Mouse Small Intestine Research Articles

The effect of mitochondrial inhibitors on Ca2+ signalling and pacemaking conductances in interstitial cells of Cajal in the mouse small intestine

Myenteric interstitial cells of Cajal (ICC‐MY) in the mouse small intestine are pacemaker cells that generate and propagate electrical slow waves. Conduction of slow waves to smooth muscle cells activates voltage‐gated Ca2+ channels and coordinates rhythmic contractions of intestinal muscles. Slow waves are activated by intracellular Ca2+ release events firing asynchronously from multiple release sites within ICC‐MY to activate the Cl− channel Ano1, the resulting depolarization of which forms slow waves. 1 Propagation of slow waves through the ICC‐MY network depends upon activation of a voltage dependent, T‐type Ca2+ conductance (Cav3.2). Previous studies have suggested that mitochondria play an important role in the Ca2+ signaling responsible for slow waves, and slow waves in mouse small intestine, guinea‐pig stomach and canine colon were blocked by pharmacological agents that inhibit the electron transport chain, reduce mitochondrial transmembrane potential and prevent mitochondrial Ca2+ uptake (FCCP, CCCP and Antimycin). 2 We used in‐situ Ca2+ imaging from the small intestines of mice expressing a genetically encoded Ca2+ indicator (GCaMP3) exclusively in ICC (GCaMP3 activated using Cre‐LoxP approach, driven off the Kit promoter) to visualize Ca2+ transients in ICC‐MY and used spatio‐temporal particle‐based analysis techniques to quantify the effects of mitochondrial inhibitors on Ca2+ transients in ICC‐MY. The protonophores FCCP (1 μM, n=6) and CCCP (1 μM, n=6), the ETC inhibitor, Antimycin (10 μM, n=5), and the mitochondrial Na+/Ca2+ exchanger inhibitor CGP‐37157 (30 μM, n=6) either abolished or drastically reduced Ca2+ transients in ICC‐MY. This effect was not due to ATP depletion, since oligomycin (1 μM, n=6) had no effect on Ca2+ transients. We also used the patch‐clamp technique to measure Ano1 and T‐type Ca2+ (Cav3.2) currents in HEK 293 cells to examine the effects of these drugs on key membrane conductances that are essential for slow wave propagation in ICC‐MY. Antimycin (1–10 μM, n=5) blocked Ano1 and reduced Cav3.2 currents (n=4). CCCP (1–3 μM, n=5) had no effect on Ano1 but significantly reduced Cav3.2 currents (n=5). Both Ano1 and Cav3.2 currents were inhibited by CGP‐37157 (10–30 μM, n=5). Thus, the inhibitory effects of mitochondrial drugs on ICC‐MY Ca2+ signalling and slow wave generation can be explained by direct antagonism of the conductances that generate and propagate slow waves and their effects are not necessarily due to mitochondrial effects. Our findings suggest that ICC pacemaker models, which emphasize a role for mitochondrial Ca2+ handling based on similar pharmacological evidence, should be interpreted with caution.Support or Funding InformationSupported by P01 DK 41315This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evidence for Altered Non‐Canonical Wnt Signaling and Increased Tight Junction Remodeling in Cftr Knockout (KO) Mouse Small Intestine

Recent investigations have shown Cftr expression and its functional activity in the Lgr5+ intestinal stem cell (ISC) population of mice where it acts as a negative regulator of proliferation (Strubberg et al., CMGH 2017). In freshly‐isolated small intestinal crypts and crypts of cultured enteroids from Cftr KO mice, loss of Cftr activity results in increased Wnt/β‐catenin signaling and ISC hyperproliferation. A persistent alkaline intracellular pHi in Cftr KO ISC increases plasma membrane association of the Wnt transducer Disheveled (Dvl) thereby facilitating binding to the Wnt receptor Frizzled 7. Dvl also acts a transducer of non‐canonical Wnt signaling involved in directed cell migration and cell polarity, a necessary accompaniment to crypt proliferation. It is known that both cystic fibrosis (CF) patients and Cftr KO mice exhibit increased intestinal permeability. We hypothesized that increased pHi in progenitor cells of the crypt transit‐amplifying (TA) zone in Cftr KO mice increases plasma membrane Dvl association, potentially driving non‐canonical Wnt signaling for active tight junction (TJ) remodeling and increased permeability. Dvl2 association at the apical membrane was measured in TA cells of live WT and Cftr KO enteroids from mice expressing Dvl2‐EGFP (Dvl2 KO/Dvl2‐EGFP rescue) and membrane Tomato (RosamT/mG) using fluorescence confocal microscopy. Activity of the noncanonical Wnt receptor Frizzled 6 (Fz6) and the Rho GTPase Cdc42, an important regulator of intestinal polarity and migration, assessed TJ remodeling. Enteroid permeability was measured by FITC‐dextran (3 kD) diffusion into the lumen. Dvl2‐EGFP intensity at the apical membrane in TA zone crypt cells was increased ~2‐fold in Cftr KO crypts as compared to WT. Increased Dvl2‐EGFP apical membrane localization could be normalized in Cftr KO crypts by two days exposure to reduced medium pH (pH 6.6). Immunolocalization indicated the Fz6 was well expressed on the basolateral membranes near TJ of both WT and Cftr KO TA zone cells. Immunolocalization revealed increased Cdc42 activity at the TJ and immunoblots showed increased Cdc42 protein amounts in both fresh and enteroid crypts in Cftr KO intestine as compared to WT. Intestinal permeability was significantly increased in Cftr KO vs. WT enteroids. We conclude that cell alkalinity in the Cftr KO crypt epithelium affects Dvl transduction of both canonical and non‐canonical Wnt signaling thereby abnormally increasing proliferation and TJ remodeling. Increased TJ remodeling may contribute to increased intestinal permeability in CF patients.Support or Funding InformationSupported by NIDDK 5R01DK048816 and the Cystic Fibrosis Foundation (CLARKE11G0 and LIU13Q0).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Comparative study of effects of assemblages AII and BIV of Giardia duodenalis on mucosa and microbiota of the small intestine in mice

AimsGiardiasis is one of the major causes of diarrhea worldwide and its symptoms vary in intensity, which can be attributed to different parasite assemblages. The goal of the present study was to compare the effects of infection caused by assemblages AII and BIV ofGiardia duodenalis on the response of the small intestine, microbiota, and behavioral parameters in mice. Main methodsSwiss mice were infected with assemblages AII and BIV of G. duodenalis for 15 days. Leucometry, pain, intestinal microbiota and histological parameters of the duodenum and jejunum were evaluated in the experimental groups. Key findingsBoth assemblages modified the composition of the intestinal microbiota. Infection with assemblage AII promoted leukocytosis, reflected by increasing number of polymorphonuclear cells, intraepithelial lymphocytes and pain-related behavior, indicating that this was the more aggressive assemblage with regard to its effects on the intestinal mucosa and duodenum. SignificanceThe specific assemblage of the parasite is an important parameter that affects symptomatology in the host.

Bile acids are important direct and indirect regulators of the secretion of appetite- and metabolism-regulating hormones from the gut and pancreas

ObjectiveBile acids (BAs) facilitate fat absorption and may play a role in glucose and metabolism regulation, stimulating the secretion of gut hormones. The relative importance and mechanisms involved in BA-stimulated secretion of appetite and metabolism regulating hormones from the gut and pancreas is not well described and was the purpose of this study. MethodsThe effects of bile acids on the secretion of gut and pancreatic hormones was studied in rats and compared to the most well described nutritional secretagogue: glucose. The molecular mechanisms that underlie the secretion was studied by isolated perfused rat and mouse small intestine and pancreas preparations and supported by immunohistochemistry, expression analysis, and pharmacological studies. ResultsBile acids robustly stimulate secretion of not only the incretin hormones, glucose-dependent insulinotropic peptide (GIP), and glucagon-like peptide-1 (GLP-1), but also glucagon and insulin in vivo, to levels comparable to those resulting from glucose stimulation. The mechanisms of GLP-1, neurotensin, and peptide YY (PYY) secretion was secondary to intestinal absorption and depended on activation of basolateral membrane Takeda G-protein receptor 5 (TGR5) receptors on the L-cells in the following order of potency: Lithocholic acid (LCA) >Deoxycholicacid (DCA)>Chenodeoxycholicacid (CDCA)> Cholic acid (CA). Thus BAs did not stimulate secretion of GLP-1 and PYY from perfused small intestine in TGR5 KO mice but stimulated robust responses in wild type littermates. TGR5 is not expressed on α-cells or β-cells, and BAs had no direct effects on glucagon or insulin secretion from the perfused pancreas. ConclusionBAs should be considered not only as fat emulsifiers but also as important regulators of appetite- and metabolism-regulating hormones by activation of basolateral intestinal TGR5.

Dietary Fructose Metabolism By Splanchnic Organs: Size Matters

The initial metabolism of fructose is thought to primarily take place in the liver. Using stable isotope labeling combined with tissue and arterio-venous sampling, Jang etal. (2018) demonstrate that in mice, the small intestine is the primary site of fructose metabolism. This raises important questions about fructose handling in humans.

Berberine ameliorates neonatal necrotizing enterocolitis by activating the phosphoinositide 3-kinase/protein kinase B signaling pathway

Neonatal necrotizing enterocolitis (NEC) is a severe acquired disease that predominantly affects the small intestine of neonates. NEC is caused by a combination of metabolic products, dysfunctions of the blood vessels, mucus and other unknown factors. Berberine may induce beneficial effects on necrotic and cardiovascular diseases due to its anti-inflammatory and anti-apoptotic effects on epithelial cells. In the present study, the therapeutic effects of berberine were investigated and the potential mechanisms by which it functions within a neonatal NEC mouse model were analyzed. Inflammation and levels of associated factors were measured in the serum of mice with NEC prior to and following treatment with berberine. Apoptotic rates in epithelial cells were analyzed following treatment with berberine. The expression of genes associated with apoptosis and apoptosis signaling were determined in epithelial cells in the small intestines of mice with NEC following treatment with berberine. The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway was investigated in epithelial cells isolated from mice following treatment with either berberine or PBS. Histology and immunohistochemistry were used to determine the area of infarction and apoptosis. Body weight and food intake were measured to evaluate the physical effects of berberine on mice with NEC. The results indicated that berberine attenuated the inflammation caused by NEC in mice after 10 days of treatment. The apoptosis rate of epithelial cells isolated from experimental mice was decreased following berberine treatment. Western blot analysis indicated that the expression of the anti-apoptotic genes c-Myc and p53 were upregulated by berberine, whereas caspase-3 and -9 levels were downregulated in epithelial cells following treatment with berberine. In addition, the expression and phosphorylation levels of PI3K and AKT were downregulated in epithelial cells following treatment with berberine. An in vitro assay indicated that treatment with PI3K alone increased the expression of AKT and promoted the apoptosis of epithelial cells. Treatment with berberine markedly increased epidermal growth factor (EGF) and Bcl-2 expression levels, the activity of epithelial cells and decreased the infarction area of the small intestine. Accordingly, the body weight and food intake of mice with NEC were increased following berberine treatment. Therefore, the results of the present study demonstrate that berberine inhibits inflammation and apoptosis via the PI3K/AKT signaling pathway and may therefore attenuate the progression of NEC. These results suggest that berberine may be a potential therapeutic agent for the treatment of patients with NEC.

The effects of mitochondrial inhibitors on Ca2+ signalling and electrical conductances required for pacemaking in interstitial cells of Cajal in the mouse small intestine

Interstitial cells of Cajal (ICC-MY) are pacemakers that generate and propagate electrical slow waves in gastrointestinal (GI) muscles. Slow waves appear to be generated by the release of Ca2+ from intracellular stores and activation of Ca2+-activated Cl− channels (Ano1). Conduction of slow waves to smooth muscle cells coordinates rhythmic contractions. Mitochondrial Ca2+ handling is currently thought to be critical for ICC pacemaking. Protonophores, inhibitors of the electron transport chain (FCCP, CCCP or antimycin) or mitochondrial Na+/Ca2+ exchange blockers inhibited slow waves in several GI muscles. Here we utilized Ca2+ imaging of ICC in small intestinal muscles in situ to determine the effects of mitochondrial drugs on Ca2+ transients in ICC. Muscles were obtained from mice expressing a genetically encoded Ca2+ indicator (GCaMP3) in ICC. FCCP, CCCP, antimycin, a uniporter blocker, Ru360, and a mitochondrial Na+/Ca2+ exchange inhibitor, CGP-37157 inhibited Ca2+ transients in ICC-MY. Effects were not due to depletion of ATP, as oligomycin did not affect Ca2+ transients. Patch-clamp experiments were performed to test the effects of the mitochondrial drugs on key pacemaker conductances, Ano1 and T-type Ca2+ (CaV3.2), in HEK293 cells. Antimycin blocked Ano1 and reduced CaV3.2 currents. CCCP blocked CaV3.2 current but did not affect Ano1 current. Ano1 and Cav3.2 currents were inhibited by CGP-37157. Inhibitory effects of mitochondrial drugs on slow waves and Ca2+ signalling in ICC can be explained by direct antagonism of key pacemaker conductances in ICC that generate and propagate slow waves. A direct obligatory role for mitochondria in pacemaker activity is therefore questionable.

Oral delivery of siRNA lipid nanoparticles: Fate in the GI tract

Oral delivery, a patient-friendly means of drug delivery, is preferred for local administration of intestinal therapeutics. Lipidoid nanoparticles, which have been previously shown to deliver siRNA to intestinal epithelial cells, have potential to treat intestinal disease. It is unknown, however, whether the oral delivery of these particles is possible. To better understand the fate of lipid nanoparticles in the gastrointestinal (GI) tract, we studied delivery under deconstructed stomach and intestinal conditions in vitro. Lipid nanoparticles remained potent and stable following exposure to solutions with pH values as low as 1.2. Efficacy decreased following exposure to “fed”, but not “fasting” concentrations of pepsin and bile salts. The presence of mucin on Caco-2 cells also reduced potency, although this effect was mitigated slightly by increasing the percentage of PEG in the lipid nanoparticle. Mouse biodistribution studies indicated that siRNA-loaded nanoparticles were retained in the GI tract for at least 8 hours. Although gene silencing was not initially observed following oral LNP delivery, confocal microscopy confirmed that nanoparticles entered the epithelial cells of the mouse small intestine and colon. Together, these data suggest that orally-delivered LNPs should be protected in the stomach and upper intestine to promote siRNA delivery to intestinal epithelial cells.

5-aminosalicylic acid improves lipid profile in mice fed a high-fat cholesterol diet through its dual effects on intestinal PPARγ and PPARα.

Obesity is associated with a series of metabolic complications, including dyslipidemia and insulin resistance (IR) that lack effective therapies. In recent years, intestinal inflammation has been suggested to contribute to obesity related metabolic syndrome and targeting gut inflammation with 5-ASA improves diet induced IR, however, its role in dyslipidemia is unknown and has never been explored. In the present study, we reported for the first time that administration of 5-ASA for 12 weeks significantly improved lipid profile by repressing plasma triglycerides and free cholesterol levels in mice fed high-fat cholesterol diet (HFC). In addition, liver lipids were significantly reduced by 5-ASA treatment in HFC-fed mice. Mechanistically, anti-inflammatory genes peroxisome proliferator-activated receptor-γ (Pparγ) and M2 marker, such as Mrc1 and Ym1, were remarkably upregulated, while pro-inflammation gene monocyte chemoattractant protein-1 (Mcp-1) were downregulated in small intestine of mice treated by 5-ASA. Further, 5-ASA improved gastrointestinal barrier by increasing the expression of the tight junction marker ZO-1. 5-ASA also enhanced cholesterol translocation by elevating genes expression of Npc1l1 and Abcg5/8. Moreover, mice fed HFC 5-ASA expressed increased Pparα in small intestinal and its target genes function in lipid oxidation and hydrolysis were remarkable elevated. Taken together, we reported a novel role of 5-ASA which may serve as a therapy target intestinal inflammation induced dyslipidemia.

Effects of ATP on Pacemaker Activity of Interstitial Cells of Cajal from the Mouse Small Intestine.

Purinergic receptors play an important role in regulating gastrointestinal (GI) motility. Interstitial cells of Cajal (ICCs) are pacemaker cells that regulate GI smooth muscle activity. We studied the functional roles of external adenosine 5′-triphosphate (ATP) on pacemaker activity in cultured ICCs from mouse small intestines by using the whole-cell patch clamp technique and intracellular Ca2+ ([Ca2+]i) imaging. External ATP dose-dependently depolarized the resting membrane and produced tonic inward pacemaker currents, and these effects were antagonized by suramin, a purinergic P2 receptor antagonist. ATP-induced effects on pacemaker currents were suppressed by an external Na+-free solution and inhibited by the nonselective cation channel blockers, flufenamic acid and niflumic acid. The removal of external Ca2+ or treatment with thapsigargin (inhibitor of Ca2+ uptake into endoplasmic reticulum) inhibited the ATP-induced effects on pacemaker currents. Spontaneous [Ca2+]i oscillations were enhanced by external ATP. These results suggest that external ATP modulates pacemaker activity by activating nonselective cation channels via external Ca2+ influx and [Ca2+]i release from the endoplasmic reticulum. Thus, it seems that activating the purinergic P2 receptor may modulate GI motility by acting on ICCs in the small intestine.

Peyer's patch-specific Lactobacillus reuteri strains increase extracellular microbial DNA and antimicrobial peptide expression in the mouse small intestine.

Specific Lactobacillus reuteri is autochthonous Lactobacillus species in humans with potential application in food production as a probiotic. The difference in colonizing Peyer's patches (PP) might decide their health-promoting properties. We aimed to investigate the difference between PP- and lumen-specific L. reuteri on antimicrobial peptide expression in this study. L. reuteri strains were isolated from PP and the lumen of C57BL/6J mice, which were used to treat mice. PP-specific L. reuteri cells stimulate RegIIIγ mRNA expression of the crypt epithelial sample. PP-specific L. reuteri induces accumulation of extracellular DNA (eDNA) in the bottom of crypts. eDNA was extracted from the small-intestinal mucus, the yield of which was significantly increased after the PP-specific L. reuteri treatment. And it increased cytokine production in RAW264.7 murine macrophages. PP-specific L. reuteri significantly increased the relative abundance of Bacteroidetes-, Lactobacillus-, and Proteobacteria-derived eDNA. However, the levels of Strentrophomonas-derived eDNA decreased. These results provide a rationale for the screening of human derived L. reuteri with an immune-modulatory property.

The ubiquitin ligase ITCH coordinates small intestinal epithelial homeostasis by modulating cell proliferation, differentiation, and migration

Maintenance of the intestinal mucosa is driven by local signals that coordinate epithelial proliferation, differentiation, and turnover in order to separate antigenic luminal contents from the host's immune system. Breaches in this barrier promote gastrointestinal pathologies ranging from inflammatory bowel disease to cancer. The ubiquitin ligase ITCH is known to regulate immune responses, and loss of function of ITCH has been associated with gastrointestinal inflammatory disorders, particularly in the colon. However, the small intestine appears to be spared from this pathology. Here we explored the physiological mechanism that underlies the preservation of mucosal homeostasis in the small intestine in mice lacking ITCH (Itcha18H/a18H). Histological analysis of the small intestines from young adult mice revealed architectural changes in animals deficient for ITCH, including villus blunting with cell crowding, crypt expansion, and thickening of the muscularis propria relative to age-matched mice sufficient for ITCH. These differences were more prominent in the distal part of the small intestine and were not dependent upon lymphoid cells. Underlying the observed changes in the epithelium were expansion of the Ki67+ proliferating transit amplifying progenitor population and increased numbers of terminally differentiated mucus-secreting goblet and anti-microbial producing Paneth cells, which are both important in controlling local inflammation in the small intestine and are known to be dysregulated in inflammatory bowel disease. Homeostasis in the small intestine of Itcha18H/a18H animals was maintained by increased cell turnover, including accelerated migration of epithelial cells along the crypt-villus axis and increased apoptosis of epithelial cells at the crypt-villus junction. Consistent with this enhanced turnover, Itcha18H/a18H mice carrying the Min mutation (Itcha18H/a18H; ApcMin/+) displayed a 76% reduction in tumor burden as compared to ApcMin/+ littermates with normal levels of ITCH. These findings highlight the role of ITCH as an important modulator of intestinal epithelial homeostasis.

Activation of intestinal GR-FXR and PPARα-UGT signaling exacerbates ibuprofen-induced enteropathy in mice.

Nonsteroidal anti-inflammatory drugs (NSAIDs)-induced small intestinal injury (enteropathy) occurs in about two-thirds of regular NSAID users. To date, there is no proven-effective treatment for NSAID enteropathy, and its underlying mechanism remains obscure. The present study showed that glucocorticoids are an important determinant of NSAID enteropathy. High dose dexamethasone (DEX, 75mg/kg) markedly exacerbated the acute toxicity of ibuprofen (IBU, 200mg/kg) in the small intestine of mice, which was not due to the pregnane-X-receptor pathway. Instead, glucocorticoid receptor (GR) mediated the effect of DEX (5mg/kg) on both the acute (200mg/kg) and 7-day repeated-dose (50mg/kg) toxicity of IBU in the small intestine. Combined treatment of DEX (5mg/kg) and IBU (50mg/kg) synergistically repressed the intestinal farnesoid X receptor (FXR)-cystathionine-γ-lyase signaling, which was accompanied with an elevation in the biliary excretion of bile acids, especially the FXR antagonist tauro-β-muricholic acid. DEX (5mg/kg) also activated intestinal peroxisome proliferator-activated receptor α (PPARα)-UDP-glucuronosyltransferase (UGT) pathway, which increased the formation and enterohepatic circulation of IBU-acyl glucuronide. Furthermore, DEX (5mg/kg) and IBU (50mg/kg) altered the intestinal microbial composition, characterized with a marked decrease in Actinobacteria. To conclude, the present study for the first time suggests that glucocorticoids play vital roles in control of IBU enteropathy via intestinal GR-FXR and PPARα-UGT signaling.

Mouse d-Amino-Acid Oxidase: Distribution and Physiological Substrates.

d-Amino-acid oxidase (DAO) catalyzes the oxidative deamination of d-amino acids. DAO is present in a wide variety of organisms and has important roles. Here, we review the distribution and physiological substrates of mouse DAO. Mouse DAO is present in the kidney, brain, and spinal cord, like DAOs in other mammals. However, in contrast to other animals, it is not present in the mouse liver. Recently, DAO has been detected in the neutrophils, retina, and small intestine in mice. To determine the physiological substrates of mouse DAO, mutant mice lacking DAO activity are helpful. As DAO has wide substrate specificity and degrades various d-amino acids, many d-amino acids accumulate in the tissues and body fluids of the mutant mice. These amino acids are d-methionine, d-alanine, d-serine, d-leucine, d-proline, d-phenylalanine, d-tyrosine, and d-citrulline. Even in wild-type mice, administration of DAO inhibitors elevates D-serine levels in the plasma and brain. Among the above d-amino acids, the main physiological substrates of mouse DAO are d-alanine and d-serine. These two d-amino acids are most abundant in the tissues and body fluids of mice. d-Alanine derives from bacteria and produces bactericidal reactive oxygen species by the action of DAO. d-Serine is synthesized by serine racemase and is present especially in the central nervous system, where it serves as a neuromodulator. DAO is responsible for the metabolism of d-serine. Since DAO has been implicated in the etiology of neuropsychiatric diseases, mouse DAO has been used as a representative model. Recent reports, however, suggest that mouse DAO is different from human DAO with respect to important properties.

Regeneration Potential of Lymphoid Tissue of Small Intestine in Mice after Exposure to Low-Intensity Radiation.

Number and size of aggregated lymphoid nodules in the small intestine of mice exposed to radiation using a γ-irradiation unit GOBO-60 with 137Cs source were measured using the methods of visual microscopy and morphometry. Morphological status of the nodules was estimated on days 4, 28, 60, and 90 after the exposure. Significant changes were observed in the lymphoid system of the small intestine after exposure to low-intensity radiation. The size of aggregated lymphoid nodules decreased and the relative number of cells with degenerative changes increased. Regenerative processes were observed as soon as on day 4 of the rehabilitation period. However, the number and sizes of aggregated lymphoid nodules (length, width, and section area), and the ratio of lymphoid nodules with germinal center were still lower than in the control. The number of cells forming lymphoid interstitial plaques was close to the control on days 28-90 of the rehabilitation process.

The impact of sesquiterpenes β-caryophyllene oxide and trans-nerolidol on xenobiotic-metabolizing enzymes in mice in vivo

1. Sesquiterpenes, constituents of plant essential oil, are popular bioactive compounds due to the positive effect on human health, but their potential toxicity and possible herb-drug interactions are often omitted. In our in vivo study, we followed up the effect of p.o. administration of two sesquiterpenes β-caryophyllene oxide (CAO) and trans-nerolidol (NER) on various xenobiotic-metabolizing enzymes in mice liver and small intestine.2. To spot the early effect of studied compounds, enzymatic activity and mRNA levels were assessed 6 and 24 h after single dose.3. CAO and NER markedly increased cytochromes P450 (CYP2B, 3A, 2C) activity and mRNA levels in both tissues. Liver also showed elevated activity of aldo-ketoreductase 1C and carbonyl reductase after treatment. Contrary, sesquiterpenes decreased NAD(P)H:quinone oxidoreductase 1 activity in small intestine. Among conjugation enzymes, only liver sulfotransferase activity was increased by sesquiterpenes.4. Our results document that single dose of sesquiterpenes modulate activities and expression of several xenobiotic-metabolizing enzymes.

Analysis of the Specificity of IgA Antibodies Produced in the Mouse Small Intestine

Analysis of the Specificity of IgA Antibodies Produced in the Mouse Small Intestine

Impact of chrysosplenetin, per se or in combination with artemisinin, on breast cancer resistance protein (Bcrp)/ABCG2 mRNA expression levels in mice small intestine

Our previous work revealed that chrysosplenetin in combination with artemisinin inhibited in vivo P-glycoprotein (P-gp, one of classic multi-drug resistance proteins) mediated digoxin transportation activity by reversing the upregulated P-gp/Mdr1 mRNA expression levels by artemisinin. Therefore, chrysosplenetin might be a potential artemisinin-resistance reversal agent as a P-gp inhibitor. But it still remains unknown if chrysosplenetin has an impact on another pivotal multi-drug resistance protein, breast cancer resistance protein (Bcrp), which is co-expressed with P-gp in apical membrane of intestinal epithelial cell and overlaps some of the substrates and inhibitors. This study, therefore, further addressed the impact of chrysosplenetin, per se or in combination with artemisin, on Bcrp/ABCG2 mRNA expression levels in mice small intestine determined by western blot and real time-quantitative polymerase chain reaction (RT-qPCR) assay. The drugs were intragastrically administrated once per day for 7 days. Novobiocin, a known Bcrp inhibitor, was observed to have no impact on Bcrp/ABCG2 levels with or without artemisinin versus vehicle. Interestingly, artemisinin alone attenuated Bcrp level while chrysosplenetin alone increased it (p<0.05). Relative mRNA level was significantly decreased when co-used with artemisinin and chrysosplenetin in ratio of 1:2 (p<0.05). The discrepant results for chrysosplenetin on Bcrp/ABCG2 mRNA expressions might be closely related to the transcriptional or posttranscriptional regulation.

Nano and bulk ZnO trigger diverse Zn-transport-related gene transcription in distinct regions of the small intestine in mice after oral exposure

The oral ingestion of ZnO nanoparticles (NPs) has attracted considerable attention because of the wide usage in food packaging and additives. The small intestine is the major absorption site for ZnO NPs. Unfortunately, studies on the absorption of ZnO NPs in the GIT were still scarce. This study evaluated the absorption characteristics of ZnO NPs (30 nm) and bulk ZnO (rod morphology with a mean size of 139–846 nm). Results showed that ZnO NPs and bulk ZnO were absorbed and redistributed in various organs of mice at 4 h after exposure. Significantly higher levels of MT1 were observed in the duodenums of bulk ZnO and ZnO NPs groups than those of the control (9.8 and 5660.11 fold increases, respectively). The MT4 levels in the bulk ZnO and ZnO NPs groups also showed 4.07 and 43.21fold increases, respectively. In addition, the transcript levels of ZIPs, ZnTs, and MTs in the jejunum of bulk ZnO group were higher than those of ZnO NPs group. And the transcript levels of ZIPs, ZnTs, and MTs were all lower in the ileum than in the jejunum. The results suggested that ZnO NPs were mainly absorbed in the duodenum in the form of particles and can be absorbed as Zn2+ in the jejunum and secondly in the ileum. By contrast, bulk ZnO was more easily absorbed as Zn2+ in the jejunum and secondly in the ileum.

Atf3 deficiency promotes genome instability and spontaneous tumorigenesis in mice.

Mice lacking genes involving in the DNA damage response (DDR) are often tumor prone owing to genome instability caused by oncogenic challenges. Previous studies demonstrate that activating transcription factor 3 (ATF3), a common stress sensor, can activate the tumor suppressor p53 and regulate expression of p53 target genes upon DNA damage. However, whether ATF3 contributes to the maintenance of genome stability and tumor suppression remains unknown. Here we report that Atf3-deficient (Atf3-/-) mice developed spontaneous tumors, and died significantly earlier than wild-type (Atf3+/+) mice. Consistent with these results, Atf3-/- mouse embryonic fibroblasts (MEFs) had more aberrant chromosomes and micronuclei, and were genetically unstable. Whereas we demonstrated that ATF3 activated p53 and promoted its pro-apoptotic activity in mouse thymi and small intestines, the chromosomal instability caused by Atf3 deficiency was largely dependent on the regulation of p53 by ATF3. Interestingly, loss of Atf3 also promoted spontaneous tumorigenesis in Trp53+/- mice, but did not affect tumor formation in Trp53-/- mice. Our results thus provide the first genetic evidence linking ATF3 to the suppression of the early development of cancer, and underscore the importance of ATF3 in the maintenance of genome integrity.