Intestinal Tight Junction Proteins Research Articles

Mechanisms of Peitu Yimu acupuncture in repairing intestinal mucosal barrier by regulating CRF/CRFR1 pathway in diarrhea-predominant irritable bowel syndrome rats.

To investigate the effect of Peitu Yimu(strengthening spleen and soothing liver) acupuncture on intestinal mucosal barrier function and corticotropin-releasing factor (CRF)/CRF receptor 1 (CRFR1) pathway in rats with diarrhea-predominant irritable bowel syndrome (IBS-D), so as to explore its underlying mechanism in alleviating IBS-D. Forty female SD rats were randomly divided into blank, model, electroacupuncture (EA), and agonist groups, with 10 rats in each group. Except for the blank group, rats in the other groups were given folium sennae infusion by gavage combined with chronic unpredictable mild stress to establish IBS-D model. Rats in the EA group received acupuncture at "Tianshu"(ST25) and EA at "Zusanli"(ST36) and "Taichong"(LR3) (2 Hz/15 Hz) on one side for 20 min, with the side chosen alternately every other day, for 14 days after modeling. Rats in the agonist group received acupuncture 30 min after intravenous injection of CRFR1 agonist urocortin, with the same manipulation method and time as the EA group. Before and after intervention, visceral pain threshold and stool Bristol scores were measured. Elevated plus maze test and open field test were used to detect anxiety and depression like behavior of rats. ELISA was used to detect the contents of CRF and CRFR1 in rats serum. Immunohistochemistry was used to detect the positive expressions of CRF, CRFR1, zonula occludens protein 1(ZO-1), occlusal protein(Occludin), and closure protein 1 (Claudin-1) in colon tissue. Compared with the blank group, the visceral pain threshold, open arm time percentage (OT%), total distance of movement in the open field test, and positive expression of ZO-1, Occludin, and Claudin-1 in colon were decreased (P<0.01, P<0.05), while Bristol stool scores, serum CRF and CRFR1 contents, and positive expressions of CRF and CRFR1 in colon were increased (P<0.01) in the model group. After intervention and compared with the model group, the visceral pain threshold, OT%, total distance of movement in the open field test, and positive expressions of ZO-1, Occludin, and Claudin-1 in colon were increased (P<0.05, P<0.01), while Bristol stool scores, serum CRF and CRFR1 contents, and positive expressions of CRF and CRFR1 in colon were decreased (P<0.01) in the EA group；the Bristol stool scores, serum CRF content, and CRF positive expression in colon were significantly decreased in the agonist group (P<0.01). Peitu Yimu acupuncture can significantly improve visceral hypersensitivity and anxiety-depression state in IBS-D rats. Its mechanism may be related to the inhibition of CRF/CRFR1 pathway and restoration of intestinal tight junction protein expressions.

Huang Lian Jie Du decoction attenuates inflammation in septic rats by activating autophagy and altering the intestinal microbiome

AimsThe aim of this study was to investigate the protective effect of HLJDD on septic rats and the underlying mechanisms. Materials and methodsAdult male Sprague–Dawley (SD) adult rats (150–180 g) were randomly divided into the following 5 groups (n = 7 per group): the Sham group, caecal ligation and puncture (CLP) group, HLJDD + CLP (Huang Lian Jie Du Decoction, HLJDD) group (1 g/mL/100 g), HLJDD + Rap + CLP (H. Rap) group (Rap: 3 mg/kg), and HLJDD+3-MA + CLP (H. 3-MA) group (3-MA: 30 mg/kg). Rapamycin (Rap) and 3-methyladenosine (3-MA) were used to activate and inhibit autophagy, respectively. HLJDD was purchased from Beijing Tong Ren Tang Guiyang Branch and verified by experts as a genuine product. We used CLP to establish an animal model of sepsis in the last four groups. Survival was analysed by the Kaplan‒Meier method. Then, we examined autophagy-related genes (Atgs) and proteins using real-time PCR and Western blotting, respectively. The microstructure of the ileum and the number of autophagosomes were observed by transmission electron microscopy (TEM). Analyses of HE-stained pathological ileum and inflammatory factor levels were examined to assess the extent of septic injury. The effect of HLJDD on the gut microbiota was analysed by 16S rRNA gene sequencing of faeces. ResultsIn this study, we identified the protective effects of HLJDD on mortality and inflammation in septic rats. Several key proteins, including LC3-II, Beclin-1 and p62, were examined and showed that HLJDD could effectively reverse the sepsis-induced decrease in autophagy. TEM was performed and the expression of Atgs was assessed to evaluate fluctuations in autophagy. Then, we examined the intestinal tight junction protein zona occludens (ZO-1), lipopolysaccharide (LPS) and inflammatory factors, and found that HLJDD effectively alleviated the increase in ZO-1 gene expression, the level of LPS and serum level of inflammatory factors caused by sepsis. These results were consistent with those obtained from pathological sectioning and TEM analysis. Moreover, autophagy activation effectively ameliorated sepsis, and autophagy inhibition exacerbated the systemic symptoms caused by infection. By examining the expression of key proteins upstream of the autophagy pathway, we found that HLJDD inhibited mTOR via the MAPK/PI3K signalling pathway to promote autophagy in septic rats. 16S rRNA sequencing revealed that HLJDD significantly affected the diversity and physiological function of the gut microbiota in septic rats. ConclusionsThe results of this study indicate that autophagy activation is a potential mechanism underlying the protective effect of HLJDD on the intestine in septic rats.

Study on the Protective Effect and Mechanism of Umbilicaria esculenta Polysaccharide in DSS-Induced Mice Colitis and Secondary Liver Injury.

This study aimed to explore the ameliorative effects and potential mechanisms of Huangshan Umbilicaria esculenta polysaccharide (UEP) in dextran sulfate sodium-induced acute ulcerative colitis (UC) and UC secondary liver injury (SLI). Results showed that UEP could ameliorate both colon and liver pathologic injuries, upregulate mouse intestinal tight junction proteins (TJs) and MUC2 expression, and reduce LPS exposure, thereby attenuating the effects of the gut-liver axis. Importantly, UEP significantly downregulated the secretion levels of TNF-α, IL-1β, and IL-6 through inhibition of the NF-κB pathway and activated the Nrf2 signaling pathway to increase the expression levels of SOD and GSH-Px. In vitro, UEP inhibited the LPS-induced phosphorylation of NF-κB P65 and promoted nuclear translocation of Nrf2 in RAW264.7 cells. These results revealed that UEP ameliorated UC and SLI through NF-κB and Nrf2-mediated inflammation and oxidative stress. The study first investigated the anticolitis effect of UEP, suggesting its potential for the treatment of colitis and colitis-associated liver disease.

Obeticholic acid attenuates the intestinal barrier disruption in a rat model of short bowel syndrome

BackgroundShort bowel syndrome (SBS) features nutrients malabsorption and impaired intestinal barrier. Patients with SBS are prone to sepsis, intestinal flora dysbiosis and intestinal failure associated liver disease. Protecting intestinal barrier and preventing complications are potential strategies for SBS treatment. This study aims to investigate the effects of farnesoid X receptor (FXR) agonist, obeticholic acid (OCA), have on intestinal barrier and ecological environment in SBS. Methods and resultsThrough testing the small intestine and serum samples of patients with SBS, impaired intestinal barrier was verified, as evidenced by reduced expressions of intestinal tight junction proteins (TJPs), increased levels of apoptosis and epithelial cell damage. The intestinal expressions of FXR and related downstream molecules were decreased in SBS patients. Then, global FXR activator OCA was used to further dissect the potential role of the FXR in a rat model of SBS. Low expressions of FXR-related molecules were observed on the small intestine of SBS rats, along with increased proinflammatory factors and damaged barrier function. Furthermore, SBS rats possessed significantly decreased body weight and elevated death rate. Supplementation with OCA mitigated the damaged intestinal barrier and increased proinflammatory factors in SBS rats, accompanied by activated FXR-related molecules. Using 16S rDNA sequencing, the regulatory role of OCA on gut microbiota in SBS rats was witnessed. LPS stimulation to Caco-2 cells induced apoptosis and overexpression of proinflammatory factors in vitro. OCA incubation of LPS-pretreated Caco-2 cells activated FXR-related molecules, increased the expressions of TJPs, ameliorated apoptosis and inhibited overexpression of proinflammatory factors. ConclusionsOCA supplementation could effectively ameliorate the intestinal barrier disruption and inhibit overexpression of proinflammatory factors in a rat model of SBS and LPS-pretreated Caco-2 cells. As a selective activator of FXR, OCA might realize its protective function through FXR activation.

Da-yuan-yin decoction alleviates ulcerative colitis by inhibiting complement activation, LPS-TLR4/NF-κB signaling pathway and NET formation

Ethnopharmacological relevanceDa-yuan-yin decoction (DYY) is a classical traditional Chinese medicine prescription for ulcerative colitis (UC). Aim of studyThis study explored the protective effects and mechanisms of DYY on UC. Materials and methodsThe mice were fed 2.5% dextran sulfate sodium (DSS) for 7 days to establish UC. On the second day, DYY (0.4 g/kg, 0.8 g/kg, 1.6 g/kg) was orally administered daily for 7 consecutive days. The colon tissues and serum were measured by histopathological examination and biochemical analysis. ResultsDYY significantly reduced the disease activity index (DAI) and severity of colon shortening and alleviated pathological changes in the colon tissue. DYY restored the protein expression of intestinal tight junction (TJ) protein (ZO-1, occludin and claudin-3). DYY remarkably decreased the level of lipopolysaccharide (LPS), Lactic acid (LA), circulating free DNA (cfDNA), complement (C3, C3a, C3c, C3aR1, C5a and C5aR1) and regulated the levels of inflammatory cytokines in serum. DYY significantly inhibited the expressions of nuclear factor kappa-B p65 (NF-κB p65) and Toll-like receptor 4 (TLR4), citrullinated histone H3 (CitH3) and myeloperoxidase (MPO), reactive oxygen species (ROS) peptidylarginine deiminase 4 (PAD4) and CD 11b, the mRNA levels of PADI4, MPO and ELANE in colon tissues. ConclusionsDYY significantly attenuated DSS-induced UC, which was related with regulating the inflammatory response by the inhibition of complement activation, the LPS-TLR4/NF-κB signaling pathway and neutrophil extracellular traps (NETs) formation. DYY is a potential therapeutic agent for UC.

A PROTEIN COMPLEX OF LIVER ORIGIN ACTIVATES A PRO-INFLAMMATORY PROGRAM THAT DRIVES HEPATIC AND INTESTINAL INJURY IN ALCOHOL-ASSOCIATED LIVER DISEASE

There is limited information on how the liver-to-gut axis contributes to alcohol-associated liver disease (AALD). We previously identified that high-mobility group box-1 (HMGB1) undergoes oxidation in hepatocytes and demonstrated elevated serum levels of oxidized HMGB1 ([O] HMGB1) in alcoholic patients. Since interleukin-1 beta (IL1B) increases in AALD, we hypothesized hepatocyte-derived [O] HMGB1 could interact with IL1B to activate a pro-inflammatory program that, besides being detrimental to the liver, drives intestinal barrier dysfunction. Alcohol-fed RageΔMye mice exhibited decreased NFκB signaling, a pro-inflammatory signature, and reduced total intestinal permeability, resulting in protection from AALD. Additionally, [O] HMGB1 bound and signaled through the receptor for advanced-glycation end-products (RAGE) in myeloid cells, driving hepatic inflammation, intestinal permeability, and increased portal blood lipopolysaccharide in AALD. We identified that [O] HMGB1 formed a complex with IL1B, which was found in the livers of patients with acute alcoholic hepatitis and mice with AALD. This complex originated from the liver, as it was absent in the intestine when hepatocytes did not produce [O] HMGB1. Mechanistically, the complex bound RAGE in Kupffer cells and macrophages induces a pro-inflammatory program. Moreover, it bound RAGE in intestinal macrophages and epithelial cells, leading to intestinal inflammation, altered intestinal epithelial cell tight junction protein expression, increased intestinal permeability, and elevated portal blood lipopolysaccharide, enhancing AALD pathogenesis. We identified a protein complex of liver origin that amplifies the pro-inflammatory feedback loop in AALD; therefore, targeting this complex could have significant therapeutic potential.

An integrated network pharmacology approach reveals that Ampelopsis grossedentata improves alcoholic liver disease via TLR4/NF-κB/MLKL pathway.

BackgroundAlcohol-related liver damage is the most prevalent chronic liver disease, which creates a heavy public health burden worldwide. The leaves of Ampelopsis grossedentata have been considered a popular tea and traditional herbal medicine in China for more than one thousand years, and possess anti-inflammatory, antioxidative, hepatoprotective, and antiviral activities. PurposeWe explored the protective effects of Ampelopsis grossedentata extract (AGE) against chronic alcohol-induced hepatic injury (alcoholic liver disease, ALD), aiming to elucidate its underlying mechanisms. MethodsFirstly, UPLC-Q/TOF-MS analysis and network pharmacology were used to identify the constituents and elucidate the potential mechanisms of AGE against ALD. Secondly, C57BL/6 mice were pair-fed the Lieber-DeCarli diet containing either isocaloric maltodextrin or ethanol, AGE (150 and 300 mg/kg/d) and silymarin (200 mg/kg) were administered to chronic ethanol-fed mice for 7 weeks to evaluate the hepatoprotective effects. Serum biochemical parameters were determined, hepatic and ileum sections were used for histologic examination, and levels of inflammatory cytokines and oxidative stress in the liver were examined. The potential molecular mechanisms of AGE in improving ALD were demonstrated by RNA-seq, Western blotting analysis, and immunofluorescence staining. ResultsTen main constituents of AGE were identified using UPLC-Q/TOF-MS and 274 potential ALD-related targets were identified. The enriched KEGG pathways included Toll-like receptor signaling pathway, NF-κB signaling pathway, and necroptosis. Moreover, in vivo experimental studies demonstrated that AGE significantly reduced serum aminotransferase levels and improved pathological abnormalities after chronic ethanol intake. Meanwhile, AGE improved ALD in mice by down-regulating oxidative stress and inflammatory cytokines. Furthermore, AGE notably repaired damaged intestinal epithelial barrier and suppressed the production of gut-derived lipopolysaccharide by elevating intestinal tight junction protein expression. Subsequent RNA-seq and experimental validation indicated that AGE inhibited NF-κB nuclear translocation, suppressed IκB-α, RIPK3 and MLKL phosphorylation and alleviated hepatic necroptosis in mice. ConclusionIn this study, we have demonstrated for the first time that AGE protects against alcoholic liver disease by regulating the gut-liver axis and inhibiting the TLR4/NF-κB/MLKL-mediated necroptosis pathway. Therefore, our present work provides important experimental evidence for AGE as a promising candidate for protection against ALD.

Shen-yan-yi-hao oral solution ameliorates IgA nephropathy via intestinal IL-17/NF-κB pathway

Ethnopharmacological relevanceImmunoglobulin A nephropathy (IgAN) is the most common primary glomerulonephritis in the world, it is one of the most common causes of kidney disease and can lead to end-stage kidney disease, however, its pathogenesis is still complicated. The Shen-yan-yi-hao oral solution (SOLI) is an effective prescription for the clinical treatment of IgAN while its specific mechanism remains to be further elucidated. Aim of the studyThis study investigates SOLI’s effects on IgAN in rats, particularly on the intestinal mucosal barrier, and identifies potential therapeutic targets through network pharmacology and molecular docking, validated experimentally. Materials and methodsTarget genes for SOLI in IgAN were identified and analyzed through molecular docking and KEGG pathway enrichment. An IgAN rat model examined SOLI’s effect on renal biomarkers and cytokines involved in specific pathways, ileum mucosal lesions, and the intestinal immune system. The IL-17 pathway’s role was studied in IEC-6 cells with SOLI in vitro. ResultRats developed increased proteinuria and kidney damage marked by IgA deposition and inflammation. SOLI treatment significantly ameliorated these symptoms, reduced galactose-deficient Ig A1 (Gd-IgA1), and decreased cytokines like IL-17, TNF-α, IL-6 and IL-1β etc. SOLI also normalized intestinal tight junction protein expression, ameliorated intestinal damage, and regulated intestinal immune response (focused on IL-17/NF-κB signal pathway). SOLI moderated the abnormally activated IL-17 pathway, which damages intestinal epithelial cells, suggesting IgAN treatment potential. ConclusionSOLI reduces proteinuria and enhances intestinal mucosal function in IgAN rats, kidney protection in the IgAN rat model may initiate from modulating the intestinal IL-17/NF-κB pathway and subsequent Gd-IgA1 accumulation.

Microbe-Derived Antioxidants Protect IPEC-1 Cells from H2O2-Induced Oxidative Stress, Inflammation and Tight Junction Protein Disruption via Activating the Nrf2 Pathway to Inhibit the ROS/NLRP3/IL-1β Signaling Pathway.

Oxidative stress can induce inflammation and tight junction disruption in enterocytes. The initiation of inflammation is thought to commence with the activation of the ROS/NLRP3/IL-1β signaling pathway, marking a crucial starting point in the process. In our previous studies, we found that microbe-derived antioxidants (MAs) showed significant potential in enhancing both antioxidant capabilities and anti-inflammatory effects. The main aim of this research was to investigate the ability of MAs to protect cells from oxidative stress caused by H2O2, to reduce inflammatory responses, and to maintain the integrity of tight junction proteins by modulating the ROS/NLRP3/IL-1β signaling pathway. IPEC-1 cells (1 × 104 cells/well) were initially exposed to 100 mg/L of MAs for 12 h, after which they were subjected to 1 mM H2O2 treatment for 1 h. We utilized small interfering RNA (siRNA) to inhibit the expression of NLRP3 and Nrf2. Inflammatory factors such as IL-1β and antioxidant enzyme activity levels were detected by ELISA. Oxidative stress marker ROS was examined by fluorescence analysis. The NLRP3/IL-1β signaling pathway, Nrf2/HO-1 signaling pathway and tight junction proteins (ZO-1 and Occludin) were detected by RT-qPCR or Western blotting. In our research, it was observed that MA treatment effectively suppressed the notable increase in H2O2-induced inflammatory markers (TNF-α, IL-1β, and IL-18), decreased ROS accumulation, mitigated the expression of NLRP3, ASC, and caspase-1, and promoted the expression of ZO-1 and Occludin. After silencing the NLRP3 gene with siRNA, the protective influence of MAs was observed to be linked with the NLRP3 inflammasome. Additional investigations demonstrated that the treatment with MAs triggered the activation of Nrf2, facilitating its translocation into the nucleus. This process resulted in a notable upregulation of Nrf2, NQO1, and HO-1 expression, along with the initiation of the Nrf2-HO-1 signaling pathway. Consequently, there was an enhancement in the activities of antioxidant enzymes like SOD, GSH-Px, and CAT, which effectively mitigated the accumulation of ROS, thereby ameliorating the oxidative stress state. The antioxidant effectiveness of MAs was additionally heightened in the presence of SFN, an activator of Nrf2. The antioxidant and anti-inflammatory functions of MAs and their role in regulating intestinal epithelial tight junction protein disruption were significantly affected after siRNA knockdown of the Nrf2 gene. These findings suggest that MAs have the potential to reduce H2O2-triggered oxidative stress, inflammation, and disruption of intestinal epithelial tight junction proteins in IPEC-1 cells. This reduction is achieved by blocking the ROS/NLRP3/IL-1β signaling pathway through the activation of the Nrf2 pathway.

HUC-MSCs therapy for Crohn’s disease: efficacy in TNBS-induced colitis in rats and pilot clinical study

The use of mesenchymal stem cells (MSCs) has recently emerged as a promising new therapeutic strategy for many diseases including perianal fistulizing Crohn's disease (CD). Whether hUC-MSCs can promote the healing of luminal ulcer in CD has not been studied so far. The model of TNBS-induced colitis in rats was used to confirm the efficacy of hUC-MSCs in the treatment of CD. Then, seventeen CD patients refractory to or unsuitable for currently available therapies were enrolled and received once submucosal local injection through colonoscopy combined with once intravenous drip on the next day. All patients received a 24-week follow-up. Clinical and laboratory assessments were monitored at baseline, week 4, 8, 12, and 24. Endoscopic evaluations were conducted at baseline and week 12. Mucosal specimens were obtained at the margin of lesions by endoscopy biopsies and used for RNA sequencing. Two hUC-MSCs co-culture systems were established invitro, one with the mucosa specimens and the other with M1 macrophages induced from THP1. The expressions of genes representing inflammation (TNFα, IL-6, and IL-1β) and intestinal barrier function (ZO1, CLAUDIN1, and CDH1) were tested by RT-PCR. hUC-MSCs treatment increased body weight and decreased disease activity index (DAI), colon macroscopic damage index (CMDI), and histopathological score (HPS) of rats with TNBS-induced colitis. The results of the clinical study also showed that this mode of hUC-MSCs application was associated with regression of intestinal ulceration. Eight patients (47%) got endoscopic responses (SES-CD improvement of ≥50% from baseline) and three patients (17.65%) got mucosal healing (SES-CD is zero), with a parallel improvement of clinical and laboratory parameters without serious adverse events. RNA sequencing showed hUC-MSCs therapy was associated with an upregulation of transcripts linked to intestinal epithelial barrier integrity and a downregulation of inflammatory signaling pathways in the intestinal mucosa, especially the TNF signaling pathway, IL-17 signaling pathway, and TLR signaling pathway. RNA expression of intestinal epithelial tight junction protein (ZO1, CLAUDIN1, and CDH1), and the RNA expression of major intestinal inflammatory factors in CD (IL-1β, IL-6, and TNFα, p<0.001 for all) were improved significantly. Moreover, hUC-MSCs could attenuate the polarization of M1 macrophage induced from THP1, thereby decreasing the mRNA expression of IL-1β, IL-6, and TNFα significantly (p<0.05 for all). TSG-6 expression was evaluated in hUC-MSCs culture supernatant after treatment with TNFα, IFNγ, and LPS for 48h. And hUC-MSCs could inhibit the phosphorylation of JAK/STAT1 in the intestinal mucosa of CD patients. hUC-MSCs transplantation alleviated TNBS-induced colitis in rats. In this pilot clinical study, preliminary data suggested that this approach to administering hUC-MSCs might have potential for clinical efficacy and manageable safety in treating refractory CD, potentially providing hope for better outcomes. No serious adverse events were observed. This work was funded by General Program of National Natural Science Foundation of China (Grant No. 82270639), the Scientific research project of Shanghai Municipal Health Committee (Grant No. 202240001), Specialty Feature Construction Project of Shanghai Pudong New Area Health Commission (Grant No. PWZzb2022-05), Shanghai East Hospital Youth Research and Cultivation Foundation program (Grant No. DFPY2022015), Peak Disciplines (Type IV) of Institutions of Higher Learning in Shanghai and Technology Development Project of Pudong Science, Technology and Economic Commission of Shanghai (Grant No. PKJ2021-Y08).

Single cell transcriptomics unveiled that early life BDE-99 exposure reprogrammed the gut-liver axis to promote a pro-inflammatory metabolic signature in male mice at late adulthood.

Polybrominated diphenyl ethers (PBDEs) are legacy flame retardants that bioaccumulate in the environment. The gut microbiome is an important regulator of liver functions including xenobiotic biotransformation and immune regulation. We recently showed that neonatal exposure to polybrominated diphenyl ether-99 (BDE-99), a human breast milk-enriched PBDE congener, up-regulated pro-inflammation- and down-regulated drug metabolism-related genes predominantly in males in young adulthood. However, the persistence of dysregulation into late adulthood, differential impact of hepatic cell types, and the involvement of the gut microbiome from neonatal BDE-99 exposure remains unknown. To address these knowledge gaps, male C57BL/6 mouse pups were orally exposed to corn oil (10 ml/kg) or BDE-99 (57 mg/kg) once daily from postnatal days 2-4. At 15 months of age, neonatal BDE-99 exposure down-regulated xenobiotic and lipid metabolizing enzymes and up-regulated genes involved in microbial influx in hepatocytes. Neonatal BDE-99 exposure also increased the hepatic proportion of neutrophils and led to a predicted increase of macrophage migration inhibitory factor signaling. This was associated with decreased intestinal tight junction protein (Tjp) transcripts, altered gut environment, and dysregulation of inflammation-related metabolites. ScRNA-seq using germ-free (GF) mice demonstrated the necessity of a normal gut microbiome in maintaining hepatic immune tolerance. Microbiota transplant to GF mice using large intestinal microbiome from adults neonatally exposed to BDE-99 down-regulated Tjp transcripts and up-regulated several cytokines in the large intestine. In conclusion, neonatal BDE-99 exposure reprogrammed cell type-specific gene expression and communication in liver towards pro-inflammation, and BDE-99-mediated pro-inflammatory signatures may be partly due to the dysregulated gut environment.

Modulation of intestinal epithelial permeability by chronic small intestinal helminth infections.

Increased permeability of the intestinal epithelial layer is linked to the pathogenesis and perpetuation of a wide range of intestinal and extra-intestinal diseases. Infecting humans with controlled doses of helminths, such as human hookworm (termed hookworm therapy), is proposed as a treatment for many of the same diseases. Helminths induce immunoregulatory changes in their host which could decrease epithelial permeability, which is highlighted as a potential mechanism through which helminths treat disease. Despite this, the influence of a chronic helminth infection on epithelial permeability remains unclear. This study uses the chronically infecting intestinal helminth Heligmosomoides polygyrus to reveal alterations in the expression of intestinal tight junction proteins and epithelial permeability during the infection course. In the acute infection phase (1 week postinfection), an increase in intestinal epithelial permeability is observed. Consistent with this finding, jejunal claudin-2 is upregulated and tricellulin is downregulated. By contrast, in the chronic infection phase (6 weeks postinfection), colonic claudin-1 is upregulated and epithelial permeability decreases. Importantly, this study also investigates changes in epithelial permeability in a small human cohort experimentally challenged with the human hookworm, Necator americanus. It demonstrates a trend toward small intestinal permeability increasing in the acute infection phase (8 weeks postinfection), and colonic and whole gut permeability decreasing in the chronic infection phase (24 weeks postinfection), suggesting a conserved epithelial response between humans and mice. In summary, our findings demonstrate dynamic changes in epithelial permeability during a chronic helminth infection and provide another plausible mechanism by which chronic helminth infections could be utilized to treat disease.

Dietary copper improves intestinal structural integrity in juvenile grass carp (Ctenopharyngodon idella) probably related to its increased intestinal antioxidant capacity and apical junction complex

This research evaluated the effects of copper (Cu) on intestinal antioxidant capacity and apical junctional complex (AJC) in juvenile grass carp. A total of 1080 healthy juvenile grass carp (11.16 ± 0.01 g) were fed six diets including different dosages of Cu, namely 0, 2, 4, 6, 8 (Cu citrate [CuCit] as Cu source) and 3 mg/kg (CuSO4·5H2O as Cu source), lasting for 9 weeks. The findings revealed that dietary optimal Cu supplementation (2.2 to 4.1 mg/kg) promoted intestinal growth, including intestinal length, intestinal length index, intestinal weight, and intestinal somatic index (P < 0.05). Furthermore, optimal Cu boosted the intestinal mucosal barrier in juvenile grass carp. On the one hand, optimal Cu reduced diamine oxidase and D-lactate levels in serum (P < 0.05), reduced levels of the oxidative damage indicators malondialdehyde, reactive oxygen species (ROS), protein carbonyl, and also elevated superoxide dismutase and catalase mRNA levels (P < 0.05), thus boosting intestinal antioxidant capacity, in which the binding protein Keap1a/1b/Nrf2 signaling pathway might be involved. Optimal Cu had no impact on glutathione peroxidase 1b (GPx1b) gene expression (P > 0.05). On the other hand, optimal Cu increased intestinal tight junction (TJ) proteins (except for claudin 15b) and adherens junction (AJ) proteins (E-cadherin, α-catenin, β-catenin, nectin and afadin) mRNA levels (P < 0.05), which could be connected to the signaling pathway formed by the Ras homolog gene family, member A (RhoA), Rho-associated protein kinase (ROCK), and myosin light chain kinase (MLCK). Finally, based on serum indicator D-lactate and intestinal oxidative damage index (ROS), Cu requirement (CuCit as Cu source) for juvenile grass carp from initial weight to final weight (from 11 to 173 g) was determined to be 4.14 and 4.12 mg/kg diet, respectively. This work may provide a theoretical foundation for identifying putative Cu regulation pathways on fish intestinal health.

Dietary supplementation of α-linolenic acid-rich flaxseed oil enhances anti-PD-1 protection against orthotopic hepatocellular carcinoma by reshaping gut homeostasis and improving anti-tumor immunity via gut-liver axis in mice

Blockade therapy of immune checkpoint with anti-programmed cell death (PD)-1 antibody has achieved tremendous success on the treatment of numerous malignant tumors, whereas there is still a problem of low response rate. In the present study, the complement of α-linolenic acid (ALA) on the impact of PD-1 immunotherapy for hepatocellular carcinoma (HCC) was investigated. We found that the ALA combined with PD-1 immunotherapy exerted superior in inhibiting tumor growth. Moreover, the combination treatment significantly changed diverse T cell subsets and ant-tumor immunity associated cytokine levels in plasma and tumor tissues. Intriguingly, gut dysbiosis was notably attenuated after the intervention by elevating the expression of intestinal tight junction proteins alonged with reducing liver lipopolysaccharide (LPS), as well as modulating gut microbiota and short-chain fatty acids (SCFAs). Collectively, these results indicated that the supplement with ALA strengthened anti-tumor immunity and reshaped the gut homeostasis to contribute to the PD-1 treatment against HCC.

Gut microbes combined with metabolomics reveal the protective effects of Qijia Rougan decoction against CCl4-induced hepatic fibrosis.

Background: The occurrence and development of Hepatic fibrosis (HF) are closely related to the gut microbial composition and alterations in host metabolism. Qijia Rougan decoction (QJ) is a traditional Chinese medicine compound utilized clinically for the treatment of HF with remarkable clinical efficacy. However, its effect on the gut microbiota and metabolite alterations is unknown. Therefore, our objective was to examine the impact of QJ on the gut microbiota and metabolism in Carbon tetrachloride (CCl4)-induced HF. Methods: 40% CCl4 was used to induce HF, followed by QJ administration for 6weeks. Serum biochemical analyses, histopathology, immunohistochemistry, RT-PCR, 16S rRNA gene sequencing, and non-targeted metabolomics techniques were employed in this study to investigate the interventional effects of QJ on a CCl4-induced HF model in rats. Results: This study demonstrated that QJ could effectively ameliorate CCl4-induced hepatic inflammation and fibrosis. Moreover, QJ upregulated the expression of intestinal tight junction proteins (TJPs) and notably altered the abundance of some gut microbes, for example, 10 genera closely associated with HF-related indicators and TJPs. In addition, metabolomics found 37 key metabolites responded to QJ treatment and strongly associated with HF-related indices and TJPs. Furthermore, a tight relation between 10 genera and 37 metabolites was found post correlation analysis. Among them, Turicibacter, Faecalibaculum, Prevotellaceae UCG 001, and unclassified Peptococcaceae may serve as the core gut microbes of QJ that inhibit HF. Conclusion: These results suggest that QJ ameliorates hepatic inflammation and fibrosis, which may be achieved by improving intestinal tight junctions and modulating gut microbiota composition as well as modulating host metabolism.

Symbiotic combination of Akkermansia muciniphila and inosine alleviates alcohol-induced liver injury by modulating gut dysbiosis and immune responses.

Alcoholic liver disease (ALD) is exacerbated by disruptions in intestinal microecology and immune imbalances within the gut-liver axis. The present study assesses the therapeutic potential of combining Akkermansia muciniphila (A. muciniphila) with inosine in alleviating alcohol-induced liver injury. Male C57BL/6 mice, subjected to a Lieber-DeCarli diet with 5% alcohol for 4 weeks, served as the alcoholic liver injury model. Various analyzes, including quantitative reverse transcription polymerase chain reaction (qRT-PCR), ELISA, immunochemistry, 16S rRNA gene sequencing, and flow cytometry, were employed to evaluate liver injury parameters, intestinal barrier function, microbiota composition, and immune responses. Compared to the model group, the A. muciniphila and inosine groups exhibited significantly decreased alanine aminotransferase, aspartate aminotransferase, and lipopolysaccharide (LPS) levels, reduced hepatic fat deposition and neutrophil infiltration, alleviated oxidative stress and inflammation, and increased expression of intestinal tight junction proteins (Claudin-1, Occludin, and ZO-1). These effects were further pronounced in the A. muciniphila and inosine combination group compared to individual treatments. While alcohol feeding induced intestinal dysbiosis and gut barrier disruption, the combined treatment reduced the abundance of harmful bacteria (Oscillibacter, Escherichia/Shigella, and Alistipes) induced by alcohol consumption, promoting the growth of butyrate-producing bacteria (Akkermansia, Lactobacillus, and Clostridium IV). Flow cytometry revealed that alcohol consumption reduced T regulatory (Treg) populations while increasing those of T-helper (Th) 1 and Th17, which were restored by A. muciniphila combined with inosine treatment. Moreover, A. muciniphila and inosine combination increased the expression levels of intestinal CD39, CD73, and adenosine A2A receptor (A2AR) along with enhanced proportions of CD4+CD39+Treg and CD4+CD73+Treg cells in the liver and spleen. The A2AR antagonist KW6002, blocked the beneficial effects of the A. muciniphila and inosine combination on liver injury in ALD mice. This study reveals that the combination of A. muciniphila and inosine holds promise for ameliorating ALD by enhancing the gut ecosystem, improving intestinal barrier function, upregulating A2AR, CD73, and CD39 expression, modulating Treg cells functionality, and regulating the imbalance of Treg/Th17/Th1 cells, and these beneficial effects are partly A2AR-dependent.

Kaempferol Ameliorated Alcoholic Hepatitis through Improving Intestinal Barrier Function by Targeting miRNA-155 Signaling

Introduction: The aim of this study was to investigate the effect and mechanism of kaempferol on alcoholic steatohepatitis. Methods: C57BL/6 N mice were utilized to establish Binge-on-Chronic alcohol exposure mice model. Kaempferol was given as the interventional drug to chronic alcohol-fed mice for 6 weeks to assess its effects. In vitro, intestinal epithelial Caco-2 cells were stimulated by alcohol, and miRNA-155 mimics were used to further study the effect of kaempferol to miRNA-155 signaling in intestinal epithelial cells. HE staining and oil red O staining were used to observe the liver and intestinal tissue damage in each group of mice, and ALT, AST, IL-1β, and TNF-α were detected by kits; lipopolysaccharide (LPS) expression was detected by ELISA kit, and the expression of IL-1β and TNF-α was assessed by qRT-PCR; Western blot was utilized to assess the excessive inflammatory response of liver and colon tissue and the related signaling pathway activation. Results: Kaempferol treatment significantly improved pathological changes such as steatosis and vacuolated lesions in liver tissue of the alcohol diet model group, and reduced serum ALT and AST enzyme activities and liver tissue interleukin-1β and tumor necrosis factor-α mRNA expression levels. Kaempferol significantly reduced the expression of miRNA-155 in the intestinal tissue of alcohol-fed mice, significantly increased their cytokine suppressor signaling 1 (SOCS1) protein expression, inhibited the activation of nuclear factor kappa-B and significantly increased the production of the intestinal tight junction proteins occludin and zonula occludens-1. More importantly, kaempferol significantly reduced serum LPS levels in alcoholic steatohepatitis mice. In vitro experiments showed that compared with the control group, kaempferol significantly inhibited the expression level of miRNA-155 in Caco-2 cells under ethanol exposure, decreased the activation of nuclear factor kappa-B, led to an increase in the expression of SOCS1 protein, and increased the production level of occludin protein in Caco-2 cells under the effect of alcohol. In contrast, overexpression of miRNA-155 significantly decreased occludin and SOCS1 protein production and increased nuclear factor kappa-B activation levels in Caco-2 cells, and the administration of kaempferol significantly inhibited this effect. Conclusion: Kaempferol improved the stability of gut barrier function to ameliorate hepatic injury induced by alcohol intake through enhancing occludin protein expression, by targeting miR-155 to inhibit the excessive inflammatory response in the intestine.

Irisin delays the onset of type 1 diabetes in NOD mice by enhancing intestinal barrier

Type 1 diabetes (T1D), a complex autoimmune disease, is intricately linked to the gut's epithelial barrier function. Emerging evidence emphasizes the role of irisin, an exercise-related hormone, in preserving intestinal integrity. This study investigates whether irisin could delay T1D onset by enhancing the colon intestinal barrier. Impaired colon intestinal barriers were observed in newly diagnosed T1D patients and non-obese diabetic (NOD) mice, worsening with age and accompanied by islet inflammation. Using an LPS-induced colonic inflammation model, a dose-dependent impact of LPS on colon cells irisin expression, secretion, and barrier function was revealed. Exogenous irisin demonstrated remarkable effects, mitigating islet insulitis, enhancing energy expenditure, and alleviating autoimmune symptoms by reducing colon intestinal permeability. Single-cell RNA sequencing (scRNA-seq) highlighted irisin's positive impact on colon epithelial cell clusters, effectively restoring the intestinal barrier. Irisin also selectively modulated bacterial composition, averting potential bacterial translocation. Mechanistically, irisin enhanced colon intestinal barrier tight junction proteins through the AMPK/PI3K/AKT pathway, with FAM120A playing a crucial role. Irisin upregulated MUC3 expression, a protector against damage and inflammation. Harnessing irisin's exercise-mimicking properties suggests therapeutic potential in clinical settings for preventing T1D progression, offering valuable insights into fortifying the colon's intestinal barrier and managing autoimmune conditions associated with T1DM.

Urolithin A Ameliorates Athletic Ability and Intestinal Microbiota in Sleep Deprivation from the Perspective of the Gut-Muscle Axis.

Urolithin A (UA), a gut-microbiota-derived metabolite of ellagic acid, presents various benefits to intestinal microecology. The presence of "gut-muscle axis" regulating the onset and progression of exercise-related physical frailty and sarcopenia has been recently hypothesized. This study aims to explore the underlying mechanism of gut-muscle axis by which UA enhances muscle strength and fatigue resistance of sleep-deprived (SD) mice. UA is gavaged to C57BL/6 mice (50mgkg-1bw) before 48-h SD. The results indicate that pretreatment of UA significantly enhances motor abilityand energy metabolism. The inflammation is suppressed, and intestinal permeability is improved after prophylactic treatment with UA. The decreased level of serum lipopolysaccharide (LPS) is concomitant with augmentation of the intestinal tight junction proteins. 16s rRNA analysis of colonic contents reveals that UA significantly reduces the abundance of Clostridia_UCG-014 and Candidatus_Saccharimonas, and upregulates Lactobacillus and Muribaculaceae. UA probably influences on gut microbial functions via several energy metabolism pathways, such as carbon metabolism, phosphotransferase system (PTS), and ATP binding cassette (ABC) transporters. The dietary intervention of UA helps to create a systemic protection, a bidirectional communication connecting the gut microbiota with muscle system, able to alleviate SD-induced mobility impairment and gut dysbiosis.

Mechanism of intestinal injury induced by acute diquat poisoning in rats

To investigate the effects of diquat (DQ) on the expression of intestinal pyroptosis-related proteins and tight junction proteins in rats,and to analyze the role of pyroptosis in the intestinal injury of rats with acute DQ poisoning. A total of 36 Wistar male rats were randomly divided into control group, and 3 hours, 12 hours, 36 hours and 3 days exposure groups, with 6 rats in each group. Each exposure group was given 1/2 median lethal dose (LD50) of 115.5 mg/kg DQ by one-time gavage. The control group was given the same amount of normal saline by gavage. The control group was anesthetized at 3 hours after DQ gavage to take jejunal tissues; each exposure group was anesthetized at 3 hours, 12 hours, 36 hours, and 3 days after DQ gavage to take jejunal tissues, respectively. The general conditions of the rats were recorded. The pathological changes of jejunum tissue were observed by hematoxylin-eosin (HE) staining. The expression of intestinal pyroptosis-related proteins [NOD-like receptor protein 3 (NLRP3), cysteine aspartate-specific protease 1 (caspase-1), Gasdemin D (GSDMD)] in the intestinal tissues was observed by immunohistochemical staining. Western blotting was used to detect the expression of intestinal pyroptosis-related proteins and intestinal tight junction proteins (Occludin and Claudin-1). Light microscopy showed that pathological changes occurred in jejunum tissue at the early stage of exposure (3 hours), and the injury was the most serious in the 12 hours exposure group, with a large number of inflammatory cells infiltrating in the tissue, and the damage was significantly reduced after 3 days exposure. Immunohistochemical results showed that NLRP3, caspase-1 and GSDMD were expressed in the jejunal mucosa of the control group and the exposure groups, and the positive cells in the control group were less expressed with light staining. The expression of the above proteins in the exposed group was increased significantly and the staining was deep. Western blotting results showed that compared with the control group, the expression of NLRP3 protein in jejunum tissues of all groups was increased, with the most significant increase in the 36 hours group (NLRP3/β-actin: 1.47±0.06 vs. 0.43±0.14, P < 0.01). Compared with the control group, the expression of GSDMD protein in the 3 hours, 12 hours and 36 hours exposure groups increased, and the expression of GSDMD protein in the 3 hours and 12 hours exposure groups increased significantly (GSDMD/β-actin: 1.04±0.40, 1.25±0.15 vs. 0.65±0.25, both P < 0.05). The expression of caspase-1 protein was increased in 36 hours exposure group compared with the control group (caspase-1/β-actin: 1.44±0.34 vs. 0.98±0.19, P > 0.05). Compared with the control group, the expression of Occludin and Claudin-1 proteins in each exposure group decreased, and the expression of Occludin proteins was significantly decreased in the 3 hours, 12 hours, and 36 hours exposure groups decreased significantly (Occludin/β-actin: 0.74±0.17, 0.91±0.20, 0.79±0.23 vs. 1.41±0.08, all P < 0.05). Although the protein expression of Claudin-1 decreased in each exposure group, the difference was not statistically significant. The intestinal injury caused by acute DQ poisoning may be related to the activation of pyroptosis pathway of small intestinal cells and the reduction of the density of intercellular junctions.