Expression Of Claudin-1 Research Articles

Use of gene expression profiling to examine association between MUC1 and claudin 18 in gastrointestinal cancers: Implications for targeted therapies.

838 Background: Gastrointestinal tract cancers exhibit considerable molecular heterogeneity, which requires a detailed understanding of their distinct genetic profiles. Understanding the gene expression of other genes along with Claudin 18 (CLDN18) could provide insights into their interactions. Methods: We conducted a retrospective analysis of NGS profiling on 1,043 GI cancer tissue samples from three cohorts: Upper and Lower Gastrointestinal Cancer Cohort (GICC; n=651), Pancreaticobiliary Cancer Cohort (PBCC; n=316), and Hepatocellular Carcinoma Cohort (HCC; n=76). Immunohistochemistry data for PD-L1, Her2 and MMR proteins, NGS based Tumor mutation burden (TMB) and gene expression profiling (GEP) of 20,802 genes including CLDN18 was examined in a subset by targeted transcriptome profiling. We explored associations of these biomarkers with expression of CLDN18. Results: In GICC, frequent mutations were seen in TP53 (68%; 442), APC (33.5%; 218) and KRAS (33.3%; 217). PBCC, showed mutations in TP53 (62%; 197), KRAS (47%; 149) and CDKN2A (16%; 51). HCC showed mutations in TP53 (42%; 32), ARID1A (18%; 14) and KRAS (9%; 7). Amplifications were predominantly observed in MYC (13%; 120), ERBB2 (5%; 49) and CCND1 (5%; 44). Fusions were infrequent. All MSI-H cases (11/462; 2.4%) were observed in GICC. High TMB (≥10 muts/mb) was seen in 23% (111/481), stratified as 28% (90/318) in GICC, 13% (18/139) in PBCC and 10% (3/30) in HCC. Out of 321 samples analyzed for gene expression profiling, CLDN18 was significantly upregulated in 65 (20%) samples. The expression of CLDN18 did not show statistically significant correlation with Her2 (n=184), PD-L1 (n=231), TMB (n=228), or MSI (n=229); (p>0.05). In subset where both CLDN18 and MUC1 expression were analyzed (n=118), all the CLDN18 positive tumors showed overexpression of MUC1. This finding was statistically significant (p<0.05). Increased expression of MUC1 promotes cancer cell mobility promoting metastases. Expression of MUC1 can play an important role in the development of resistance to chemotherapy. MUC1 expression in cancer cells is associated with avoidance of immune defenses. Thus, this finding of CLDN18 and MUC1 association is especially relevant as the CLDN18 targeted therapies may be the potential avenue for the aggressive and chemo-resistant MUC1 expressing tumors. Conclusions: This study identifies potential association between MUC1 and CLDN18. GEP could enhance biomarker detection and support personalized treatment approaches. Future research should integrate these findings with clinical data to improve precision in GI cancer care.

Bacillus coagulans alleviates intestinal barrier injury induced by Klebsiella pneumoniae in rabbits by regulating the TLR4/MyD88/NF-κB signalling pathway.

Probiotics effectively alleviate host diarrhoea, but the specific mechanism is not clear. Therefore, we explored the protective mechanism of Bacillus coagulans (BC) on intestinal barrier injury induced by Klebsiella pneumoniae (K. pneumoniae) in rabbits by HE, immunofluorescence and 16S rRNA. The results showed that BC pretreatment alleviated the changes in average daily gain, average daily feed intake and FCR caused by K. pneumoniae in rabbits. Moreover, BC alleviated the inflammatory cell infiltration, intestinal villus reduction, crypt deepening and goblet cell reduction caused by K. pneumoniae in rabbits. Further research revealed that BC improved the intestinal barrier by improving the mechanical barrier, chemical barrier, immune barrier and microbial barrier. Specifically, BC improved the intestinal mechanical barrier by improving the intestinal structure, increasing the protein expression of PCNA, increasing the number of goblet cells, and altering the expression of occludin, claudin-1 and ZO-1. BC improved the intestinal chemical barrier by regulating the expression of MUC1 and MUC2 and inhibited the TLR4/MyD88/NF-κB signalling pathway by altering the expression levels of the inflammatory factors IL-1β, IL-6 and TNF-α, thus optimizing the intestinal immune barrier. In addition, adding BC to the diet improved the intestinal microbial barrier of rabbits by reducing the abundance of harmful bacteria and increasing the abundance of beneficial bacteria. In summary, BC protects against K. pneumoniae-induced intestinal barrier damage by improving intestinal morphology, mitigating the inflammatory response and regulating the microbial composition. Among the pretreatments, the pretreatment effect of 1 × 106 CFU/g was the best. This study provides a theoretical basis for the use of BC to prevent and treat diarrhoea caused by K. pneumoniae in rabbits.

Effect of the IL-6 trans-signaling pathway in the absence or presence of TGF-β2 on Schlemm's canal endothelial cells.

Intraocular pressure (IOP) is regulated through the balance of production and drainage of aqueous humor. The main route of aqueous-humor outflow comprises the trabecular meshwork (TM) and Schlemm's canal (SC). We reported that IL-6 trans-signaling can inhibit TGF-β signaling in TM cells and may affect regulation of IOP. However, the function of IL-6 trans-signaling in SC cells remains unclear. Therefore, we investigated the role of IL-6 trans-signaling in monkey SC cells. Simultaneous treatment with IL-6 and soluble IL-6 receptor (sIL-6R) significantly decreased the trans-endothelial electrical resistance (TER) of SC cells and reduced aqueous-humor outflow resistance. Moreover, activation of IL-6 trans-signaling significantly reduced expression of fibronectin, ZO-1 and claudin-5, and increased that of several matrix metalloproteinases. We also investigated the effect of IL-6 trans-signaling on TGF-β2-induced changes in SC cells. Simultaneous treatment with IL-6 and sIL-6R significantly suppressed the TGF-β2-induced increase in the TER of SC cells but did not affect the activity of the TGF-β2 signaling pathway. By contrast, the TGF-β2-induced increases in the expression of fibronectin and collagen type I were significantly decreased upon simultaneous treatment with IL-6 and sIL-6R. The results show that IL-6 trans-signaling suppressed TGF-β2-induced increase in outflow resistance.

Ion permeability profiles of renal paracellular channel-forming claudins.

Members of the claudin protein family are the major constituents of tight junction strands and determine the permeability properties of the paracellular pathway. In the kidney, each nephron segment expresses a distinct subset of claudins that form either barriers against paracellular solute transport or charge- and size-selective paracellular channels. It was the aim of the present study to determine and compare the permeation properties of these renal paracellular ion channel-forming claudins. MDCK II cells, in which the five major claudins had been knocked out (claudin quintupleKO), were stably transfected with individual mouse Cldn2, -4, -8, -10a, -10b, or -15, or with dog Cldn16 or -19, or with a combination of mouse Cldn4 and Cldn8, or dog Cldn16 and Cldn19. Permeation properties were investigated in the Ussing chamber and claudin interactions by FRET assays. Claudin-4 and -19 formed barriers against solute permeation. However, at low pH values and in the absence of HCO3 -, claudin-4 conveyed a weak chloride and nitrate permeability. Claudin-8 needed claudin-4 for assembly into TJ strands and abolished this anion preference. Claudin-2, -10a, -10b, -15, -16+19 formed highly permeable channels with distinctive permeation profiles for different monovalent and divalent anions or cations, but barriers against the permeation of ions of opposite charge and of the paracellular tracer fluorescein. Paracellular ion permeabilities along the nephron are strictly determined by claudin expression patterns. Paracellular channel-forming claudins are specific for certain ions and thus lower transepithelial resistance, yet form barriers against the transport of other solutes.

The role of CLDN11 in promotion of granulosa cell proliferation in polycystic ovary syndrome via activation of the PI3K-AKT signalling pathway

Polycystic ovary syndrome (PCOS) is a complex gynecological endocrinological condition that significantly impacts women’s fertility during their reproductive lifespan. The causes of PCOS are multifaceted, and its pathogenesis is not yet clear. This study established a rat model of PCOS and, in conjunction with clinical samples and database data, analysed the role of claudin 11 (CLDN11) in follicular granulosa cells (GCs) in regulating the proliferation of GCs. Our findings revealed a notable decrease in the protein expression of CLDN11 within the follicular GCs of individuals with PCOS. In vitro rat cell experiments revealed that interference with CLDN11 significantly inhibited viability and increased the apoptosis of GCs. Additional research has illuminated the mechanism by which CLDN11 regulates the expression levels of CCND1 and PCNA through the PI3K/AKT signalling pathway, significantly influencing the proliferation of rat follicular GCs. Furthermore, overexpression of CLDN11 via an adeno-associated virus (AAV) vector was found to reverse the PCOS-like phenotype induced in rats by letrozole. Our findings suggest that CLDN11 stimulates the proliferation of these cells by activating the PI3K/AKT pathway, thereby increasing the expression of CCND1 and PCNA. These discoveries underscore the critical function of CLDN11 in regulating the functionality of follicular GCs, which offers novel insights into the fundamental mechanisms governing PCOS.

Disrupting of IGF2BP3-stabilized CLDN11 mRNA by TNF-α increases intestinal permeability in obesity-related severe acute pancreatitis

BackgroundObesity is a significant risk factor for severe acute pancreatitis (SAP) and is typically associated with increased intestinal permeability. Understanding the role of specific molecules can help reduce the risk of developing SAP. Claudin 11 (CLDN11), a member of the Claudin family, regulates the permeability of various internal barriers. However, the role and mechanism of CLDN11 in the intestinal permeability of obesity-related SAP remain unclear.MethodsWe evaluated intestinal permeability and the expression of CLDN11 in experimental obesity-related SAP. A recombinant adeno-associated virus carrying CLDN11 was used to treat experimental obesity-related SAP. The interaction between CLDN11 mRNA and insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) protein was predicted through bioinformatics analysis and validated by RNA immunoprecipitation and RNA pull-down assay. Additionally, tumor necrosis factor-α (TNF-α) treatment in Caco-2 cells was conducted, and the IGF2BP3/CLDN11 axis was detected. Moreover, we conducted anti-TNFα therapy and evaluated intestinal permeability and pancreatic inflammation in experimental obesity-related SAP.ResultsDownregulation of CLDN11 was observed in the intestinal epithelial cells of experimental obesity-related SAP. When the expression of CLDN11 in intestinal epithelial cells of experimental obesity-related SAP was increased exogenously, intestinal epithelial permeability and pancreatic inflammation were relieved. Overexpression of CLDN11 reduced the paracellular permeability of Caco-2 monolayer cells, while knockdown of CLDN11 increased it. IGF2BP3 bound to and regulated the stability of CLDN11 mRNA. TNF-α treatment downregulated IGF2BP3 and CLDN11 in vitro. Anti-TNFα therapy reduced intestinal permeability, alleviated pancreatitis, and improved the expression of IGF2BP3 and CLDN11 in intestinal epithelial cells in experimental obesity-related SAP.ConclusionCLDN11 regulates intestinal permeability in obesity-related SAP. Mechanistically, an increase in TNF-α impaired the stability of IGF2BP3-dependent CLDN11 mRNA in obesity-related SAP.

P0183 GSDMD regulates interactions between macrophages and intestinal epithelial cells in inflammatory bowel disease

Abstract Background Our previous studies have demonstrated the significant role of the GSDMD-mediated pyroptotic pathway in inflammatory bowel disease (IBD). However, there remains some controversy surrounding the research findings concerning GSDMD in IBD. Methods Employing lentiviral transduction techniques, we manipulated GSDMD expression levels in THP-1 and HT-29 cells, assessing the impact of GSDMD on macrophage M1 polarization and epithelial barrier function. Further, co-culturing transduced THP-1 and HT-29 cells enabled the examination of changes in the intestinal barrier. Moreover, we generated conditional knockout mice with Gsdmdflox/floxLyz2-Cre and Gsdmdflox/floxVillin1-Cre, inducing experimental colitis with dextran sodium sulfate (DSS) to evaluate mucosal macrophage phenotypes and intestinal barrier integrity. Additionally, primary macrophages and intestinal epithelial cells were co-cultured, and downstream inflammatory factors were screened through transcriptome sequencing. Results Knockdown of GSDMD in THP-1 cells significantly reduces CD86 expression, indicating a decrease in M1 polarization efficiency, whereas overexpression of GSDMD yields opposite results. In HT-29 cells, overexpression of GSDMD followed by LPS treatment leads to a significant decrease expression of Claudin-1, Occludin, and Zo-1, whereas knockdown of GSDMD shows the opposite outcome. ELISA results demonstrate a correlation between GSDMD expression levels and levels of LDH, IL-1β, IL-18, TNF-α, and IL-6. In the inflammatory co-culture model of THP-1 and HT-29 cells with double knockdown of GSDMD, the severity of intestinal barrier damage and release of inflammatory factors is minimal. Conversely, in the inflammatory co-culture model of THP-1 and HT-29 cells with double overexpression of GSDMD, the intestinal barrier damage and release of inflammatory factors are maximal. Following DSS-induced colitis, compared to the Gsdmdflox/flox group, two conditional knockout mice exhibit reduced inflammatory evaluation. Furthermore, the protein expression of Zo-1, Occludin, and Claudin-1 in two conditional knockout mice is significantly higher than that in the Gsdmdflox/flox group. Flow cytometry results show that the proportion of M1 macrophages in the mucosal lamina propria is significantly lower in two conditional knockout mice compared to the Gsdmdflox/flox group. Additionally, primary macrophage and intestinal epithelial cell co-culture experiments, coupled with transcriptomic sequencing, revealed IL-1β as a downstream mediator of GSDMD-mediated macrophage-epithelial cell interplay. Conclusion It was confirmed that GSDMD serves as a key target in the pathological interplay between macrophages and epithelial cells.

P0145 Role of PPARg on anti-inflammatory effects mediated by AhR ligands in a Caco-2/THP-1 in vitro “inflamed gut” model

Abstract Background Inflammatory Bowel Diseases (IBD) lack therapeutic options capable of inducing long-term remission in a cost-efficient manner. Repurposing strategies have allowed already established receptors and ligands to be tested as alternative pharmacotherapy for treatment of IBD. Agonists of the aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor traditionally associated with xenobiotic toxicity, have been explored as potential anti-inflammatory drugs to treat IBD, but specific mechanisms have yet to be elucidated. Like AhR, the peroxisome proliferator-activated receptor gamma (PPARg), responsible for control of lipid metabolism, has been evidenced as a key player in control of inflammation. In this study, we have explored a hypothetical crosstalk between PPARg and AhR in attenuating intestinal inflammation. Methods Bioinformatics analyses of a GEO RNAseq dataset of gut macrophages from IBD patients were used to assess correlations between PPARg and AhR. In vitro, macrophage-differentiated THP-1 cells and Caco-2 gut epithelial cells were seeded in Transwell® inserts to generate a gut-like co-culture model. Cells were treated with 100 ng/mL lipopolysaccharide (LPS) for 24 h in order to mimic a phenotype of intestinal inflammation, and also with combinations of AhR/PPARg agonists and antagonists at concentrations of 10 µM each. Proprietary AhR agonists were also tested. AhR translocation and cytokine secretion in macrophages as well as monolayer integrity and expression of tight junction proteins (e.g., claudin-2) in epithelial cells were assessed. Results Principal component analysis and Pearson correlation tests of the RNAseq dataset revealed that PPARg and AhR correlate with each other during intestinal homeostasis, but their expression patterns diverge during gut inflammation. In our co-culture model, blocking PPARg in THP-1 macrophages with a specific antagonist (GW9662) prevented AhR agonist 6-formylindolo[3,2-b]carbazole (FICZ) from inducing AhR nuclear translocation and secreting interleukin (IL)-6. In Caco-2 gut epithelial cells, GW9662 blocked the anti-inflammatory effects of FICZ on monolayer disruption and claudin-2 expression. These effects of FICZ were confirmed to be AhR-dependent, as they were prevented under treatment with the AhR antagonist CH223191. Proprietary AhR ligands also inhibited IL-6 secretion, which was shown to be the most dominant parameter in terms of converging anti-inflammatory effects of both PPARg and AhR. Conclusion Our data points to a pivotal cross-talk between AhR and PPARg, where PPARg is likely recruited downstream of AhR activation and contributes to the anti-inflammatory effects of FICZ, especially inhibition of IL-6 secretion.

P0153 The role of the proglucagon-derived peptide GLP-2 in resection-triggered growth and barrier response in mice

Abstract Background Ulcerative colitis (UC) and Crohn’s disease (CD) of the colon may cause significant malnutrition by “inflammation-dependent”, and “inflammation-independent” mechanisms. Glucagon-like peptide 2 (GLP-2) is secreted by ileocolonic L cells in response to nutrient intake and promotes small intestinal barrier function. GLP-2 secretion is enhanced after small bowel resection. We hypothesized that loss of GLP-2 secretion by a) genetic ablation, and b) inflammatory ablation through severe DSS-colitis, impairs small intestinal barrier dysfunction in the mouse mid small bowel resection model. Methods The impact of GLP-2 loss on small intestinal barrier function was evaluated using two models. In the first model, gcg knockout (KO) and wild-type (WT) mice underwent 50% mid-small bowel resection (MSBR). In the second model, male C57BL/6J mice were divided into the following groups: non-operated controls +/- DSS, MSBR alone, MSBR with dextran sulfate sodium (MSBR+DSS), and MSBR with DSS plus teduglutide (GLP-2 analogue). Body weight changes, mucosal height, tight junction mRNA expression, and FITC-4kDa-dextran flux were assessed using Ussing chambers. Results The gcg-WT mice did not develop diarrhea during the experimental period, while gcg-KO mice showed significantly increased stool water content two days post-surgery (p < 0.05). Although no other major clinical differences were observed between gcg-WT and KO mice after MSBR, gcg-KO mice exhibited reduced mucosal height and increased FITC-dextran flux, indicating impaired barrier function (p < 0.05). DSS-colitis was less severe in the MSBR-model, compared to non-operated DSS-colitis. However, in the inflammatory model, MSBR+DSS mice experienced significant body weight loss, colon shortening, all of which were significantly mitigated by teduglutide treatment. Teduglutide also improved villus length (p < 0.001) and reduced FITC-dextran permeability (p < 0.05). Claudin-2 expression, which was markedly elevated in MSBR+DSS mice, was significantly reduced with teduglutide treatment. Conclusion Our findings demonstrate that DSS-induced colitis is less severe in the 50% MSBR model. Furthermore, genetic knockout of gcg, i.e. complete ablation of GLP-2 secretion capacity, diminishes resection-triggered barrier- and growth response, while DSS-colitis, i.e. incomplete ablation of GLP-2 secretion capacity, diminishes resection-triggered barrier-, but not growth response. Thus, GLP-2-deficiency with small intestinal barrier dysfunction may be an inflammation-independent mechanism ultimately contributing to malnutrition in severe colitis.

DOP107 Real-time intestinal barrier assessment by endocytoscopy and confocal laser endomicroscopy uniquely correlates with multiple barrier protein expression and reflects the gut-brain axis

Abstract Background The intestinal barrier is gaining recognition as a key indicator of mucosal healing in IBD. Nonetheless, its assessment remains challenging. This study evaluates advanced endoscopic tools for real-time gut barrier assessment, examining their correlation with automated epithelial and vascular barrier markers and exploring their potential to elucidate the gut-brain axis. Methods IBD patients undergoing endoscopic assessment or surveillance and healthy controls were included. The intestinal mucosa was assessed with advanced imaging, i.e. ultra-high magnification endocytoscopy (ECS) or confocal laser endomicroscopy (CLE), using newly developed scores for barrier evaluation.1,2 Barrier healing was defined as ECS ≤ 1 -ileum and colon- and CLE ≤ 4 -colon- or ≤ 3 -ileum-. Biopsies were collected from different bowel segments, including representative inflamed and non-inflamed areas. Epithelial and vascular barrier markers, including ZO-1, Claudin-2, E-cadherin, PV-1 and CD-31, were analysed through confocal microscopy, and their expression was automatically quantified using QuPath. The validated IBD disk, focusing on energy, sleep and emotional components, was employed to evaluate neuropsychological functional impairment and indirectly assess gut-brain interaction.3 A per-biopsy-based linear regression model was used for inferential analysis using R software. Results A cohort of 33 IBD patients (15 CD and 18 UC) and 2 healthy controls were recruited (65 biopsies; 3.8 X106 cells). 9/16 (56%) patients who underwent ECS in the colon and 6/9 (67%) in the ileum had ECS scores indicative of barrier healing. CLE assessment revealed features suggestive of barrier healing in 3/11 patients (27%) in the colon and 1/14 (7%) in the ileum. ECS and CLE sub- and total scores demonstrated unique correlation with epithelial and vascular barrier proteins. For the epithelial barrier, CLE total scores in colon and ileum and villi architecture at ECS significantly correlated with Claudin-2 mean expression (p<0.01). Regarding the vascular barrier, a significant correlation was found between ECS-assessed vascular architecture in ileum and colon and PV-1 mean expression (p<0.01). In contrast, CLE-assessed blood flow in the colon was significantly correlated with CD-31 expression (p=0.03). Interestingly, patients with higher levels of sleeping difficulties based on IBD disk showed higher Claudin-2 and lower E-cadherin expression, while those with altered CD-31 expression reported lower energy levels. Conclusion CLE and ECS may offer a unique ability for real-time assessment of barrier impairment in IBD, showing promises for targeting real-time barrier healing and unravelling the complexities of gut-brain axis. References 1Iacucci M, Jeffery L, Acharjee A, et al. Computer-Aided Imaging Analysis of Probe-Based Confocal Laser Endomicroscopy With Molecular Labeling and Gene Expression Identifies Markers of Response to Biological Therapy in IBD Patients: The Endo-Omics Study. Inflamm Bowel Dis. 2023;29(9):1409-1420. doi:10.1093/ibd/izac233 2Majumder S, Santacroce G, Maeda Y, et al. Endocytoscopy with automated multispectral intestinal barrier pathology imaging for assessment of deep healing to predict outcomes in ulcerative colitis. Gut. 2024;73(10):1603-1606. Published 2024 Sep 9. doi:10.1136/gutjnl-2024-332894 3Le Berre C, Flamant M, Bouguen G, et al. VALIDation of the IBD-Disk Instrument for Assessing Disability in Inflammatory Bowel Diseases in a French Cohort: The VALIDate Study. J Crohns Colitis. 2020;14(11):1512-1523. doi:10.1093/ecco-jcc/jjaa100

Probiotic administration aggravates dextran sulfate sodium salt-induced inflammation and intestinal epithelium disruption in weaned pig

BackgroundA. muciniphila (AKK) has attracted extensive research interest as a potential next-generation probiotics, but its role in intestinal pathology is remains unclear. Herein, this study was conducted to investigate the effects of A. muciniphila DSM 22,959 on growth performance, intestinal barrier function, microecology and inflammatory response of weaned piglets stimulated by dextran sulfate sodium salt (DSS).MethodTwenty-four Duroc × Landrace × Yorkshire (DLY) weaned piglets used for a 2 × 2 factorial arrangement of treatments were divided into four groups with six piglets in each group. From 1 to 15 d, the CA and DA groups were orally fed with 1.0 × 1011 colony-forming units A. muciniphila per day, while the CON and DCON groups were received gastric infusion of anaerobic sterile saline per day. The pigs were orally challenged (DCON, DA) or not (CON, CA) with DSS from day 9 to the end of the experiment and slaughtered on day 16.ResultsPresence of A. muciniphila in DSS-challenged weaned pigs resulted in numerically increased diarrhea rate, blood neutrophilic granulocyte, serum C-reactive protein and immunoglobulin M levels, and numerically reduced final weight, average daily feed intake and average daily gain. The decrease in intestinal villus height, villous height: crypt depth ratio and digestibility was accompanied by lower expression of ZO1, ZO2, Claudin1, DMT1, CAT1, SGLT1 and PBD114 genes, as well as decreased enzyme activities of intestinal alkaline phosphatase, lactase, sucrase and maltase of piglets in DA group compared to piglets in DCON group. The abundance of Bifdobacterium, Lactobacillus, A. muciniphila, Ruminococcus gnavus was numerically higher in digesta of pigs in DA group than those in DCON group. The inflammatory responses of piglets were dramatically changed by the simultaneous presence of A. muciniphila and DSS: expression level of IL17A, IL17F, IL23, RORγt, Stat3 was elevated in DA pigs compared to the other pig groups.ConclusionsOur result showed that the oral A. muciniphila aggravates DSS-induced health damage of weaned piglet, which may attribute to the deteriorating intestinal morphology, dysbiosis of microbiota and inflammatory response disorders.

Effect of Gardeniae Fructus Powder on Growth Performance, Antioxidant Capacity, Intestinal Barrier Function, and Colonic Microbiota of Weaned Piglets.

The present study aimed to explore the effect of GF powder on the growth performance, diarrhea rate, antioxidant and immune capacity, and intestinal health of weaned piglets. A total of 144 weaned piglets (8.29 ± 0.11 kg) at 21 d old were randomly assigned to four groups, with each treatment consisting of six replicate pens, with six piglets per pen, and each pen containing three barrows and three gilts. The piglets were fed a basal diet supplement with 0%, 0.4%, 0.6%, and 0.8% GF powder (n = 36). Our results indicated that compared with the basal diet, the F/G and diarrhea rate were remarkably decreased in the 0.8% GF group (p < 0.05). Serum biochemical parameters showed that supplementation with GF significantly increased the content of HDL-C (0.6 and 0.8% levels), IL-6 (0.8% level), IL-10 (0.4, 0.6, and 0.8% levels), Ig G (0.4% level), and Ig A (0.8% level) compared with the basal diet (p < 0.05). The index of antioxidant capacity showed that compared with a basal diet, supplementation with GF significantly decreased serum MDA content (0.4% and 0.8% levels) and jejunal and ileal MDA content (0.4%, 0.6%, and 0.8% levels) (p < 0.05). Additionally, compared with the basal diet, supplementation with GF significantly increased serum and ileal T-AOC content (0.4%, 0.6%, and 0.8% levels), serum T-SOD content (0.4% and 0.8% levels), ileal T-SOD content (0.4%, 0.6%, and 0.8% levels), CAT content (0.4%, 0.6%, and 0.8% levels), and jejunal GSH-Px content (0.8% level) (p < 0.05). The results of gene expression indicate that compared with the basal diet, supplementation with GF significantly increased Nrf 2 (0.4% level), NQO (0.4% level), SOD 1 (0.4% and 0.8% levels), and GCLC (0.4% level) and GCLM (0.8% level) abundance in jejunal mucosa; supplementation with GF significantly increased Nrf 2 (0.4%, 0.6%, and 0.8% levels), HO-1 (0.4% level), NQO (0.8% level), SOD 1 (0.4% and 0.8% levels), and GCLC (0.4% level) and GCLM (0.8% level) abundance in ileal mucosa (p < 0.05). Ulteriorly, the present results indicate that supplementation with GF at the 0.8% level significantly increased the villus height in the jejunum and ileum as well as the villus/crypt ratio in the ileum compared with the basal diet (p < 0.05). Compared with the basal diet, 0.4% GF significantly increased Occludin gene expression in ileal mucosa (p < 0.05), 0.6% GF significantly increased ZO-1, Claudin-1, and Occludin gene expression in jejunal mucosa (p < 0.05), and 0.8% GF significantly increased ZO-1 and Occludin gene expression in jejunal mucosa along with Occludin expression in ileal mucosa (p < 0.05). Furthermore, colonic microbiota composition showed that Shannon, observed species, and Chao 1 indices were significantly increased in the 0.8% GF group compared with the basal diet (p < 0.05). At the phylum level, in comparison with the basal diet, the relative abundance of Firmicutes significantly decreased in the 0.4%, 0.6%, and 0.8% GF groups, and Bacteroidetes increased in the 0.8% GF group (p < 0.05). At the genus level, compared with the basal diet, 0.6% and 0.8% GF significantly increased Prevotella abundance, and 0.6% GF significantly decreased Coprococcus abundance (p < 0.05). At the species level, compared with the basal diet, 0.8% GF significantly increased Prevotella copri abundance, and 0.4%, 0.6%, and 0.8% GF significantly decreased Blautia obeum abundance (p < 0.05). In summary, a dietary supplement with 0.8% Gardeniae Fructus powder significantly decreased the F/G and diarrhea rate and improved antioxidant capacity and intestinal barrier function, which may be associated with the improvement of the relative abundance of Prevotella copri. These findings indicate that Gardeniae Fructus powder may be used as a feed additive in swine weaning.

Claudin 18.2: An attractive marker in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) is a highly aggressive tumor with limited treatment options. Zolbetuximab, a monoclonal antibody against the tight junction protein Claudin 18.2 has recently been developed. At present, few and conflicting data have been reported regarding the clinical-pathological features of Claudin 18.2 expression in PDA. The present study investigated the expression of Claudin 18.2 in histological samples from PDA patients with the aim of verifying its utility as a therapeutic biomarker. Claudin 18.2 immunoreactivity was assessed by immunohistochemical staining on 70 formalin-fixed, paraffin-embedded PDA specimens (28 surgical specimens and 42 fine needle aspiration biopsies). The results obtained were associated with the clinicopathological characteristics and the survival rate of patients. Claudin 18.2 was detected only in neoplastic cells, not in normal pancreatic tissue. Claudin 18.2 was positive in 50% of samples and a higher expression was associated with well- and moderately-differentiated tumors and lymph node-negative status. The high expression of Claudin 18.2 in neoplastic tissue and absence in normal cells suggested that this protein had an attractive role in PDA as both a diagnostic and a prognostic-therapeutic marker. High expression of Claudin 18.2 in neoplastic tissue was associated with more favorable prognostic parameters and the high percentage of positive samples obtained suggests that Zolbetuximab may be suitable for a large number of patients.

Berberine alleviates enterotoxigenic Escherichia coli-induced intestinal mucosal barrier function damage in a piglet model by modulation of the intestinal microbiome.

Enterotoxic Escherichia coli (ETEC) is the main pathogen that causes diarrhea, especially in young children. This disease can lead to substantial morbidity and mortality and is a major global health concern. Managing ETEC infections is challenging owing to the increasing prevalence of antibiotic resistance. Berberine, categorized as a substance with similarities in "medicine and food," has been used in China for hundreds of years to treat gastrointestinal disorders and bacteria-induced diarrhea. This study investigated the preventive effect of dietary berberine on the intestinal mucosal barrier induced by ETEC and the microbial community within the intestines of weaned piglets. Twenty-four piglets were randomly divided into four groups. Piglets were administered either a standard diet or a standard diet supplemented with berberine at concentrations of 0.05 and 0.1%. and orally administered ETEC or saline. Dietary supplementation with berberine reduced diamine oxidase, d-lactate, and endotoxin levels in piglets infected with ETEC (P < 0.05). Berberine increased jejunal villus height, villus/crypt ratio, mucosal thickness (P < 0.05), and goblet cell numbers in the villi and crypts (P < 0.05). Furthermore, berberine increased the optical density of mucin 2 and the mucin 2, P-glycoprotein, and CYP3A4 mRNA expression levels (P < 0.05). Berberine increased the expressions of zonula occludins-1 (ZO-1), zonula occludins-2 (ZO-2), Claudin-1, Occludin, and E-cadherin in the ileum (P < 0.05). Moreover, berberine increased the expression of BCL2, reduced intestinal epithelial cell apoptosis (P < 0.05) and decreased the expression of BAX and BAK in the duodenum and jejunum, as well as that of CASP3 and CASP9 in the duodenum and ileum (P < 0.05). Berberine decreased the expression of IL-1β, IL-6, IL-8, TNF-α, and IFN-γ (P < 0.05) and elevated total volatile fatty acids, acetic acid, propionic acid, valeric acid, and isovaleric acid concentrations (P < 0.05). Notably, berberine enhanced the abundance of beneficial bacteria including Enterococcus, Holdemanella, Weissella, Pediococcus, Muribaculum, Colidextribacter, Agathobacter, Roseburia, Clostridium, Fusicatenibacter, and Bifidobacterium. Simultaneously, the relative abundance of harmful and pathogenic bacteria, such as Prevotella, Paraprevotella, Corynebacterium, Catenisphaera, Streptococcus, Enterobacter, and Collinsella, decreased (P < 0.05). Berberine alleviated ETEC-induced intestinal mucosal barrier damage in weaned piglets models. This is associated with enhancement of the physical, chemical, and immune barrier functions of piglets by enhancing intestinal microbiota homeostasis.

A claudin5-binding peptide enhances the permeability of the blood-brain barrier in vitro.

The blood-brain barrier (BBB) maintains brain homeostasis but also prevents most drugs from entering the brain. No paracellular diffusion of solutes is allowed because of tight junctions that are made impermeable by the expression of claudin5 (CLDN5) by brain endothelial cells. The possibility of regulating the BBB permeability in a transient and reversible fashion is in strong demand for the pharmacological treatment of brain diseases. Here, we designed and tested short BBB-active peptides, derived from the CLDN5 extracellular domains and the CLDN5-binding domain of Clostridium perfringens enterotoxin, using a robust workflow of structural modeling and in vitro validation techniques. Computational analysis at the atom level based on solubility and affinity to CLDN5 identified a CLDN5-derived peptide not reported previously called f1-C5C2, which was soluble in biological media, displayed efficient binding to CLDN5, and transiently increased BBB permeability. The peptidomimetic strategy described here may have potential applications in the pharmacological treatment of brain diseases.

Cellular Senescence Contributes to the Dysfunction of Tight Junctions in Submandibular Glands of Aging Mice.

The current mechanism by which aging reduces salivary secretion is unknown. This study investigates the mechanism of aging-related submandibular (SMG) dysfunction and evaluates the therapeutic potential of dental pulp stem cell-derived exosomes (DPSC-exos). We found that the stimulated salivary flow rate was significantly reduced in naturally aging and D-galactose-induced aging mice (D-gal mice) compared to control mice. Acinar atrophy and periductal fibrosis in SMGs and parotid glands (PGs) were observed in naturally aging and D-gal mice, whereas sublingual glands (SLGs) had no notable alterations. We observed the accumulation of senescent cells in the SMGs, along with a decrease in claudin-3 (Cldn-3) expression and alterations in the distribution of Cldn1 and Cldn3. Additionally, after D-gal-induced senescence of SMG-C6 cells, there was a decrease in paracellular pathway permeability, reduced expression of Cldn3 and occludin, and changes in the distribution of Cldn1, 3, 4, and 7. Furthermore, injecting DPSC-exos into the SMGs of D-gal mice improved stimulated salivary flow rate, reduced acinar atrophy, and decreased SA-β-gal activity. Our study identified that increased senescence of SMGs in aging mice can cause a decrease in salivary secretion by disrupting the expression and distribution of tight junction molecules, and injection of DPSC-exos ameliorates SMG secretory dysfunction. These findings may provide new clues to novel therapeutic targets for aging-related dysfunction of SMGs.

The high efficacy of claudin18.2-targeted CAR-T cell therapy in advanced pancreatic cancer with a strategy to ensure the safety of patients.

Pancreatic cancer (PC) is one of the most lethal digestive system tumors. Claudin18.2 is highly expressed in PC tissue and could serve as a suitable target for CAR-T therapy. In the present study, we reported the utilization of tEGFR-expressing claudin18.2-targeted CAR-T cells to treat 3 patients with advanced PC. Intriguingly, all 3 patients achieved disease remission after CAR-T cell infusion, with 1 complete remission (CR) and 2 partial remissions (PR). However, gastric mucosal injury was observed, which was recognized as on-target off-tumor toxicity (OTOT) and may be due to the expression of claudin18.2 on normal gastric tissues. To control the severe OTOT in patient 3, cyclophosphamide and cetuximab were administered to deplete CAR-T cells, and they successfully controlled OTOT. Single cell transcriptome and TCR sequencing revealed the objective alterations of CAR-T cell clones after cetuximab treatment. Collectively, the present study showed the robust anti-tumor activity of claudin18.2-targeted CAR-T cells against PC, and reported the feasibility of antibody-dependent safety switch strategy to control the OTOT caused by CAR-T cells in patients. Our study may pave the way for the development of a novel strategy to treat patients with advanced PC in the future.

The major roles of intestinal microbiota and TRAF6/NF-κB signaling pathway in acute intestinal inflammation in mice, and the improvement effect by Hippophae rhamnoides polysaccharide.

Acute enteritis, an intestinal disease with intestinal inflammation and injury as the main pathological manifestations. Inhibiting the inflammatory response is critical to the treatment of acute enteritis. Previous studies have shown that the Hippophae rhamnoides polysaccharide (HRP) has strong immune-enhancing effects. However, their functions regarding the intestines and the underlying mechanism are still unclear. In this study, the role of HRP in lipopolysaccharide (LPS)-induced acute enteritis in mice and its related mechanisms are discussed from two aspects: intestinal inflammation and intestinal microbiota. Kunming mice were inoculated with LPS to establish animal models of acute enteritis. The results showed that HRP attenuated the histological damage and maintained the intestine mucosal barrier via up-regulating the expression of occludin, claudin-1, and zona occludens-1 (ZO-1), and suppressing the levels of pro-inflammatory cytokines (tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β)). The relative mRNA and protein levels of nuclear factor-kappa B p65 (NF-κBp65) and tumor necrosis factor-receptor-associated factor 6 (TRAF6) in the intestine tissues of LPS-induced acute enteritis mice significantly increased, whereas these adverse changes were alleviated in the HRP intervention groups. Notably, HRP may regulate the expression of the TRAF6/NF-κB signaling pathway by affecting the diversity of the intestinal microbiota. Microbiota analysis showed that HRP promoted the proliferation of beneficial bacteria, including Clostridia_UCG-014, Candidatus_Saccharimonas, Lachnospiraceae_NK4A136_group, Bacteroidota, Deferribacterota, and reduced the abundance of Atopostipes, Corynebacterium, Actinobacteriota, and Desulfobacterota. The studies conformed that the gut microbiota is crucial in HRP-mediated immunity regulation. HRP shows great potential as an immune enhancer and a natural medicine for treating intestinal inflammatory diseases.

Limosilactobacillus reuteri ZY15 Alleviates Intestinal Inflammation and Barrier Dysfunction via AKT/mTOR/HIF-1α/RORγt/IL-17 Signaling and the Gut Microbiota in ETEC K88-Challenged Mice.

Limosilactobacillus reuteri, a recognized probiotic, improves intestinal health in animals, but the mechanism remains unclear. This study investigates the mechanisms by which L. reuteri ZY15, isolated from healthy pig feces, mitigates intestinal barrier damage and inflammation caused by oxidative stress in Enterotoxigenic Escherichia coli (ETEC) K88-challenged mice. The results indicated that L. reuteri ZY15 increased antioxidant capacity by reducing serum reactive oxygen species (ROS) and superoxide dismutase (SOD) levels. L. reuteri ZY15 enhanced the intestinal barrier by upregulating mucin 1, mucin 2, occludin, zonula occludens-1 (ZO-1), and claudin-1 expressions in protein and mRNA levels. It significantly alleviated intestinal inflammation by reducing the proinflammatory cytokines interleukin-1β (IL-1β), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-17 (IL-17) mRNA and protein levels. Notably, L. reuteri ZY15 suppressed intestinal inflammation by inhibiting AKT/mTOR/HIF-1α/RORγt/IL-17 pathway activation. Additionally, it significantly altered the structure of gut microorganisms by enriching Akkermansia and Clostridia_UCG.014, and thereby re-establishing colonization resistance and alleviating ETEC K88-induced intestinal barrier damage and inflammation in mice. Taken together, our findings reveal the protective mechanism of L. reuteri ZY15 in mice challenged with ETEC K88 by regulating AKT/mTOR/HIF-1α/RORγt/IL-17 signaling and microbial imbalance. Leveraging these properties, live L. reuteri ZY15 offers a promising alternative treatment for Escherichia coli-induced diarrhea in weaned piglets.

Estrogen Alleviates Oxidative Bowel Injury and Neuroinflammation in Necrotizing Enterocolitis.

High mortality and morbidity of neonates with necrotizing enterocolitis (NEC) necessitates the investigation of novel therapies to improve outcomes. It was aimed to elucidate the potential therapeutic effect of estrogen receptor agonists on NEC-induced intestinal and brain injury in rats. Sprague-Dawley pups of both sexes were separated from their mothers at postnatal 5th d. Feeding with formula along with a single session of hypoxia was applied to induce NEC, while control pups were kept with their mothers. The NEC rats received either vehicle, estrogen receptor α (ERα) agonist propyl pyrazole triol (1mg/kg/day), ERβ agonist diarylpropionitrile (1mg/kg/day), or 17β-estradiol (1mg/kg/day) during maternal separation. All pups were decapitated on postnatal 9th d to collect intestinal and brain tissue samples. Elevation in proinflammatory cytokines, apoptosis, and microscopically and biochemically evident oxidative injury in both the intestinal and brain tissues were observed in NEC-induced pups. In both the intestinal and brain tissues, nerve growth factor and brain-derived neurotrophic factor protein levels were depleted, expressions of both the ESR1 and ESR2 genes were downregulated, while treatment with 17β-estradiol or ER agonists alleviated extent of oxidative injury of the intestines and brain tissue, upregulated nerve growth factor, brain-derived neurotrophic factor, and ER gene expressions, abolished NEC-induced decrease in claudin-3 expression, increased the survival rates, improved the clinical states of the survived pups at varying degrees. Activation of estrogen signaling by receptor agonists alleviated NEC-induced intestinal and cerebral injury, implicating that estrogen agonists could be regarded as promising preventive/therapeutic agents for NEC.