Levels Of Tight Junction Proteins Research Articles

Streptococcus salivarius ameliorates the destructive effect on the epithelial barrier by inhibiting the growth of Prevotella melaninogenica via metabolic acid production.

Oral lichen planus (OLP) is one of the most common oral mucosal diseases, exhibiting a higher prevalence in women than men, but its pathogenesis is still unclear. Current research suggests that microbial dysbiosis may play an important role in the pathogenesis of OLP. Our previous research has found that the increase of Prevotella melaninogenica and decrease of Streptococcus salivarius have been identified as a potential pathogenic factor in OLP. Consequently, the objective of this study is to examine whether S. salivarius can counteract the detrimental effects of P. melaninogenica on the integrity of the epithelial barrier function. Epithelial barrier disruption was induced by P. melaninogenica in human keratinocytes (HaCaT cells). HaCaT cells were pretreated with S. salivarius(MOI=20) or cell-free supernatant for 3h, followed by treatment with P. melaninogenica (MOI=5) for 3h. The epithelial barrier integrity of HaCaT cells was detected by FD4 permeability. The mRNA level of tight junction protein was detected by quantitative real-time polymerase chain reaction (PCR). Immunofluorescence and Western Blot were used to detect the protein expression of zonula occludin-1 (ZO-1). The serial dilution-spotting assay was applied to monitor the viability of P. melaninogenica at the end of 8 and 24h incubation. Challenge by P. melaninogenica decreased the levels of tight junction proteins, including occludin, ZO-1, and claudin in HaCaT cells. S. salivarius or its cell-free supernatant inhibited the down-regulation of ZO-1 mRNA and protein expression levels induced by P. melaninogenica and thus improved the epithelial barrier function. The inhibitory effect of the cell-free supernatant of S. salivarius on the growth of P. melaninogenica is associated with metabolic acid production rather than with bacteriocins and hydrogen peroxide. These results suggest that live S. salivarius or its cell-free supernatant significantly ameliorated the disruption of epithelial tight junctions induced by P. melaninogenica, likely through the inhibition of P. melaninogenica growth mediated by metabolic acid production.

Bergapten enhances mitophagy to regulate intestinal barrier and Th17/Treg balance in mice with Crohn's disease-like colitis via PPARγ/NF-κB signaling pathway.

This study aimed to investigate whether bergapten (BG), a furanocoumarin phytohormone, holds promise for Crohn's disease (CD)-like colitis treatment and to preliminarily explore its potential mechanisms. 2,4,6-Trinitrobenzenesufonic acid (TNBS)-treated mice were applied to establish an in vivo research model, and BG was administered with different concentrations. The status of mice in each group was evaluated by disease activity index (DAI), and the severity was evaluated by pathological sections. The intestinal barrier was assessed by measuring in vivo intestinal permeability, peripheral blood intestinal fatty acid-binding protein (I-FABP) levels, epithelial resistance values, and tight junction protein levels. Markers were then used to assess Th17/Treg levels, mitophagy, and the peroxisome proliferator-activated receptor (PPAR)γ/ nuclear factor kappa B (NF-κB) signaling pathway. BG significantly reduced colon tissue damage in a concentration-dependent manner. DAI scores showed that the loose feces, occult blood, and weight loss of mice in the BG treatment were significantly reduced, and pathological section results revealed reduced inflammatory infiltration and fibrosis. Reduced serum FITC-dextran and I-FABP and increased levels of epithelial resistance and tight junction proteins support that the intestinal barrier was protected upon BG. The proportion of Th17 in mesenteric lymph nodes increased while Treg decreased in the model group. BG treatment effectively reduced the conversion of Treg to Th17. Additionally, BG was found to enhance mitophagy and activate the PPARγ/NF-κB signaling. BG demonstrates promising effects in ameliorating intestinal barrier damage and Th17/Treg imbalance in a murine model of CD-like colitis, while also promoting intracellular mitophagy. The PPARγ/NF-κB signaling pathway may serve as a key mediator of BG's regulatory mechanisms.

Biochanin a ameliorates DSS-induced ulcerative colitis by improving colonic barrier function and protects against the development of spontaneous colitis in the Muc2 deficient mice

There is an increasing appreciation that colonic barrier function is closely related to the development and progression of colitis. The mucus layer is a crucial component of the colonic barrier, responsible for preventing harmful bacteria from invading the intestinal epithelium and causing inflammation. Furthermore, a defective mucus barrier is also a significant characteristic of ulcerative colitis (UC). Biochanin A (BCA), an isoflavonoid, has garnered increasing interest due to its significant biological activities. However, the impact of BCA on UC has not been reported yet. In this study, we used a dextran sodium sulfate (DSS)-induced ulcerative colitis model and the Muc2 deficient (Muc2−/−) mice spontaneous colitis model to explore the mechanisms of BCA in the treatment of UC. Here, we verified that DSS-induced UC was observably attenuated and spontaneous colitis in Muc2−/− mice was relieved by BCA. Treatment with BCA improved colitis-related symptoms and reduced intestinal permeability by upregulating the levels of goblet cells and tight junction (TJ) proteins. In addition, we confirmed that BCA promotes autophagy through the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/Unc-51-like kinase 1 (ULK1) pathway, thereby alleviating DSS-induced UC. In addition, the administration of BCA was able to reduce apoptosis and promote proliferation by suppressing Cleaved Caspase-3 (Cleaved Cas-3) expression, and increasing PCNA and Ki67 levels. Further research revealed that BCA treatment ameliorated spontaneous colitis and alleviated epithelial damage in Muc2−/− mice by restoring the intestinal barrier and promoting autophagy. Our results demonstrated that BCA alleviated UC by enhancing intestinal barrier function and promoting autophagy. These findings indicate that BCA may be a novel treatment alternative for UC.

Unveiling the role of PD-L1 in vascular endothelial dysfunction: Insights into the mtros/NLRP3/caspase-1 mediated pyroptotic pathway

BackgroundProgrammed death ligand-1(PD-L1) has been postulated to play a crucial role in the regulation of barrier functions of the vascular endothelium, yet how this novel molecule mediates dysfunction in endothelial cells (ECs) during acute lung injury (ALI) remains largely unknown. MethodsPD-L1 siRNA and plasmids were synthesized and applied respectively to down- or up-regulate PD-L1 expression in human lung microvascular endothelial cells (HMVECs). RNA sequencing was used to explore the differentially expressed genes following PD-L1 overexpression. The expression levels of tight junction proteins (ZO-1 and occludin) and the signaling pathways of NLRP-3/caspase-1/pyroptosis were analyzed. A mouse model of indirect ALI was established through hemorrhagic shock (HEM) followed by cecal ligation and puncture (CLP), enabling further investigation into the effects of intravenous delivery of PD-L1 siRNA. ResultsA total of 1502 differentially expressed genes were identified, comprising 532 down-regulated and 970 up-regulated genes in ECs exhibiting PD-L1overexpression. Enrichment of PD-L1-correlated genes were observed in the NOD-like receptor signaling pathway and the TNF signaling pathway. Western blot assays confirmed that PD-L1 overexpression elevated the expression of NLRP3, cleaved-caspase-1, ASC and GSDMD, and concurrently diminished the expression of ZO-1 and occludin. This overexpression also enhanced mitochondrial oxidative phosphorylation and mitochondrial reactive oxygen species (mtROS) production. Interestingly, mitigating mitochondrial dysfunction with mitoQ partially countered the adverse effects of PD-L1 on the functionality of ECs. Furthermore, intravenous administration of PD-L1 siRNA effectively inhibited the activation of the NLRP3 inflammasome and pyroptosis in pulmonary ECs, subsequently ameliorating lung injury in HEM/CLP mice. ConclusionPD-L1-mediated activation of the inflammasome contributes significantly to the disruption of tight junction and induction of pyroptosis in ECs, where oxidative stress associated with mitochondrial dysfunction serves as a pivotal mechanism underpinning these effects.

Extract of Scutellaria baicalensis and Lonicerae flos improves growth performance, antioxidant capacity, and intestinal barrier of yellow-feather broiler chickens against Clostridium perfringens

In this study, we aimed to investigate the effect of Scutellaria baicalensis and Lonicerae Flos (SL) extract on the growth performance and intestinal health of yellow-feather broilers following a Clostridium perfringens challenge. In total, 600 one-day-old yellow-feather broilers were divided into five treatments (6 replicate pens of 20 birds per treatment), including a control (Con) group fed a basal diet and the infected group (iCon) fed a basal diet and infected with Clostridium perfringens, the other 3 groups receiving different doses of SL (150, 300, and 450 mg/kg) and infected with Clostridium perfringens. The total experimental period was 80 d. When the birds were 24-days-old, a subclinical necrotizing enteritis model was induced by orally inoculating the birds with 11,000 oocysts of mixed Eimeria species on d 24, followed by C. perfringens (108 CFU/mL) from d 28 to 30. The birds were evaluated for parameters such as average weight gain (AWG), average daily feed intake (ADFI), mortality, feed conversion ration (FCR), intestinal lesion score, intestinal C. perfringens counts, and villus histomorphometry. Results indicated that C. perfringens infection led to reduced AWG and the levels of tight junction proteins, increased the FCR, ileum E. coli load, and intestinal permeability, causing damage to the intestinal mucosal barrier (P < 0.05). Compared with the infected group, supplementing 300 mg/kg of SL significantly increased AWG at 43 to 80 d, the ratio of villus height to crypt depth in the jejunum and ileum at 35 d, and the activity of superoxide dismutase (SOD) in serum. It also significantly reduced the FCR at 22 to 42 d, intestinal lesion score, and the amount of C. perfringens in the ileum (P < 0.05). Additionally, compared with the infected group, the addition of 300 mg/kg SL significantly increased mRNA levels of claudin-2, claudin-3, mucin-2, and toll-like receptor 2 (TLR-2) in the ileum of infected birds at 35 d of age. In conclusion, supplementation with SL extract could effectively mitigate the negative effects of C. perfringens challenge by improving intestinal barrier function and histomorphology, positively influencing the growth performance of challenged birds.

428 Promoting Infant Gut Barrier Development Through Culturally Relevant Adoption of Fruit and Vegetable Intake.

OBJECTIVES/GOALS: To determine in vitro mechanisms by which fruits and vegetables (FV) contribute to colon barrier development in Latin American infants. We hypothesize that simulated colonic fermentation of FVs will stimulatein vitro cell barrier function by activating the hypoxia-inducible factor (HIF) pathway in colonocytes. METHODS/STUDY POPULATION: FVs consumed by US-based Latin American infants 6-12 months old (identified from NHANES-What We Eat in America Surveys) will be combined with human breast-milk samples from women self-identified as Hispanic or non-Hispanic, and then subjected to in vitro digestion and anaerobic colonic fermentation using human feces. FV fermenta will be incubated with Caco2 monolayers to measure in vitro cell permeability and protein levels of cellular tight junction, metabolic, and HIF signaling enzymes. To examine their effects in vivo, FVs identified to modulate in vitro barrier function, will be fed (5% freeze dried powder) to wild-type mice and the above parameters will be examined. If in vivo effects are found, intestinal specific HIF knockout mice will be used to examine the role of HIF signaling in mediating these effects. RESULTS/ANTICIPATED RESULTS: We expect that fermenta derived from human milk and FVs will reduce in vitro gut permeability in Caco2 monolayers by increasing gene and protein expression of the HIF signaling complex relative to fermenta of human milk alone. This will be reflected with higher cellular trans-epithelial resistance and greater expression levels of tight junction proteins. We expect FV powder consumption will similarly increase in vivo gut permeability and expression of related genes in mice as compared to mice fed diets without FVs. As we expect an increase in HIF signaling in the colon, we expect that FV powder consumption will not enhance in vivo gut permeability in mice colons with an intestinal specific knockout of HIF. DISCUSSION/SIGNIFICANCE: Data from this study will provide mechanistic evidence to help clinicians promote relevant FVs recommendations for Latin American infants and families. Due to the link between gut permeability and obesity, our next step will be to conduct a dietary intervention in this population.

Anti-Inflammatory Effects of a Novel Acetonitrile-Water Extract of Lens Culinaris against LPS-Induced Damage in Caco-2 Cells.

Natural compounds like flavonoids preserve intestinal mucosal integrity through their antioxidant, anti-inflammatory, and antimicrobial properties. Additionally, some flavonoids show prebiotic abilities, promoting the growth and activity of beneficial gut bacteria. This study investigates the protective impact of Lens culinaris extract (LE), which is abundant in flavonoids, on intestinal mucosal integrity during LPS-induced inflammation. Using Caco-2 cells as a model for the intestinal barrier, the study found that LE did not affect cell viability but played a cytoprotective role in the presence of LPS. LE improved transepithelial electrical resistance (TEER) and tight junction (TJ) protein levels, which are crucial for barrier integrity. It also countered the upregulation of pro-inflammatory genes TRPA1 and TRPV1 induced by LPS and reduced pro-inflammatory markers like TNF-α, NF-κB, IL-1β, and IL-8. Moreover, LE reversed the LPS-induced upregulation of AQP8 and TLR-4 expression. These findings emphasize the potential of natural compounds like LE to regulate the intestinal barrier and reduce inflammation's harmful effects on intestinal cells. More research is required to understand their mechanisms and explore therapeutic applications, especially for gastrointestinal inflammatory conditions.

Therapeutic potential of Latilactobacillus curvatus CCFM1268 in colitis treatment: Insights from in vitro and in vivo studies

Latilactobacillus curvatus is a candidate probiotic that is known to have antioxidant, immunomodulatory, anti-obesity, and colitis-relieving beneficial effects. Here, we evaluated the effects of Lt. curvatus CCFM1268 on dextran sulfate sodium (DSS)-induced Caco-2 cell damage and colitis in mice. Our results demonstrate that Lt. curvatus CCFM1268 significantly restored cell viability and promoted the expression of barrier factors in vitro. In vivo, Lt. curvatus CCFM1268 significantly restored weight loss and colon damage in mice and modulated the levels of inflammatory factors and tight junction proteins in the colon. Latilactobacillus curvatus CCFM1268 also reduced the abundance of conditionally pathogenic bacteria including Escherichia_shigella and Alistipes, enriched the abundance of Lactobacillus, Parabacteroides, Enterococcus and Faecalibaculum, and significantly increased the concentrations of acetic, propionic, and butyric acid in the gut. These results indicate that Lt. curvatus CCFM1268 can alleviate colitis by inhibiting colonic inflammation, repairing the gut barrier, regulating the gut microbiota composition, and secreting metabolites, which was demonstrated both in vivo and in vitro.

Lotus Leaf Extract Alleviates Lipopolysaccharide-Induced Intestinal Injury in Mice by Regulating Oxidative Stress and Inflammation.

Inflammatory bowel disease, a disease featured by intestinal epithelial barrier destruction and dysfunction, has been a constant threat to animal health. The primary objective of this research was to assess the impact of the extract derived from lotus leaves (LLE) on lipopolysaccharide (LPS) induced damage to the intestines in mice, as well as to investigate the fundamental mechanism involved. The LLE was prepared using ultrasonic extraction in this experiment, and the LLE total flavonoid content was 117.02 ± 10.73 mg/g. The LLE had strong antioxidant activity in vitro, as assessed by 2, 2-diphenyl-1-picrylhydrazyl, ferric reducing antioxidant power, and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) methods. In the vivo experiment, different doses of LLE (50, 100, and 200 mg/kg) were administered for 2 weeks before LPS treatment in mice. The results revealed that LLE alleviates intestinal tissue damage in LPS-induced mice. In the jejunum tissue, LLE significantly upregulated mRNA and protein expression levels of tight junction proteins, such as ZO-1, occludin, and claudin-1, and decreased the contents of the inflammatory cytokines, interleukin (IL)-1β, IL-6, and tumor necrosis factor-α. Furthermore, the malondialdehyde and lactate dehydrogenase contents increased by LPS in the liver were significantly reduced after administration of LLE, and the total antioxidant capacity, superoxide dismutase, and reduced glutathione decreased by LPS were remarkably increased by LLE. It was found that LLE could relieve LPS-induced oxidative stress by upregulating mRNA and protein expression of Nrf2 and HO-1 in jejunum tissue. In conclusion, LLE alleviates LPS-induced intestinal damage through regulation of the Nrf2/HO-1 signal pathway to alleviate oxidative stress, reducing inflammatory factors and increasing the expression of tight junction proteins in mice.

[Restraint stress induces blood-brain barrier injury in rat amygdala by activating the Rho/ROCK signaling pathway].

To investigate the role of Rho/ROCK signaling pathway in mediating restraint stress-induced blood-brain barrier (BBB) injury in the amygdala of rats. Sixty male SD rats were randomized equally into control group (with food and water deprivation for 6 h per day), restraint stress group (with restraint for 6 h per day), stress + fasudil treatment (administered by intraperitoneal injection at 1 mg/100 g 30 min before the 6-h restraint) group, and fasudil treatment alone group. The elevated plus-maze test was used to detect behavioral changes of the rats, serum corticosterone and S100B levels were determined with ELISA, and Evans Blue leakage in the brain tissue was examined to evaluate the changes in BBB permeability. The changes in expression levels of tight junction proteins in the amygdala were detected using immunofluorescence assay and Western blotting, and Rho/ROCK pathway activation was detected by Pull-down test and Western blotting. Ultrastructural changes of the cerebral microvascular endothelial cells were observed using transmission electron microscopy. Compared with those in the control group, the rats in restrain stress group and stress+fasudil group showed obvious anxiety-like behavior with significantly increased serum corticosterone level (P<0.001). Compared with those in the control group and stress+fasudil group, the rat models of restrain stress showed more obvious Evans Blue leakage and higher S100B expression (P<0.01) but lower expressions of tight junction proteins in the amygdala. Pull-down test and Western blotting confirmed that the expression levels of RhoA-GTP, ROCK2 and P-MLC 2 were significantly higher in stress group than in the control group and stress + fasudil group (P<0.05). Transmission electron microscopy revealed obvious ultrastructural changes in the cerebral microvascular endothelial cells in the rat models of restrain stress. Restraint stress induces BBB injury in the amygdala of rats by activating the Rho/ROCK signaling pathway.

Resveratrol ameliorates ulcerative colitis by upregulating Nrf2/HO‑1 pathway activity: Integrating animal experiments and network pharmacology.

Ulcerative colitis (UC) is a chronic idiopathic inflammatory condition affecting the rectum and colon. Inflammation and compromisation of the intestinal mucosal barrier are key in UC pathogenesis. Resveratrol (Res) is a naturally occurring polyphenol that exhibits anti‑inflammatory and antioxidant properties. Nuclear factor erythroid‑2‑related factor 2/heme oxygenase 1 (Nrf2/HO‑1) pathway regulates occurrence and development of numerous types of diseases through anti‑inflammatory and antioxidant activity. However, it is not clear whether Nrf2/HO‑1 pathway is involved in the treatment of Res in UC. Therefore, the present study aimed to investigate whether Res modulates the Nrf2/HO‑1 signaling pathway to attenuate UC in mice. Dextran sulfate sodium (DSS) was used to induce experimental UC in male C57BL/6J mice. Disease activity index (DAI) and hematoxylin eosin (H&E) staning was used to assessed the magnitude of colonic lesions in UC mice. ELISA) was utilized to quantify inflammatory cytokines (IL‑6, IL‑1β, TNF‑α and IL‑10) in serum and colon tissues. Immunohistochemistry and Western blot were used to evaluate the expression levels of tight junction (TJ) proteins [zonula occludens (ZO)‑1 and Occludin] in colon tissues. Pharmacokinetic (PK) parameters of Res were derived from TCMSP database. Networkpharmacology was employed to identify the biological function and pharmacological mechanism of Res in the process of relieving UC, and the key target was screened. The binding ability of Res and key target was verified by molecular docking. Finally, the effectiveness of key target was substantiated by Western blot. Res decreased DAI, ameliorated histopathological changes such as crypt loss, disappeatance of the mucosal epithelium, and inflammatory infiltration in mice. Additionally, Res decreased expression of pro‑inflammatory cytokines IL‑6, IL‑1β and TNF‑α and increased anti‑inflammatory factor IL‑10 expression. Res also restored the decreased protein expression of ZO‑1 and occludin after DSS treatment, increasing the integrity of the intestinal mucosal barrier. The PK properties of Res suggested that Res possesses the therapeutic potential for oral administration. Network pharmacology revealed that Res alleviated UC through anti‑inflammatory and antioxidant pathways, and confirmed that Nrf2 has a high binding affinity with Res and is a key target of Res against UC. Western blotting demonstrated that Res treatment increased the protein levels of Nrf2 and HO‑1. In conclusion, Res treatment activated the Nrf2/HO‑1 pathway to decrease clinical symptoms, inflammatory responses, and intestinal mucosal barrier damage in experimental UC mice.

Kinase PIM1 governs ferroptosis to reduce retinal microvascular endothelial cell dysfunction triggered by high glucose.

Previous studies have implicated targeting Pim-1 proto-oncogene, serine/threonine kinase (PIM1) as a preventive measure against high glucose-induced cellular stress and apoptosis. This study aimed to reveal the potential role and regulatory mechanism of PIM1 in diabetic retinopathy. Human retinal microvascular endothelial cells (hRMECs) underwent high glucose induction, and fluctuations in PIM1 levels were assessed. By overexpressing PIM1, its effects on the levels of inflammatory factors, oxidative stress indicators, migration and tube formation abilities, tight junction protein expression levels, and ferroptosis in hRMECs were identified. Afterwards, hRMECs were treated with the ferroptosis-inducing agent erastin, and the effect of erastin on the above PIM1 regulatory functions was focused on. PIM1 was downregulated upon high glucose, and its overexpression inhibited the inflammatory response, oxidative stress, cell migration, and tube formation potential in hRMECs, whereas elevated tight junction protein levels. Furthermore, PIM1 overexpression reduced intracellular iron ion levels, lipid peroxidation, and levels of proteins actively involved in ferroptosis. Erastin treatment reversed the impacts of PIM1 on hRMECs, suggesting the mediation of ferroptosis in PIM1 regulation. The current study has yielded critical insights into the role of PIM1 in ameliorating high glucose-induced hRMEC dysfunction through the inhibition of ferroptosis.

Cholangiocyte organoids to study drug-induced injury.

Drug induced bile duct injury is a frequently observed clinical problem leading to a wide range of pathological features. During the past decades, several agents have been identified with various postulated mechanisms of bile duct damage, however, mostly still poorly understood. Here, we investigated the mechanisms of chlorpromazine (CPZ) induced bile duct injury using advanced in vitro cholangiocyte cultures. Intrahepatic cholangiocyte organoids (ICOs) were driven into mature cholangiocyte like cells (CLCs), which were exposed to CPZ under cholestatic or non-cholestatic conditions through the addition of a bile acid cocktail. CPZ caused loss of monolayer integrity by reducing expression levels of tight junction protein 1 (TJP1), E-cadherin 1 (CDH1) and lysyl oxidase homolog 2 (LOXL2). Loss of zonula occuludens-1 (ZO-1) and E-cadherin was confirmed by immunostaining after exposure to CPZ and rhodamine-123 leakage further confirmed disruption of the cholangiocyte barrier function. Furthermore, oxidative stress seemed to play a major role in the early damage response by CPZ. The drug also decreased expression of three main basolateral bile acid transporters, ABCC3 (ATP binding cassette subfamily C member 3), SLC51A/B (solute carrier family 51 subunit alpha/beta) and multidrug resistance transporter ABCB1 (ATP binding cassette subfamily B member 1), thereby contributing to bile acid accumulation. CPZ did not induce an inflammatory response by itself, but addition of TNFα revealed a synergistic effect. These results show that ICOs present a model to identify toxic drugs affecting the bile ducts while providing mechanistic insights into hepatotoxicity.

Intestinal epithelial Krüppel-like factor 4 alleviates endotoxemia and atherosclerosis through improving NF-κB/miR-34a-mediated intestinal permeability.

Maintenance of intestinal barrier function contributes to gastrointestinal homeostasis and therefore cardiovascular diseases. A number of studies show that intestinal permeability is affected by excessive inflammatory responses. Krüppel-like factor (KLF) 4 is one of the critical transcriptional factors, which controls multiple immune responses. In this study we investigated the role of KLF4 in regulating intestinal inflammation and permeability during the atherosclerotic process. Atherosclerotic model was established in ApoE-/- mice by feeding a high fat high cholesterol (HFHC) diet. We showed that colon expression levels of KLF4 and tight junction proteins were significantly decreased whereas inflammatory responses increased in atherosclerotic mice. Overexpression of colon epithelial Klf4 decreased atherosclerotic plaque formation and vascular inflammation in atherosclerotic mice, accompanied by remarkable suppression of intestinal NF-κB activation. We found that overexpression of epithelial Klf4 in atherosclerotic mice significantly increased intestinal tight junction expression and ameliorated endotoxemia, whereas replenishment of LPS abolished these benefits. Overexpression of Klf4 reversed LPS-induced permeability and downregulation of ZO-1 and Occludin in Caco-2 cells in vitro. HFHC diet stimulated the expression of epithelial microRNA-34a, whereas silence of epithelial Klf4 abolished the benefits of microRNA-34a sponge, a specific miR-34a inhibitor, on intestinal permeability and atherosclerotic development. A clinical cohort of 24 atherosclerotic patients supported colon KLF4/NF-κB/tight junction protein axis mediated intestine/cardiovascular interaction in patients with atherosclerosis. Taken together, intestinal epithelial KLF4 protects against intestinal inflammation and barrier dysfunction, ameliorating atherosclerotic plaque formation.

Ameliorating effect of 2ʹ-Fucosyllactose and 6ʹ-Sialyllactose on lipopolysaccharide-induced intestinal inflammation

Human milk oligosaccharides (HMO) affect gut microbiota during neonatal development, particularly with respect to the immune system. Bovine milk-based infant formulas have low oligosaccharide contents. Thus, efforts to fortify infant formulas with HMO are being undertaken. Two major HMO, 2'-fucosyllactose (2'-FL) and 6'-sialyllactose (6'-SL), exert anti-inflammatory effects; however, the associations between anti-inflammatory effects induced by 2'-FL and 6'-SL co-treatment and gut microbiota composition and metabolite modulation remain unclear. Therefore, in this study, we evaluated the effects of a mixture of these HMO. To determine the optimal HMO ratio for anti-inflammatory effects and elucidate its mode of action, LPS-induced inflammatory HT-29 epithelial cells and intestinal inflamed suckling mice were treated with various mixtures of 2'-FL and 6'-SL. 2'-FL:6'-SL ratio of 5:1 was identified as the most effective pre-treatment HMO mixture in vitro; thus, this ratio was selected and used for low, middle, and high-dose treatments for subsequent in vivo studies. In vivo, high-dose HMO treatment restored LPS-induced inflammation symptoms, such as body weight loss, colon length reduction, histological structural damage, and intestinal gene expression related to inflammatory responses. High-dose HMO was the only treatment that modulated the major phyla Bacteroidetes and Firmicutes and the genera Ihubacter, Mageeibacillus, and Saccharofermentans. These changes in microbial composition were correlated with intestinal inflammation-related gene expression and short-chain fatty acid production. To our knowledge, our study is the first to report the effects of Ihubacter, Mageeibacillus, and Saccharofermentans on short chain fatty acid levels, which can subsequently affect inflammatory cytokine and tight junction protein levels. Conclusively, the HMO mixture exerted anti-inflammatory effects through changes in microbiota and metabolite production. These findings suggested that supplementation of infant formula with HMO may benefit formula-fed infants by forming unique microbiota contributing to neonatal development.

Heat-Killed Saccharomyces boulardii Alleviates Dextran Sulfate Sodium-Induced Ulcerative Colitis by Restoring the Intestinal Barrier, Reducing Inflammation, and Modulating the Gut Microbiota.

Ulcerative colitis (UC) is a global intestinal disease, and conventional therapeutic drugs often fail to meet the needs of patients. There is an urgent need to find efficient and affordable novel biological therapies. Saccharomyces boulardii has been widely used in food and pharmaceutical research due to its anti-inflammatory properties and gut health benefits. However, there is still a relatively limited comparison and evaluation of different forms of S. boulardii treatment for UC. This study aimed to compare the therapeutic effects of S. boulardii, heat-killed S. boulardii, and S. boulardii β-glucan on UC, to explore the potential of heat-killed S. boulardii as a new biological therapy. The results demonstrate that all three treatments were able to restore body weight, reduce the disease activity index (DAI), inhibit splenomegaly, shorten colon length, and alleviate histopathological damage to colonic epithelial tissues in DSS-induced colitis mice. The oral administration of S. boulardii, heat-killed S. boulardii, and S. boulardii β-glucan also increased the levels of tight junction proteins (Occludin and ZO-1), decreased the levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in the serum, and suppressed the expressions of TNF-α, IL-1β, and IL-6 mRNA in the colon. In particular, in terms of gut microbiota, S. boulardii, heat-killed S. boulardii, and S. boulardii β-glucan exhibited varying degrees of modulation on DSS-induced dysbiosis. Among them, heat-killed S. boulardii maximally restored the composition, structure, and functionality of the intestinal microbiota to normal levels. In conclusion, heat-killed S. boulardii showed greater advantages over S. boulardii and S. boulardii β-glucan in the treatment of intestinal diseases, and it holds promise as an effective novel biological therapy for UC. This study is of great importance in improving the quality of life for UC patients and reducing the burden of the disease.

An Inulin-Type Fructan CP-A from Codonopsis pilosula Alleviated 5-Fluorouracil-Induced Intestinal Mucositis via the ERK/MLCK/MLC2 Pathway and Regulation of Gut Microbiota.

Intestinal mucositis (IM) is a common adverse effect of chemotherapy, limiting its clinical application. Codonopsis pilosula-derived CP-A (an inulin-type fructan) is an edible Chinese medicine with anti-inflammatory and gastrointestinal protective effects, which may be useful for treating IM. Here, we explored CP-A's role in ameliorating IM induced by 5-fluorouracil (5-FU) and investigated the underlying mechanism using in vitro experiments and rat models. Western blotting, immunohistochemistry (IHC), and real-time PCR (RT-PCR) analyses were used to assess protein expression related to the extracellular-regulated protein kinases (ERK)/myosin light chain kinase (MLCK)/myosin light chain 2 (MLC2) signaling pathway and tight junction proteins. Inflammatory factors were quantified using enzyme-linked immunosorbent assays (ELISAs), and 16S rRNA amplicon sequencing was employed for cecum content analysis. The results indicated that CP-A restored body weight and food intake and reversed histopathological changes in IM rats. Further, abnormal MLCK activation induced by 5-FU was attenuated by CP-A via the ERK/MLCK/MLC2 pathway. CP-A treatment improved tight junction protein levels and reduced inflammatory factor expression. Moreover, CP-A intervention regulated the intestinal microbiota community structure, increasing the abundance of Lactobacillus and decreasing the abundance of Shigella. In conclusion, CP-A mitigates 5-FU-induced IM by inhibiting the ERK/MLCK/MLC2 pathway, reducing the expression of inflammatory factors, improving the intestinal mucosal barrier, and regulating the intestinal microbial community. This study highlights CP-A's therapeutic potential in IM treatment and provides insights for future research.

MUC13 negatively regulates tight junction proteins and intestinal epithelial barrier integrity via protein kinase C.

Glycosylated mucin proteins contribute to the essential barrier function of the intestinal epithelium. The transmembrane mucin MUC13 is an abundant intestinal glycoprotein with important functions for mucosal maintenance that are not yet completely understood. We demonstrate that in human intestinal epithelial monolayers, MUC13 localized to both the apical surface and the tight junction (TJ) region on the lateral membrane. MUC13 deletion resulted in increased transepithelial resistance (TEER) and reduced translocation of small solutes. TEER buildup in ΔMUC13 cells could be prevented by addition of MLCK, ROCK or protein kinase C (PKC) inhibitors. The levels of TJ proteins including claudins and occludin were highly increased in membrane fractions of MUC13 knockout cells. Removal of the MUC13 cytoplasmic tail (CT) also altered TJ composition but did not affect TEER. The increased buildup of TJ complexes in ΔMUC13 and MUC13-ΔCT cells was dependent on PKC. The responsible PKC member might be PKCδ (or PRKCD) based on elevated protein levels in the absence of full-length MUC13. Our results demonstrate for the first time that a mucin protein can negatively regulate TJ function and stimulate intestinal barrier permeability.

Modified Gegen Qinlian Decoction Ameliorates DSS-Induced Ulcerative Colitis in Mice by Inhibiting Ferroptosis via Nrf2/GPX4 Pathway

Objective. Ferroptosis, a form of programmed cell death, is considered a novel target for the treatment of ulcerative colitis (UC). The aim of this study was to explore whether modified Gegen Qinlian decoction (MGQD) ameliorates UC in mice via mediating ferroptosis. Methods. Mice with dextran sulfate sodium- (DSS-) induced colitis were administered with MGQD for seven days. Subsequently, iron, malondialdehyde (MDA), glutathione (GSH), and reactive oxygen species (ROS) were measured. ELISA and immunohistochemistry were used to evaluate the levels of proinflammatory cytokines and tight junction proteins, respectively. Transmission electron microscopy was used to reveal mitochondrial morphology. Western blot and qRT-PCR analyses were used to assess the expression levels of the proteins of Nrf2/GPX4 pathway. The docking affinity of MGQD and Nrf2 was assessed using AutoDock Vina 1.1.2. Results. Ferroptosis was identified in mice with UC, as demonstrated by mitochondrial disruption, MDA and ROS production, iron overload, decrease in GSH level, and abnormal expression of core marker proteins of ferroptosis. After MGQD treatment, these characteristic changes of ferroptosis were significantly reversed, along with concomitant activation of the Nrf2/GPX4 pathway. Furthermore, molecular docking analysis revealed that MGQD had a high affinity for Nrf2. Conclusion. MGQD may ameliorate UC by inhibiting ferroptosis via the activation of Nrf2/GPX4 pathway. This study provided new insights into the application of MGQD complementary therapy for UC.

Transcriptional Levels of Intercellular Junction Proteins in an Alveolar Epithelial Cell Line Exposed to Irradiation or Bleomycin.

We performed a comparative study of the effects of X-ray irradiation and bleomycin on the mRNA levels of E-cadherin and tight junction proteins (claudin-3, claudin-4, claudin-18, ZO-2, and occludin) in an alveolar epithelial cell line L2. Irradiation decreased claudin-4 levels and increased occludin levels, while the levels of other mRNAs remained unchanged. Bleomycin increased the expression levels of all proteins examined except claudin-3. Irradiation and bleomycin have different effects on the expression level of intercellular junction proteins, indicating different reactions triggered in alveolar epithelial cells and a great prospects of further comparative studies.