Intestinal Permeability Assay Research Articles

Cadmium modulates intestinal Wnt/β-catenin signaling ensuing intestinal barrier disruption and systemic inflammation

The intricate architecture of the intestinal epithelium, crucial for nutrient absorption, is constantly threatened by environmental factors. The epithelium undergoes rapid turnover, which is essential for maintaining homeostasis, under the control of intestinal stem cells (ISCs). The central regulator, Wnt/β-catenin signaling plays a key role in intestinal integrity and turnover. Despite its significance, the impact of environmental factors on this pathway has been largely overlooked. This study, for the first time, investigates the influence of Cd on the intestinal Wnt signaling pathway using a mouse model. In this study, male BALB/c mice were administered an environmentally relevant Cd dose (0.98 mg/kg) through oral gavage to investigate the intestinal disruption and Wnt signaling pathway. Various studies, including histopathology, immunohistochemistry, RT-PCR, western blotting, ELISA, intestinal permeability assay, and flow cytometry, were conducted to study Cd-induced changes in the intestine. The canonical Wnt signaling pathway experienced significant downregulation as a result of sub-chronic Cd exposure, which caused extensive damage throughout the small intestine. Increased intestinal permeability and a skewed immune response were also observed. To confirm that Wnt signaling downregulation is the key driver of Cd-induced gastrointestinal toxicity, mice were co-exposed to LiCl (a recognized Wnt activator) and Cd. The results clearly showed that the harmful effects of Cd could be reversed, which is strong evidence that Cd mostly damages the intestine through the Wnt/β-catenin signalling axis. In conclusion, this research advances the current understanding of the role of Wnt/β catenin signaling in gastrointestinal toxicity caused by diverse environmental pollutants.

Role of transient receptor potential vanilloid 4 channels in an ovalbumin-induced murine food allergic model.

The prevalence of food allergy (FA) has increased worldwide but an effective therapeutic strategy has not been established. Transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive nonselective cation channel, is mainly expressed in the epithelium of various organs. The present study investigated the role of TRPV4 in the pathogenesis of an ovalbumin (OVA)-induced FA model in mice. Wild-type (WT) and TRPV4-deficient (TRPV4KO) mice were sensitized and challenged by OVA to establish FA model. Intestinal tissue samples were processed for biochemical, molecular, and image analyses. Intestinal permeability and antigen uptake assay were conducted using FITC-dextran and OVA-FITC, respectively. TRPV4 was expressed in the colonic epithelium in normal and OVA-treated WT mice. Repeated oral administration of OVA to mice induced systemic allergic symptoms, diarrhea, upregulation of T helper 2 cytokines, OVA-specific immunoglobulin, and FA-related inflammatory cells. These responses were significantly augmented in TRPV4KO mice compared with WT mice. After the induction of FA, the intestinal permeability was significantly increased in TRPV4KO mice compared with WT mice. The expressions of the tight junction protein occludin and adherence junction protein E-cadherin in the colon were significantly lower in TRPV4KO mice compared with WT mice under normal and FA conditions. In addition, the uptake of OVA by CD11c-positive cells was significantly increased in TRPV4KO mice compared with WT mice under FA conditions. These results suggest that epithelial TRPV4 protects against OVA-induced FA symptoms by suppressing the penetration of allergens by maintaining epithelial barrier functions.

A preventative role of nitrate for hypoxia-induced intestinal injury

BackgroundStudying effective interventions for hypoxia-induced injury is crucial, particularly in high-altitude areas. Symptoms stemming from intestinal injuries have a significant impact on the health of individuals transitioning from plains to plateau regions. This research explores the effects and mechanisms of nitrate supplementation in preventing hypoxia-induced intestinal injury. MethodsA hypoxia survival mouse model was established using 7% O2 conditions. The intervention with 4 mM sodium nitrate (NaNO3) in drinking water commenced 7 days prior to hypoxia exposure. Weight monitoring, hematoxylin and eosin (HE) staining, transmission electron microscopy (TEM), and intestinal permeability assays were employed for physiological, histological, and functional analyses. Quantitative PCR (qPCR), Western blot, and immunofluorescence were utilized to analyze the levels of tight junction (TJ) proteins and hypoxia-inducible factor 1α (Hif 1α). RNA sequencing (RNA-seq) identified nitrate's target, and chromatin immunoprecipitation (ChIP) verified the transcriptional impact of Hif 1α on TJ proteins. Villin-cre mice infected with AAV9-FLEX-EGFP-Hif 1α were used for mechanism validation. ResultsThe results demonstrated that nitrate supplementation significantly alleviated small intestinal epithelial cell necrosis, intestinal permeability, disruption of TJs, and weight loss under hypoxia. Moreover, the nitrate-triggered enhancement of TJs is mediated by Hif 1α nuclear translocation and its subsequent transcriptional function. The effect of nitrate supplementation on TJs was largely attributed to the stimulation of the EGFR/PI3K/AKT/mTOR/Hif 1α signaling pathways. ConclusionNitrate serves as a novel approach in preventing hypoxia-induced intestinal injury, acting through Hif 1α activation to promote the transcription of TJ proteins. Furthermore, our study provides new and compelling evidence for the protective effects of nitrate in hypoxic conditions, especially at high altitudes.

Gut-liver interaction study on an all-polydimethylsiloxane microfluidic device integrating intestinal paracellular permeability assay

Microphysiological systems (MPSs) have attracted increasing attention as a method for simulating in vitro drug efficiency. In particular, the interaction between liver and intestine tissues is one of the primary targets since they are closely involved in drug absorption and metabolism. However, most of the intestine-liver MPSs reported previously require pumps, electrodes, and porous membranes for co-culture of cells and evaluation of intestinal permeability (i.e., Trans-Epithelial Electrical Resistance, TEER), requiring complex manufacturing processes and operations. In this study, we report an all-polydimethylsiloxane (PDMS) co-culture microfluidic device, connecting microchamber-based paracellular transport assay on gut microtissues to liver tissues matured on the same device. On one side of the device, HepaRG cells are confined within thin parallel grooves that induce their differentiation into hepatocytes. The other side of the device is connected with microchannels to the liver side and includes the gut tissues, organized above microchambers. Such microchambers allow the evaluation of paracellular permeability by fluorescence imaging. Thanks to the microfluidic device we investigated changes in intestinal permeability induced by differentiated hepatocyte excretion and found that Caco-2 permeability was decreased when co-culture with HepaRG. Due to its simplicity and straightforward implementation, this method is anticipated as an innovative and efficient approach to assess tissue barrier function in multi-organ on-chip experiments.

Atheroprotective CD40-TRAF6 inhibitors: translational pipeline towards a clinical application

Abstract Background The co-stimulatory CD40–CD40 ligand axis is central in atherogenesis. Upon activation, CD40 recruits tumor necrosis factor receptor-associated factors (TRAFs) to induce downstream signaling. Murine studies with mutated CD40-TRAF binding sites in macrophages have shown that the CD40-TRAF6, rather than CD40-TRAF2/3/5, axis is crucial for atherosclerosis. We thus synthesized the small molecule inhibitor TRAF-STOP 6877002, that selectively blocks CD40-TRAF6 signaling. 6877002 successfully reduced the onset and development of atherosclerosis by reducing monocyte recruitment and macrophage activation in ApoE-/- mice without having immunosuppressive side effects. Purpose To assess whether 6877002 is a suitable candidate to pass the translational pipeline towards a clinical application. Methods Elaborate in vitro and in vivo assays evaluating the pharmacological properties and potential toxicity of 6877002. Results Together with Cyprotex, we performed in vitro assays examining the ADME profile of 6877002. In an intestinal permeability assay, 6877002 had low to moderate resorption with no significant efflux, suggesting it is not a substrate for the efflux transporters Pgp/BCRP or MRP2. A plasma protein binding assay showed very low unbound 6877002 fractions, indicating that it is highly bound to plasma proteins. The potential to inhibit cytochrome P450 enzymes was assessed using human liver microsomes in the presence of enzyme-specific probe substrates. While 6877002 inhibited CYP1A2 (IC50=4.01 µM), no potential for inhibiting other CYP isoforms was detected, suggesting low drug-drug interaction potential. To screen for putative cardiovascular risks, hERG ion channel inhibition was assessed and no IC50 could be determined. Hepatic clearance was rapid in mice, rats and dogs, while minipig and human hepatocytes had lower intrinsic clearance. Together with Aptuit, we developed suitable formulations for oral and i.v. administration. In vivo pharmacokinetics studies in rats showed low bioavailability (8.43%) and rapid clearance of orally administered 6877002 compared to i.v. injected 6877002, likely – and in line with our in vitro data – due to fast first-past effect in mouse hepatocytes and uptake by macrophages. To explain the high efficacy of 6877002 despite its rapid metabolism, we assessed whether 6877002 has active metabolites that bind the CD40-TRAF6 pocket and performed an LC-MS/MS-based metabolite profiling. We detected 17 metabolites. Further MD simulations confirmed that Metabolite (M)4, the most abundant metabolite, binds the CD40-TRAF6 pocket, reflected by the highest binding mode (-19.6; defined as Binding Free Energy derived from 40-50 ns of MD run as compared to -16.5 for 6877002). Similar to 6877002, M4 reduced atherosclerosis by 54% in Western diet-fed ApoE-/- mice, indicative of M4 being an active metabolite. Conclusion CD40-TRAF6 inhibitors are an attractive strategy to combat atherosclerosis with high translational potential.

25 Measuring Intestinal Permeability When Urine is not an Option

Abstract The need to understand intestinal health is of increasing importance as the livestock industry seeks to maintain performance efficiency while eliminating prophylactic antibiotic usage. Intestinal permeability is a biomarker of intestinal health as it measures the movement of an indigestible marker across the intestinal barrier with excretion in the urine as the primary biological specimen collected for measurement. The adoption of these methods into poultry has been slow due to the lack of urine excretion. Therefore, the objective of this study is to compare differences in intestinal permeability assays in serum of poultry to determine if these assays could be a reliable method to detect changes in intestinal permeability in poultry under a known stressor. To compare the two commonly used methods [fluorescein isothiocyanate-dextran (FITC-D) method and lactulose/mannitol (LMS) dual sugar method], 2 independent trials were conducted with 40 pens split across 2 fed status (fed or fasted for 12 hours) and 4 sugar treatments (no sugar, FITC-D, LMS, FITC-D+LMS). All pens were sampled at 3 timepoints (14, 28, and 42 days of age) with 5 birds sampled per timepoint. Serum FITC-D was determined through fluorescent spectrophotometry, while mannitol and lactulose concentrations were determined using ion chromatography. Data were analyzed using PROC Glimmix. Fed status, sugar treatment, age, and all 2-and 3-way interactions were included as fixed effects. Trial was included in the model as a random effect with pen included as a repeated effect. Serum lactulose and FITC-D increased in fasted compared with fed birds (P < 0.006). Surprisingly, mannitol, which was expected to be similar across fed and fasted, was increased in fed compared with fasted birds (P < 0.001). The increased concentrations of mannitol in fed birds and measurable concentrations of mannitol in birds not given mannitol suggest another molecule eluted at the same rate as mannitol. Therefore, we propose under these conditions that mannitol, and its ratio with lactulose, are not appropriate methods for measuring intestinal permeability in poultry. Lactulose and FITC-D were increased in serum when the birds were provided those sugars and serum concentration of both sugars decreased at 28 days compared with 14 and 42 days (P < 0.003). FITC-D had a significant 2-way interaction for the sugar by fed status (P < 0.05) whereas other 2-and 3-way interactions were similar across treatments (P > 0.202). Serum lactulose concentrations were affected by all 2-and 3-way interactions with elevated serum lactulose concentrations in broilers provided lactulose and fasted (P < 0.001). Birds that did not receive lactulose had very low to undetectable concentrations of lactulose. This reduced background noise for the detection of serum lactulose and the ability statistically to detect small changes in intestinal permeability make lactulose a potential serum biomarker for intestinal permeability in poultry.

Traditional Medicine Pien Tze Huang Suppresses Colorectal Tumorigenesis Through Restoring Gut Microbiota and Metabolites

Pien Tze Huang (PZH) is a well-established traditional medicine with beneficial effects against inflammation and cancer. We aimed to explore the chemopreventive effect of PZH in colorectal cancer (CRC) through modulating gut microbiota. CRC mouse models were established by azoxymethane plus dextran sulfate sodium treatment or in Apcmin/+ mice treated with or without PZH (270 mg/kg and 540 mg/kg). Gut barrier function was determined by means of intestinal permeability assays and transmission electron microscopy. Fecal microbiota and metabolites were analyzed by means of metagenomic sequencing and liquid chromatography mass spectrometry, respectively. Germ-free mice or antibiotic-treated mice were used as models of microbiota depletion. PZH inhibited colorectal tumorigenesis in azoxymethane plus dextran sulfate sodium-treated mice and in Apcmin/+ mice in a dose-dependent manner. PZH treatment altered the gut microbiota profile, with an increased abundance of probiotics Pseudobutyrivibrio xylanivorans and Eubacterium limosum, while pathogenic bacteria Aeromonas veronii, Campylobacter jejuni, Collinsella aerofaciens, and Peptoniphilus harei were depleted. In addition, PZH increased beneficial metabolites taurine and hypotaurine, bile acids, and unsaturated fatty acids, and significantly restored gut barrier function. Transcriptomic profiling revealed that PZH inhibited PI3K-Akt, interleukin-17, tumornecrosis factor, and cytokine-chemokine signaling. Notably, the chemopreventive effect of PZH involved both microbiota-dependent and -independent mechanisms. Fecal microbiota transplantation from PZH-treated mice to germ-free mice partly recapitulated the chemopreventive effects of PZH. PZH components ginsenoside-F2 and ginsenoside-Re demonstrated inhibitory effects on CRC cells and primary organoids, and PZH also inhibited tumorigenesis in azoxymethane plus dextran sulfate sodium-treated germ-free mice. PZH manipulated gut microbiota and metabolites toward a more favorable profile, improved gut barrier function, and suppressed oncogenic and pro-inflammatory pathways, thereby suppressing colorectal carcinogenesis.

Comparison of Intestinal Permeability Methods in Broilers over a 6-Week Growth Period

The adoption of methods detecting intestinal permeability in poultry has been slow due to the lack of urine availability in avian species. The objective of this study was to examine intestinal permeability assays in broilers using serum. Fluorescein isothiocyanate-dextran (FITC-D) and lactulose/mannitol/sucralose (LMS), indigestible sugars, were used to detect intestinal permeability across two fed states (fed or fasted) and four sugar treatments (Control, FITC-D, LMS, or FITC-D+LMS). Broilers housed in pens were assigned one of eight treatments and sampled on 14, 28, and 42 days of age. Data were analyzed using PROC Glimmix for fed state, sugar treatment, age, and all interactions. Serum lactulose and FITC-D increased in fasted compared to fed birds (p < 0.006), whereas mannitol increased in fed compared to fasted birds (p < 0.001). Serum lactulose and FITC-D decreased on day 28 compared to other timepoints (p < 0.003). Serum FITC-D only had a significant sugar by fed state interaction (p < 0.05) with elevated concentrations in fasted and fed birds that received FITC-D. Serum lactulose was significant for all interactions with elevated concentrations in broilers provided lactulose and fasted (p < 0.001). The ability to detect a three-way interaction with serum lactulose suggests an increased sensitivity; however, additional studies are needed.

Role of SIRT2 in intestinal barrier under cold exposure

Prolonged cold exposure causes body stress and damages health. The intestinal environment is complex and variable, and direct contact with the external environment can easily cause stress, damage and even lead to diseases such as diarrhea. AimsThis study aimed to reveal the role of cold exposure on ileum damage and the role of SIRT2 in this process. Main methodsC57BL6 mice and SIRT2 knockout mice were used to construct a chronic cold exposure model (21 days, random 4 °C exposure for 3 h per day), which was tested by various methods, including intestinal permeability assays, morphological assays, ultrastructural assays, western blotting, and fluorescence staining. In vitro assays were performed on the mouse small intestinal epithelial cell line MODE-K to investigate the role of endoplasmic reticulum stress, SIRT2 knockout, and autophagy on tight junctions. Key findingsThe results showed that chronic cold exposure damaged the ileal epithelial barrier, with endoplasmic reticulum stress. Knockout of SIRT2 alleviates ileal injury via enhanced autophagy under cold exposure. And autophagy can restore the expression of ZO-1 under stress. SignificanceThis study can provide potential target and basic data for the treatment of IBD and other disorders of the intestinal barrier. Autophagy may be an important means of restoring damage to the intestinal barrier.

Antibiotic use during pregnancy is linked to offspring gut microbial dysbiosis, barrier disruption, and altered immunity along the gut-lung axis.

Antibiotic use during pregnancy is associated with increased asthma risk in children. Since approximately 25% of women use antibiotics during pregnancy, it is important to identify the pathways involved in this phenomenon. We investigate how mother-to-offspring transfer of antibiotic-induced gut microbial dysbiosis influences immune system development along the gut-lung axis. Using a mouse model of maternal antibiotic exposure during pregnancy, we immunophenotyped offspring in early life and after asthma induction. In early life, prenatal-antibiotic exposed offspring exhibited gut microbial dysbiosis, intestinal inflammation (increased fecal lipocalin-2 and IgA), and dysregulated intestinal ILC3 subtypes. Intestinal barrier dysfunction in the offspring was indicated by a FITC-dextran intestinal permeability assay and circulating lipopolysaccharide. This was accompanied by increased T-helper (Th)17 cell percentages in the offspring's blood and lungs in both early life and after allergy induction. Lung tissue additionally showed increased percentages of RORγt T-regulatory (Treg) cells at both time points. Our investigation of the gut-lung axis identifies early-life gut dysbiosis, intestinal inflammation, and barrier dysfunction as a possible developmental programming event promoting increased expression of RORγt in blood and lung CD4+ T cells that may contribute to increased asthma risk.

Ex vivo intestinal permeability assay (X-IPA) for tracking barrier function dynamics

The intestinal epithelial barrier facilitates homeostatic host–microbiota interactions and immunological tolerance. However, mechanistic dissections of barrier dynamics following luminal stimulation pose a substantial challenge. Here, we describe an ex vivo intestinal permeability assay, X-IPA, for quantitative analysis of gut permeability dynamics at the whole-tissue level. We demonstrate that specific gut microbes and metabolites induce rapid, dose-dependent increases to gut permeability, thus providing a powerful approach for precise investigation of barrier functions.

Erk5 functions in modulation of zebrafish intestinal permeability.

The intestine of zebrafish consists of mucosa, muscularis and serosa. Intestinal epithelial cells (IECs) act as a physical and biochemical barrier to protect against invasion by external commensal bacteria. Cell junction is one of the crucial basis of the barrier function. When cell junctions were disrupted, intestinal permeability would be naturally impeded. Extracellular signal-regulated kinase 5 (ERK5), belonging to the Mitogen-activated protein kinase (MAPK) family, is involved in the normal physiological development of the cardiovascular system and nervous system. But the role of erk5 in intestinal morphogenesis and intestinal function is yet to know. Here, we showed that knockout of the erk5 in zebrafish larvae resulted in intestinal wall hypoplasia, including the thinned intestinal wall, reduced intestinal folds, and disrupted cell junctions. In addition, the intestinal permeability assay demonstrated that knockout of erk5 resulted in increased intestinal permeability. All of these showed that erk5 plays an essential role in the maintenance of intestinal barrier function. Thus, our data indicate that erk5 is a critical effector in intestinal morphogenesis and intestinal function, and dysfunction of erk5 would lead to intestinal diseases.

Carbon monoxide-releasing molecule 2 inhibits inflammation associated with intestinal ischemia-reperfusion injury in a rat model of hemorrhagic shock.

Intestinal ischemia-reperfusion injury (IRI) occurs in multiple clinical settings and contributes to systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS). Due to the innate inflammatory immune nature, T cells play a crucial role in the pathogenesis of IRI, which includes not only CD4+T cells, but also CD8+and γδ T cells. Carbon monoxide (CO) plays an important role in regulating CD4+T cell responses and has been proven to be a novel therapeutic target in a variety of inflammatory disease models. This study aimed to assess whether pretreatment with carbon monoxide-releasing molecule-2 (CORM-2) could ameliorate inflammation by regulating differentiation of CD4+T cells in intestinal mucosa of rats undergoing hemorrhagic shock. A hemorrhagic shock model was established to study intestinal IRI. Morphological changes were investigated using light microscopes. Fluorescein isothiocyanate-dextran (FITC-dextran) was used as an indicator of intestinal paracellular permeability. Transcription factors involved in differentiation of CD4+T cells in intestinal mucosa were detected by immunofluorescence, and the expression levels of related cytokines were determined by Western blotting. The results of hematoxylin-eosin (H-E) staining and FITC-dextran intestinal paracellular permeability assay revealed that CORM-2 maintained the integrity of intestinal mucosal barrier and inhibited the changes of intestinal mucosal permeability. In addition, activation of T helper type 1 (Th1) and T helper type 17 (Th17) cells, and the increased expression levels of proinflammatory cytokines, such as interleukin-17 (IL-17) and interferon-gamma (IFN-γ), were observed in intestinal IRI process. In contrast, pretreatment with CORM-2 weakened changes of the abovementioned observations, in which inhibited activation of Th1 and Th17 cells. However, CORM-2 did not influence differentiation of regulatory T (Treg) cells in intestinal IRI progress. Notably, CORM-2 significantly upregulated the expression level of interleukin-10 (IL-10) protein and downregulated the expression levels of IL-17 and IFN-γ proteins in ileal tissues of rats. CORM-2 possessed anti-inflammatory effects in the progress of intestinal IRI by inhibiting activation of Th1 and Th17 cells in rats undergoing hemorrhagic shock.

Basal diet composition contributes to differential performance, intestinal health, and immunological responses to a microalgae-based feed ingredient in broiler chickens

Novel feed ingredients may improve poultry health, but functionality of these ingredients may vary across basal diet formulations. This study evaluated a proprietary algae ingredient's effects on broiler performance, intestinal health, systemic immunity, and metabolic/immune kinotypes between corn- or wheat-based diets. Ross 308 broilers were housed in 80 floor pens (14 birds/pen) and assigned to 1 of 4 corn or wheat-based diets ± 0.175% algae ingredient for 42 d. At the end of each 14 d starter, grower, and finisher period, 10 birds/treatment were euthanized for tissue collection to assess intestinal histomorphology, systemic immune cell populations by flow cytometry and kinotypes by peptide arrays. On d 28 and 29, forty-three birds/treatment underwent a 12 h feed restriction challenge followed by a fluorescein isothiocyanate-dextran intestinal permeability assay. For the entire 42 d study, wheat-based diets improved feed conversion rate (FCR) by 5 points compared to corn-based diets (P < 0.0001). Performance benefits related to algae inclusion were diet dependent, with algae inclusion improving 42 d FCR by 6 points only in corn-based diets (P=0.006). Birds fed wheat-based diets had reduced splenic monocyte/macrophage, CD1.1+, and T cell populations in the first 14 d (P < 0.0001) and reduced serum fluorescence on d 28/29 (P=0.0002). Algae inclusion in the corn-based diet increased villus height in the duodenum on d 28 and jejunum on d 42, while reducing splenic CD3+CD8α+ cytotoxic T cells 13.4 to 27.5% compared to the corn-based control at the same timepoints (P < 0.0001). Kinome results showed a significant innate immune toll-like receptor (TLR) response via MyD88 at d 14 in the small intestine of birds fed corn-based diets with algae that shifted to a more growth factor and adaptive immune-oriented response by d 42. Concurrent with immune changes, signaling changes indicative of lipid metabolism in the small intestine, ceca, and liver were seen in birds fed the corn-based diet with algae. The observed differential responses to basal diet composition and algae inclusion emphasize the need to comparatively evaluate feed ingredients in various diet formulations.

Low-grade endotoxemia and NOX2 in patients with coronary microvascular angina.

Endothelial dysfunction and oxidative stress were hypothesized to be involved in the pathogenesis of coronary microvascular angina (MVA). NADPH oxidase-2 (NOX2) activation could provoke increased oxidative stress and endothelial dysfunction, but data on MVA have not been provided yet. This study aimed to evaluate the interaction among NOX2 activation, serum lipopolysaccharide (LPS) levels, as well as oxidative stress production as potential causes of endothelial dysfunction in MVA patients. In this study, we wanted to compare serum levels of soluble NOX2-dp (sNOX2-dp), H2O2 production, hydrogen peroxide breakdown activity (HBA), nitric oxide (NO) bioavailability, endothelin 1 (ET-1), serum zonulin (as intestinal permeability assay), and LPS in 80 consecutive subjects, including 40 MVA patients and 40 controls (CT), matched for age and sex. Compared with CT, MVA patients had significantly higher values of sNOX2-dp, H2O2, ET-1, LPS, and zonulin. Conversely HBA and NO bioavailability were significantly lower in MVA patients. Simple linear regression analysis showed that sNOX2 was associated with serum LPS, serum zonulin, H2O2, and ET-1. Furthermore, an inverse correlation between sNOX2, HBA, and nitric oxide bioavailability was observed. Multiple linear regression analysis showed that LPS and zonulin emerged as the only independent predictive variables associated with sNOX2. This study provides the first report attesting that patients with MVA have high LPS levels, NOX2 activation, and an imbalance between pro-oxidant and antioxidant systems, in favor of the oxidizing molecules that could be potentially implicated in the endothelial dysfunction and vasoconstriction of this disease.

Schisantherin A alleviates non-alcoholic fatty liver disease by restoring intestinal barrier function

BackgroundNon-alcoholic fatty liver disease (NAFLD) is intricately linked to dysregulation of the gut–liver axis, and correlated with intestinal inflammation and barrier disruption.ObjectivesTo investigate the protective effects and possible molecular mechanism of Schisantherin A (Sin A) in a high-fat diet (HFD) induced NAFLD mouse model.MethodsHFD-fed NAFLD mice were treated with the vehicle and 80 mg/kg Sin A every day for 6 weeks. The gut permeability of the NAFLD mice was assessed by intestinal permeability assays in vivo and transepithelial electrical resistance (TEER) measurements in vitro were also used to evaluate the function of the gut barrier. TLR4 inhibitor was then used to investigate the impact of Sin A in the LPS- TLR4 signaling pathway. Alternatively, the composition of the microbiome was assessed using 16S rRNA amplification. Finally, the experiment of antibiotic treatment was performed to elucidate the roles of the gut microbiome mediating Sin A induced metabolic benefits in the NAFLD mice.ResultsWe found that Sin A potently ameliorated HFD-induced hepatic steatosis and inflammation, alleviated gut inflammation, and restored intestinal barrier function. We also observed that Sin A improved gut permeability and reduced the release of lipopolysaccharide (LPS) into circulation and further found that Sin A can suppress LPS-TLR4 signaling to protect against HFD-induced NAFLD. Sin A treatment altered the composition of the microbiome in NAFLD mice compared to vehicle controls.ConclusionsSin A is an effective and safe hepatoprotective agent against HFD-induced NAFLD by partly ameliorating gut inflammation, restoring intestinal barrier function, and regulating intestinal microbiota composition.

Treatment with TLR7/8 agonist compromises intestinal epithelium integrity in a lupus prone mouse model.

Abstract Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies leading to chronic inflammation and subsequent organ damage. Both spontaneous and induced mouse models have been central to the dissection of disease mechanisms. Mounting evidence suggests that increased gut permeability, or leaky gut, and the resulting translocation of pathobionts or their metabolites can impact SLE pathogenesis. Resiquimod (R848), a TLR7/8 agonist has been shown to induce lupus-like autoimmunity in various non-autoimmune mouse strains. Here, we examined the effect of the epicutaneous administration of R848 in the B6.Sle1.Sle2.Sle3 triple congenic (TC) spontaneous mouse model of lupus as compared to their non-autoimmune B6 controls. R848 induced a much stronger autoimmune activation in pre-autoimmune TC mice than in B6 controls. Unexpectedly, R848 decreased gut barrier integrity in TC but not in B6 mice. Immunofluorescence staining of the ileum showed a reduced expression of the tight junction protein claudin-1 and an increased infiltration of immune cells in R848-treated TC as compared to either R848-treated B6 or untreated control mice. The reduced intestinal epithelial integrity was functionally confirmed with a FITC-dextran intestinal permeability assay. These results demonstrate for the first time that TLR7/8 activation, which plays an essential role in autoimmune activation, also induces a leaky gut in lupus-prone mice. Ongoing studies are expected to unveil the causal relationships between TLR7/8 activation, gut barrier integrity, and microbial dysbiosis in SLE. Supported by R01 AI143313 from the NIH to LM

Beneficial Effects of Celastrol on Immune Balance by Modulating Gut Microbiota in Experimental Ulcerative Colitis Mice

Ulcerative colitis (UC) is a chronic inflammatory bowel disease caused by many factors including colonic inflammation and microbiota dysbiosis. Previous studies have indicated that celastrol (CSR) has strong anti-inflammatory and immune-inhibitory effects. Here, we investigated the effects of CSR on colonic inflammation and mucosal immunity in an experimental colitis model, and addressed the mechanism by which CSR exerts the protective effects. We characterized the therapeutic effects and the potential mechanism of CSR on treating UC using histological staining, intestinal permeability assay, cytokine assay, flow cytometry, fecal microbiota transplantation (FMT), 16S rRNA sequencing, untargeted metabolomics, and cell differentiation. CSR administration significantly ameliorated the dextran sodium sulfate (DSS)-induced colitis in mice, which was evidenced by the recovered body weight and colon length as well as the decreased disease activity index (DAI) score and intestinal permeability. Meanwhile, CSR down-regulated the production of pro-inflammatory cytokines and up-regulated the amount of anti-inflammatory mediators at both mRNA and protein levels, and improved the balances of Treg/Th1 and Treg/Th17 to maintain the colonic immune homeostasis. Notably, all the therapeutic effects were exerted in a gut microbiota-dependent manner. Furthermore, CSR treatment increased the gut microbiota diversity and changed the compositions of the gut microbiota and metabolites, which is probably associated with the gut microbiota-mediated protective effects. In conclusion, this study provides the strong evidence that CSR may be a promising therapeutic drug for UC.

Preventive Effect of a Postbiotic and Prebiotic Mixture in a Rat Model of Early Life Rotavirus Induced-Diarrhea.

Rotavirus (RV) is the main cause of gastroenteritis in children. Prebiotics and, more recently, postbiotics are used for preventing and treating gastrointestinal infections. The aim of this study was to analyze the effects of a LactofidusTM, short-chain galacto-oligosaccharides (scGOS) and long-chain fructo-oligosaccharides (lcFOS) mixture, and their combination on RV infection, in a rat model, for early life diarrhea. Fifteen litters of suckling rats were intragastrically administered daily with the vehicle, the prebiotic mixture, the postbiotic or the combination. The RV was inoculated on day 5 and then fecal samples were clinically evaluated daily. Viral shedding, intestinal permeability assay, in vitro blocking assay, immunoglobulin profiles, and anti-RV response were assessed at day 8 and 16 of life. Cecal microbiota composition, intestinal gene expression, and short chain fatty acids (SCFAs) were analyzed at day 16. The incidence and severity of diarrhea were significantly reduced by all the supplementations. Moreover, they showed blocking activity, changes in the immunoglobulin profiles, in gut microbiota, and in the intestinal gene expression. The prebiotic mixture reduced gut permeability and changed the SCFA profile, whereas the postbiotic enhanced the expression of Toll-like receptors (TLRs). The combination preserved most of the individual observed effects, and furthermore, complementary effects, such as an increase in white blood cells and lymphocytes recruitment, as well as upregulation of TLR7 and TLR9 gene expression.

The combination of four main components in Xuebijing injection improved the preventive effects of Cyclosporin A in acute graft-versus-host disease mice by protecting intestinal microenvironment

Acute graft-versus-host disease (aGVHD) is a major life-threatening complication after Allogeneic Hematopoietic Stem Cell Transplant (allo-HSCT). Although a series of immunosuppressant agents are routinely used as the first-line prevention, the morbidity and mortality rate remains high in allo-HSCT recipients. Our previous work indicated that combining Xuebijing (XBJ) with Cyclosporin A (CSA) is superior to CSA alone in preventing aGVHD. However, it was not clear which compounds in XBJ may prevent aGVHD. Whether the effective compounds in XBJ can be safely combined with CSA to prevent GVHD remain to be evaluated. Here, we accessed whether the combination of four main components in XBJ (C0127) had the same efficacy as XBJ in preventing aGVHD. In addition, the effectiveness of a novel combination therapy (C0127 + CSA) on aGVHD prophylaxis was evaluated using 16 s rRNA sequencing and RNA sequencing approaches in vitro and in vivo. In aGVHD mice, C0127 enhanced the preventive effects of CSA including decreasing mortality, maintaining weight, reducing GVHD score and reducing the expression of IL-6 and TNF-α in serum. Fatal GVHD is a frequent consequence of intestinal tract damage. We found combining C0127 with CSA alleviated the gut damage and maintained the normal physiological function of intestine by H&E staining, intestinal permeability and short chain fatty acid (SCFA) assays. Next, 16 S sequencing analysis of feces showed the combination treatment maintained the intestinal microbial diversity, normalized the intestinal microorganism and prevented flora disorder by reducing the relative abundances of Escherichia coli and Enterococcus. Further, RNA-seq analysis of colonic epithelium revealed C0127 combined with CSA chiefly regulated chemokines and cytokines in IL-17 signaling pathway. The combination treatment reduced the expression of G-CSF and its effector STAT3 (an axis that aggravated gut inflammation and flora disorder) in gut epithelium on mRNA and protein level. These findings indicated that C0127 improved the prevention of CSA in aGVHD mice partially by protecting the gut from damage through normalizing G-CSF signaling, which regulates the intestinal microbiota and the integrity of the epithelial barrier.