Mouse Jejunum Research Articles

Lactobacillus acidophilus BIOTECH 1900 decreases the transepithelial mucosal-to-serosal transport of colchicine in an ex vivo non-everted gut sac model.

Colchicine (COL) is a permeability-glycoprotein (P-gp) substrate drug used for familial Mediterranean fever, acute pericarditis, and the management of acute gout. It has a narrow therapeutic index which implies that a small change in the drug's absorption profile may lead to either toxicity or therapeutic failure. Absorption can be altered by modulating the function of P-gp via the concomitant use of drugs, herbal medicines, or food supplements such as probiotics. Here, we investigated the effect of probiotic Lactobacillus acidophilus BIOTECH 1900 on COL's transepithelial mucosal-to-serosal transport in the jejunum of ICR mice. A high-performance liquid chromatography-photodiode array (HPLC-PDA) method for the assay of COL was developed and validated. The HPLC-PDA method was applied in an ex vivo non-everted gut sac model to measure COL's cumulative mucosal-to-serosal transport and apparent permeability (Papp). Treatment of L. acidophilus BIOTECH 1900 resulted to a significantly lower COL transport and Papp value compared to the control group. Additionally, the activity of L. acidophilus BIOTECH 1900 was found to be similar to dexamethasone, a known P-gp inducer. We report that L. acidophilus BIOTECH 1900 decreases the transepithelial mucosal-to-serosal transport of COL, suggesting possible P-gp induction. Further studies are recommended to substantiate this transporter-based drug-probiotic interaction.

Transcytosis of Galectin-3 in Mouse Intestine.

The GlycoLipid-Lectin (GL-Lect) hypothesis provides a conceptual framework to explain how endocytic pits are built in processes of clathrin-independent endocytosis. According to this hypothesis, oligomeric cellular or pathogenic lectins interact with glycosylated plasma membrane lipids in a way such as to drive the formation of tubular endocytic pits that then detach to generate clathrin-independent endocytic carriers for the cellular uptake of cellular or pathogenic products. This process operates in a complementary manner to the conventional clathrin pathway for biological function linked to cell polarity. Up to date, the premises of the GL-Lect hypothesis have been based on model membrane and cell culture experiments. It has therefore become urgent to extend its exploration to complex organisms. In the current protocol, we describe methods to study the endocytosis and transcytosis of a key driver of the GL-Lect mechanism, the cellular galectin-3, and of one of its cargoes, lactotransferrin, in enterocytes of the intact jejunum of mice. In a step-by-step manner, we present the generation of fluorescent endocytic ligands, tissue preparation for cellular uptake measurements, binding and internalization assays, tissue fixation and preparation for sectioning, light and electron microscopical observations, and quantification of data by image processing. Pitfalls are discussed to optimize the chances of success with the described methods.

Prehabilitation modulates cell protection proteins expressions of gut in mice

Rationale: Our previous study revealed that a preoperative repeated treadmill running improves survival after gut ischemia reperfusion (gut I/R) in mice. However, the mechanism underlying amelioration of host response to gut I/R has not been clarified. Herein, we examined the expressions of cell protection proteins in mice jejunum.

Clostridium butyricum Alleviates Enterotoxigenic Escherichia coli K88-Induced Oxidative Damage Through Regulating the p62-Keap1-Nrf2 Signaling Pathway and Remodeling the Cecal Microbial Community.

Clostridium butyricum (CB) can enhance antioxidant capacity and alleviate oxidative damage, but the molecular mechanism by which this occurs remains unclear. This study used enterotoxigenic Escherichia coli (ETEC) K88 as a pathogenic model, and the p62-Keap1-Nrf2 signaling pathway and intestinal microbiota as the starting point to explore the mechanism through which CB alleviates oxidative damage. After pretreatment with CB for 15 d, mice were challenged with ETEC K88 for 24 h. The results suggest that CB pretreatment can dramatically reduce crypt depth (CD) and significantly increase villus height (VH) and VH/CD in the jejunum of ETEC K88-infected mice and relieve morphological lesions of the liver and jejunum. Additionally, compared with ETEC-infected group, pretreatment with 4.4×106 CFU/mL CB can significantly reduce malondialdehyde (MDA) level and dramatically increase superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels in the serum. This pretreatment can also greatly increase the mRNA expression levels of tight junction proteins and genes related to the p62-Keap1-Nrf2 signaling pathway in the liver and jejunum in ETEC K88-infected mice. Meanwhile, 16S rDNA amplicon sequencing revealed that Clostridium disporicum was significantly enriched after ETEC K88 challenge relative to the control group, while Lactobacillus was significantly enriched after 4.4×106 CFU/mL CB treatment. Furthermore, 4.4×106 CFU/mL CB pretreatment increased the short-chain fatty acid (SCFA) contents in the cecum of ETEC K88-infected mice. Moreover, we found that Lachnoclostridium, Roseburia, Lactobacillus, Terrisporobacter, Akkermansia, and Bacteroides are closely related to SCFA contents and oxidative indicators. Taken together, 4.4×106 CFU/mL CB pretreatment can alleviate ETEC K88-induced oxidative damage through activating the p62-Keap1-Nrf2 signaling pathway and remodeling the cecal microbiota community in mice.

Assessment of the oxidative status and goblet cell response during eimeriosis and after treatment of mice with magnesium oxide nanoparticles

Magnesium nanoparticles have been the focus of study over the past few years because of its functionality in the body. Assessment of the impact of magnesium oxide nanoparticles (MgNPs) on eimeriosis has yet to be conducted. The goal of this study was to see how MgNPs affected the parasite Eimeria papillata infected jejunum. To induce eimeriosis, mice were infected with sporulated oocysts. For treatment, 5 mg / Kg MgNPs was used for 5 consecutive days. The infection reduced the number of intestinal goblet cells and their associated genes MUC2 and MUC4, as well as increasing oxidative damage in the jejunum. MgNPs significantly reduced the oocyst production in the feces by about 77 %. After treatment, the number of goblet cells per villus increased from 4.17% to 7.40.6%. Moreover, the MgNPs were able to upregulate the expression of MUC2 and MUC4-mRNA. MgNPs significantly increased the activity of catalase and superoxide dismutase, as well as the extent of glutathione, by day 5 after infection with the parasite. On contrary, MgNPs decreased the level of malondialdehyde and nitric oxide. The findings suggested that MgNPs could be an effective anti-eimeriosis agent due to their anti-eimerial and anti-oxidant roles, as well as the regulatory effect on the goblet cell mucin genes in the jejunum of mice.

Arecoline Hydrobromide Enhances Jejunum Smooth Muscle Contractility via Voltage-Dependent Potassium Channels in W/Wv Mice

Gastrointestinal motility was disturbed in W/Wv, which were lacking of interstitial cells of Cajal (ICC). In this study, we have investigated the role of arecoline hydrobromide (AH) on smooth muscle motility in the jejunum of W/Wv and wild-type (WT) mice. The jejunum tension was recorded by an isometric force transducer. Intracellular recording was used to identify whether AH affects slow wave and resting membrane potential (RMP) in vitro. The whole-cell patch clamp technique was used to explore the effects of AH on voltage-dependent potassium channels for jejunum smooth muscle cells. AH enhanced W/Wv and WT jejunum contractility in a dose-dependent manner. Atropine and nicardipine completely blocked the excitatory effect of AH in both W/Wv and WT. TEA did not reduce the effect of AH in WT, but was sufficient to block the excitatory effect of AH in W/Wv. AH significantly depolarized the RMP of jejunum cells in W/Wv and WT. After pretreatment with TEA, the RMP of jejunum cells indicated depolarization in W/Wv and WT, but subsequently perfused AH had no additional effect on RMP. AH inhibited the voltage-dependent K+ currents of acutely isolated mouse jejunum smooth muscle cells. Our study demonstrate that AH enhances the contraction activity of jejunum smooth muscle, an effect which is mediated by voltage-dependent potassium channels that acts to enhance the excitability of jejunum smooth muscle cells in mice.

Underestimated health risks: polystyrene micro- and nanoplastics jointly induce intestinal barrier dysfunction by ROS-mediated epithelial cell apoptosis

BackgroundMicro- and nanoplastic pollution has become a global environmental problem. Nanoplastics in the environment are still hard to detect because of analysis technology limitations. It is believed that when microplastics are found in the environment, more undetected nanoplastics are around. The current “microplastic exposure” is in fact the mixture of micro- and nanoplastic exposures. Therefore, the biological interaction between organisms among different sizes of micro- and nanoplastics should not be neglected.ResultsWe measured the biodistribution of three polystyrene (PS) particles (50 nm PS, PS50; 500 nm PS, PS500; 5000 nm PS, PS5000) under single and co-exposure conditions in mice. We explored the underlying mechanisms by investigating the effects on three major components of the intestinal barrier (the mucus layer, tight junctions and the epithelial cells) in four intestine segments (duodenum, jejunum, ileum and colon) of mice. We found that the amounts of both PS500 and PS5000 increased when they were co-exposed with PS50 for 24 h in the mice. These increased amounts were due primarily to the increased permeability in the mouse intestines. We also confirmed there was a combined toxicity of PS50 and PS500 in the mouse intestines. This manifested as the mixture of PS50 and PS500 causing more severe dysfunction of the intestinal barrier than that caused by PS50 or PS500 alone. We found that the combined toxicity of PS micro- and nanoplastics on intestinal barrier dysfunction was caused primarily by reactive oxygen species (ROS)-mediated epithelial cell apoptosis in the mice. These findings were further confirmed by an oxidants or antioxidants pretreatment study. In addition, the combined toxicity of PS micro- and nanoplastics was also found in the mice after a 28-day repeated dose exposure.ConclusionsThere is a combined toxicity of PS50 and PS500 in the mouse intestines, which was caused primarily by ROS-mediated epithelial cell apoptosis in the mice. Considering that most recent studies on PS micro- and nanoplastics have been conducted using a single particle size, the health risks of exposure to PS micro- and nanoplastics on organisms may be underestimated.

CBP22, a Novel Bacteriocin Isolated from Clostridium butyricum ZJU-F1, Protects against LPS-Induced Intestinal Injury through Maintaining the Tight Junction Complex.

A novel bacteriocin secreted by Clostridium butyricum ZJU-F1 was isolated using ammonium sulfate fractionation, cation exchange chromatography, affinity chromatography, and reverse-phase high-performance liquid chromatography (RP-HPLC). The bacteriocin, named CBP22, contained 22 amino acids with the sequence PSAWQITKCAGSIAWALGSGIF. Analysis of its structure and physicochemical properties indicated that CBP22 had a molecular weight of 2264.63 Da and a +1 net charge. CBP22 showed activity against E. col K88, E. coli ATCC25922, and S. aureus ATCC26923. The effects and potential mechanisms of bacteriocin CBP22 on the innate immune response were investigated with a lipopolysaccharide- (LPS-) induced mouse model. The results showed that pretreatment with CBP22 prevented LPS-induced impairment in epithelial tissues and significantly reduced serum levels of IgG, IgA, IgM, TNF-α, and sIgA. Moreover, CBP22 treatment increased the expression of the zonula occludens and reduced permeability as well as apoptosis in the jejunum in LPS-treated mice. In summary, CBP22 inhibits the intestinal injury and prevents the gut barrier dysfunction induced by LPS, suggesting the potential use of CBP22 for treating intestinal damage.

Succinate‐Induced Tuft Cell Hyperplasia Enhances FFA2/5‐HT Mediated Anion Secretion in Mouse Duodenum

Tuft cells are one of luminal chemosensing components in the small intestine, while the role of tuft cell in mucosal secretory responses to luminal chemicals such as short-chain fatty acids (SCFA) has not been elucidated. We thus hypothesized that tuft cell hyperplasia may increase the secretory responses to luminal SCFA due to the increased cholinergic signals since tuft cells express choline acetyltransferase (ChAT). Tuft cell hyperplasia was induced by oral sodium succinate (NaSuc, 100 mM) in the drinking water for 7 days. Sodium propionate (NaP, 10 mM, m), the selective free fatty acid receptor 2 (FFA2) agonist 4-CMTB (10 µM, s), or the TRPA1 agonist allyl isothiocyanate (AITC, 1 mM, m) were applied to the mucosal (m) or serosal (s) baths of an Ussing chamber mounted with muscle-stripped mucosa-submucosa preparations of mouse proximal duodenum or jejunum. The density of tuft cells (doublecortin-like kinase 1; DCLK1), enteroendocrine L cells (proglucagon, glucagon-like peptide-2), and enterochromaffin (EC) cells (5-HT, tryptophan hydroxylase 1; TPH1) were assessed by real-time PCR and immunostaining. NaSuc treatment significantly increased the number of DCLK1-positive tuft cells in the duodenal and jejunal villi. mRNA expression of DCLK1 and FFA2 were increased in the duodenal and jejunal mucosa, whereas TPH1 was increased only in the duodenal mucosa, and ChAT was increased in the jejunal mucosa, but proglucagon was unchanged in both. 5-HT-positive mast cell density rather than EC cell density was increased in the duodenum. Isc in response to luminal NaP was enhanced in the jejunum, which was atropine sensitive. In contrast, Isc responses to serosal 4-CMTB and luminal AITC were enhanced in the duodenum, which were mediated by 5-HT3 and 5-HT4 receptors. These results suggest that NaSuc differentially enhances secretory responses, with 5-HT signals in the duodenum and with cholinergic signals in the jejunum. These data may be implicated in the pathogenesis of functional dyspepsia (FD), since increased duodenal mast cell density and enhanced 5-HT signals are present in FD patients.

799 INTESTINAL RBM47 DELETION PROMOTES SPONTANOUS POLYPOSIS THROUGH UP-REGULATED EPITHELIAL PROLIFERATION, ALTERNATIVE SPLICING OF TJP1 BUT MITIGATES COLITIS-ASSOCAITED TUMORIGENESIS VIA ANTI-OXIDATIVE ADAPTATION AND REDUCED INFLAMMATION

Background RNA-binding motif protein 47 (RBM47) is a requisite cofactor in APOBEC1-dependent C-to-U RNA editing whose RNA targets are unknown. Aims Because Apobec-1 knockout mice are protected against polyposis in the ApcMin background, we asked if intestine-specific Rbm47 knockout mice (Rbm47IKO) exhibit altered tumor susceptibility, either spontaneously or in different murine cancer models. Results 86% (6/7) of 12-month old chow-fed Rbm47IKOmice developed spontaneous intestinal and colonic polyps (Fig 1A), compared to 10% (1/10) aged Rbm47floxcontrols. RNA-seq showed upregulated proliferation, glutathione metabolism, and anti-oxidative pathways in Rbm47 IKOintestine. QPCR revealed reduced abundance of the long Tjp1 mRNA isoform (Tjp1+) in Rbm47 IKOmice (Fig 1B), which was further reduced in polyp vs uninvolved tissue (11.8±0.2 vs 15.1±1.2, P=.03) in aged Rbm47 IKOmice. In support, we observed decreased Rbm47 mRNA (–DCT 0.97±2.79 vs 3.47±2.58, P Conclusion Rbm47IKOmice demonstrate spontaneous polyposis through upregulating proliferation and modifying Tjp1 alternative RNA splicing, with increased proximal intestinal polyposis in the ApcMin/+ background. By contrast, Rbm47IKOmice are protected against AOM-DSS-driven colitis-associated colon tumorogenesis by augmenting antioxidant capacity, over-expression of Il-33, and decreasing inflammatory activity. These contrasting phenotypes suggest that RBM47 exerts pathway-specific effects on intestinal tumorigenesis as a genetic modifier of growth, proliferation and inflammatory pathways. Download : Download high-res image (81KB) Download : Download full-size image Figure 1 . A: dissecting microscope images (left) and hematoxylin and eosin staining sections (right) of spontaneous polypogenesis in small intestine and colon of 12-month old chowfed Rbm47 IKOmice. B: Relative abundance of long Tjp1 mRNA isoform (Tjp1+) by quantitative RT-PCR in young adult mice jejunum. C: Rbm47 mRNA expression and relative abundance of Tjp1+ isoform by quantitative RT-PCR in human colorectal cancer tissue vs paired normal tissue. D: Comparison of overall survival among colorectal cancer patients with different levels of Rbm47 mRNA expression in tumor tissue using the cancer genome atlas (TCGA) database. Download : Download high-res image (71KB) Download : Download full-size image Figure 2 . A: Macroscopic illustration of colitis-associated colon tumors in mice treated with AOM)-DSS. B: left; the HE stained microscopic sections illustrate abundance of crypt abscess formation (yellow arrowheads) within dysplastic lesions of colon in AOM-DSS treated mice. Middle; F4/80 immunohistochemical staining of dysplastic lesions of colon in AOM-DSS treated mice. Right: Relative mRNA expression of inflammatory genes by quantitative RTPCR in dysplastic lesions of AOM-DSS treated mice. C: Relative mRNA expression of genes related to interleukin 33 by quantitative RT-PCR in dysplastic lesions of AOM-DSS treated mice. D: Macroscopic illustration of proximal small intestine tumors in mice with Apc Min/+background.

Upregulation of Vitamin C Transporter Functional Expression in 5xFAD Mouse Intestine.

The process of obtaining ascorbic acid (AA) via intestinal absorption and blood circulation is carrier-mediated utilizing the AA transporters SVCT1 and SVCT2, which are expressed in the intestine and brain (SVCT2 in abundance). AA concentration is decreased in Alzheimer’s disease (AD), but information regarding the status of intestinal AA uptake in the AD is still lacking. We aimed here to understand how AA homeostasis is modulated in a transgenic mouse model (5xFAD) of AD. AA levels in serum from 5xFAD mice were markedly lower than controls. Expression of oxidative stress response genes (glutathione peroxidase 1 (GPX1) and superoxide dismutase 1 (SOD1)) were significantly increased in AD mice jejunum, and this increase was mitigated by AA supplementation. Uptake of AA in the jejunum was upregulated. This increased AA transport was caused by a marked increase in SVCT1 and SVCT2 protein, mRNA, and heterogeneous nuclear RNA (hnRNA) expression. A significant increase in the expression of HNF1α and specific protein 1 (Sp1), which drive SLC23A1 and SLC23A2 promoter activity, respectively, was observed. Expression of hSVCT interacting proteins GRHPR and CLSTN3 were also increased. SVCT2 protein and mRNA expression in the hippocampus of 5xFAD mice was not altered. Together, these investigations reveal adaptive up-regulation of intestinal AA uptake in the 5xFAD mouse model.

CBP/P300 Inhibitors Mitigate Radiation-Induced GI Syndrome by Promoting Intestinal Stem Cell-Mediated Crypt Regeneration.

Radiation-induced gastrointestinal syndrome (RIGS) is currently the main cause of death for people exposed to a high dose of irradiation during nuclear incidents, and there is currently no approved effective therapy. Here, we found that CBP/P300 inhibitors, with high efficacy and low toxicity, might be promising radiation mitigators that can cure RIGS. Exvivo 3D organoid cultures derived from mouse jejunum and human ileum and colon were used to examine the radio-mitigative effects of CBP/P300 inhibitors. The radio-mitigative effect was evaluated by quantifying the survival rate and size of organoids after radiation. SGC-CBP30 (50 mg/kg body weight), an inhibitor of CBP/P300, was intraperitoneally injected into C57B/6J mice 24 hours after subtotal-body irradiation or whole-body irradiation. The regenerated crypts and animal survival were determined by microcolony assay and the Kaplan-Meier method, respectively. Lgr5-lacZ mice were used to evaluate the survival of intestinal stem cells after treatments. We found that CBP/P300 inhibitors were effective mitigators that could be used to treat RIGS. CBP/P300 inhibition promoted the regeneration of intestinal organoids invitro and of crypts invivo. Remarkably, the administration of CBP/P300 inhibitors to mice 24 hours after lethal irradiation promoted Lgr5+ intestinal stem cell and crypt recovery, resulting in improved mouse survival. Moreover, our data show that CBP/P300 inhibitors rescued irradiated mice from RIGS by delaying intestinal epithelial cell cycle progression after radiation. These data demonstrate that CBP/P300 inhibitors are effective medical countermeasures to mitigate gastrointestinal toxicity from radiation.

A further study on a disturbance of intestinal epithelial cell population and kinetics in APC1638T mice.

Adenomatous polyposis coli (APC), a well-known anti-oncogene, is considered to have multiple functions through its several binding domains. We have continuingly studied APC1638T/1638T mice (APC1638T mice) to elucidate the functions of APC other than tumor suppression. A distinctive feature of the APC1638T mice is they are tumor free and live as long as APC+/+ mice (WT mice). Previously, we found the length of crypt-villus axis in the jejunum was significantly elongated in APC1638T mice compared with that of WT mice. The populations of goblet cells, Paneth cells, and enteroendocrine cells were also disordered in APC1638T mice. Here, we further analyzed the intestinal dyshomeostasis in APC1638T mice, focusing on the proliferation and differentiation of intestinal stem cell (ISC) lineages, and apoptotic cell shedding at the villus tips. We found that the proliferation of ISC lineages was normally controlled; however, the shedding process of apoptosis cells was significantly delayed in the APC1638T mouse jejunum. Furthermore, the number of microfold cells (M cells) was significantly increased in the APC1638T mouse jejunum. Our data suggested both differentiation process of ISCs and turnover process of intestinal epithelia were disturbed in APC1638T mice, and that contributed to the villus elongation in the APC1638T mouse jejunum.

Steroid Receptor Coactivator-3 Is Required for Inhibition of the Intestinal Muscularis Inflammatory Response of Postoperative Ileus in Mice.

Inflammation theory has suggested that the pathogenesis of postoperative ileus (POI) involves the steroid receptor coactivator-3 (SRC-3). Therefore, we investigated the role of SRC-3 in the muscles of the small intestine using a mouse POI model. Here, we reported that intestinal manipulation (IM) significantly reduced the extent of phenol red migration in the entire gastrointestinal tract, and the calculated geometric center (GC) value in wild-type (WT) mice at 24 h after surgery was higher than that in the knockout (KO) mice and in the sham-operated control group. The expression of SRC-3 was upregulated in the mouse intestinal muscularis at 24 h after surgical manipulation, and the mRNA and protein levels of inflammatory cytokines were upregulated compared with those in the control group. At 24 h after IM, the number of neutrophils in the experimental group was significantly higher than that in the control group; in the IM group, the number of neutrophils in the SRC-3-/- mice was markedly higher than that in the WT mice. At 24 h after IM, the myeloperoxidase (MPO) activity in the experimental group was significantly higher than that in the control group. In the IM group, the MPO activity of the SRC-3-/- mice was markedly higher than that of the WT mice. In summary, proinflammatory cytokines, the number of neutrophils, and the MPO activity were significantly increased in the muscularis of the jejunum and ileum of KO mice after IM compared with those of the WT mice, indicating that SRC-3 might play a protective role in POI.

Acute blockade of endogenous melatonin by Luzindole, with or without peripheral LPS injection, induces jejunal inflammation and morphological alterations in Swiss mice.

This study investigated the acute blockade of endogenous melatonin (MLT) using Luzindole with or without systemic lipopolysaccharide (LPS) challenge and evaluated changes in inflammatory and oxidative stress markers in the mouse jejunum. Luzindole is an MT1/MT2 MLT receptor antagonist. Both receptors occur in the small intestine. Swiss mice were treated with either saline (0.35 mg/kg, ip), Luzindole (0.35 mg/kg, ip), LPS (1.25 mg/kg, ip), or Luzindole+LPS (0.35 and 1.25 mg/kg, ip, respectively). Jejunum samples were evaluated regarding intestinal morphometry, histopathological crypt scoring, and PAS-positive villus goblet cell counting. Inflammatory Iba-1, interleukin (IL)-1β, tumor necrosis factor (TNF)-α, nuclear factor (NF)-kB, myeloperoxidase (MPO), and oxidative stress (NP-SHs, catalase, MDA, nitrate/nitrite) markers were assessed. Mice treated with Luzindole, LPS, and Luzindole+LPS showed villus height shortening. Crypt damage was worse in the LPS group. Luzindole, LPS, and Luzindole+LPS reduced the PAS-goblet cell labeling and increased Iba-1-immunolabelled cells compared to the saline group. Immunoblotting for IL-1β, TNF-α, and NF-kB was greater in the Luzindole group. The LPS-challenged group showed higher MPO activity than the saline and Luzindole groups. Catalase was reduced in the Luzindole and Luzindole+LPS groups compared to saline. The Luzindole group showed an increase in NP-SHs, an effect related to compensatory GSH activity. The acute blockade of endogenous MLT with Luzindole induced early changes in inflammatory markers with altered intestinal morphology. The other non-detectable deleterious effects of Luzindole may be balanced by the unopposed direct action of MLT in immune cells bypassing the MT1/MT2 receptors.

Increased intestinal phosphate absorption, an often-overlooked effect of vitamin D.

Increased intestinal phosphate absorption, an often-overlooked effect of vitamin D.

Short-Term Lincomycin Exposure Depletion of Murine Microbiota Affects Short-Chain Fatty Acids and Intestinal Morphology and Immunity.

Lincomycin, as one of the most commonly used antibiotics, may cause intestinal injury, enteritis and other side effects, but it remains unknown whether these effects are associated with microbial changes and the effects of different doses of lincomycin on infants. Here, 21-day old mice were exposed to 1 and 5 g/L lincomycin to explore the effects of lincomycin on the gut microbiota, metabolites and inflammation. Compared to the control mice, 1 g/L lincomycin exposure decreased the body weight gain of mice (p < 0.05). Both 1 and 5 g/L lincomycin exposure reduced the diversity and microbial composition of mice (p < 0.05). Furthermore, 1 and 5 g/L lincomycin reduced the relative concentrations of acetate, propionate, butyrate, valerate, isobutyric acid and isovaleric acid in the colon chyme of mice (p < 0.05). In addition, 5 g/L lincomycin exposure reduced the villus height, crypt depth, and relative expression of TLR2, TLR3, TLR4, IL-18, TNF-α, and p65 in the jejunum of mice (p < 0.05), while 1 g/L lincomycin exposure reduced the relative expression of TLR2, TLR3, TNF-α, and p65 (p < 0.05). Collectively, these results highlight the depletion effect of short-term lincomycin exposure on microbiota and the further regulatory effect on intestinal morphology and immunosuppression in infant mice.

1,25(OH)2 vitamin D3 stimulates active phosphate transport but not paracellular phosphate absorption in mouse intestine.

Intestinal absorption of phosphate proceeds via an active/transcellular route mostly mediated by NaPi-IIb/Slc34a2 and a poorly characterized passive/paracellular pathway. Intestinal phosphate absorption and expression of NaPi-IIb are stimulated by 1,25(OH)2 vitamin D3 but whether NaPi-IIb is the only target under hormonal control remains unknown. We report that administration of 1,25(OH)2 vitamin D3 to wild-type mice resulted in the expected increase in active transport of phosphate in jejunum, without changing paracellular fluxes. Instead, the same treatment failed to alter phosphate transport in intestinal-depleted Slc34a2-deficient mice. In both genotypes, 1,25(OH)2 vitamin D3 induced similar hyperphosphaturic responses and changes in the plasma levels of FGF23 and PTH. While urinary phosphate loss induced by administration of 1,25(OH)2 vitamin D3 did not alter plasma phosphate, further studies should investigate whether chronic administration would lead to phosphate imbalance in mice with reduced active intestinal absorption. Intestinal absorption of phosphate is stimulated by 1,25(OH)2 vitamin D3. At least two distinct mechanisms underlie phosphate absorption in the gut, an active transcellular transport requiring the Na+ /phosphate cotransporter NaPi-IIb/Slc34a2, and a poorly characterized paracellular passive pathway. 1,25(OH)2 vitamin D3 stimulates NaPi-IIb expression and function, and loss of NaPi-IIb reduces intestinal phosphate absorption. However, it is remains unknown whether NaPi-IIb is the only target for hormonal regulation by 1,25(OH)2 vitamin D3 . Here we compared the effects of intraperitoneal administration of 1,25(OH)2 vitamin D3 (2days, once per day) in wild-type and intestinal-specific Slc34a2-deficient mice, and analysed trans- vs. paracellular routes of phosphate absorption. We found that treatment stimulated active transport of phosphate only in jejunum of wild-type mice, though NaPi-IIb protein expression was upregulated in jejunum and ileum. In contrast, 1,25(OH)2 vitamin D3 administration had no effect in Slc34a2-deficient mice, suggesting that the hormone specifically regulates NaPi-IIb expression. In both groups, 1,25(OH)2 vitamin D3 elicited the expected increase of plasma fibroblast growth factor 23 (FGF23) and reduction of parathyroid hormone (PTH). Treatment resulted in hyperphosphaturia (and hypercalciuria) in both genotypes, though mice remained normophosphataemic. While increased intestinal absorption and higher FGF23 can trigger the hyperphosphaturic response in wild types, only higher FGF23 can explain the renal response in Slc34a2-deficient mice. Thus, 1,25(OH)2 vitamin D3 stimulates intestinal phosphate absorption by acting on the active transcellular pathway mostly mediated by NaPi-IIb while the paracellular pathway appears not to be affected.

Pharmacological activation of REV-ERBα improves nonalcoholic steatohepatitis by regulating intestinal permeability

Background and objectivesThe gut–liver axis plays an important role in the pathogenesis of nonalcoholic steatohepatitis (NASH), and increased intestinal permeability causes transfer of endotoxin to the liver, which activates the immune response, ultimately leading to hepatic inflammation. Nuclear receptor Rev-erbα is a critical regulator of circadian rhythm, cellular metabolism, and inflammatory responses. However, the role and mechanism of Rev-erbα in gut barrier function and NASH remain unclear. In the present study, we investigated the involvement of Rev-erbα in the regulation of intestinal permeability and the treatment of NASH. Methods and resultsThe expression of tight junction-related genes and Rev-erbs decreased in the jejunum, ileum and colon of mice with high cholesterol, high fat diet (CL)-induced NASH. Chromatin immunoprecipitation analysis indicated that REV-ERBα directly bound to the promoters of tight junction genes to regulate intestinal permeability. Pharmacological activation of REV-ERBα by SR9009 protected against lipopolysaccharide-induced increased intestinal permeability both in vitro and in vivo, and these effects were associated with the activation of autophagy and decreased apoptotic signaling of epithelial cells. In addition, the chronopharmacological effects of SR9009 were more potent at Zeitgeber time 0 (ZT0) than at ZT12, which was contrary to the rhythm of Rev-erbs in the gastrointestinal tract. The administration of SR9009 attenuated hepatic lipid accumulation, insulin resistance, inflammation, and fibrosis in mice with CL diet-induced NASH, which might be partly attributed to the enhancement of intestinal barrier function. ConclusionChronopharmacological activation of REV-ERBα might be a potential strategy to treat intestinal barrier dysfunction-related disorders and NASH.

Identification of intrinsic primary afferent neurons in mouse jejunum

The gut is the only organ system with intrinsic neural reflexes. Intrinsic primary afferent neurons (IPANs) of the enteric nervous system initiate intrinsic reflexes, form gut-brain connections, and undergo considerable neuroplasticity to cause digestive diseases. They remain inaccessible to study in mice in the absence of a selective marker. Advillin is used as a marker for primary afferent neurons in dorsal root ganglia. The aim of this study was to test the hypothesis that advillin is expressed in IPANs of the mouse jejunum. Advillin expression was assessed with immunohistochemistry and using transgenic mice expressing an inducible Cre recombinase under the advillin promoter were used to drive tdTomato and the genetically encoded calcium indicator GCaMP5. These mice were used to characterize the morphology and physiology of advillin-expressing enteric neurons using confocal microscopy, calcium imaging, and whole-cell patch-clamp electrophysiology. Advillin is expressed in about 25% of myenteric neurons of the mouse jejunum, and these neurons demonstrate the requisite properties of IPANs. Functionally, they demonstrate calcium responses following mechanical stimuli of the mucosa and during antidromic action potentials. They have Dogiel type II morphology with neural processes that mostly remain within the myenteric plexus, but also project to the mucosa and express NeuN and calcitonin gene-related peptide (CGRP), but not nNOS. Advillin marks jejunal IPANs providing accessibility to this important neuronal population to study and model digestive disease.