Intestinal Mucosal Tissue Research Articles

Oxidative Stress in Poultry and the Therapeutic Role of Herbal Medicine in Intestinal Health.

The intensive broiler farming model has accelerated the development of the poultry farming industry. However, it has also inevitably brought about many stressors that lead to oxidative stress in the organism. The intestine is the leading site of nutrient digestion, absorption, and metabolism, as well as a secretory and immune organ. Oxidative stress in animal production can harm the intestine, potentially leading to significant losses for the farming industry. Under conditions of oxidative stress, many free radicals are produced in the animal's body, attacking the intestinal mucosal tissues and destroying the barrier integrity of the intestinal tract, leading to disease. Recently, herbs have been shown to have a favorable safety profile and promising application in improving intestinal oxidative stress in poultry. Therefore, future in-depth studies on the specific mechanisms of herbs and their extracts for treating intestinal oxidative stress can provide a theoretical basis for the clinical application of herbs and new therapeutic options for intestinal oxidative stress injury during poultry farming. This review focuses on the causes and hazards of oxidative stress in the intestinal tract of poultry, and on herbs and their extracts with therapeutic potential, to provide a reference for developing and applying new antioxidants.

Development of an intestinal mucosa ex vivo co-culture model to study viral infections.

Studying viral infections necessitates well-designed cell culture models to deepen our understanding of diseases and develop effective treatments. In this study, we present a readily available ex vivo 3D co-culture model replicating the human intestinal mucosa. The model combines fully differentiated human intestinal epithelium (HIE) with human monocyte-derived macrophages (hMDMs) and faithfully mirrors the in vivo structural and organizational properties of intestinal mucosal tissues. Specifically, it mimics the lamina propria, basement membrane, and the air-exposed epithelial layer, enabling the pioneering observation of macrophage migration through the tissue to the site of viral infection. In this study, we applied the HIE-hMDMs model for the first time in viral infection studies, infecting the model with two globally significant viruses: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human norovirus GII.4. The results demonstrate the model's capability to support the replication of both viruses and show the antiviral role of macrophages, determined by their migration to the infection site and subsequent direct contact with infected epithelial cells. In addition, we evaluated the production of cytokines and chemokines in the intestinal niche, observing an increased interleukin-8 production during infection. A parallel comparison using a classical in vitro cell line model comprising Caco-2 and THP-1 cells for SARS-CoV-2 experiments confirmed the utility of the HIE-hMDMs model in viral infection studies. Our data show that the ex vivo tissue models hold important implications for advances in virology research.IMPORTANCEThe fabrication of intricate ex vivo tissue models holds important implications for advances in virology research. The co-culture model presented here provides distinct spatial and functional attributes not found in simplified models, enabling the evaluation of macrophage dynamics under severe acute respiratory syndrome coronavirus 2 and human norovirus (HuNoV) infections in the intestine. Moreover, these models, comprised solely of primary cells, facilitate the study of difficult-to-replicate viruses such as HuNoV, which cannot be studied in cell line models, and offer the opportunity for personalized treatment evaluations using patient cells. Similar co-cultures have been established for the study of bacterial infections and different characteristics of the intestinal tissue. However, to the best of our knowledge, a similar intestinal model for the study of viral infections has not been published before.

Research progress of probiotics regulating intestinal micro-ecological environment in obese patients after bariatric surgery

Bariatric surgery may cause intestinal microecological environment imbalance due to changes in gastrointestinal anatomy. Some patients may have compli-cations, even regain weight. Probiotics can act on intestinal mucosa, epithelium and gut-associated lymphoid tissue to improve the intestinal microecological environment of obese patients after bariatric surgery. Probiotics can promote the production of short-chain fatty acids, stimulate intestinal cells to release glucagon-like peptide-1, peptide tyrosine-tyrosine, insulin and other endocrine hormones, affect the function of the central nervous system through the gut-brain axis, make patients after bariatric surgery feel full, and reduce blood sugar at the same time. Probiotics can produce lactic acid, acetic acid and lactase, to inhibit the growth of harmful bacteria and to improve gastrointestinal symptoms of patients after bariatric surgery. Probiotics can activate the AMP-activated protein kinase signaling pathway, improve lipid metabolism, and promote the recovery of symptom indicators of nonalcoholic fatty liver disease after bariatric surgery. Probiotics can regulate the release of neurotransmitters or metabolites by the microbiota through the gut-brain axis to affect brain activity and behavior, thus helping patients improve negative emotions after bariatric surgery. This article describes the intestinal microecological environment of obese patients and mechanism of the change after bariatric surgery and summarizes the effects and possible mechanisms of probiotics in improving the intestinal microecological environment of obese patients after bariatric surgery, to provide references for promoting the clinical application of probiotics.

The mechanism by which intestinal flora metabolites regulate ILC2s transformation and intestinal immunity through the metabolite-sensitive receptor Ffar2.

The objective of this article was to investigate how the metabolites produced by the intestinal flora regulate the transformation of ILC2s and intestinal immunity via the receptor Ffar2, which is sensitive to metabolites. Forty male C57BL/6 mice with wild-type characteristics (6-7 weeks in age) and 20 FFAR2-/- mice were acquired from The Jackson Laboratory. The mice were kept in a controlled environment without any disease-causing agents, with the help of air conditioning and a 12-hour cycle of light and darkness. Throughout the experiment, every mouse was provided with unrestricted availability of both food and water. All protocols were performed following the Regulations of Animal Welfare and the recommendations for Animal Testing. According to the research protocol, the mice were categorized into three groups, each consisting of 20 mice. FFAR2-/- mice in the FFAR2-/- group were reared in conventional environments. Male C57BL/6 mice with wild-type characteristics were reared in conventional conditions in the wild-type group. In the group where wild-type mice were inhibited with Ffar2, a total of 20 mice with the wild-type phenotype were chosen to receive intraperitoneal injection of an FFAR2 antagonist at a dosage of 5 mg/kg per day for a continuous period of 3 weeks. qRT-PCR was used to detect the expression of FFAR2 mRNA in the mucosal tissue of the mouse colon. High-throughput sequencing was used to conduct an examination of metabolites in the intestinal tract of mice. Flow cytometry was used to test the quantity of ILC2s. ELISA was used to measure the levels of IL-5 and IL-13 in cellular mouse intestinal mucosal tissues. Flow cytometry was used to detect the quantity of CD4+ and CD8+ T cells. CCL20 and CCL25 expression was analyzed by Western blotting. The level of FFAR2 mRNA expression was higher in the wild-type group than the FFAR2-/- group (P < 0.05), while it was lower in the wild-type + Ffar2 inhibition group than the wild-type group (P < 0.05). No variations were observed in the composition of metabolites and levels of major SCFAs in the gut microbiota of mice across all groups (P > 0.05). The quantity of CRTH2+ and ST2+ cells in the wild-type category exceeded that in the FFAR2-/- category (P < 0.05), whereas the quantity of CRTH2+ and ST2+ cells in the wild-type + Ffar2 inhibition category was lower than that in the wild-type category (P < 0.05). The concentrations of IL-5 and IL-13 were higher in the wild-type group compared with the FFAR2-/- group (P < 0.05), while the wild-type + Ffar2 inhibition group exhibited lower levels of IL-5 and IL-13 than the wild-type group (P < 0.05). The count of CD4+ T cells and CD8+ T cells in the wild-type group showed an increase in comparison to the FFAR2-/- group (P < 0.05), whereas the count of CD4+ T cells and CD8+ T cells in the wild-type + Ffar2 inhibition group exhibited a decrease in comparison to the wild-type group (P < 0.05). The levels of protein expression for CCL20 and CCL25 were higher in the wild-type group compared with the FFAR2-/- group (P < 0.05), whereas the wild-type + Ffar2 inhibition group exhibited lower protein expression of CCL20 and CCL25 than the wild-type group (P < 0.05). Ffar2 has a significant regulatory function in the conversion of ILC2s, consequently impacting the immune response in the intestines. Ffar2, a crucial receptor for metabolites produced by the intestinal flora, plays a vital function in controlling the conversion of ILC2s and the overall immune response in the intestines.

Characterization of faecal microbiota and serum inflammatory markers in dogs diagnosed with chronic enteropathy or small-cell lymphoma: a pilot study

Dogs diagnosed with chronic enteropathy (CE) or small-cell lymphoma (SCL) exhibit marked differences in faecal microbiota and organic acid profiles compared with healthy dogs, as well as immune abnormalities in intestinal mucosal tissue. However, few studies have analysed trace organic acids, such as succinic acid, which have been suggested to be associated with IBD in humans. Therefore, in this study, we compared the faecal microbiota and organic acid profiles as well as serum inflammatory markers between dogs with disease (n = 11; 6 with CE and 5 with SCL) and healthy controls (n = 16). We also performed machine learning and correlation analysis to obtain more detailed insights into the characteristics of affected dogs. These results revealed that dogs with CE and SCL had lower levels of Erysipelotrichaceae (e.g. Turicibacter and Allobaculum), exhibited abnormalities in the succinic acid metabolism (i.e. succinic acid accumulation and decreased levels of Phascolarctobacterium as succinic acid-utilising bacteria) and increased levels of pathobiont bacteria such as Escherichia-Shigella. Additionally, the presence of Dubosiella was significantly negatively correlated with Canine Inflammatory Bowel Disease Activity Index scores. These findings are expected to aid the development of microbiome-based medications and/or supplements, although further verification is needed.

Comprehensive analysis of disulfidptosis-related genes reveals the effect of disulfidptosis in ulcerative colitis

Ulcerative colitis (UC) is a chronic inflammatory condition of the intestinal tract. Various programmed cell death pathways in the intestinal mucosa are crucial to the pathogenesis of UC. Disulfidptosis, a recently identified form of programmed cell death, has not been extensively reported in the context of UC. This study evaluated the expression of disulfidptosis-related genes (DRGs) in UC through public databases and assessed disulfide accumulation in the intestinal mucosal tissues of UC patients and dextran sulfate sodium (DSS)-induced colitis mice via targeted metabolomics. We utilized various bioinformatics techniques to identify UC-specific disulfidptosis signature genes, analyze their potential functions, and investigate their association with immune cell infiltration in UC. The mRNA and protein expression levels of these signature genes were confirmed in the intestinal mucosa of DSS-induced colitis mice and UC patients. A total of 24 DRGs showed differential expression in UC. Our findings underscore the role of disulfide stress in UC. Four UC-related disulfidptosis signature genes—SLC7A11, LRPPRC, NDUFS1, and CD2AP—were identified. Their relationships with immune infiltration in UC were analyzed using CIBERSORT, and their expression levels were validated by quantitative real-time PCR and western blotting. This study provides further insights into their potential functions and explores their links to immune infiltration in UC. In summary, disulfidptosis, as a type of programmed cell death, may significantly influence the pathogenesis of UC by modulating the homeostasis of the intestinal mucosal barrier.

Rapamycin promotes the intestinal barrier repair in ulcerative colitis via the mTOR/PBLD/AMOT signaling pathway

Intestinal barrier dysfunction characterized by the functional loss of the intestinal epithelium's tight junction (TJ) barrier is a key factor in the pathogenesis of ulcerative colitis (UC). Although rapamycin, an mTOR (mechanistic target of rapamycin) inhibitor, has shown promise in inducing clinical remission and mucosal healing in inflammatory bowel disease, its underlying mechanism remains elusive. Thus, this study investigated the role of the mTOR pathway in regulating the intestinal barrier. To investigate the molecular mechanism regulating the intestinal barrier, specific intestinal epithelial phenazine biosynthesis-like domain-containing protein (PBLD)-deficient (PBLDIEC−/−) mice and control wild-type (WT) mice were intraperitoneally injected with rapamycin or MHY1485. To determine the relevance of the findings for UC, we analyzed transcriptome data and single-cell expression profiles from public databases and intestinal mucosal tissues obtained from patients with active UC or colon cancer. We observed that mTOR activation in the intestinal epithelium of patients with active UC. Moreover, in vivo, rapamycin markedly increased the expressions of PBLD and TJ proteins and reduced intestinal inflammation in mice with dextran sulfate sodium-induced enteritis. However, the therapeutic efficacy of rapamycin was notably reduced in PBLDIEC−/− mice. In vitro, rapamycin influenced PBLD expression by modulating the nuclear transcription of transcription factor EB (TFEB). Angiomotin (AMOT) could directly bind to PBLD, and rapamycin could not effectively increase the expression of TJ proteins after the knockdown of PBLD or AMOT. In summary, the administration of rapamycin is a potential treatment for UC, and targeting the mTOR/PBLD/AMOT axis is a potential novel approach for UC treatment.

Shenfu injection alleviate gut ischemia/reperfusion injury after severe hemorrhagic shock through improving intestinal microcirculation in rats

BackgroundShenfu (SF) injection, a traditional Chinese medication, would improve microcirculation in cardiogenic shock and infectious shock. This study was aimed to explore the therapeutic potential of the SF injection in gut ischemia-reperfusion (I/R) injury after severe hemorrhagic shock (SHS) and resuscitation. Furthermore, we also investigated the optimal adm？ inistration timing. MethodsTwenty-four male SD rats were randomly divided into four groups: Sham group (sham, n = 6), Control group (n = 6), SF injection group (SF, n = 6), and Delayed Shenfu injection administration group (SF-delay, n = 6). In SHS and resuscitation model, rats were induced by blood draw to a mean arterial pressure (MAP) of 40 ± 5 mmHg within 1 h and then maintained for 40 min; HR, MAP ‘were recorded, microcirculation index [De Backer score, perfused small vessel density (PSVD), total vessel density (TVD), microcirculation flow index score (MFI), flow heterogeneity index (HI)] were analyzed. The blood gas index was detected, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), diamine oxidase (DAO), malondialdehyde (MDA) were measured by ELISA; ZO-1, and claudin-1 were measured by Western blotting. In addition, hematoxylin-eosin (HE) and periodic acid schiff (PAS) staining pathological sections of the intestinal mucosal tissues were also performed. ResultsSF injection increased the MAP, relieved the metabolic acidosis degree associated with the hypoperfusion, and improved the intestinal microcirculatory density and perfusion quality after I/R injury. The expression of DAO, MDA in intestinal tissue, and plasma IL-6, TNF-α significantly decreased in the SF injection group compared to the control group. The concentration of ZO-1 and claudin-1 is also higher in the SF injection group. In addition, the HE and PAS staining results also showed that SF injection could decrease mucosal damage and maintain the structure. In the SF-delay group, the degree of intestinal tissue damage was intermediate between that of the control group and SF injection group. ConclusionsSF injection protect the intestine from I/R injury induced by SHS and resuscitation, the mechanism of which might be through improving intestinal microcirculation, reducing the excessive release of inflammatory factors and increasing intestinal mucosal permeability. Furthermore, the protection effect is more pronounced if administration during the initial resuscitation phase.

MiR-141-3p attenuates RSL3-induced ferroptosis and intestinal epithelial-mesenchymal transition via directly inhabits ZEB1 in intestinal Behçet's syndrome.

The aims of this study were to investigate whether the ferroptosis is involved in intestinal Behçet's syndrome (IBS), and to identify if miR-141-3p could attenuate RAS-selective lethal 3 (RSL3)-induced ferroptosis and intestinal epithelial to mesenchymal transition (EMT) via directly inhabits zinc fnger E-box binding homeobox 1 (ZEB1). The expressions of ferroptosis-related proteins in the intestinal tissues of patients with IBS were investigated by immunohistochemistry and quantitative real-time PCR (qRT-PCR). Malondialdehyde (MDA) contents of the intestinal tissues and cells were detected. Serum from IBS patients and RSL3 were co-cultured with intestinal epithelial cells in vitro. In order to investigate whether RSL3-induced ferroptosis can be ameliorated by miR-141-3p, the intestinal epithelial cells were firstly stimulated with RSL3 and then incubated with miR-141-3p mimics. Western blot was used to measure the expression of EMT and ferroptosis-related proteins. Expression of GPX4 (22.51% ± 2.05%, 51.75% ± 3.47%, t = - 7.77, p = 0.000) and xCT (17.49% ± 1.57%, 28.73% ± 1.75%, t = - 4.38, p = 0.003) were significantly lower in intestinal mucosal tissues of patients with IBS compared with HC group. Compared with the HC samples, the IBS specimens had significantly higher MDA (t = 4.32, p = 0.01). Moreover, the relative mRNA levels of ferritin light chain (FTL) (t = 4.07, p = 0.02) and ferritin heavy chain (FTH) (t = 8.82, p = 0.001) in the intestinal tissues were significant higher in IBS patients than in HC group. Serum from IBS patients could induce intestinal epithelial cell ferroptosis in vitro. Moreover, miR-141-3p could attenuate intestinal epithelial cell ferroptosis-induced by RSL3 and intestinal EMT via targeting ZEB1 in vitro. Ferroptosis were induced in patients with IBS. Moreover, the serum from IBS patients could induce ferroptosis in vitro. miR-141-3p could attenuate intestinal epithelial cell ferroptosis and intestinal EMT via targeting ZEB1. Therefore, miR-141-3p may open new avenues for the treatment of IBS in the future. Key Points • Ferroptosis in IBS is first reported in this study. • In this study, we explored that the serum from IBS patients could induce ferroptosis in vitro and miR-141-3p could attenuate intestinal epithelial cell ferroptosis and intestinal EMT via targeting ZEB1.

Diagnostic Utility of Cytomegalovirus (CMV) DNA Quantitation in Ulcerative Colitis.

Cytomegalovirus (CMV) colitis is a critical condition associated with severe complications in ulcerative colitis (UC). This study aimed to investigate the diagnostic value of the presence of CMV DNA in intestinal mucosa tissue and blood samples in patients with active UC. This study included 81 patients with exacerbated symptoms of UC. Patient data were obtained from the Hospital Information Management System. CMV DNA in colorectal tissue and plasma samples were analyzed using a real-time quantitative PCR assay. CMV markers were detected using immunohistochemistry and hematoxylin-eosin staining. Immunohistochemistry positivity was observed in tissue samples from eight (9.9%) patients. Only one (1.2%) patient showed CMV-specific intranuclear inclusion bodies. CMV DNA was detected in 63.0% of the tissues (median: 113 copies/mg) and in 58.5% of the plasma samples (median: 102 copies/mL). For tissues, sensitivity and the negative predictive value (NPV) for qPCR were excellent (100.0%), whereas specificity and the positive predictive value (PPV) were low (41.9% and 15.7%, respectively). For plasma, sensitivity and NPV were high (100.0%) for qPCR, whereas specificity and PPV were low (48.6% and 24.0%, respectively). CMV DNA ≥392 copies/mg in tissue samples (sensitivity 100.0% and specificity 83.6%) and ≥578 copies/mL (895 IU/mL) in plasma samples (sensitivity 66.7% and specificity 100.0%) provided an optimal diagnosis for this test. The qPCR method improved patient management through the early detection of CMV colitis in patients with UC. However, reliance on qPCR positivity alone can lead to overdiagnosis. Quantification of CMV DNA can improve diagnostic specificity, although standardization is warranted.

Upregulating KLF11 ameliorates intestinal inflammation in mice with 2, 4, 6-trinitrobenesulfonic acid-induced colitis by inhibiting the JAK2/STAT3 signaling pathway

To investigate the expression level of Kruppel-like transcription factor family member KLF11 in intestinal mucosal tissues of Crohn's disease (CD) and its regulatory effect on intestinal inflammation in CD-like colitis. We examined KLF11 expression levels in diseased and normal colon mucosal tissues from 12 CD patients and 12 patients with colorectal cancer using immunofluorescence staining. KLF11 expression was also detected in the colon mucosal tissues of a mouse model of 2, 4, 6-trinitrobenesulfonic acid (TNBS)-induced colitis. A recombinant adenoviral vector was used to upregulate KLF11 expression in the mouse models and the changes in intestinal inflammation was observed. A Caco-2 cell model with stable KLF11 overexpression was constructed by lentiviral infection. The effect of KLF11 overexpression on expressions of JAK2/STAT3 signaling pathway proteins was investigated using immunoblotting in both the mouse and cell models. The mouse models were treated with coumermycin A1, a JAK2/STAT3 signaling pathway agonist, and the changes in intestinal inflammatory responses were observed. The expression level of KLF11 was significantly lowered in both the clinical specimens of diseased colon mucosal tissues and the colon tissues of mice with TNBS-induced colitis (P < 0.05). Adenovirus-mediated upregulation of KLF11 significantly improved intestinal inflammation and reduced the expression levels of inflammatory factors in the intestinal mucosa of the colitis mouse models (P < 0.05). Overexpression of KLF11 significantly inhibited the expression levels of p-JAK2 and p-STAT3 in intestinal mucosal tissues of the mouse models and in Caco-2 cells (P < 0.05). Treatment with coumermycin A1 obviously inhibited the effect of KLF11 upregulation for improving colitis and significantly increased the expression levels of inflammatory factors in the intestinal mucosa of the mouse models (P < 0.05). KLF11 is downregulated in the intestinal mucosa in CD, and upregulation of KLF11 can improve intestinal inflammation and reduce the production of inflammatory factors probably by inhibiting the JAK2/STAT3 signaling pathway.

MURAMYL DIPEPTIDE CAUSES MITOCHONDRIAL DYSFUNCTION AND INTESTINAL INFLAMMATORY CYTOKINE RESPONSES IN RATS.

Introduction: Intestinal flora and the translocation of its products, such as muramyl dipeptide (MDP), are common causes of sepsis. MDP is a common activator of the intracellular pattern recognition receptor NOD2, and MDP translocation can cause inflammatory damage to the small intestine and systemic inflammatory responses in rats. Therefore, this study investigated the effects of MDP on the intestinal mucosa and distant organs during sepsis and the role of the NOD2/AMPK/LC3 pathway in MDP-induced mitochondrial dysfunction in the intestinal epithelium. Methods: Fifty male Sprague Dawley rats were randomly divided into five treatment groups: lipopolysaccharide (LPS) only, 1.5 and 15 mg/kg MDP+LPS, and 1.5 and 15 mg/kg MDP+short-peptide enteral nutrition (SPEN)+LPS. The total caloric intake was the same per group. The rats were euthanized 24 h after establishing the model, and peripheral blood and small intestinal mucosal and lung tissues were collected. Results: Compared to the LPS group, both MDP+LPS groups had aggravated inflammatory damage to the intestinal mucosal and lung tissues, increased IL-6 and MDP production, increased NOD2 expression, decreased AMPK and LC3 expression, increased mitochondrial reactive oxygen species production, and decreased mitochondrial membrane potential. Compared to the MDP+LPS groups, the MDP+SPEN+LPS groups had decreased IL-6 and MDP production, increased AMPK and LC3 protein expression, and protected mitochondrial and organ functions. Conclusions: MDP translocation reduced mitochondrial autophagy by regulating the NOD2/AMPK/LC3 pathway, causing mitochondrial dysfunction. SPEN protected against MDP-induced impairment of intestinal epithelial mitochondrial function during sepsis.

Identifying functional dysregulation of NOD2 variant Q902K in patients with Yao syndrome

Background and objectivesThe study investigated the pathogenesis of Yao syndrome (YAOS), a rare systemic autoinflammatory disease associated with the nucleotide-binding oligomerization domain containing 2 (NOD2) gene variants.MethodsRNA sequencing analyses were used to detect transcriptomic profile changes. Immunoblot and immunohistochemistry were used to examine the NOD2-mediated inflammatory signaling pathways and ELISA was used to detect cytokines.ResultsTranscriptome analysis of YAOS revealed NOD-like receptor signaling pathway enrichment. Compared with HCs, P-RIP2, p-p65, p-p38, p-ERK, and p-JNK notably increased in PBMCs of a patient with YAOS. P-RIP2, p-p65, and p-p38 elevated in small intestinal mucosa tissues. P-p65 and p-p38 in synovial tissues from YAOS were higher than those in patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Serum interleukin (IL)-6 level along with tumor necrosis factor (TNF)-α and IL-6 secreted from PBMCs were markedly higher in patients with YAOS in comparison to healthy controls (HCs). The supernatants of synovial cells from a patient with YAOS showed substantially higher IL-1β and IL-6 levels than those of RA and OA. Canakinumab therapy of a Q902K heterozygous patient with YAOS resulted in notable clinical improvement.ConclusionOverproduction of pro-inflammatory cytokines and the hyperactivation of NOD2-mediated signaling pathways were found in the NOD2 variant Q902K patient with YAOS. NOD2-RIP2-MAPK pathway might play a pivotal role in the pathogenesis of YAOS. These results provide new perspectives for targeted therapies in YAOS.

The recovery of intestinal barrier function and changes in oral microbiota after radiation therapy injury.

Colorectal cancer (CRC) is the third most common malignant tumor, and neoadjuvant chemo-radiotherapy is usually recommended for advanced stage colorectal cancer. Radiotherapy can cause damage to intestinal mucosal barrier, which may be related to perioperative complications. Intestinal microbiota is one of the constituents of the intestinal mucosal biological barrier, and literature reports that patients with CRC have changes in corresponding oral microbiota. This study aims to analyze the levels of immunoglobulin SIgA, inflammatory factors, lymphocyte subsets quantity, and proportion in surgical specimens of intestinal mucosa at different time intervals after radiotherapy, in order to seek investigation for the optimal surgical time after radiotherapy and to provide evidence for finding probiotics or immunomodulators through high-throughput sequencing of bacterial 16s rRNA in patients' saliva microbiota. Ultimately, this may provide new ideas for reducing perioperative complications caused by radiotherapy-induced intestinal damage. We selected intestinal mucosal tissue and saliva samples from over 40 patients in our center who did not undergo radiotherapy and underwent surgery at different time intervals after radiotherapy. Detection of SIgA was performed using ELISA assay. Western Blotting was used to detect IL-1β, IL-6, and IL-17 in the intestinal mucosal tissue. Flow cytometry was used to detect CD4 and CD8. And the microbial community changes in saliva samples were detected through 16s rRNA sequencing. After radiotherapy, changes in SIgA, various cytokines, CD4CD8 lymphocyte subsets, and oral microbiota in the intestinal mucosal tissue of rectal cancer patients may occur. Over time, this change may gradually recover. In colorectal cancer, oncological aspects often receive more attention, while studies focusing on the intestinal mucosal barrier are less common. This study aims to understand the repair mechanisms of the intestinal mucosal barrier and reduce complications arising from radiotherapy-induced damage. The relationship between oral microbiota and systemic diseases has gained interest in recent years. However, the literature on the oral microbiota after radiotherapy for rectal cancer remains scarce. This study addresses this gap by analysing changes in the salivary microbiota of rectal cancer patients before and after radiotherapy, shedding light on microbiota changes. It aims to lay the groundwork for identifying suitable probiotics or immunomodulators to alleviate perioperative complications and improve the prognosis of CRC.

P1080 Real-world Study on Early Predictors of Infliximab Treatment Efficacy in Crohn's disease

Abstract Background Infliximab (IFX) can effectively induce and maintain the clinical remission of Crohn's disease (CD). However, a substantial number of CD patients experience primary or secondary nonresponse to IFX during treatment. Therefore, it is essential to identify early predictors of IFX treatment efficacy. Methods This study included 147 CD patients. Based on clinical outcomes, patients were categorized into IFX nonresponse and response group. We collected and compared laboratory data on blood routine examination, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), fecal calprotectin and post-induction IFX drug concentration and antibody levels in both groups. Logistic regression models were employed to identify potential factors associated with the risk of IFX nonresponse. Machine learning using random forest analysis was utilized to quantitatively assess the predictive features for IFX treatment efficacy and ROC curves was used to evaluate the model’s accuracy. Additionally, we performed HE staining and double immunofluorescence staining for CD64 and CD206 on intestinal biopsy tissues obtained before IFX treatment to compare the difference of macrophage subtypes between the nonresponse and response groups. Results Data from both cohorts revealed that patients in the IFX nonresponse group had lower drug concentration (P &amp;lt; 0.001), higher antibody levels (P &amp;lt; 0.001), and increasing ESR during the induction therapy (P &amp;lt; 0.001). Univariate and multivariate Logistic regression models demonstrated that IFX drug concentration and the ratio of ESR before and after induction therapy were associated with the risk of nonresponse. After the induction period, for each unit increase in drug concentration (1 μg/ml), the risk of IFX nonresponse decreased by 23% (RR= 0.77, 95% CI = 0.68-0.89), while each doubling of the ESR ratio after induction was associated with a 1.43-fold increase in the risk of nonresponse (RR= 2.43, 95% CI = 1.48-4.00). After combining data from the two cohorts, random forest machine learning analysis revealed that drug concentration below 1.5 μg/ml and an increase in ESR during induction could predict IFX nonresponse, with ROC curve areas of 0.740 and 0.651, respectively. Furthermore, immunofluorescence staining of intestinal mucosal biopsy tissues before IFX treatment showed a higher proportion of M1-type macrophages in the nonresponse group compared to the response group (P &amp;lt; 0.05). Conclusion Our study suggests that lower post-induction IFX drug concentrations and an increase in ESR during the induction phase are predictive of IFX nonresponse. Additionally, a higher proportion of M1-type macrophages in the intestinal mucosa before IFX treatment is associated with IFX nonresponse.

S100A8-Mediated Inflammatory Signaling Drives Colorectal Cancer Progression via the CXCL5/CXCR2 Axis.

Background: S100A8/S100A9 belong to the S100 calcium-binding protein family and play an essential role in the progression of chronic inflammation in diseases. It also regulates various biological processes such as tumor cell survival, apoptosis, and invasive metastasis. The extracellular form of S100A8/S100A9 functions by modulating cellular oxidative metabolism and facilitating inflammation-to-cancer progression. This modulation occurs through specific binding to receptors like RAGE and TLR4 and activation of signaling pathways including STAT3 and NF-κB. In tumor cells, S100A8 and S100A9 induce phenotypic changes by influencing calcium ion concentrations and other pathways. However, the precise function of high S100A8/S100A9 expression in colorectal cancer cells remains unclear. Methods: To explore the role of S100A8/S100A9 in colorectal cancer, we used immunohistochemistry and data from GEO and TCGA databases to analyze its expression in colorectal cancer cells, normal intestinal mucosa, and adjacent tissues. Functional models of high S100A8/S100A9 expression in colorectal cancer cells were established through transfection with overexpression plasmids. Protein microarrays, enzyme-linked immunosorbent assays (ELISAs), and real-time PCR were employed to assess the expression and secretion of 40 cytokines. MTT and Transwell invasion assays were conducted to evaluate changes in cell proliferation, invasion, and chemotaxis. Finally, tail vein and subcutaneous tumorigenesis assays assessed cell proliferation and migration in vivo. Results: We observed significantly higher expression of S100A8/S100A9 in colorectal cancer epithelial cells compared to normal intestinal mucosa and adjacent tissues. Overexpression of S100A8/S100A9 in mouse colon cancer cells CT26.WT led to differential increases in the secretion levels of various cytokines (CXCL5, CXCL11, GM-CSF, G-CSF, IL1a, IL1b, sTNF RI, and CCL3). Additionally, this overexpression activated signaling pathways such as STAT3, NF-κB, and ERK-MAPK. The synthesis and secretion of inflammatory factors could be inhibited by using NF-κB and ERK-MAPK pathway inhibitors. Moreover, S100A8 promotes the proliferation and invasion of colon cancer cells. Notably, the CXCR2 inhibitor (SB265610) effectively reversed the phenotypic changes induced by the CXCL5/CXCR2 biological axis. Conclusions: Our findings indicate that increased expression of S100A8 and S100A9 in colon cancer epithelial cells enhances the secretion of inflammatory factors by activating NF-κB, ERK-MAPK, and other signaling pathways. S100A8 facilitates colon cancer cell proliferation, invasion, and metastasis through the CXCL5/CXCR2 biological axis.

Correlation Between DCAMKL-1 Protein Expression and K-ras Gene Mutation in Colorectal Cancer.

To investigate the correlation between doublecortin and CaM kinase-like-1 (DCAMKL-1) protein expression, K-ras gene mutation, and their impact on patient prognosis in colorectal cancer (CRC). Immunohistochemistry was used to detect the expression of DCAMKL-1 protein in 60 cases of colorectal adenoma, 82 cases of CRC (including 65 cases of lymph node metastasis) and paraffin-embedded paracancerous intestinal mucosal tissue. K-ras gene mutations in primary CRC lesions were detected using an amplification-refractory mutation system and fluorescent polymerase chain reaction. The relationship between DCAMKL-1 protein expression and K-ras gene mutations with the clinicopathological characteristics of patients with CRC was analyzed. Univariate Kaplan‒Meier survival analysis and multivariate Cox regression analysis were performed using follow-up data. The mutation rate of the K-ras gene in 82 cases of CRC was 48.8% (40/82). The positivity rate for the presence of DCAMKL-1 protein in CRC was 70.7% (58/82), significantly higher than that for colorectal adenomas (53.3%; 32/60) and paracancerous intestinal mucosa (0%; 0/82) (P<0.05). The positive expression rate for the presence of DCAMKL-1 protein in 65 patients with lymph node metastasis was higher in the primary lesions (69.2%; 45/65) than in the lymph node metastases (52.3%; 34/65) (χ2=12.087, P=0.001). The K-ras gene mutation status was positively correlated with DCAMKL-1 protein expression (r=0.252, P=0.022). In this study, a potential positive correlation between K-ras gene mutation and DCAMKL-1 protein expression was identified in CRC tissues. The assessment of K-ras gene mutation status and DCAMKL-1 protein expression holds promise for augmenting early diagnosis and prognosis evaluation in CRC. This approach may improve the overall prognosis and survival outcomes for CRC patients.

The role of TLR4/MyD88/NF-κB in the protective effect of ulinastatin on the intestinal mucosal barrier in mice with sepsis

ObjectiveTo investigate the effect of the TLR4/MyD88/NF-κB (Toll-like receptor 4/myeloid differentiation factor/nuclear factor kappa B) signalling pathway on the protective effect of ulinastatin on the intestinal mucosal barrier in mice with sepsis.MethodsA mouse model of sepsis was established by classical caecal ligation and perforation. Forty-four SPF C57BL/6 mice were randomly divided into the following four groups with 11 mice in each group: the control group (Con group), ulinastatin group (Uti group), Uti + LPS (lipopolysaccharide, LPS) group (Uti + LPS group) and LPS group. Mice in the Con group and Uti group received saline or ulinastatin injected 2 h after modelling; Mice in the Uti + LPS group received LPS injected 0 h after modelling, other procedures were the same as in the Uti group; Mice in the LPS group received LPS only. At 48 h after surgery, the levels of TNF-α (tumour necrosis factor-α, TNF-α), IL-6 (interleukin-6, IL-6) and IL-1β (interleukin-1β, IL-1β) in vein, and the expression of TLR4, MyD88 and NF-κB mRNA in small intestinal mucosa tissues using ELISA and RT‒PCR.ResultsThe pathological specimens showed increased inflammatory injury in the Con and LPS groups, while these injuries and changes improved in the Uti group. The scores of intestinal mucosal injury at 48 h of Uti injection were significantly lower than those of the Con group (P < 0.001), while the scores of intestinal mucosal injury of Uti + LPS were significantly higher than those of the Uti group (P = 0.044). The expression of TNF-α, IL-6 and IL-1β in the Uti decreased significantly at 48 h after surgery than that in the Con group (P = 0.001, P = 0.014, P = 0.004), while the expression of TNF-α, IL-6 and IL-1β in the Uti + LPS group increased significantly after surgery than that in the Uti group (P = 0.026, P = 0.040, P = 0.039). The expression of TLR4, MyD88 and NF-κB mRNA in the Uti group decreased significantly compared with that in the Con group (P = 0.001, P = 0.021, P = 0.007), while the expression of TLR4, MyD88 and NF-κB mRNA in the Uti + LPS group was higher than that in the Uti group (P = 0.023, P = 0.040, P = 0.045).ConclusionThese findings indicate that the protective effect of ulinastatin on the intestinal mucosal barrier against sepsis may be mediated through the TLR4/MyD88/NF-κB pathway.

Stigmasterol: Remodeling gut microbiota and suppressing tumor growth through Treg and CD8+ T cells in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC), the most primary malignant liver tumor and is ranked as the fifth most common malignancy worldwide. Despite various therapeutic approaches being used in clinical practice, the overall effectiveness remains insufficient. Stigmasterol, a compound known for its anti-tumor properties and ability to induce apoptosis in tumor cells, has been found to influenced the composition of the intestinal microbiota. However, the mechanism through which stigmasterol influences the intestinal microbial-host crosstalk in HCC remains elusive. PurposeThis study was to investigate whether stigmasterol can remodel gut microbiota, and suppress tumor volume by regulating Treg and IFN-γ+ CD8+ cell in the host with HCC. MethodStigmasterol (at dosages of 0, 50, 100, or 200 mg/kg) was orally administered to Balb/c mice with subcutaneous tumor once every 2 days for 3 weeks. ResultsWe first found that tumors volume in the group treated with 100 mg/kg stigmasterol were significantly decreased compared with those in the control group (P < 0.05), which exhibited a similar effect as the sorafenib treatment in mice with HCC. This resulted in a significant upregulation of Caspase3, Bax, and P53 expressions, as well as a decrease in Cyclin D1 expression, ultimately leading to a reduction in tumor volume. Additionally, stigmasterol can alter the α and β diversity of the intestinal flora and significantly increase the abundance of Lactobacillus_johnsonii, Lactobacillus_murinus, and Lactobacillus_reuteri (P<0.05), which can lead to a decrease in the ratio of regulatory T cells (Tregs) to CD8+ T cells in the intestinal tract and tumor tissue, and consequently enhance immune response in the tumor microenvironment (TME) in the host with HCC. ConclusionIn this study, we initially utilized different dosages of stigmasterol to intervene in mice with HCC and confirmed its inhibitory effects on tumor growth in vivo, and discovered that stigmasterol affected Lactobacillus johnsonii, Lactobacillus murinus, and Lactobacillus reuteri, resulting in an increased proportion of IFN-γ+ CD8+ T cells and Treg cells in both the intestinal mucosa and tumor tissues, and ultimately leading to increased levels of apoptotic proteins and the subsequent death of tumor cells, which shed light on the effect of stigmasterol on host intestinal tissue and intratumoral immune cells by reshaping the intestinal microbiota, and provide a theoretical foundation for the potential clinical application of stigmasterol in the treatment of HCC.

High-dimensional mass cytometry reveals systemic and local immune signatures in necrotizing enterocolitis.

Patients with necrotizing enterocolitis display severe gastrointestinal complications of prematurity, but the mechanism driving this clinical profile remains unknown. We used mass cytometry time-of-flight to characterize and compare immune cell populations in the blood and intestine tissue from patients with and without (controls) necrotizing enterocolitis at single-cell resolution. We completed a deep mapping of the immune system of the peripheral blood mononuclear cells and intestinal mucosa tissue using mass cytometry to evaluate immune cell types, which revealed global immune dysregulation characteristics underlying necrotizing enterocolitis. Compared with controls, natural killer cells display signs of heightened activation and increased cytotoxic potential in the peripheral blood and mucosa of patients with necrotizing enterocolitis. Furthermore, CD4+ T effector memory cells, non-classical monocytes, active dendritic cells, and neutrophils were specifically enriched in the mucosa, suggesting trafficking from the periphery to areas of inflammation. Moreover, we mapped the systemic and local distinct immune signatures suggesting patterns of cell localization in necrotizing enterocolitis. We used mass cytometry time-of-flight technology to identify immune cell populations specific to the peripheral blood and intestinal mucosa tissue from patients with necrotizing enterocolitis and controls. This information might be used to develop precise diagnosis and therapies that target specific cell populations in patients with necrotizing enterocolitis.