Biochemical Modulators of Tight Junctions (TJs): Occludin, Claudin-2 and Zonulin as Biomarkers of Intestinal Barrier Leakage in the Diagnosis and Assessment of Inflammatory Bowel Disease Progression.

BACKGROUND: Considering the increasing worldwide prevalence of inflammatory bowel disease (IBD), the early diagnosis of this disease is extremely important. However, non-invasive diagnostic methods remain limited, while invasive techniques are the most commonly used in daily practice. Therefore, there is a serious need to find new non-invasive biomarkers of IBD. METHODS: The serum profiles of occludin, claudin-2, and zonulin were assessed in IBD patients using the ELISA method. The levels of the analyzed biomarkers were measured before and after a year of anti-inflammatory treatment, which was a tumor necrosis factor α (TNF-α) inhibitor (adalimumab) in patients with ulcerative colitis (UC) and conventional therapy in patients with Crohn's disease (CD). RESULTS: In IBD patients, the serum level of occludin (p < 0.001) decreased compared to healthy individuals, while the level of claudin-2 (p < 0.001) increased. Additionally, zonulin (p < 0.01) concentration increased in CD patients compared to the control group. The highest diagnostic ability was presented by occludin measurements with the area under the curve (AUC) of 0.959 (95% CI 0.907-1) in UC and 0.948 (95% CI 0.879-1) in CD. Claudin-2 also demonstrated very good ability in diagnosing UC and CD with AUC values of 0.864 (95% CI 0.776-0.952) and 0.896 (95% CI 0.792-0.999), respectively. The ability of zonulin to diagnose CD was estimated as good with an AUC of 0.74 (95% CI 0.598-0.881). Moreover, a significant correlation was identified between C-reactive protein (CRP), claudin-2 (r = -0.37; p < 0.05), and zonulin (r = -0.44; p < 0.05) in UC patients. Treatment with adalimumab improved the level of occludin, claudin-2, and zonulin in UC patients, while anti-inflammatory conventional therapy decreased the concentration of zonulin in CD. CONCLUSIONS: Occludin and claudin-2 measurements present significant utility in diagnosing both UC and CD, while zonulin assessments may be useful in CD diagnosis. Additionally, claudin-2 and zonulin measurements may be helpful in evaluating the intensity of the inflammatory process. Anti-TNF-α treatment improved the value of occludin, claudin-2, and zonulin, indicating its beneficial effect on the integrity of tight junctions in UC.

[Sanguinarine alleviates ulcerative colitis in mice by regulating the Nrf2/NF-κB pathway].

OBJECTIVE: To investigate the mechanism of sanguinarine (SA) for alleviating ulcerative colitis (UC) induced by dextran sodium sulfate (DSS) in mice. METHODS: Male C57BL/6 mouse models of 3.5% DSS-induced UC were randomized for treatment with 1, 5 and 10 mg/kg SA by gavage, 400 mg/kg sulfasalazine by gavage, or 10 mg/kg SA combined with intraperitoneal injection of 30 mg/kg ML385 (a Nrf2 inhibitor). The changes in intestinal inflammation was assessed by monitoring weight changes, disease activity index (DAI) score, colon length measurement, and HE staining. After the treatments, the colon tissues were collected for detection of malondialdehyde (MDA) content using colorimetry, mRNA expressions of inflammatory factors using RT-qPCR, and the expressions of Nrf2, HO-1, Keap-1, p-p65, p65, occludin, and ZO-1 proteins were detected using Western blotting. RESULTS: SA treatment obviously alleviated weight loss, colon length shortening and DAI score increase and ameliorated structural destruction of the colon glands and colonic crypts in mice with DSSinduced UC. SA intervention significantly decreased the levels of TNF-α, IL-1ß and IL-6 mRNA and lowered ROS and MDA levels in the colon tissue of UC mice. The mouse models receiving SA treatment showed significantly increased expressions of Nrf2, HO-1, occludin and ZO-1 and lowered expressions of Keap-1 and P-P65 in the colon tissue without significant changes of p65 expression, and these changes were SA dose-dependent. Treatment with ML385 obviously attenuated the effect of highdose SA for improving UC in the mouse models. CONCLUSION: SA can improve UC-like enteritis in mice possibly by activating the Nrf2 pathway and inhibiting the NF-κB pathway in the colon tissue.

Disulfiram attenuates cell and tissue damage and blood‒brain barrier dysfunction after intracranial haemorrhage by inhibiting the classical pyroptosis pathway.

No single treatment significantly reduces the mortality rate and improves neurological outcomes after intracerebral haemorrhage (ICH). New evidence suggests that pyroptosis-specific proteins are highly expressed in the perihaematomal tissues of patients with ICH and that the disulfiram (DSF) inhibits pyroptosis. An ICH model was established in C57BL/6 mice by intracranial injection of collagenase, after which DSF was used to treat the mice. Cell model of ICH was constructed, and DSF was used to treat the cells. HE, TUNEL, Nissl, FJC and IF staining were performed to evaluate the morphology of brain tissues; Western blotting and ELISA were performed to measure the protein expression of NOD-like receptor protein 3 (NLRP3)/Caspase-1/gasdermin D (GSDMD) classical pyroptosis pathway and Toll-likereceptor4 (TLR4)/nuclear factor-kappaB (NF-κB) inflammatory signaling pathway and blood‒brain barrier-associated factoes, and the wet/dry weight method was used to determine the brain water content. The expression of proteins related to the NLRP3/Caspase-1/GSDMD pathway and the TLR4/NF-κB pathway was upregulated in tissues surrounding the haematoma compared with that in control tissues; Moreover, the expression of the blood-brain barrier structural proteins occludin and zonula occludens-1 (ZO-1) was downregulated, and the expression of Aquaporin Protein-4 (AQP4) and matrix metalloprotein 9 (MMP-9) was upregulated. DSF significantly inhibited these changes, reduced the haematoma volume, decreased the brain water content, reduced neuronal death and degeneration and improved neurological function after ICH. ICH activated the classical pyroptosis pathway and TLR4/NF-κB inflammatory pathway, disruped the expression of blood-brain barrier structural proteins, and exacerbated brain injury and neurological dysfunction. DSF inhibited these changes and exerted the therapeutic effects on pathological changes and dysfunction caused by ICH.

Protective effects of sodium humate on the intestinal barrier damage of Salmonella Typhimurium-challenged broilers.

Salmonella Typhimurium (S. Typhimurium) infections can lead to severe intestinal damage and reduce growth performance in broilers. Thus, this study examined the potential mitigating impact of sodium humate (HNa) on intestinal barrier damage resulting from S. Typhimurium infection in broilers. A total of 320 1-day-old Arbor Acres broilers were randomly assigned into 5 treatments with 8 replicates. On d 22-24, broilers in the CON group were challenged with 1 ml of PBS, while broilers in the other groups were challenged with 1 ml of 3 × 109 CFU/ml S. Typhimurium, daily. Dietary administration with 4 g/kg of HNa increased (P < 0.05) the final body weight, jejunal secretory immunoglobulin A (sIgA), total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), and catalase (CAT) levels as compared with the MOD group broilers. Furthermore, HNa alleviated intestinal barrier damage by increasing villus height (VH), upregulating protein expression of Occludin, Claudin-1, and zonula occludens-1 (ZO-1), inhibiting toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) signaling pathway activation, and decreasing the secretion of inflammatory cytokines (P < 0.05). Collectively, the present study showed that HNa mitigated intestinal barrier damage induced by S. Typhimurium infection in broilers.

[Effect of Modified Xiaoyao Powder on intestinal barrier and intestinal flora in mice with metabolic associated fatty liver disease based on "gut-liver axis"].

This study explores the effects and mechanisms of Modified Xiaoyao Powder on the intestinal barrier and intestinal flora in mice with metabolic associated fatty liver disease(MAFLD) based on the " gut-liver axis". Sixty male C57BL/6 mice were randomly divided into the normal group, model group, bifidobacterium tetrad tablet group(SQ), and Modified Xiaoyao Powder groups with low,medium and high doses(XL, XM, XH), with 10 mice in each group. All the mice were administrated with a high-fat diet to build the MAFLD model except the normal group and then treated with related drugs for 12 weeks. Body mass, liver wet weight, and liver index were detected. Serum aspartate aminotransferase(AST), alanine aminotransferase(ALT), total cholesterol(TC), triacylglycerol(TG), low density lipoprotein cholesterol(LDL-C), high density lipoprotein cholesterol(HDL-C), and lipopolysaccharide(LPS)levels were detected using the biochemical kits. The contents of tumor necrosis factor-α(TNF-α) and interleukin(IL-6) in the liver were tested simultaneously. The morphological changes of the liver and intestine were observed using hematoxylin-eosin(HE) staining and oil red O staining. The goblet cells in the ileum were detected by periodic acid Schiff and alcian blue stain(AB-PAS) staining.The expression of zonula occludens-1(ZO-1), recombinant occludin(occludin), and recombinant claudin 1(claudin-1) in ileum and colon were detected by immunohistochemistry and Western blot. The changes of intestinal flora in mice were analyzed by 16S rRNA gene sequencing. The results showed that compared with the normal group, body weight, liver wet weight and liver index in the model group increased. The contents of TC, TG, ALT, AST, LDL-C, and LPS in the serum of the model group increased, while HDL-C decreased. Meanwhile, the contents of TNF-α and IL-6 in liver tissue increased and liver lipid accumulation increased, indicating successful model induction. Compared with the model group, body weight, liver wet weight, and liver index were decreased in XM,XH groups and SQ group. Serum levels of TC, TG, LDL-C, ALT and AST in XM group and SQ group were significantly decreased,and HDL-C levels were increased. The levels of IL-6, TNF-α in liver tissue and serum LPS in the XL, XM groups and SQ group were significantly decreased. The protein expression of claudin-1, occludin and ZO-1 in XL, XM groups and SQ group were increased. The analysis of intestinal flora showed that compared with the model group, Modified Xiaoyao Powder with a medium dose could significantly improve the richness and diversity of intestinal flora in mice. At the phylum level, the Firmicutes/Bacteroidetes(F/B) ratio decreased; at the genus level, Lactobacillus, Brautella, Bacteroides, and Ackermannia increased, while Prevotella, Desulfovibrio and Turicibacter decreased. The main differential species were Odorbacteraceaeae and Peptostreptococcaceae. In conclusion, Modified Xiaoyao Powder could inhibit inflammation, regulate intestinal flora homeostasis, and promote the repair of the intestinal mucosal barrier in mice with MAFLD.

Astragalus Polysaccharide improves immunogenicity of influenza vaccine as well as modulate gut microbiota in BALB/c mice.

BACKGROUND: Vaccination is the best way to prevent influenza virus infection, and insufficient antibodies make it difficult to resist influenza virus invasion. Astragalus Polysaccharide (APS) has a boosting effect on immunity, so we evaluate the effect of APS as an immune adjuvant for H1N1 influenza vaccines in this study. METHODS: The mice were immunized twice with influenza A (H1N1) vaccine and APS. Subsequently, the serum antibody levels were assessed using enzyme-linked immunosorbent assay (ELISA). The frequency of peripheral immune T cells was determined by flow cytometry. Following this, the immunized mice were exposed to a lethal dose of the virus, and changes in body weight and survival rates were recorded. Hematoxylin-eosin staining was employed to observe pathological alterations in lung and intestinal tissues. Western blot analysis was conducted to detect the expression of intestinal barrier function proteins (Occludin and Claudin-1). ELISA was utilized to measure the expression level of serum inflammatory cytokine TNF-α. Fresh mouse feces were collected after the initial immunization as well as after viral infection for 16S rRNA analysis aimed at detecting alterations in gut microbiota. RESULTS: Compared to the Hemagglutinin (HA) group, the APS group demonstrated higher levels of immunoglobulin G (IgG), IgG1, and IgG3, as well as neutralizing antibody levels. Additionally, it increased the frequency of CD8+ cells to enhance resistance against lethal infection. On day 14 post-infection, the high-dose APS group exhibited a higher survival rate (71.40 %) compared to the HA group (14.28 %), along with faster weight recovery. Furthermore, APS was found to ameliorate alveolar damage in lung tissue and rectify intestinal structural disorder. It also upregulated the expression levels of tight junction proteins Occludin and Claudin-1 in intestinal tissue while reducing serum TNF-α expression levels. In addition, populations of Colidextribacter, Peptococcaceae, and Ruminococcaceae were the dominant gut microbiota in the APS group after viral infection. CONCLUSION: APS has an immune-enhancing effect and is expected to be a novel adjuvant in the H1N1 influenza vaccine.

Age-related retinal degeneration resulting from the deletion of Shp2 tyrosine phosphatase in photoreceptor neurons.

Shp2, a critical SH2-domain-containing tyrosine phosphatase, is essential for cellular regulation and implicated in metabolic disruptions, obesity, diabetes, Noonan syndrome, LEOPARD syndrome, and cancers. This study focuses on Shp2 in rod photoreceptor cells, revealing its enrichment, particularly in rods. Deletion of Shp2 in rods leads to age-dependent photoreceptor degeneration. Shp2 targets occludin (OCLN), a tight junction protein, and its deletion reduces OCLN expression in the retina and retinal pigment epithelium (RPE). The isolation of actively translating mRNAs from rods lacking Shp2, followed by RNA sequencing, reveals alterations in cell cycle regulation. Additionally, altered retinal metabolism is observed in retinal cells lacking Shp2. Our studies indicate that Shp2 is crucial for maintaining the structure and function of photoreceptors.

Capsaicin Reduces Obesity by Reducing Chronic Low-Grade Inflammation.

Chronic low-grade inflammation (CLGI) is associated with obesity and is one of its pathogenetic mechanisms. Lipopolysaccharide (LPS), a component of Gram-negative bacterial cell walls, is the principal cause of CLGI. Studies have found that capsaicin significantly reduces the relative abundance of LPS-producing bacteria. In the present study, TRPV1-knockout (TRPV1-/-) C57BL/6J mice and the intestinal epithelial cell line Caco-2 (TRPV1-/-) were used as models to determine the effect of capsaicin on CLGI and elucidate the mechanism by which it mediates weight loss in vivo and in vitro. We found that the intragastric administration of capsaicin significantly blunted increases in body weight, food intake, blood lipid, and blood glucose in TRPV1-/- mice fed a high-fat diet, suggesting an anti-obesity effect of capsaicin. Capsaicin reduced LPS levels in the intestine by reducing the relative abundance of Proteobacteria such as Helicobacter, Desulfovibrio, and Sutterella. Toll-like receptor 4 (TLR4) levels decreased following decreases in LPS levels. Then, the local inflammation of the intestine was reduced by reducing the expression of tumor necrosis factor (TNF)-α and interleukin (IL)-6 mediated by TLR4. Attenuating local intestinal inflammation led to the increased expression of tight junction proteins zonula occludens 1 (ZO-1) and occludin and the restoration of the intestinal barrier function. Capsaicin increased the expression of ZO-1 and occludin at the transcriptional and translational levels, thereby increasing trans-endothelial electrical resistance and restoring intestinal barrier function. The restoration of intestinal barrier function decreases intestinal permeability, which reduces the concentration of LPS entering the circulation, and reduced endotoxemia leads to decreased serum concentrations of inflammatory cytokines such as TNF-α and IL-6, thereby attenuating CLGI. This study sheds light on the anti-obesity effect of capsaicin and its mechanism by reducing CLGI, increasing our understanding of the anti-obesity effects of capsaicin. It has been confirmed that capsaicin can stimulate the expression of intestinal transmembrane protein ZO-1 and cytoplasmic protein occludin, increase the trans-epithelial electrical resistance value, and repair intestinal barrier function.

Targeting monocytic Occludin impairs transendothelial migration and HIV neuroinvasion.

Transmigration of circulating monocytes from the bloodstream to tissues represents an early hallmark of inflammation. This process plays a pivotal role during viral neuroinvasion, encephalitis, and HIV-associated neurocognitive disorders. How monocytes locally unzip endothelial tight junction-associated proteins (TJAPs), without perturbing impermeability, to reach the central nervous system remains poorly understood. Here, we show that human circulating monocytes express the TJAP Occludin (OCLN) to promote transmigration through endothelial cells. We found that human monocytic OCLN (hmOCLN) clusters at monocyte-endothelium interface, while modulation of hmOCLN expression significantly impacts monocyte transmigration. Furthermore, we designed OCLN-derived peptides targeting its extracellular loops (EL) and show that transmigration of treated monocytes is inhibited in vitro and in zebrafish embryos, while preserving vascular integrity. Monocyte transmigration toward the brain is an important process for HIV neuroinvasion and we found that the OCLN-derived peptides significantly inhibit HIV dissemination to cerebral organoids. In conclusion, our study identifies an important role for monocytic OCLN during transmigration and provides a proof-of-concept for the development of mitigation strategies to prevent monocyte infiltration and viral neuroinvasion.

Shenling Baizhu Decoction treats ulcerative colitis of spleen-deficiency and dampness obstruction types by targeting 'gut microbiota and galactose metabolism-bone marrow' axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Shenlin Baizhu Decoction (SLBZD), which comes from 'Taiping Huimin Heji Ju Fang', belongs to a classical prescription for treating spleen deficiency and dampness obstruction (SQDDS)-type ulcerative colitis (UC) in traditional Chinese medicine. However, the mechanism of SLBZD in treating UC with SQDDS remains unclear. AIM OF THE STUDY: This study aims to investigate the mechanism of SLBZD against SQDDS-type UC of based on the "gut microbiota and metabolism - bone marrow" axis to induce endogenous bone marrow mesenchymal stem cells (BMSCs) homing. MATERIALS AND METHODS: Ultra-performance liquid chromatography-mass spectrometry was used to analysis of SLBZD qualitatively. The efficacy of SLBZD in SQDDS-type UC was evaluated based on the following indicators: the body weight, colon length, disease activity index (DAI) score, Haemotoxylin and Eosin (H&E) pathological sections, and intestinal permeability proteins (occluding and ZO-1). 16S rRNA gene sequencing and non-target metabolomics were performed to identify gut microbiota changes and its metabolites in feces, respectively. BMSCs in each group was collected, cultured, and analyzed. Optimal passaged BMSCs were injected by tail vein into UC rats of SQDDS types. BMSCs homing to the colonic mucosal tissue was observed by immunofluorescent. Finally, the repairing effect of BMSCs homing to the colonic mucosal tissue after SLBZD treatment was analyzed by transmission electron microscopy, qRT-PCR, and immunohistochemistry. RESULTS: SLBZD effectively improved the colonic length and the body weight, reduced DAI and H&E scores, and increased the expression of the intestinal permeability proteins, including occluding and ZO-1, to treat SQDDS-type UC. After SLBZD treatment, the α-diversity and ß-diversity of the gut microbiota were improved. The differential microbiota was screened as Aeromonadaceae, Lactobacillaceae, and Clostridiaceae at the family level, and Aeromonas, Lactobacillus, Clostridium_sensu_stricto_1 at the genus level. Meanwhile, the main metabolic pathway was the galactose metabolism pathway. SLBZD treatment timely corrected the aberrant levels of ß-galactose in peripheral blood and bone marrow, senescence-associate-ß-galactosidase in BMSCs, and galactose kinase-2, galactose mutase, and galactosidase beta-1 in peripheral blood to further elevate the expression levels of senescence-associated (SA) proteins (p16, p53, p21, and p27) in BMSCs. The Spearman's correlation analysis demonstrated the relationship between microbiota and metabolism, and the relationship between the galactose metabolism pathway and SA proteins. After BMSCs in each group injection via the tail vein, the pharmacodynamic effects were consistent with those of SLBZD in SQDDS-type UC rats. Furthermore, BMSCs have been homing to colonic mucosal tissue. BMSCs from the SLBZD treatment group had stronger restorative effects on intestinal permeability function due to increasing protein and mRNA expressions of occludin and ZO-1, and decreasing the proteins and mRNA expressions of SDF-1 and CXCR4 in colon. CONCLUSIONS: SLBZD alleviated the damaged structure of gut microbiota and regulated their metabolism, specifically the galactose metabolism, to treat UC of SDDOS types. SLBZD treatment promotes endogenous BMSCs homing to colonic mucosal tissue to repaire the intestinal permeability. The current exploration revealed an underlying mechanism wherein SLBZD activates endogenous BMSCs by targeting 'the gut microbiota and its metabolism-bone marrow' axis and repairs colonic mucosal damage to treat SDDOS-type UC.

Paeoniae Decoction restores intestinal barrier dysfunction by promoting the interaction between ILC3 and gut flora.

BACKGROUND: Intestinal barrier dysfunction is a significant contributor to the recurrence and refractory of ulcerative colitis (UC). Promoting the interaction between group 3 innate lymphoid cells (ILC3s) and gut flora is a valuable strategy for mucosal repair. Paeoniae decoction (PD) is a compound commonly used in clinical treatment of UC, but its exact mechanism remains unclear. PURPOSE: We aimed to investigate the protective effect of PD on intestinal mucosal injury induced by dextran sulfate sodium (DSS) in chronic colitis, as well as to elucidate its potential mechanism. METHODS: C57BL/6 mice were induced with chronic colitis by 2 % DSS and divided into four groups: control group, model group, PD low dose (4 g/kg), and high dose (8 g/kg) group. The effectiveness of PD in treating chronic colitis mice was evaluated based on changes in body weight, colon length, colon pathological tissue scores, and the mRNA levels of inflammatory factors IL-6 and IL-1ß. The expressions of intestinal epithelial tight junction proteins (ZO-1 and Occludin), IL-22, and MUC2 were observed using immunofluorescence and RT-PCR. Additionally, the proportion of ILC3 and natural cytotoxicity receptor (NCR)+ ILC3 in the colon were detected using flow cytometry. Furthermore, UHPLC-QE-MS was utilized to identify chemical components of PD and network pharmacology was employed to predict potential pathways for PD intervention in UC. Subsequently, MNK-3 cells (ILC3 in vitro cell line) and NCM460 cells were used to verify the network pharmacology results. Finally, the effects of PD on UC gut flora have been explored using in vitro fermentation and 16S rDNA techniques. RESULTS: The results showed that PD significantly restored body weight and colon length in mice with chronic colitis, while also reducing colon inflammatory cell infiltration and the expression of IL-6 and IL-1ß. Additionally, PD notably promoted the expression of MUC2, ZO-1, Occludin, and IL-22, as well as increasing the ratio of ILC3 and NCR+ILC3. UHPLC-QE-MS analysis identified 443 components of PD, and network pharmacology suggested that PD could target the aryl hydrocarbon receptor (AHR) signaling pathway, which was confirmed by MNK-3 cells and in vitro fermentation experiments. Furthermore, MNK-3-conditioned medium (CM) increased the expression of ZO-1 and Occludin in NCM460 cells. In addition, 16S rDNA results indicated that PD promoted the abundance of Lactobacillales, thus contributing to mucosal damage repair by activating the AHR signal in ILC3s. CONCLUSION: In summary, our study demonstrates that PD repairs intestinal mucosal damage in chronic colitis by regulating the interaction of gut flora with ILC3, and the specific mechanism is related to the activation of AHR signaling pathway.

Sanfeng Tongqiao Dripping Pills alleviate House Dust Mite-induced allergic rhinitis in mice by inhibiting Th2 differentiation and repairing the nasal epithelial barrier.

BACKGROUND: Sanfeng Tongqiao Dripping Pills (SFTQ) has clinically demonstrated a promising therapeutic effect on allergic rhinitis (AR). However, the active ingredients and underlying mechanisms of SFTQ remain unclear. PURPOSE: Exploring the effects, mechanisms, and active ingredients of SFTQ in the treatment of AR is valuable. STUDY DESIGN: The mechanisms of SFTQ and its active ingredients in treating AR were investigated through in vivo and in vitro studies. METHODS: A HDM-induced AR model was established in BALB/c mice. The effects of SFTQ in treating AR were evaluated by AR-like symptoms, EOS count, and pathological changes in the nasal tissue in vivo. The effects of SFTQ active components on epithelial cells (ECs) were evaluated in Poly(I:C) and TNF-α co-stimulated human nasal ECs (RPMI-2650). Additionally, the effects of SFTQ active components on splenocytes proliferation and Th cell differentiation were assessed. A co-culture system of ECs and T lymphocytes was established to investigate the impact of Th2 cells on the structure and function of ECs. The effects of SFTQ ingredients on ECs, T lymphocytes, and the HDM-induced AR model were further confirmed through in vivo and in vivo studies, respectively. RESULTS: SFTQ significantly alleviated AR-like symptoms and pathological changes in the nasal tissue of AR mice. The treatment elevated the expression of Occludin and E-cadherin in the nasal epithelium and reduced the percentage of Th2 cells in cervical lymph nodes (CLN). Among the active compounds of SFTQ, L-Menthone and Pulegone notably downregulated IL-33 levels in activated ECs, while Hesperetin significantly decreased TSLP and IL-33 levels. In the co-culture system of ECs and Th2 cells, exposure to Baicalin, Wogonin, and Pulegone increased the TEER value of ECs, while notably inhibiting the production of TSLP and IL-33. Furthermore, in HDM-induced AR mice, treatments with Baicalin, Luteolin, and Hesperetin effectively inhibited AR-like symptoms. Additionally, Luteolin and Hesperetin significantly reduced the inflammatory cells infiltration and the population of Th2 cells in AR mice. CONCLUSION: SFTQ and its active ingredients effectively alleviated HDM-induced AR in mice by inhibiting Th2 cell differentiation and repairing the nasal epithelial barrier. Our study can provide a scientific basis for SFTQ to be used in clinical treatment of AR.

Study on the mechanism of mitigating radiation damage by improving the hematopoietic system and intestinal barrier with Tenebrio molitor peptides.

Research on plant and animal peptides has garnered significant attention, but there is a lack of studies on the functional properties of Tenebrio molitor peptides, particularly in relation to their potential mitigating effect on radiation damage and the underlying mechanisms. This study aims to explore the protective effects of Tenebrio molitor peptides against radiation-induced damage. Mice were divided into five groups: normal, radiation model, and low-, medium-, and high-dose Tenebrio molitor peptide (TMP) groups (0.15 g per kg BW, 0.30 g per kg BW, and 0.60 g per kg BW). Various parameters such as blood cell counts, bone marrow DNA content, immune organ indices, serum levels of D-lactic acid, diamine oxidase (DAO), endotoxin (LPS), and inflammatory factors were assessed at 3 and 15 days post gamma irradiation. Additionally, the intestinal tissue morphology was examined through H&E staining, RT-qPCR experiments were conducted to analyze the expression of inflammatory factors in the intestine, and immunohistochemistry was utilized to evaluate the expression of tight junction proteins ZO-1 and Occludin in the intestine. The findings revealed that high-dose TMP significantly enhanced the hematopoietic system function in mice post radiation exposure, leading to increased spleen index, thymus index, blood cell counts, and bone marrow DNA production (p < 0.05). Moreover, TMP improved the intestinal barrier integrity and reduced the intestinal permeability. Mechanistic insights suggested that these peptides may safeguard intestinal barrier function by downregulating the gene expression of inflammatory factors TNF-α, IL-1ß, and IL-6, while upregulating the expression of tight junction proteins ZO-1 and Occludin (p < 0.05). Overall, supplementation with TMP mitigates radiation-induced intestinal damage by enhancing the hematopoietic system and the intestinal barrier, offering valuable insights for further investigations into the mechanisms underlying the protective effects of these peptides against ionizing radiation.

Attenuated replication and damaging effects of SARS-CoV-2 Omicron variants in an intestinal epithelial barrier model.

Many COVID-19 patients suffer from gastrointestinal symptoms and impaired intestinal barrier function is thought to play a key role in Long COVID. Despite its importance, the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on intestinal epithelia is poorly understood. To address this, we established an intestinal barrier model integrating epithelial Caco-2 cells, mucus-secreting HT29 cells and Raji cells. This gut epithelial model allows efficient differentiation of Caco-2 cells into microfold-like cells, faithfully mimics intestinal barrier function, and is highly permissive to SARS-CoV-2 infection. Early strains of SARS-CoV-2 and the Delta variant replicated with high efficiency, severely disrupted barrier function, and depleted tight junction proteins, such as claudin-1, occludin, and ZO-1. In comparison, Omicron subvariants also depleted ZO-1 from tight junctions but had fewer damaging effects on mucosal integrity and barrier function. Remdesivir, the fusion inhibitor EK1 and the transmembrane serine protease 2 inhibitor Camostat inhibited SARS-CoV-2 replication and thus epithelial barrier damage, while the Cathepsin inhibitor E64d was ineffective. Our results support that SARS-CoV-2 disrupts intestinal barrier function but further suggest that circulating Omicron variants are less damaging than earlier viral strains.

Red raspberry (Rubus idaeus) preserves intestinal barrier integrity and reduces oxidative stress in Caco-2 cells exposed to a proinflammatory stimulus.

Growing evidence showed the capacity of (poly)phenols to exert a protective role on intestinal health. Nevertheless, the existing findings are still heterogeneous and the underlying mechanisms remain unclear. This study investigated the potential benefits of a red raspberry (Rubus idaeus) powder on the integrity of the intestinal barrier, focusing on its ability to mitigate the effects of tumor necrosis factor-α (TNF-α)-induced intestinal permeability. Human colorectal adenocarcinoma cells (i.e., Caco-2 cells) were used as a model to assess the impact of red raspberry on intestinal permeability, tight junction expression, and oxidative stress. The Caco-2 cells were differentiated into polarized monolayers and treated with interferon-γ (IFN-γ) (10 ng mL-1) for 24 hours, followed by exposure to TNF-α (10 ng mL-1) in the presence or absence of red raspberry extract (1-5 mg mL-1). The integrity of the intestinal monolayer was evaluated using transepithelial electrical resistance (TEER) and fluorescein isothiocyanate-dextran (FITC-D) efflux assay. Markers of intestinal permeability (claudin-1, occludin, and zonula occludens-1 (ZO-1)) and oxidative stress (8-hydroxy-2-deoxyguanosine (8-OHdG) and protein carbonyl) were assessed using ELISA kits. Treatment with red raspberry resulted in a significant counteraction of TEER value loss (41%; p < 0.01) and a notable reduction in the efflux of FITC-D (-2.5 times; p < 0.01). Additionally, red raspberry attenuated the levels of 8-OHdG (-48.8%; p < 0.01), mitigating the detrimental effects induced by TNF-α. Moreover, red raspberry positively influenced the expression of the integral membrane protein claudin-1 (+18%; p < 0.01), an essential component of tight junctions. These findings contribute to the growing understanding of the beneficial effects of red raspberry in the context of the intestinal barrier. The effect of red raspberry against TNF-α-induced intestinal permeability observed in our in vitro model suggests, for the first time, its potential as a dietary strategy to promote gastrointestinal health.

[Bmal1 mediates the neuroprotective effect of sodium butyrate in a mouse model of Parkinson's disease].

OBJECTIVE: To investigate the mechanisms that mediate the neuroprotective effect of the intestinal microbial metabolite sodium butyrate (NaB) in a mouse model of Parkinson's disease (PD) via the gut-brain axis. METHODS: Thirty-nine 7-week-old male C57BL/6J mice were randomized equally into control group, PD model group, and NaB treatment group. In the latter two groups, PD models were established by intraperitoneal injection of 30 mg/kg 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) once daily for 5 consecutive days, and normal saline was injected in the control group. After modeling, the mice received daily gavage of NaB (300 mg/kg) or an equal volume of saline for 14 days. Behavioral tests were carried out to assess the changes in motor function of the mice, and Western blotting was performed to detect the expressions of tyrosine hydroxylase (TH) and α-synuclein (α-syn) in the striatum and nuclear factor-κB (NF-κB), tumor necrosis factor (TNF-α), interleukin 6 (IL-6), and the tight junction proteins ZO-1, Occludin, and Claudinin the colon. HE staining was used to observe inflammatory cell infiltration in the colon of the mice. RNA sequencing analysis was performed to identify the differentially expressed genes in mouse colon tissues, and their expressions were verified using qRT-PCR and Western blotting. RESULTS: The mouse models of PD with NaB treatment showed significantly increased movement speed and pulling strength of the limbs with obviously upregulated expressions of TH, Occludin, and Claudin and downregulated expressions of α-syn, NF-κB, TNF-α, and IL-6 (all P < 0.05). HE staining showed that NaB treatment significantly ameliorated inflammatory cell infiltration in the colon of the PD mice. RNA sequencing suggested that Bmal1 gene probably mediated the neuroprotective effect of NaB in PD mice (P < 0.05). CONCLUSION: NaB can improve motor dysfunction, reduce dopaminergic neuron loss in the striatum, and ameliorate colonic inflammation in PD mice possibly through a mechanism involving Bmal1.

Heterogenous Gene Expression of Bicellular and Tricellular Tight Junction-Sealing Components in the Human Intestinal Tract.

A major site for the absorption of orally administered drugs is the intestinal tract, where the mucosal epithelium functions as a barrier separating the inside body from the outer environment. The intercellular spaces between adjacent epithelial cells are sealed by bicellular and tricellular tight junctions (TJs). Although one strategy for enhancing intestinal drug absorption is to modulate these TJs, comprehensive gene (mRNA) expression analysis of the TJs components has never been fully carried out in humans. In this study, we used human biopsy samples of normal-appearing mucosa showing no endoscopically visible inflammation collected from the duodenum, jejunum, ileum, colon, and rectum to examine the mRNA expression profiles of TJ components, including occludin and tricellulin and members of the claudin family, zonula occludens family, junctional adhesion molecule (JAM) family, and angulin family. Levels of claudin-3, -4, -7, -8, and -23 expression became more elevated in each segment along the intestinal tract from the upper segments to the lower segments, as did levels of angulin-1 and -2 expression. In contrast, expression of claudin-2 and -15 was decreased in the large intestine compared to the small intestine. Levels of occludin, tricellulin, and JAM-B and -C expression were unchanged throughout the intestine. Considering their segment specificity, claudin-8, claudin-15, and angulin-2 appear to be targets for the development of permeation enhancers in the rectum, small intestine, and large intestine, respectively. These data on heterogenous expression profiles of intestinal TJ components will be useful for the development of safe and efficient intestinal permeation enhancers.

Study on the mechanism of hirudin multi target delaying renal function decline in chronic kidney disease based on the "gut-kidney axis" theory.

The disorder of the "gut-kidney axis" exacerbates renal function decline in chronic kidney disease (CKD), and current CKD therapy is insufficient to address this issue. Hirudin has a palliative effect on the decline of renal function. However, whether hirudin can delay CKD by regulating the "intestinal renal axis" disorder remains unclear. Unilateral ureteral ligation (UUO) induced CKD rat model, and the rats were treated with bifidobacterium and hirudin for 36 days. After 14 and 36 days of modeling, kidney and colon tissues were collected for pathology, western blot (WB) assay, and quantitative real-time PCR (qPCR) detection. Serum samples were collected for renal function testing. Fecal samples were used for 16S rRNA sequencing and research on fecal bacterial transplantation. Lipopolysaccharide combine with adenosine 5'-triphosphate (LPS + ATP)-induced intestinal epithelial cell injury was treated with a nod-like receptor pyrin domain-associated protein 3 (NLRP3) inhibitor and hirudin. Protein expression was detected using WB and qPCR. The kidneys and colons of the CKD rats exhibited varying degrees of lesions. Creatinine (CRE), blood urea nitrogen (BUN), N-acetyl-ß-D-glucosidase (NAG) enzyme, and serum uremic toxins were elevated. The expression of claudin-1 and occludin was decreased, NLRP3 inflammatory-related proteins were increased, and the gut microbiota was disrupted. These pathological changes were more pronounced after 36 days of modeling. Meanwhile, high-dose hirudin treatment significantly improved these lesions and restored the intestinal flora to homeostasis in CKD rats. In vitro, hirudin demonstrated comparable effects to NLRP3 inhibitors by upregulating claudin-1 and occludin expression, and downregulating NLRP3 inflammatory-related proteins expression. The dysbiosis of the gut microbiota and impaired intestinal epithelial barrier function in CKD are associated with renal dysfunction in CKD. Hirudin delays the progression of CKD by regulating the disorder of the "gut-kidney axis" and inhibiting the activation of the NLRP3-ASC-caspase-1 pathway.

Insights into the effects of chronic combined chromium-nickel exposure on colon damage in mice through transcriptomic analysis and in vitro gastrointestinal digestion assay.

Heavy metals interact with each other in a coexisting manner to produce complex combined toxicity to organisms. At present, the toxic effects of chronic co-exposure to heavy metals hexavalent chromium [Cr(VI)] and divalent nickel [Ni(II)] on organisms are seldom studied and the related mechanisms are poorly understood. In this study, we explored the mechanism of the colon injury in mice caused by chronic exposure to Cr or/and Ni. The results showed that, compared with the control group, Cr or/and Ni chronic exposure affected the body weight of mice, and led to infiltration of inflammatory cells in the colon, decreased the number of goblet cells, fusion of intracellular mucus particles and damaged cell structure of intestinal epithelial. In the Cr or/and Ni exposure group, the activity of nitric oxide synthase (iNOS) increased, the expression levels of MUC2 were significantly down-regulated, and those of ZO-1 and Occludin were significantly up-regulated. Interestingly, factorial analysis revealed an interaction between Cr and Ni, which was manifested as antagonistic effects on iNOS activity, ZO-1 and MUC2 mRNA expression levels. Transcriptome sequencing further revealed that the expression of genes-related to inflammation, intestinal mucus and tight junctions changed obviously. Moreover, the relative contents of Cr(VI) and Ni(II) in the Cr, Ni and Cr+Ni groups all changed with in-vitro gastrointestinal （IVG）digestion, especially in the Cr+Ni group. Our results indicated that the chronic exposure to Cr or/and Ni can lead to damage to the mice colon, and the relative content changes of Cr(VI) and Ni(II) might be the main reason for the antagonistic effect of Cr+Ni exposure on the colon damage.

Epithelial-mesenchymal Transition (EMT) and the Effect of Atorvastatin on it in ARPE-19 cells.

Proliferative vitreoretinopathy (PVR) develops after an unsuccessful or complicated recovery from rhegmatogenous retinal detachment (RRD) surgery. Intraocular scar formation with the contribution of epithelial-mesenchymal transition (EMT) in RPE cells is prominent in the pathology of PVR. In the present study, the EMT process was experimentally induced in human retinal pigment epithelium (RPE; ARPE-19) cells, and the effect of atorvastatin on the process was studied. The mRNA and protein levels of mesenchymal markers actin alpha 2 (ACTA2) / alpha-smooth muscle actin (α-SMA) and fibronectin (FN), and epithelial markers occludin (OCLN) and zonula occludens-1 (ZO-1) were measured using quantitative real-time PCR (qRT-PCR) and western blot methods, respectively. In addition, α-SMA and FN were visualized using immunofluorescence staining. Cells were photographed under a phase contrast light microscope. Changes in the functionality of cells following the EMT process were studied using the IncuCyte scratch wound cell migration assay and the collagen cell invasion assay with confocal microscopy. The induction of EMT in ARPE-19 cells increased the expression of mesenchymal markers ACTA2/α-SMA and fibronectin and reduced the expression of epithelial marker OCLN both at mRNA and protein levels. The mRNA levels of ZO-1 were lower after EMT, as well. Increased levels of α-SMA and FN were confirmed by immunofluorescence staining. Atorvastatin further increased the mRNA levels of mesenchymal markers ACTA2 and FN as well as the protein levels of α-SMA and reduced the mRNA levels of epithelial markers OCLN and ZO-1 under the EMT process. EMT promoted wound closure and cell invasion into the 3D collagen matrix when compared to untreated control cells. These data present cellular changes upon the induction of the EMT process in ARPE-19 cells and the propensity of atorvastatin to complement the effect. More studies are needed to confirm the exact influence of the EMT process and atorvastatin treatment on the PVR development after RRD surgery.