Expression Of Tight Junction Proteins Research Articles

Lactobacillus paracasei Jlus66 relieves DSS-induced ulcerative colitis in a murine model by maintaining intestinal barrier integrity, inhibiting inflammation, and improving intestinal microbiota structure.

Ulcerative colitis (UC) is a serious health problem with increasing morbidity and prevalence worldwide. The pathogenesis of UC is complex, currently believed to be influenced by genetic factors, dysregulation of the host immune system, imbalance in the intestinal microbiota, and environmental factors. Currently, UC is typically managed using aminosalicylates, immunosuppressants, and biologics as adjunctive therapies, with the risk of relapse and development of drug resistance upon discontinuation. Therefore, further research into the pathogenesis of UC and exploration of potential treatment strategies are necessary to improve the quality of life for affected patients. According to previous studies, Lactobacillus paracasei Jlus66 (Jlus66) reduced inflammation and may help prevent or treat UC. We used dextran sulfate sodium (DSS) to induce a mouse model of UC to assess the effect of Jlus66 on the progression of colitis. During the experiment, we monitored mouse body weight, food and water consumption, as well as rectal bleeding. Hematoxylin-eosin staining was performed to assess intestinal pathological damage. Protein imprinting and immunohistochemical methods were used to evaluate the protein levels of nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and tight junction (TJ) proteins in intestinal tissues. Fecal microbiota was analyzed based on partial 16S rRNA gene sequencing. Jlus66 supplementation reduced the degree of colon tissue damage, such as colon shortening, fecal occult blood, colon epithelial damage, and weight loss. Supplementation with Jlus66 reduced DSS-induced upregulation of cytokine levels such as TNF-α, IL-1β, and IL-6 (p < 0.05). The NF-κB pathway and MAPK pathway were inhibited, and the expression of TJ proteins (ZO-1, Occludin, and Claudin-3) was upregulated. 16S rRNA sequencing of mouse cecal contents showed that Jlus66 effectively regulated the structure of the intestinal biota. In conclusion, these data indicate that Jlus66 can alter the intestinal biota and slow the progression of UC, providing new insights into potential therapeutic strategies for UC.

Impact of Probiotic Lactiplantibacillus plantarum ATCC 14917 on atherosclerotic plaque and its mechanism.

Atherosclerosis is viewed as not just as a problem of lipid build-up in blood vessels, but also as a chronic inflammatory disease involving both innate and acquired immunity. In atherosclerosis, the inflammation of the arterial walls is the key characteristic that significantly contributes to both the instability of plaque and the occlusion of arteries by blood clots. These events ultimately lead to stroke and acute coronary syndrome. Probiotics are living microorganisms that, when consumed in the right quantities, offer advantages for one's health. The primary objective of this study was to investigate the influence of Lactiplantibacillus plantarum ATCC 14917 (ATCC 14917) on the development of atherosclerotic plaques and its underlying mechanism in Apo lipoprotein E-knockout (Apoe-/-mice). In this study, Apoe-/-mice at approximately 8weeks of age were randomly assigned to three groups: a Normal group that received a normal chow diet, a high fat diet group that received a gavage of PBS, and a Lactiplantibacillus plantarum ATCC 14917 group that received a high fat diet and a gavage of 0.2ml ATCC 14917 (2 × 109CFU/mL) per day for a duration of 12weeks. Our strain effectively reduced the size of plaques in Apoe-/-mice by regulating the expression of inflammatory markers, immune cell markers, chemokines/chemokine receptors, and tight junction proteins (TJPs). Specifically, it decreased the levels of inflammatory markers (ICAM-1, CD-60 MCP-1, F4/80, ICAM-1, and VCAM-1) in the thoracic aorta, (Ccr7, cd11c, cd4, cd80, IL-1β, TNF-α) in the colon, and increased the activity of ROS-scavenging enzymes (SOD-1 and SOD-2). It also influenced the expression of TJPs (occludin, ZO-1, claudin-3, and MUC-3). In addition, the treatment of ATCC 14917 significantly reduced the level of lipopolysaccharide in the mesenteric adipose tissue. The findings of our study demonstrated that our strain effectively decreased the size of atherosclerotic plaques by modulating inflammation, oxidative stress, intestinal integrity, and intestinal immunity.

Forsythiaside A attenuates lipopolysaccharide-induced mouse mastitis by activating autophagy and regulating gut microbiota and metabolism

Mastitis is an inflammatory disease of the mammary gland with a high incidence in lactating animals, significantly impacting their health and breastfeeding. Moreover, mastitis adversely affects milk quality and yield, resulting in substantial economic losses for the dairy farming industry. Forsythiaside A (FTA), a phenylethanol glycoside analog extracted from Forsythia, exhibits notable anti-inflammatory and antioxidant properties. However, its protective effects and specific mechanisms against mastitis remain unclear. In this study, a lipopolysaccharide (LPS)-induced mouse mastitis model was used to investigate the protective effect of FTA on LPS-induced mastitis and its potential mechanism using histological assays, Western blot, qRT-PCR, FITC-albumin permeability test, 16s rRNA gene sequencing analysis and non-targeted metabolomics assays to investigate the protective effect of FTA on LPS-induced mastitis model and its potential mechanism. The results demonstrated that FTA significantly mitigated LPS-induced mouse mastitis by reducing inflammation and apoptosis levels, modulating the PI3K/AKT/mTOR signaling pathways, inducing autophagy, and enhancing antioxidant capacity and the expression of tight junction proteins. Furthermore, FTA increased the abundance of beneficial microbiota while decreasing the levels of harmful microbiota in mice, thus counteracting the gut microbiota disruption induced by LPS stimulation. Intestinal metabolomics analysis revealed that FTA primarily regulated LPS-induced metabolite alterations through key metabolic pathways, such as tryptophan metabolism. This study confirms the anti-inflammatory and antioxidant effects of FTA on mouse mastitis, which are associated with key metabolic pathways, including the restoration of gut microbiota balance and the regulation of tryptophan metabolism. These findings provide a novel foundation for the treatment and prevention of mammalian mastitis using FTA.

Weissella koreensis KJ, Which Increases Gut Tight Junction Protein Expression, Alleviates TNBS-Induced Colitis by Suppressing Inflammatory Cytokines

Inflammatory bowel disease (IBD), a chronic inflammatory disease, results from dysregulation of the immune responses. The IBD prevalence rate was 321.2 per 100,000 people in 2021 and, compared with that in 2006 (200 per 100,000 people), had increased at a rate of +46%. Therefore, the development of a safe and new treatment for IBD is urgently needed. Weissella koreensis, a strain of lactic acid bacteria (LABs), was isolated from kimchi and shown to inhibit a pro-inflammatory cytokine, tumor necrosis factor-alpha (TNF-α). Its anti-inflammatory effect was further assessed using a mouse model of colitis induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS). The administration of TNBS significantly increased myeloperoxidase (MPO) expression, macroscopic score, and colonic shortening. Oral administration of W. koreensis KJ suppressed the TNBS-induced response and significantly inhibited the expression of the pro-inflammatory cytokines TNF-α, interleukin (IL)-1β, and IL-6 in the intestinal tissues. In particular, W. koreensis KJ reversed the TNBS-induced decrease in the expression of these tight junction proteins. Therefore, since W. koreensis KJ isolated from kimchi, which increases gut tight junction proteins, attenuating colitis by suppressing inflammatory cytokines, it can be used as a therapeutic candidate for treating colitis such as IBD.

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.

Poria cocos Attenuated DSS-Induced Ulcerative Colitis via NF-κB Signaling Pathway and Regulating Gut Microbiota.

Ulcerative colitis (UC), as a chronic inflammatory disease, presents a global public health threat. However, the mechanism of Poria cocos (PC) in treating UC remains unclear. Here, LC-MS/MS was carried out to identify the components of PC. The protective effect of PC against UC was evaluated by disease activity index (DAI), colon length and histological analysis in dextran sulfate sodium (DSS)-induced UC mice. ELISA, qPCR, and Western blot tests were conducted to assess the inflammatory state. Western blotting and immunohistochemistry techniques were employed to evaluate the expression of tight junction proteins. The sequencing of 16S rRNA was utilized for the analysis of gut microbiota regulation. The results showed that a total of fifty-two nutrients and active components were identified in PC. After treatment, PC significantly alleviated UC-associated symptoms including body weight loss, shortened colon, an increase in DAI score, histopathologic lesions. PC also reduced the levels of inflammatory cytokines TNF-α, IL-6, and IL-1β, as evidenced by the suppressed NF-κB pathway, restored the tight junction proteins ZO-1 and Claudin-1 in the colon, and promoted the diversity and abundance of beneficial gut microbiota. Collectively, these findings suggest that PC ameliorates colitis symptoms through the reduction in NF-κB signaling activation to mitigate inflammatory damage, thus repairing the intestinal barrier, and regulating the gut microbiota.

Casein-quaternary chitosan complexes induced the soft assembly of egg white peptide and curcumin for ulcerative colitis alleviation

Soft assembly of peptide and curcumin (Cur) molecules enables functional integration by finding dynamic equilibrium states through non-covalent interactions. Herein, we developed two soft assembly systems, curcumin-egg white peptides (Cur-EWP) aggregations (AGs) and Cur-EWP-casein-quaternary chitosan (Cur-EWP-CA-QC) nanoparticles (NPs) to comparatively investigate their therapeutic effects on ulcerative colitis in mice and elucidate their underlying mechanism. Results revealed that Cur-EWP AGs, despite gastrointestinal tract instability, exhibited a propensity for swift accumulation within the colorectal region, enriching mucus-associated and short-chain fatty acid (SCAF)-producing bacteria, restoring the intestinal barrier damage. Whereas, Cur-EWP-CA-QC NPs, benefiting from their remarkable stability and exceptional mucosal adsorption properties, not only enhanced permeability of Cur and EWP in the small intestine to activate the immune response and boost tight junction protein expression but also, in their unabsorbed state, regulated the intestinal flora, exerting potent anti-inflammatory activity. Soft assembly of peptides and hydrophobic nutraceuticals could synergize biological activities to modulate chronic diseases.

185 Maternal live yeast supplementation protects newly weaned piglets against lipopolysaccharide challenge by regulating immune-associated milk proteins

Abstract Sow milk serves as a crucial source of nutritional, immunological, and growth-promoting components for the piglets. Our previous studies established that maternal live yeast (LY) supplementation altered the expression of antioxidant and immune-regulatory genes in the intestinal mucosa of suckling piglets. The current study aimed to investigate the effects of maternal LY supplementation during late gestation and lactation on milk proteomic profile in dams and health of offspring under lipopolysaccharide (LPS) challenge. On d 77 of gestation, 40 sows were allocated to 2 dietary treatments: without (CON) or with LY supplementation at 0.05% of diet during gestation and 0.1% during lactation. Milk samples were collected on d 0, 10, and 18 post-partum (n = 6), respectively. Within 24 h postweaning, piglets from the CON and LY groups were intraperitoneally injected with sterile saline or LPS [25 μg/kg body weight (BW)] and divided into four groups (n = 8): 1) saline + CON (CS), 2) saline + LY (YS), 3) LPS + CON (CLPS), and 4) LPS + LY (YLPS). Data were analyzed in a 2 × 2 factorial arrangement with main effects of maternal diet and immunological challenge. Rectal temperature was measured hourly for 4 h post-injection after which piglets were euthanized. Results showed that LPS increased concentrations of serum tumor necrosis factor (TNF) -α and interlekin (IL) -10 (P &amp;lt; 0.05) and tended to increase IL-8 in the ileal mucosa of piglets (P &amp;lt; 0.10). At 3 h post-injection, YLPS piglets tended to have a lower rectal temperature than CLPS piglets (P &amp;lt; 0.10). YLPS piglets also had decreased protein levels of TNF-α in the ileal mucosa (P &amp;lt; 0.05), with a tendency for greater protein abundance of E-cadherin but decreased mRNA abundance of catalase (P &amp;lt; 0.10) in the jejunal mucosa at 4 h post-injection (P &amp;lt; 0.05). In addition, YLPS piglets had less mRNA abundance of TLR2 and TLR4 in the liver than CLPS piglets (P &amp;lt; 0.05). With shotgun proteomics analysis, we found that milk of LY sows had more abundant proteins associated with immunity, including immunoglobulin and complement protein (Ig-like and C1q domain-containing proteins) on d 0 and d 10. Insulin-like growth factor binding protein (IGFBP) 5 was also more abundant in the d 0 milk of LY sows, whereas prostaglandin synthase was greater in d 10 and d 18 milk of CON sows. Additionally, pathway analysis showed that proteins identified in the milk of CON sows have roles in neutrophil degranulation and IL-12 signaling and production, whereas pathways associated with regulation of complement system, IGF and DHCR24 signaling, were enriched in the milk of LY sows. These results suggest that maternal LY supplementation could protect newly weaned piglets from bacteria endotoxin effect by suppressing inflammatory response, improving expression of tight junction proteins in offspring, and altering immune-associated proteins in sow milk.

149 Development of a leaky gut model: Impact of 24-h feed restriction on ileal expression of tight junction proteins in weaned pigs

Abstract Weanling barrows, 26 to 28 d old [n = 90; Duroc terminal line Fast genetics, initial body weight (BW)= 5.63 ± 0.43 kg] were used to evaluate the effect of 24-h complete feed restriction on growth and intestinal integrity. Upon arrival at the NutriQuest modeling center (Fergus Falls, MN), pigs were weighed, blocked by BW, and randomly assigned to 1 of 30 pens. Five treatments were randomly assigned to 6 pens: 1) Positive Control (PC), and 4 treatments that had 24-hr feed restriction (FR); 2) Negative control (NC), 3) Direct Fed Microbial (DFM) A, 4) DFM B, and 5) DFM C. Feed intake and BW were determined weekly and before and after FR. All pigs were fed a common diet. Fifteen pigs were removed prior to FR due to enteric disease. On d 18, feed was removed from pigs on treatments 2 to 5 for 24 h. After 24 h, 8 pigs per treatment were harvested for tissue collection. Feed access was restored to the remaining pigs who were harvested 3 d later. Ileal samples were collected and immediately frozen. Plasma and fecal samples were collected upon arrival, at FR, and 24 h and 4-d post-FR. Ileal expression of claudin-3, occludin, and tight-junction-1 genes was determined using QuantStudio 3 Real Time PCR system. Fold-change in gene expression compared with NC was calculated for each treatment. Plasma samples were analyzed for LPS binding protein (LBP) concentration, and fecal samples were analyzed for myeloperoxidase (MPO) concentration. Pig (n = 75) was the experimental unit for BW, LBP, MPO, and gene expression data. Pen (n = 30) was the experimental unit for feed intake. Data were analyzed using PROC GLIMMIX of SAS. Preplanned orthogonal contrasts were performed to determine the effect of PC vs. 4 FR treatments. To determine the relationship of tight junction gene expression and LBP and MPO, PROC REG of SAS was used. Data are described as PC vs. FR treatments to focus on model development. Final BW for pigs on PC was greater compared with the FR treatments (P &amp;lt; 0.05; Table 1). From 1 to 4 d post-FR, average daily gain (ADG) for FR treatments was greater than PC (P &amp;lt; 0.01). There was no effect of treatment on feed intake or feed efficiency. Plasma LBP and fecal MPO concentrations were greater 24 h post-FR for pigs on PC compared with FR treatments (P &amp;lt; 0.01). Fold change in occludin and claudin-3 expression was greater for PC compared with FR treatments (P ≤ 0.05). For pigs euthanized 24 h post-FR, there was a positive relationship between LBP concentrations and occludin expression (P &amp;lt; 0.02). Twenty-four hour FR in weaned pigs alters ileal tight junction gene expression and plasma LBP concentrations; therefore, a timed FR event may serve as a model for leaky gut.

44 Investigation of the nutritional and functional roles of mannanase on intestinal health and growth of newly weaned pigs fed a low-cost formulated diet

Abstract This study aimed to investigate the nutritional and functional roles of mannanase on the intestinal health and growth of newly weaned pigs fed a typical or low-cost formulated diet reducing the inclusion of highly digestible protein ingredients. Twenty-four newly weaned pigs at 6.2 ± 0.5 kg initial body weight (BW) were allotted to three dietary treatments based on a randomized complete block design with sex and BW as blocks. Pigs were fed for 37 d in three phases (P1, P2, and P3) and weighed at the end of each phase. Low-cost formulation was achieved by reducing the use of animal protein (fish meal, poultry meal, and blood plasma) and processed soybean meal whereas increasing the use of soybean meal from 13% in typical diets to 28% in P1, from 15% to 32% in P2, and 20.5% to 35% in P3. Treatments were 1) control diet (CON, typical nursery feed), 2) low-cost formulated diet (LCD), and 3) LCD + mannanase inclusion (0 vs. 100 g/t of Natupulse, BASF, providing 800 Thermostable Mannanase Unit, TMU, per ton of feed). On d 37 of feeding, all pigs were euthanized and jejunal mucosal tissues were taken to analyze intestinal health parameters, and gene expression of tight junction proteins and mucins. Pigs fed LCD had reduced (P &amp;lt; 0.05) average daily gain (ADG) and average daily feed intake (ADFI) during the overall period and increased (P &amp;lt; 0.05) fecal score during P3. The LCD decreased (P &amp;lt; 0.05) malondialdehyde and the gene expression of Zona occludens-1 (ZO-) in the jejunum and increased (P &amp;lt; 0.05) serum insulin. The inclusion of mannanase in LCD tended to increase (P = 0.059) BW at d 37, increased (P &amp;lt; 0.05) ADFI during P1, increased (P &amp;lt; 0.05) ADG and gain to feed ratio (G:F) during the overall period, reduced (P &amp;lt; 0.05) fecal score during the overall period, and decreased (P &amp;lt; 0.05) the viscosity of jejunal digesta. However, no effects were observed on intestinal immune response at d 37. In conclusion, feeding LCD negatively affected growth performance and intestinal health of nursery pigs. The inclusion of mannanase to the LCD (800 TMU/t feed) mitigated the negative effects of lowering feed quality observed in growth performance by reducing digesta viscosity and fecal score.

A Direct Link Implicating Loss of SLC26A6 to Gut Microbial Dysbiosis, Compromised Barrier Integrity and Inflammation

BackgroundPutative anion transporter-1 (PAT1, SLC26A6) plays a key role in intestinal oxalate and bicarbonate secretion. PAT1 knockout (PKO) mice exhibit hyperoxaluria and nephrolithiasis. Notably, diseases such as inflammatory bowel diseases (IBD) are also associated with higher risk of hyperoxaluria and nephrolithiasis. However, the potential role of PAT1 deficiency in gut barrier integrity and susceptibility to colitis is currently elusive. MethodsAge-matched PKO and wild-type (WT) littermates were administered 3.5%-DSS in drinking water for 6 days. Ileum and colon of control and treated mice were harvested. mRNA and protein expression of tight junction (TJ) proteins were determined by RT-PCR and western blotting. Severity of inflammation was assessed by measuring diarrheal phenotype, cytokine expression and H&E staining. Gut microbiome and associated metabolome were analyzed by 16S rRNA sequencing and mass spectrometry, respectively. ResultsPKO mice exhibited significantly higher loss of body weight, gut permeability, colonic inflammation, and diarrhea in response to DSS treatment. Additionally, PKO mice showed microbial dysbiosis and significantly reduced levels of butyrate and butyrate-producing microbes compared to controls. Cohousing WT and PKO mice for 4 weeks resulted in PKO-like signatures on the expression of TJ proteins in the colon of WT mice. ConclusionOur data demonstrate that loss of PAT1 disrupts gut microbiome and related metabolites, decreases gut barrier integrity, and increases host susceptibility to intestinal inflammation. These findings, thus, highlight a novel role of the oxalate transporter PAT1 in promoting gut barrier integrity and its deficiency appears to contribute to the pathogenesis of IBD.

WITHDRAWN: Recombinant osteopontin stabilizes the blood brain barrier following subarachnoid hemorrhage in rats

Background and PurposeRecombinant osteopontin (rOPN) decreases neuronal cell death and brain edema and improves the neurological scores following subarachnoid hemorrhage (SAH) in rats. The molecular mechanisms of the preservation of blood-brain barrier of rOPN following SAH in rats were examined in this study. MethodsAdult rats were divided into 3 groups: sham, vehicle, and treatment groups. Each animal in the SAH group underwent a surgical procedure to induce SAH, and the brains was harvested at 24 hours for analysis. For the mechanism part of the study two additional groups were operated and treated with Fib-14 and Wortmannin additional to rOPN. rOPN was administered via the nasal route and neurological scores as well as brain water content were evaluated at 24 hours after SAH induction. The blood brain barrier (BBB) permeability was evaluated using Evans Blue dye extravasation. The expressions of tight junction proteins claudin-3, claudin-5, occludin and the phosphorylation of β-catenin were examined using Western blot analysis. ResultsrOPN improved neurological scores and reduced brain edema at 24 hours after SAH. The BBB preservation action of rOPN is mediated in part through stabilizing β-catenin and increasing expression of tight junction proteins, claudin-3, claudin-5, and occludin. rOPN is thought to exert its effects through FAK activated PI3K-Akt pathway. ConclusionSince BBB disruption is one of the major pathophysiology of EBI after SAH, rOPN may be a promising drug to maintain BBB integrity and functions in SAH treatment.

Adjudin protects blood–brain barrier integrity and attenuates neuroinflammation following intracerebral hemorrhage in mice

Secondary brain injury exacerbates neurological dysfunction and neural cell death following intracerebral hemorrhage (ICH), targeting the pathophysiological mechanism of the secondary brain injury holds promise for improving ICH outcomes. Adjudin, a potential male contraceptive, exhibits neuroprotective effects in brain injury disease models, yet its impact in the ICH model remains unknown. In this study, we investigated the effects of adjudin on brain injury in a mouse ICH model and explored its underlying mechanisms. ICH was induced in male C57BL/6 mice by injecting collagenase into the right striatum. Mice received adjudin treatment (50 mg/kg/day) for 3 days before euthanization and the perihematomal tissues were collected for further analysis. Adjudin significantly reduced hematoma volume and improved neurological function compared with the vehicle group. Western blot showed that Adjudin markedly decreased the expression of MMP-9 and increased the expression of tight junctions (TJs) proteins, Occludin and ZO-1, and adherens junctions (AJs) protein VE-cadherin. Adjudin also decreased the blood–brain barrier (BBB) permeability, as indicated by the reduced albumin and Evans Blue leakage, along with a decrease in brain water content. Immunofluorescence staining revealed that adjudin noticeably reduced the infiltration of neutrophil, activation of microglia/macrophages, and reactive astrogliosis, accompanied by an increase in CD206 positive microglia/macrophages which exhibit phagocytic characteristics. Adjudin concurrently decreased the generation of proinflammatory cytokines, such as TNF-α and IL-1β. Additionally, adjudin increased the expression of aquaporin 4 (AQP4). Furthermore, adjudin reduced brain cell apoptosis, as evidenced by increased expression of anti-apoptotic protein Bcl-2, and decreased expression of apoptosis related proteins Bax, cleaved caspase-3 and fewer TUNEL positive cells. Our data suggest that adjudin protects against ICH-induced secondary brain injury and may serve as a potential neuroprotective agent for ICH treatment.

A human pluripotent stem cell-derived in vitro model of the blood–brain barrier in cerebral malaria

BackgroundBlood–brain barrier (BBB) disruption is a central feature of cerebral malaria (CM), a severe complication of Plasmodium falciparum (Pf) infections. In CM, sequestration of Pf-infected red blood cells (Pf-iRBCs) to brain endothelial cells combined with inflammation, hemolysis, microvasculature obstruction and endothelial dysfunction mediates BBB disruption, resulting in severe neurologic symptoms including coma and seizures, potentially leading to death or long-term sequelae. In vitro models have advanced our knowledge of CM-mediated BBB disruption, but their physiological relevance remains uncertain. Using human induced pluripotent stem cell-derived brain microvascular endothelial cells (hiPSC-BMECs), we aimed to develop a novel in vitro model of the BBB in CM, exhibiting enhanced barrier properties.MethodshiPSC-BMECs were co-cultured with HB3var03 strain Pf-iRBCs up to 9 h. Barrier integrity was measured using transendothelial electrical resistance (TEER) and sodium fluorescein permeability assays. Localization and expression of tight junction (TJ) proteins (occludin, zonula occludens-1, claudin-5), cellular adhesion molecules (ICAM-1, VCAM-1), and endothelial surface markers (EPCR) were determined using immunofluorescence imaging (IF) and western blotting (WB). Expression of angiogenic and cell stress markers were measured using multiplex proteome profiler arrays.ResultsAfter 6-h of co-culture with Pf-iRBCs, hiPSC-BMECs showed reduced TEER and increased sodium fluorescein permeability compared to co-culture with uninfected RBCs, indicative of a leaky barrier. We observed disruptions in localization of occludin, zonula occludens-1, and claudin-5 by IF, but no change in protein expression by WB in Pf-iRBC co-cultures. Expression of ICAM-1 and VCAM-1 but not EPCR was elevated in hiPSC-BMECs with Pf-iRBC co-culture compared to uninfected RBC co-culture. In addition, there was an increase in expression of angiogenin, platelet factor-4, and phospho-heat shock protein-27 in the Pf-iRBCs co-culture compared to uninfected RBC co-culture.ConclusionThese findings demonstrate the validity of our hiPSC-BMECs based model of the BBB, that displays enhanced barrier integrity and appropriate TJ protein localization. In the hiPSC-BMEC co-culture with Pf-iRBCs, reduced TEER, increased paracellular permeability, changes in TJ protein localization, increase in expression of adhesion molecules, and markers of angiogenesis and cellular stress all point towards a novel model with enhanced barrier properties, suitable for investigating pathogenic mechanisms underlying BBB disruption in CM.

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.

Tracking the therapeutic efficacy of a ketone mono ester and β-hydroxybutyrate for ulcerative colitis in rats: New perspectives

Ulcerative colitis (UC) is an inflammatory condition that affects the colon's lining and increases the risk of colon cancer. Despite ongoing research, there is no identified cure for UC. The recognition of NLRP3 inflammasome activation in the pathogenesis of UC has gained widespread acceptance. Notably, the ketone body β-hydroxybutyrate inhibits NLRP3 demonstrating its anti-inflammatory properties. Additionally, BD-AcAc 2 is ketone mono ester that increases β-hydroxybutyrate blood levels. It has the potential to address the constraints associated with exogenous β-hydroxybutyrate as a therapeutic agent, including issues related to stability and short duration of action. However, the effects of β-hydroxybutyrate and BD-AcAc 2 on colitis have not been fully investigated. This study found that while both exogenous β-hydroxybutyrate and BD-AcAc 2 produced the same levels of plasma β-hydroxybutyrate, BD-AcAc 2 demonstrated superior effectiveness in mitigating dextran sodium sulfate-induced UC in rats. The mechanism of action involves modulating the NF-κB signaling, inhibiting the NLRP3 inflammasome, regulating antioxidant capacity, controlling tight junction protein expression and a potential to inhibit apoptosis and pyroptosis. Certainly, BD-AcAc 2's anti-inflammatory effects require more than just increasing plasma β-hydroxybutyrate levels and other factors contribute to its efficacy. Local ketone concentrations in the gastrointestinal tract, as well as the combined effect of specific ketone bodies, are likely to have contributed to the stronger protective effect observed with ketone mono ester ingestion in our experiment. As a result, further investigations are necessary to fully understand the mechanisms of BD-AcAc 2 and optimize its use.

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.

Ketogenic Diet Protects from Experimental Colitis in a Mouse Model Regardless of Dietary Fat Source.

While ketogenic diets (KDs) may have potential as adjunct treatments for gastrointestinal diseases, there is little knowledge on how the fat source of these diets impacts intestinal health. The objective of this study was to investigate how the source of dietary fat of KD influences experimental colitis. We fed nine-week-old male C57BL/6J mice (n = 36) with a low-fat control diet or KD high either in saturated fatty acids (SFA-KD) or polyunsaturated linoleic acid (LA-KD) for four weeks and then induced colitis with dextran sodium sulfate (DSS). To compare the diets, we analyzed macroscopic and histological changes in the colon, intestinal permeability to fluorescein isothiocyanate-dextran (FITC-dextran), and the colonic expression of tight junction proteins and inflammatory markers. While the effects were more pronounced with LA-KD, both KDs markedly alleviated DSS-induced histological lesions. LA-KD prevented inflammation-related weight loss and the shortening of the colon, as well as preserved Il1b and Tnf expression at a healthy level. Despite no significant between-group differences in permeability to FITC-dextran, LA-KD mitigated changes in tight junction protein expression. Thus, KDs may have preventive potential against intestinal inflammation, with the level of the effect being dependent on the dietary fat source.

Expression and Localization Profiles of Tight Junction Proteins in Immune Cells Depend on Their Activation Status.

The ability of the immune system to combat pathogens relies on processes like antigen sampling by dendritic cells and macrophages migrating through endo- and epithelia or penetrating them with their dendrites. In addition, other immune cell subtypes also migrate through the epithelium after activation. For paracellular migration, interactions with tight junctions (TJs) are necessary, and previous studies reported TJ protein expression in several immune cells. Our investigation aimed to characterize, in more detail, the expression profiles of TJ proteins in different immune cells in both naïve and activated states. The mRNA expression analysis revealed distinct expression patterns for TJ proteins, with notable changes, mainly increases, upon activation. At the protein level, LSR appeared predominant, being constitutively present in naïve cell membranes, suggesting roles as a crucial interaction partner. Binding experiments suggested the presence of claudins in the membrane only after stimulation, and claudin-8 translocation to the membrane occurred after stimulation. Our findings suggest a dynamic TJ protein expression in immune cells, implicating diverse functions in response to stimulation, like interaction with TJ proteins or regulatory roles. While further analysis is needed to elucidate the precise roles of TJ proteins, our findings indicate important non-canonical functions of TJ proteins in immune response.

Effect of arginine supplementation on the growth performance, intestinal health, and immune responses of broilers during necrotic enteritis challenge

The objective of this study was to evaluate the effect of 25% arginine supplementation as a functional amino acid in partially alleviating the detrimental effects of necrotic enteritis (NE) on the growth performance, serum biochemistry, gut integrity, and the relative gene expression of tight junction proteins and inflammatory cytokines in broilers during NE. Three hundred and sixty 1-day-old chicks were randomly allocated to 4 treatments in a 2 × 2 factorial arrangement -basal diet and 125% arginine diet, with or without NE challenge. NE was induced by inoculating 1 × 104Eimeria maxima sporulated oocysts on d 14 and 1 × 108 CFU/bird C. perfringens on d 19, 20, and 21. The NE challenge had a significant effect on the BWG (p < 0.05), FCR (p < 0.05), serum AST (p < 0.05), GLU (p < 0.05), and K+ (p < 0.05) levels, and intestinal permeability (p < 0.05) and jejunal lesion score (p < 0.05). A significant challenge × diet interaction effect was observed in the cecal tonsil CD8+: CD4+ T-cell ratio on d 21 (p < 0.05) and 28 (p < 0.05) and spleen CD8+: CD4+ T-cell ratio on d 21 (p < 0.05) and 35 (p < 0.05). Arginine supplementation significantly increased the CD8+: CD4+ T-cell ratio in uninfected birds but decreased the CD8+: CD4+ T-cell ratio in infected birds. On d 21, a significant interaction effect was observed on the relative expression of the iNOS gene (p < 0.05). Arginine supplementation significantly downregulated the expression of the iNOS gene in infected birds. A significant effect of the challenge (p < 0.05) was observed on the relative gene expression of the ZO-1 gene in the jejunum. NE challenge significantly downregulated the expression of the ZO-1 gene on d 21. In conclusion, arginine supplementation did not alleviate the depression in growth performance and disease severity during the NE challenge. However, arginine downregulated the expression of inflammatory cytokines and enzymes, preventing inflammatory injury to the tissues during NE. Hence, arginine might be supplemented with other alternatives to downregulate inflammatory response during NE in poultry.