions. Thus, the finding indicated that conformational stability and reversibility of ORF8 are key properties related to function in oppressive environments.

Colonoscopy is the gold standard for detecting earlier stages of CRC, although screening of patients is difficult because of invasiveness, low compliance and procedural health risks. Therefore, the need for new screening methods for CRC is rising. Previous studies have demonstrated the diagnostic ability of serum BAs; however, the results have been inconsistent. In this study, we conducted a comprehensive analysis of serum BAs from patients with CRC and verified their diagnostic ability to detect CRC.A total of 56 CRC patients (Free CA, 3epi-DCACDCA, 3-dehydro CA, GCA and TCA were extracted as principal component (PC) 1 and free 3-dehydroDCA as PC 2 by canonical discriminant function coefficients. The verification of discriminability using cross-validation method revealed that the correct classification rate was 66.3% for original data and 52.6% for cross-validation data.A combined analysis using comprehensive serum BA concentration can be an efficient method for screening CRC.

Higher consumption of trans fatty acid (TFA) is a risk factor for several inflammatory diseases including inflammatory bowel disease (IBD). However, the detailed mechanisms by which TFA intake affects IBD pathology remain unclear. We demonstrate here that elaidate, a trans-isomer of oleate, enhances interleukin (IL)-1β production through the activation of NLRP3 inflammasome in mouse bone marrow-derived macrophages (BMDMs). Oleate has no effect on IL-1β production. Elaidate also induces oxidative stress and activates endoplasmic reticulum stress in BMDMs. The elaidate-induced IL-1β production is suppressed by co-treatments with antioxidants and a chemical chaperone. Furthermore, we analyze the effects of elaidate administration on intestinal inflammation using 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis model in mice. Increased colonic damage and myeloperoxidase activity after TNBS treatment are elevated by elaidate administration. Also, TNBS treatment induces IL-1β production in colonic mucosa; elaidate administration enhances the induction. We believe that these data reveal some mechanisms by which the TFA intake is associated with increased risk for IBD.

The inhalation of carbon monoxide (CO) gas and the administration of CO-releasing molecules were shown to inhibit the development of intestinal inflammation in a murine colitis model. However, it remains unclear whether CO promotes intestinal wound healing. Herein, we aimed to evaluate the therapeutic effects of the topical application of CO-saturated saline enemas on intestinal inflammation and elucidate the underlying mechanism. Acute colitis was induced with trinitrobenzene sulfonic acid (TNBS) in male Wistar rats. A CO-saturated solution was prepared via bubbling 50% CO gas into saline and was rectally administrated twice a day after colitis induction; rats were sacrificed 3 or 7 days after induction for the study of the acute or healing phases, respectively. The distal colon was isolated, and ulcerated lesions were measured. In vitro wound healing assays were also employed to determine the mechanism underlying rat intestinal epithelial cell restitution after CO treatment. CO solution rectal administration ameliorated acute TNBS-induced colonic ulceration and accelerated ulcer healing without elevating serum CO levels. The increase in thiobarbituric acid-reactive substances and myeloperoxidase activity after induction of acute TNBS colitis was also significantly inhibited after CO treatment. Moreover, the wound healing assays revealed that the CO-saturated medium enhanced rat intestinal epithelial cell migration via the activation of Rho-kinase. In addition, the activation of Rho-kinase in response to CO treatment was confirmed in the inflamed colonic tissue. Therefore, the rectal administration of a CO-saturated solution protects the intestinal mucosa from inflammation and accelerates colonic ulcer healing through enhanced epithelial cell restitution. CO may thus represent a novel therapeutic agent for the treatment of inflammatory bowel disease.

Gut microbiota have profound effects on bile acid metabolism by promoting deconjugation, dehydrogenation, and dehydroxylation of primary bile acids in the distal small intestine and colon. High-fat diet-induced dysbiosis of gut microbiota and bile acid dysregulation may be involved in the pathology of steatosis in patients with non-alcoholic fatty liver disease. Epigallocatechin-3-gallate (EGCG), the most abundant polyphenolic catechin in green tea, has been widely investigated for its inhibitory or preventive effects against fatty liver. The aim of the present study was to investigate the effects of EGCG on the abundance of gut microbiota and the composition of serum bile acids in high-fat diet-fed mice and determine the specific bacterial genera that can improve the serum bile acid dysregulation associated with EGCG anti-hepatic steatosis action. Male C57BL/6N mice were fed with the control diet, high-fat diet, or high-fat diet + EGCG at a concentration of 0.32% for 8 weeks. EGCG significantly inhibited the increases in weight, the area of fatty lesions, and the triglyceride content in the liver induced by the high-fat diet. Principal coordinate analysis revealed significant differences in microbial structure among the groups. At the genus level, EGCG induced changes in the microbiota composition in high-fat diet-fed mice, showing a significantly higher abundance of Adlercreutzia, Akkermansia, Allobaculum and a significantly lower abundance of Desulfovibrionaceae. EGCG significantly reversed the decreased population of serum primary cholic acid and β-muricholic acid as well as the increased population of taurine-conjugated cholic acid, β-muricholic acid and deoxycholic acid in high-fat diet-fed mice. Finally, the correlation analysis between bile acid profiles and gut microbiota demonstrated the contribution of Akkermansia and Desulfovibrionaceae in the improvement of bile acid dysregulation in high-fat diet-fed mice by treatment with EGCG. In conclusion, the present study suggests that EGCG could alter bile acid metabolism, especially taurine deconjugation, and suppress fatty liver disease by improving the intestinal luminal environment.

A probiotic is considered a live microbial feed supplement that has beneficial effects on the host. In this study, the probiotic property by which

Cancer cachexia and the associated skeletal muscle wasting are considered poor prognostic factors, although effective treatment has not yet been established. Recent studies have indicated that the pathogenesis of skeletal muscle loss in age-related sarcopenia may involve dysbiosis of the gut microbiota and the accompanying chronic inflammation or altered metabolism, the so-called ‘gut-muscle axis’. However, limited evidence is available on the relationship between cancer-related sarcopenia and the gut environment. Dietary fiber, a non-digestible nutrient including polysaccharides which modulate composition and activity of the gut microbiota, are now noticed for its beneficial effects. In this study, we evaluated the possible effects of modifying the gut microenvironment with partially hydrolyzed guar gum (PHGG), a soluble dietary fiber, on cancer-related muscle wasting and its mechanism using a colon-26 murine cachexia model. Compared to a fiber-free (FF) diet, PHGG contained fiber-rich (FR) diet attenuated body weight and skeletal muscle loss in cachectic mice by suppressing the elevation of the major muscle-specific ubiquitin ligases Atrogin-1 and MuRF1, as well as the autophagy markers LC3 and Bnip3. Although tight junction markers were partially reduced in both FR and FF diet-fed cachectic mice, the abundance of Bifidobacterium, Akkermansia, and unclassified S24-7 family increased by FR diet, contributing to the significant retention of the colonic mucus layer. The reinforcement of the gut barrier function resulted in the controlled entry of pathogens into the host system and reduced circulating levels of lipopolysaccharide-binding protein (LBP) and IL-6, which in turn led to the suppression of proteolysis by downregulating the ubiquitin-proteasome system and autophagy pathway. Moreover, cecal short chain fatty acid (SCFA) levels were relatively low in cachectic mice, although the reduction was mitigated when PHGG supplementation was given. To explore the effects of SCFAs on skeletal muscle mass, we used a well-established in vitro myotube culture model. Exposure of C2C12 myotubes to LPS induced a significant decrease in the diameter, but was reversed on administering a cocktail of SCFAs by suppressing the elevation of Atrogin-1, and upregulating the expression of PGC1α and Tfam, markers of mitochondrial biogenesis. Taken together, our findings suggest that dietary fiber may have the potential to alleviate skeletal muscle loss in cancer cachexia, providing new insights for developing effective strategies in the future. Citation Format: Tomoki Sakakida, Takeshi Ishikawa, Toshifumi Doi, Ryuichi Morita, Yasuko Hirai, Katsura Mizushima, Yasuki Higashimura, Ken Inoue, Tetsuya Okayama, Kazuhiko Uchiyama, Tomohisa Takagi, Aya Abe, Ryo Inoue, Yoshito Itoh, Yuji Naito. Modulation of the gut microenvironment by water-soluble dietary fiber alleviates cancer-induced muscle wasting in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5953.

Intestinal fibrosis with stricture formation is a severe complication of Crohn's disease (CD). Though new therapeutic targets to enable the prevention or treatment of intestinal fibrosis are needed, markers of this condition and the basic mechanisms responsible have not been established. NADPH oxidase (NOX) 4 has already been reported to play a key role in models of fibrogenesis, including that of the lung. However, its importance in intestinal fibrogenesis remains unclear. In this study, we examined the role of NOX4 in collagen production by intestinal myofibroblasts stimulated with transforming growth factor (TGF)-β1. Using LmcMF cells, an intestinal subepithelial myofibroblast (ISEMF) line, we first examined the induction of collagen production by TGF-β1. Subsequently, we investigated the role of NOX4 in TGF-β1-induced collagen I production in these cells using SB525334 (an SMAD2/3 inhibitor), diphenyleneiodonium (an NOX inhibitor), and Nox4 small interfering RNA (siRNA). Production of collagen was assessed with Sirius red staining, and Nox4 expression was measured by quantitative real-time PCR. Reactive oxygen species (ROS) production was determined using DCFDA and fluorescent microscopy. We observed that TGF-β1 induced collagen production via NOX4 activation and ROS generation in LmcMF cells. Nox4 siRNA and inhibitors of TGF-β1 receptor and NOX significantly reduced TGF-β1-induced ROS and collagen production. Thus, in the present study, we revealed that collagen production in ISEMFs is induced via an NOX4-dependent pathway. This work supports a function for NOX4 in intestinal fibrogenesis and identifies it as a potential therapeutic target in recalcitrant fibrotic disorders of CD patients.

Background and aim Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycol that regulates cell proliferation, tissue repair, and tumorigenesis. Despite evidence linking SPARC to inflammation, the mechanisms are unclear. Accordingly, the role of SPARC in intestinal inflammation was investigated. Methods Colitis was induced in wild-type (WT) and SPARC knockout (KO) mice using trinitrobenzene sulfonic acid (TNBS). Colons were assessed for damage; leukocyte infiltration; Tnf, Ifng, Il17a, and Il10 mRNA expression; and histology. Cytokine profiling of colonic lamina propria mononuclear cells (LPMCs) was performed by flow cytometry. Naive CD4+ T cells were isolated from WT and SPARC KO mouse spleens, and the effect of SPARC on Th17 cell differentiation was examined. Recombination activating gene 1 knockout (RAG1 KO) mice reconstituted with T cells from either WT or SPARC KO mice were investigated. Results Trinitrobenzene sulfonic acid exposure significantly reduced bodyweight and increased mucosal inflammation, leukocyte infiltration, and Il17a mRNA expression in WT relative to SPARC KO mice. The percentage of IL17A-producing CD4+ T cells among LPMCs from KO mice was lower than that in WT mice when both groups were exposed to TNBS. Th17 cell differentiation was suppressed in cells from SPARC KO mice. In the T cell transfer colitis model, RAG1 KO mice receiving T cells from WT mice were more severely affected than those reconstituted with cells from SPARC KO mice. Conclusions Secreted protein acidic and rich in cysteine accelerates colonic mucosal inflammation via modulation of IL17A-producing CD4+ T cells. SPARC is a potential therapeutic target for conditions involving intestinal inflammation.

Mucin is produced and secreted by epithelial goblet cells and is a key component of the innate immune system, acting as a barrier in the intestinal tract. However, no studies have been conducted investigating the increase in mucin secretion to enhance the intestinal barrier function. The present study investigated whether rebamipide (Reb) acts as a secretagogue of intestinal mucin and the underlying mechanisms involved, thereby focusing on the effect on goblet cells. The LS174T cell line was used as goblet cell‑like cells. Using Reb‑treated LS174T cells, the level of mucin content was assessed by periodic acid‑Schiff (PAS) staining, and mucin 2, oligomeric mucus/gel‑forming (MUC2) mRNA expression was assessed using quantitative polymerase chain reaction (PCR). Furthermore, MUC2 secretion in the supernatant was quantified by the dot blot method. The present study additionally investigated the involvement of the epidermal growth factor receptor/Akt serine/threonine kinase 1 (Akt) pathway in mucin secretion by western blotting. The results suggested that Reb strongly enhanced the positivity of PAS staining in LS174T cells, thereby suggesting increased intracellular mucin production. The PCR results indicated that Reb significantly increased MUC2 mRNA in whole cell lysate of LS174T cells. In order to assess the subsequent secretion of mucin by LS174T, MUC2 protein expression in the supernatant was assessed using the dot blot method and it was demonstrated that Reb significantly increased the secretion of MUC2 in a concentration‑dependent manner. The p‑Akt was significantly increased by Reb treatment, and an Akt inhibitor specifically suppressed MUC2 secretion. Overall, Reb increased mucin secretion directly via p‑Akt. Reb‑increased mucin may act as a strong non‑specific barrier against pathogenic stimulants in various intestinal diseases.

Background and Aims Patients with inflammatory bowel disease [IBD], especially Crohn’s disease, often develop zinc deficiency. However, the precise mechanisms by which zinc deficiency affects IBD pathology, particularly intestinal macrophage function, remain unclear. We studied the effects of zinc deficiency on the development and progression of colitis in mice. Methods To induce colitis, mice were treated with 2,4,6-trinitrobenzene sulphonic acid. Rag1−/− mice were then given injections of naïve CD4+CD62L+ T cells. The respective degrees of mucosal injury of mice that had received a zinc chelator (TPEN; N,N,N′,N′-tetrakis [2-pyridylmethyl]ethylenediamine) and of control mice were subsequently compared. Colonic lamina propria mononuclear cells were isolated by enzymatic digestion and were examined using flow cytometry. To generate mouse bone marrow-derived macrophages [BMDMs], bone marrow cells were stimulated with mouse macrophage-colony stimulating factor. Results Zinc deficiency aggravates colonic inflammation through the activation of type 17 helper T [Th17] cells in mice. Flow cytometric analysis revealed that zinc deficiency significantly increases the proportion of pro-inflammatory [M1] macrophages in colonic lamina propria mononuclear cells obtained from inflamed colon. Interferon-γ plus lipopolysaccharide-mediated M1 skewing alters the expression of zinc transporters in BMDMs and thereby decreases the intracellular free zinc. TPEN treatment mimicking the effects of the M1 skewing up-regulates IL-23p19 expression, which is strongly related to Th17 development. Furthermore, the nuclear accumulation of interferon-regulatory factor 5 is closely involved in IL-23p19 induction in zinc-deficient macrophages. Conclusions Zinc deficiency aggravates colonic inflammation through activation of the IL-23/Th17 axis. This activation is controlled by subcellular distribution of interferon-regulatory factor 5.

AIM Measuring trans-epithelial electrical resistance (TEER) in vitro is a method to evaluate barrier function. The first aim of this study is to verify a real-time TEER monitoring system in Caco-2 monolayer. The second aim is to investigate the therapeutic effect of partially hydrolyzed guar gum (PHGG), a dietary fiber, on interferon (IFN)-γ-induced intestinal barrier dysfunction using this monitoring system. METHODS We measured TEER using real-time monitoring system and evaluated epithelial paracellular permeability using fluorescein isothiocyanate-conjugated dextran (4 kDa; FD4) in Caco-2 monolayers treated by IFN-γ for 48 h. The expression and distribution of tight junction (TJ)-associated proteins, ZO-1 and occludin, were analyzed by Western blotting and immunocytochemistry respectively. In some experiments, PHGG was added prior to IFN-γ treatment in order to investigate its protective effect on barrier function. RESULTS Using this system, we could evaluate the dynamic TEER change induced by IFN-γ precisely. In this study, IFN-γ treatment significantly decreased TEER and increased FD4 flux across Caco-2 monolayers, indicating the great influence of IFN-γ on intestinal epithelial paracellular permeability. In contrast, the pretreatment of PHGG significantly reduced the IFN-γ-induced increment of FD4 flux without affecting TEER. IFN-γ or PHGG treatment did not affect the expression of TJ-associated proteins, while immunocytochemistry showed that the IFN-γ-induced re-distribution of occludin was clearly restored by PHGG. CONCLUSIONS Real-time TEER monitoring enabled us to evaluate the dynamic change of barrier function. PHGG might have a protective effect against IFN-γ-induced barrier dysfunction by attenuating the paracellular hyper-permeability; thus, its promotion as a functional food is anticipated.

Partially hydrolysed guar gum (PHGG), a water-soluble dietary fibre produced by the controlled partial enzymatic hydrolysis of guar gum beans, has various physiological roles. This study aimed to elucidate the beneficial effects of PHGG on colonic mucosal damage in a murine 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis model. Acute colitis was induced in male C57BL/6 mice with TNBS after 2 weeks of pre-feeding with PHGG (5 %). The colonic mucosal inflammation was evaluated using macroscopic damage scores, and neutrophil infiltration was assessed by measuring tissue-associated myeloperoxidase (MPO) activity in the colonic mucosa. TNF-αexpression in the colonic mucosa was measured by ELISA and real-time PCR. Moreover, the intestinal microbiota and production of SCFA were assessed by real-time PCR and HPLC, respectively. Colonic damage due to TNBS administration was significantly ameliorated by PHGG treatment. Furthermore, PHGG significantly inhibited increases in MPO activity and TNF-αprotein and mRNA expression in the colonic mucosa in TNBS-induced colitis. On analysis of intestinal microbiota, we found that the concentration of theClostridium coccoidesgroup (Clostridiumcluster XIVa), theClostridium leptumsubgroup (Clostridiumcluster IV) and theBacteroides fragilisgroup had significantly increased in PHGG-fed mice. On analysis of SCFA, we found that the caecal content of acetic acid, propionic acid and butyric acid had significantly increased in PHGG-fed mice. Together, these results suggest that chronic ingestion of PHGG prevents the development of TNBS-induced colitis in mice by modulating the intestinal microbiota and SCFA, which may be significant in the development of therapeutics for inflammatory bowel disease.

Estrogen receptor α (ERα) functions as a transcription factor for genes involved in estrogen-dependent development of breast cancer cells. We demonstrate here that knockdown of hypoxia-inducible factor (HIF)-2α, but not of HIF-1α, increases endogenous ERα protein expression in normoxia and hypoxia. The von Hippel-Lindau protein (pVHL)-dependent degradation of HIF-2α participates in the regulation of ERα expression. Additionally, HIF-2α forms a protein complex with ERα, and amino acids 396-823 of HIF-2α physically interact with the ligand-binding domain of ERα. These results indicate that HIF-2α functions as a negative regulator of ERα expression in breast cancer, especially in normoxia.

A relationship between Bifidobacterium and defecation has previously been reported. Our hypothesis on the effectiveness of alkaline electrolyzed water (AEW) proposes that ingestion of AEW, considered possessing antioxidative properties, increases the number of Bifidobacteria and improves stool hardness and gastrointestinal symptoms. A double-blind, randomized study was conducted to evaluate the connection between stool consistency and change in gut microbiota composition induced by drinking hydrogen-dissolved AEW. The participants drank 500 mL of purified tap water or AEW every day for 2 weeks. In this study, drinking AEW did not drastically change gut microbiota, but it appeared to act on a specific bacterial species. Drinking AEW was confirmed to cause an increase in Bifidobacterium. The AEW group also saw stool consistency significantly converge to Bristol stool scale Type 4 ("normal"). Therefore, it is highly likely that the gut microbiota will be changed by drinking AEW. This study was retrospectively registered in University Hospital Medical Information Network (UMIN) Clinical Trials Registry (UMIN ID: UMIN000039507) on February 18, 2020, and was approved by the Ethics Committee of University of Yamanashi (approval No. H30-25) on January 9, 2018.

Summary Betalains are plant pigments primarily produced by plants of the order Caryophyllales. Because betalain possesses anti‐inflammatory and anticancer activities, it may be useful as a pharmaceutical agent and dietary supplement. Recent studies have identified the genes involved in the betalain biosynthesis of betanin. Amaranthin and celosianin II are abundant in the quinoa (Chenopodium quinoa Willd.) hypocotyl, and amaranthin comprises glucuronic acid bound to betanin; therefore, this suggests the existence of a glucuronyltransferase involved in the synthesis of amaranthin in the quinoa hypocotyl. To identify the gene involved in amaranthin biosynthesis, we performed a BLAST analysis and phylogenetic tree analysis based on sequences homologous to flavonoid glycosyltransferase, followed by expression analysis on the quinoa hypocotyl to obtain three candidate proteins. Production of amaranthin in a transient Nicotiana benthamiana expression system was evaluated for these candidates and one was identified as having the ability to produce amaranthin. The gene encoding this protein was quinoa amaranthin synthetase 1 (CqAmaSy1). We also created a transgenic tobacco bright yellow‐2 (BY‐2) cell line wherein four betalain biosynthesis genes were introduced to facilitate amaranthin production. This transgenic cell line produced 13.67 ± 4.13 μm (mean ± SEM) amaranthin and 26.60 ± 1.53 μm betanin, whereas the production of isoamaranthin and isobetanin could not be detected. Tests confirmed the ability of amaranthin and betanin to slightly suppress cancer cell viability. Furthermore, amaranthin was shown to significantly inhibit HIV‐1 protease activity, whereas betanin did not.

This study evaluated the immunostimulative effect on bone marrow-derived dendritic cells (DCs) of adjuvant-active exopolysaccharide (EPS) produced by Leuconostoc mesenteroides strain NTM048. EPS stimulation increased IL-6, IL-10, IL-12, and retinal dehydrogenase (RALDH) gene expression levels and induced retinoic acid-synthesizing RALDH-active DCs, which play a crucially important role in controlling adaptive immune responses in mucosa.

Increasing evidence indicates that molecular hydrogen-dissolved alkaline electrolyzed water (AEW) has various physiological activities such as antioxidative activity. Gut microbiota are deeply associated with our health through a symbiotic relationship. Recent reports have described that most gastrointestinal microbial species encode the genetic capacity to metabolize molecular hydrogen, meaning that molecular hydrogen might affect the gut microbial composition. Nevertheless, AEW effects on gut microbiota remain unknown. This study investigated AEW effects on the intestinal environment in mice, including microbial composition and short-chain fatty acid contents. After mice were administered AEW for 4 weeks, 16S rRNA gene sequencing analyses revealed their fecal microbiota profiles. Organic acid concentrations in cecal contents were measured using an HPLC system. Compared to the control group, AEW administration mice had significantly lower serum low-density lipoprotein cholesterol level and alanine aminotransferase activity. Organic acid concentrations of propionic, isobutyric, and isovaleric acids were higher in AEW-administered mice. Results of 16S rRNA gene sequencing analyses showed that the relative abundances of 20 taxa differed significantly in AEW-administered mice. Although the definitive role of gut microbes of AEW-administered mice remains unknown, our data demonstrate the possibility that AEW administration affects the gut microbial composition and that it has beneficial health effects in terms of cholesterol metabolism and liver protection.

High-fat diet (HFD)-induced alteration in the gut microbial composition, known as dysbiosis, is increasingly recognized as a major risk factor for various diseases, including colon cancer. This report describes a comprehensive investigation of the effect of agaro-oligosaccharides (AGO) on HFD-induced gut dysbiosis, including alterations in short-chain fatty acid contents and bile acid metabolism in mice. C57BL/6N mice were fed a control diet or HFD, with or without AGO. Terminal restriction fragment-length polymorphism (T-RFLP) analysis produced their fecal microbiota profiles. Profiles of cecal organic acids and serum bile acids were determined, respectively, using HPLC and liquid chromatography-tandem mass spectrometry systems. T-RFLP analyses showed that an HFD changed the gut microbiota significantly. Changes in the microbiota composition induced by an HFD were characterized by a decrease in the order Lactobacillales and by an increase in the Clostridium subcluster XIVa. These changes of the microbiota community generated by HFD treatment were suppressed by AGO supplementation. As supported by the data of the proportion of Lactobacillales order, the concentration of lactic acid increased in the HFD + AGO group. Data from the serum bile acid profile showed that the level of deoxycholic acid, a carcinogenic secondary bile acid produced by gut bacteria, was increased in HFD-receiving mice. The upregulation tended to be suppressed by AGO supplementation. Finally, results show that AGO supplementation suppressed the azoxymethane-induced generation of aberrant crypt foci in the colon derived from HFD-treated mice. Our results suggest that oral intake of AGO prevents HFD-induced gut dysbiosis, thereby inhibiting colon carcinogenesis.

Background and aims: Healing of the intestinal mucosal epithelium was found to be a critical factor in the treatment of inflammatory bowel disease (IBD). In this study, we provide further evidence that partially hydrolyzed dietary fiber (PHGG) enhances colonic epithelial cell wound healing, and partially characterize the mechanism that governs this process. Materials and methods: Young adult mouse colonic (YAMC) epithelial cells were scraped with a 10 μl micro-pipette tip to denude a round of the monolayer and were incubated with PHGG. The area of cell migration was measured using Image J software. Meanwhile, Rho activation assays were utilized to monitor Rho activation levels. To assess in vivo effects, C57B6 mice were treated with DSS for 7 days and then provided food supplemented with PHGG for 8 days. Results: YAMC cells treated with PHGG exhibited significantly enhanced wound healing compared to the control cells; however, this enhancement was inhibited by both Y-27632 (RhoA inhibitor) and U0126 (ERK1/2 inhibitor). Likewise, there was a PHGG-dependent increase in F-actin accumulation and Rho kinase activity that was blocked by U0126. Meanwhile, PHGG-dependent ERK1/2 activity was not inhibited by Y-27632. In the DSS-induced mouse colitis model, animals that received food supplemented with PHGG exhibited significant recovery of the colonic mucosa. Conclusions: In this study, we demonstrate that PHGG promotes colonic epithelial cell wound healing via activation of RhoA, which occurs downstream of ERK1/2 activation. These findings indicate that PHGG could be utilized as a therapeutic agent for patients with intestinal mucosal damage such as those with IBD.

Background Acetyl salicylic acid (ASA) is a useful drug for the secondary prevention of cerebro-cardiovascular diseases, but it has adverse effects on the small intestinal mucosa. The pathogenesis and prophylaxis of ASA-induced small intestinal injury remain unclear. In this study, we focused on the intestinal mucus, as the gastrointestinal tract is covered by mucus, which exhibits protective effects against various gastrointestinal diseases. Materials and Methods ASA was injected into the duodenum of rats, and small intestinal mucosal injury was evaluated using Evans blue dye. To investigate the importance of mucus, Polysorbate 80 (P80), an emulsifier, was used before ASA injection. In addition, rebamipide, a mucus secretion inducer in the small intestine, was used to suppress mucus reduction in the small intestine of P80-administered rats. Results The addition of P80 reduced the mucus and exacerbated the ASA-induced small intestinal mucosal injury. Rebamipide significantly suppressed P80-reduced small intestinal mucus and P80-increased intestinal mucosal lesions in ASA-injected rats, demonstrating that mucus is important for the protection against ASA-induced small intestinal mucosal injury. These results provide new insight into the mechanism of ASA-induced small intestinal mucosal injury. Conclusion Mucus secretion-increasing therapy might be useful in preventing ASA-induced small intestinal mucosal injury.

Colorectal cancer (CRC) is one of the most predominant types of cancer, and it is the fourth most common cause of cancer-related death and it is important to diagnose CRC in early stage to decrease the mortality by CRC. In our previous study, we identified a combination of five peptides as a biomarker candidate to diagnose CRC by BLOTCHIP®-MS analysis using a set of healthy control subjects and CRC patients (stage II–IV). The aim of the present study was to validate the serum biomarker peptides reported in our previous study using a second cohort and to establish their potential usefulness in CRC diagnosis. A total of 56 patients with CRC (n = 14 each of stages I–IV), 60 healthy controls, and 60 patients with colonic adenoma were included in this study. The five peptides were extracted and analyzed by selected reaction monitoring using ProtoKey® Colorectal Cancer Risk Test Kit (Protosera, Inc., Amagasaki, Japan). The results clearly showed that the four CRC groups, stages I–IV, could be sufficiently discriminated from the control group and colonic polyp group. This five-peptide set could identify CRC at each stage compared to the control population in this validation cohort, including those with early-stage disease. The AUC values for each stage of CRC compared to the control population were 0.779, 0.946, 0.852, and 0.973 for stages I, II, III, and IV, respectively. In this case–control validation study, we confirmed high diagnostic performance for CRC using five peptides that were identified in our previous study as serum biomarker candidates for the detection of CRC.

Gut microbiota are deeply associated with the prevalence of obesity. Agarose is hydrolyzed easily to yield oligosaccharides, designated as agaro-oligosaccharides (AGO). This study evaluated the effects of AGO on obese phenotype and gut microbial composition in mice. Mice were administered AGO in drinking water (AGO-receiving mice). 16S rRNA gene sequencing analyses revealed their fecal microbiota profiles. Serum bile acids were ascertained using a LC-MS/MS system. Compared to the control group, AGO administration significantly reduced epididymal adipose tissue weights and serum non-esterified fatty acid concentrations, but the cecal content weights were increased. Data from the serum bile acid profile show that concentrations of primary bile acids (cholic acid and chenodeoxycholic acid), but not those of secondary bile acids (deoxycholic acid, lithocholic acid, and ursodeoxycholic acid), tended to increase in AGO-receiving mice. 16S rRNA gene sequencing analyses showed that the relative abundances of 15 taxa differed significantly in AGO-receiving mice. Of these, the relative abundances of Rikenellaceae and Lachnospiraceae were found to be positively correlated with epididymal adipose tissue weight. The relative abundances of Bacteroides and Ruminococcus were correlated negatively with epididymal adipose tissue weight. Although the definitive role of gut microbes of AGO-received mice is still unknown, our data demonstrate the possibility that AGO administration affects the gut microbial composition and inhibits obesity in mice.

Background and Aim Non-steroidal anti-inflammatory drugs (NSAIDs), which are commonly used in clinical medicine, cause erosion, ulcers, and bleeding in the gastrointestinal tract. No effective agent for the prevention and treatment of small intestinal injury by NSAIDs has been established. This study investigates the effects of agaro-oligosaccharides (AGOs) on NSAID-induced small intestinal injury in mice. Methods Mice were treated with indomethacin, an NSAID, to induce intestinal injury. The respective degrees of mucosal injury of mice that received AGO and control mice were compared. Heme oxygenase-1 (HO-1) expression using quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, and immunohistochemistry were measured. The expression of keratinocyte chemoattractant (KC) was measured using qRT-PCR and enzyme-linked immunosorbent assay. Results AGO administration induced HO-1 expression in mouse small intestinal mucosa. Induction was observed mainly in F4/80 positive macrophages. The increased ulcers score, myeloperoxidase activity, and KC expression by indomethacin were inhibited by AGO administration. Conversely, HO inhibitor cancelled AGO-mediated prevention of intestinal injury. In mouse peritoneal macrophages, AGOs enhanced HO-1 expression and suppressed lipopolysaccharide-induced KC expression. Furthermore, AGOs enhanced the expressions of alternatively activated macrophage markers arginase-1, mannose receptor-1, and chitinase 3-like 3. Conclusions Results suggest that oral administration of AGOs prevents NSAID-induced intestinal injury.

Background/Aims: Although the cytotoxicity of aspirin against the intestinal epithelium is a major clinical problem, little is known about its pathogenesis. We assessed the involvement of Multi Drug Resistance (MDR) 1 in intestinal epithelial cell injury caused by aspirin using MDR1 gene-transfected Caco2 cells. Methods: Caco2 cells were treated with various concentrations of aspirin for 24 h. After treatment of Caco2 cells with verapamil, a specific inhibitor of MDR1, we assessed the extent of cell injury using a WST-8 assay at 24 h after aspirin-stimulation. We performed the same procedure in MDR1 gene-transfected Caco2 cells. To determine the function of MDR1 in the metabolism of aspirin, flux study was performed using 14 C-labeled aspirin. Results: The level of aspirin-induced cell injury was higher in verapamil-treated Caco2 cells than in control cells and was less serious in MDR1-transfected Caco2 cells than in control vector-transfected cells. The efflux of 14 C-labeled aspirin was higher in verapamil-treated Caco2 cells than in control cells. Conclusion: These data suggest that aspirin efflux occurs through the MDR1 transporter and that the MDR1 transporter is involved in the pathogenesis of aspirin-induced cell injury.

Background The ratio of colonic anti-inflammatory CD11c+ macrophages (MPs) to inflammatory CD103- dendritic cells (DCs) plays pivotal roles in intestinal inflammation. Little is known about how the ratio is regulated by lactic acid bacteria (LAB) and bifidobacteria (Bif). We investigated the contribution of LAB/Bif to this ratio. Methods We established an in vitro experimental system using human myeloblastic KG-1 cells, which differentiate into CD11c+ MP-like (CD11c+ MPL) and CD103- DC-like (CD103- DCL) cells, and explored effective LAB/Bif strains. The selected strain's effect on the colonic CD11c+ MP/CD103- DC ratio and intestinal inflammation was examined in mice, and the strain's underlying mechanisms were investigated in vitro. Results We screened 19 strains of LAB/Bif, and found that Lactobacillus brevis KB290 (KB290) increased the CD11c+ MPL/CD103- DCL cell ratio only in the presence of a vitamin A (VA) metabolite, retinoic acid (RA). Supplementation of KB290 with VA increased the CD11c+ MP/CD103- DC ratio in healthy mouse and prevented the disruption of the ratio during colitis. Supplementation of KB290 with pro-VA (β-carotene) also increased the ratio in healthy mouse and ameliorated the development of colitis. The ratio was increased by reduction of CD103- DCs (or CD103- DCL cells). Our in vitro data suggested that KB290 induced cell death in CD103- DCL cells in the presence of RA signaling. Conclusions Supplementation of KB290 with VA increases the colonic CD11c+ MP/CD103- DC ratio associated with the amelioration of murine colitis, suggesting a possible way to control intestinal inflammation by LAB.

Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycol that regulates cell proliferation, tissue repair, and tumorigenesis. Despite evidence linking SPARC to inflammation, the mechanisms are unclear. Accordingly, the role of SPARC in intestinal inflammation was investigated.Colitis was induced in wild-type (WT) and SPARC knockout (KO) mice using trinitrobenzene sulfonic acid (TNBS). Colons were assessed for damage; leukocyte infiltration; Tnf, Ifng, Il17a, and Il10 mRNA expression; and histology. Cytokine profiling of colonic lamina propria mononuclear cells (LPMCs) was performed by flow cytometry. Naïve CD4Trinitrobenzene sulfonic acid exposure significantly reduced bodyweight and increased mucosal inflammation, leukocyte infiltration, and Il17a mRNA expression in WT relative to SPARC KO mice. The percentage of IL17A-producing CD4Secreted protein acidic and rich in cysteine accelerates colonic mucosal inflammation via modulation of IL17A-producing CD4

Background: Irritable bowel syndrome (IBS) causes chronic abdominal pain and abnormal bowel movements. The etiology involves complicated interactions among visceral hypersensitivity, disorders related to bowel movements, and stress. Changes in the microbiota affect the IBS pathophysiology. We investigated changes in colorectal motility, structure, and microbiota in response to stress due to maternal separation (MS) and corticotropin-releasing hormone (CRH) administration in rats. Summary: Neonatal rats were separated from their mothers for 3 h daily from postnatal day (PND) 2 to PND14. The control group included rats of the same age that were not separated. After MS, the rats were undisturbed for 5 weeks. At 8 weeks of age, 10 µg of CRH or saline was intravenously administered to MS and control groups. Two hours later, the number of fecal pellets was counted. Three hours after CRH or saline administration, the rats were sacrificed and colorectal tissue samples and cecal contents were collected to analyze the fecal microbiota. The number of fecal pellets was significantly greater in MS with the CRH group. Both stressors altered the microbiota composition. Key Messages: Among rats that received CRH, MS increased the colorectal motility. Stress due to MS altered the gut microbiota composition.

Background and Aim Although non-steroidal anti-inflammatory drugs can induce intestinal injury, the mechanisms are not fully understood, and treatment has yet to be established. Heme oxygenase-1 (HO-1) has recently gained attention for anti-inflammatory and cytoprotective effects. This study aimed to investigate the effects of hemin, an HO-1 inducer, on indomethacin-induced enteritis in mice. Methods Enteritis was induced by single subcutaneous administration of indomethacin (10 mg/kg) in male C57BL/6 mice. Hemin (30 mg/kg) was administered by intraperitoneal administration 6 h before indomethacin administration. Mice were randomly divided into four groups: (i) sham + vehicle; (ii) sham + hemin; (iii) indomethacin + vehicle; or (iv) indomethacin + hemin. Enteritis was evaluated by measuring ulcerative lesions. Myeloperoxidase activity was measured as an index of neutrophil accumulation. The mRNA expression of inflammatory cytokines and chemokines, such as tumor necrosis factor-α, monocyte chemoattractant protein-1, macrophage inflammatory protein-1α, and keratinocyte chemoattractant, were analyzed by real-time polymerase chain reaction. Results The area of ulcerative lesions, myeloperoxidase activity, and mRNA expression of inflammatory cytokines and chemokines were significantly increased in mice administrated with indomethacin compared with vehicle-treated sham mice. Development of intestinal lesions, increased levels of myeloperoxidase activities, and mRNA expressions of inflammatory cytokines and chemokines were significantly suppressed in mice treated with hemin compared with vehicle-treated mice. Protective effects of hemin were reversed by co-administration of tin protoporphyrin, an HO-1 inhibitor. Conclusions Induction of HO-1 by hemin inhibits indomethacin-induced intestinal injury through upregulation of HO-1. Pharmacological induction of HO-1 may offer a novel therapeutic strategy to prevent indomethacin-induced small intestinal injury.