Colonic Epithelial Cells Research Articles

Astragalus mongholicus Bunge extract improves ulcerative colitis by promoting PLCB2 to inhibit colonic epithelial cell pyroptosis

Ethnopharmacological relevanceAstragalus mongholicus Bunge (AM) and its active ingredients are mainly used for anti-inflammatory, antiviral, antioxidant, immune regulation, cardiovascular and nervous system protection, anti-cancer, anti-tumor and so on. Aim of the studyTo explore the Astragalus mongholicus Bunge extract pharmacological mechanisms and biology processes which improves ulcerative colitis (UC). Materials and methodsDextran sulfate sodium (DSS)-induced UC models in C57BL/6 mice were established, and the mice were treated with Astragalus mongholicus Bunge extract or salazosulfapyridine (SASP). DSS-induced mice- and human-derived colonic epithelial cell lines were used to reveal the inflammatory environment of UC. After treatment with Astragalus mongholicus Bunge extract, the expression of phospholipase C-β 2 (PLCB2) in the cells was detected by quantitative real-time PCR (qRT-PCR), and cell proliferative activity was detected by cell counting kit 8 (CCK-8) assay. Finally, the levels of pyroptosis-related inflammatory factors in cell culture supernatants was detected by ELISA. ResultsTreatment of UC mice with Astragalus mongholicus Bunge extract do significantly improved DAI scores and histopathological damage scores, and decreased the levels of Eotaxin, GCSF, KC, MCP-1, TNF-α, and IL-6. Besides, Astragalus mongholicus Bunge extract inhibited the expression of nucleotide-binding oligomerization segment-like receptor family 3 (NLRP3), cleaved Caspase-1, and GSDMD-N in the colonic tissues, and reduced the levels of inflammation-related factors IL-1β and IL-18 in serum and tissues. In vitro, Astragalus mongholicus Bunge extract partially reversed the DSS-induced reduction of PLCB2 expression in CP-M030 and NCM460, promoted cell proliferative activity, and reduced the levels of IL-1β and IL-18. ConclusionsIn DDS-induced UC mice, Astragalus mongholicus Bunge extract improves ulcerative colitis by inhibiting colonic epithelial cell pyroptosis through PLCB2 promotion.

Hypoglycemic Effect of Polysaccharides from Physalis alkekengi L. in Type 2 Diabetes Mellitus Mice.

Type 2 diabetes mellitus (T2DM) is a common metabolic disease that adversely impacts patient health. In this study, a T2DM model was established in ICR mice through the administration of a high-sugar and high-fat diet combined with the intraperitoneal injection of streptozotocin to explore the hypoglycemic effect of polysaccharides from Physalis alkekengi L. After six weeks of treatment, the mice in the high-dosage group (800 mg/kg bw) displayed significant improvements in terms of fasting blood glucose concentration, glucose tolerance, serum insulin level, insulin resistance, and weight loss (p < 0.05). The polysaccharides also significantly regulated blood lipid levels by reducing the serum contents of total triglycerides, total cholesterol, and low-density lipoproteins and increasing the serum content of high-density lipoproteins (p < 0.05). Furthermore, they significantly enhanced the hepatic and pancreatic antioxidant capacities, as determined by measuring the catalase and superoxide dismutase activities and the total antioxidant capacity (p < 0.05). The results of immunohistochemistry showed that the P. alkekengi polysaccharides can increase the expression of GPR43 in mice colon epithelial cells, thereby promoting the secretion of glucagon-like peptide-1. In summary, P. alkekengi polysaccharides can help to regulate blood glucose levels in T2DM mice and alleviate the decline in the antioxidant capacities of the liver and pancreas, thus protecting these organs from damage.

In vitro anticancer, antioxidant, cytotoxic activity and phytochemical characterization of Inula viscosa L. essential oil against colorectal cancer cell line HCT-116

Human colorectal cancer cell line (HCT116) cells are adherent epithelial cells derived from the human colorectal carcinoma cell line commonly used to study inflammatory responses in colonic epithelial cells. This study aimed to evaluate the protective effects of Inula viscosa essential oil (IVEO), an effective antioxidant compound with a radical scavenging activity in vitro using HCT-116 cells. The chemical composition of hydrodistilled IVEO was analyzed by GC-FID and GC-MS systems. The major compounds of essential oil were borneol acetate (51.54%) and borneol (21.17%). The cytotoxicity was determined using the colorimetric MTT assay for 24 h, and 48 h. Malondialdehyde (MDA) level, some enzymes activities including catalase (CAT) and superoxide dismutase (SOD) were measured in the HCT 116 cells both with and without IVEO treatment. In this study, there was a significant decrease in activities of cellular SOD and CAT, increased in the MDA levels in the cells treated with IVEO. These result indicate that IVEO can serve as an agent with strong potential to be utilized for the development of colorectal cancer chemoprevention. However, further molecular investi-gation is required to understand the molecular bearings of IVEO.

Grifola frondosa polysaccharide's therapeutic potential in oxazolone-induced ulcerative colitis

Grifola frondosa polysaccharide (GFP) is a consumable fungus recognized for its potential health advantages. The present study aimed to investigate the development and potential etiologies of ulcerative colitis (UC) utilizing oxazolone (OXZ) as an inducer in mice, along with assessing the therapeutic effects of GFP at varying doses in UC mice, with sulfasalazine (SASP) serving as the positive control. The obtained results indicated that OXZ intervention in mice induced numerous physical manifestations of UC, including increased disease activity index (DAI), decreased goblet cell division, enhanced fibrosis, reduced expression of Claudin1 and Zona encludens protein1 (ZO-1), decreased proliferative activity of colonic mucosal epithelial cells, disturbed oxidation balance, and alterations in intestinal flora. Nonetheless, GFP intervention significantly ameliorated or even resolved these abnormal indicators to a considerable extent. Consequently, this study suggests that GFP might serve as a prebiotic to regulate intestinal flora, mitigate enterotoxin production, restore oxidative balance, thereby reducing the generation of inflammatory mediators, restoring the intestinal barrier, and ultimately improving OXZ-induced UC in mice. GFP demonstrates promising potential as a candidate drug for colitis treatment and as a dietary supplement for alleviating intestinal inflammatory issues.

Optimizing single-cell RNA sequencing methods for human colon biopsies: droplet-based vs. picowell-based platforms.

Single-cell RNA sequencing (scRNA) has empowered many insights into gastrointestinal microenvironments. However, profiling human biopsies using droplet-based scRNA (D-scRNA) is challenging since it requires immediate processing to minimize epithelial cell damage. In contrast, picowell-based (P-scRNA) platforms permit short-term frozen storage before sequencing. We compared P- and D-scRNA platforms on cells derived from human colon biopsies. Endoscopic rectosigmoid mucosal biopsies were obtained from two adults and conducted D-scRNA (10X Chromium) and P-scRNA (Honeycomb HIVE) in parallel using an individual's pool of single cells (> 10,000 cells/participant). Three experiments were performed to evaluate 1) P-scRNA with cells under specific storage conditions (immediately processed [fresh], vs. frozen at -20C vs. -80C [2 weeks]); 2) fresh P-scRNA versus fresh D-scRNA; and 3) P-scRNA stored at -80C with fresh D-scRNA. Significant recovery of loaded cells was achieved for fresh (80.9%) and -80C (48.5%) P-scRNA and D-scRNA (76.6%), but not -20C P-scRNA (3.7%). However, D-scRNA captures more typeable cells among recovered cells (71.5% vs. 15.8% Fresh and 18.4% -80C P-scRNA), and these cells exhibit higher gene coverage at the expense of higher mitochondrial read fractions across most cell types. Cells profiled using D-scRNA demonstrated more consistent gene expression profiles among the same cell type than those profiled using P-scRNA. Significant intra-cell-type differences were observed in profiled gene classes across platforms. Our results highlight non-overlapping advantages of P-scRNA and D-scRNA and underscore the need for innovation to enable high-fidelity capture of colonic epithelial cells. The platform-specific variation highlights the challenges of maintaining rigor and reproducibility across studies that use different platforms.

Ferulic Acid Inhibits Arsenic-Induced Colon Injury by Improving Intestinal Barrier Function.

The prolonged exposure to arsenic results in intestinal barrier dysfunction, which is strongly concerned with detrimental processes such as oxidative stress and the inflammatory response. Ferulic acid (FA), as a phenolic acid, possesses the capability to mitigate arsenic-induced liver damage and cardiotoxic effects dependent on inhibition of oxidative stress and inflammatory responses. FA can mitigate testicular tissue damage and alveolar epithelial dysfunction, the mechanism of which may rely on nuclear factor erythroid 2-related factor 2/heme oxygenase 1 (Nrf2/HO-1) activation and nuclear factor-kappa B (NF-κB) pathway blocking. Based on the antioxidant and anti-inflammatory properties of FA, we speculated that FA might have the potential to inhibit arsenic-induced intestinal damage. To confirm this scientific hypothesis, mice exposed to sodium arsenite were treated with FA to observe colonic histopathology and TJ protein levels, and oxidative stress and TJ protein levels in Caco-2 cells exposed to sodium arsenite were assessed after FA intervention. In addition, molecular levels of NF-κB and Nrf2/HO-1 pathway in colon and Caco-2 cells were also detected. As shown in our data, FA inhibited arsenic-induced colon injury, which was reflected in the improvement of mucosal integrity, the decrease of down-regulated expression of tight junction (TJ) proteins (Claudin-1, Occludin, and ZO-1) and the inhibition of oxidative stress. Similarly, treatment with FA attenuated the inhibitory effect of arsenic on TJ protein expression in Caco-2 cells. In addition to suppressing the activation of NF-κB pathway, FA retrieved the activation of Nrf2/HO-1 pathway in colon and intestinal epithelial cells induced by arsenic. In summary, our findings propose that FA has the potential to mitigate arsenic-induced intestinal damage by preserving the integrity of intestinal epithelial TJs and suppressing oxidative stress. These results lay the groundwork for the potential use of FA in treating colon injuries caused by arsenic.

Effect of vitamin E δ-tocotrienol and aspirin on Wnt signaling in human colon cancer stem cells and in adenoma development in APCmin/+ mice.

In this study, we evaluated the effects of vitamin E δ-tocotrienol (DT3) and aspirin on Wnt signaling in human colon cancer stem cells (CCSCs) and in the prevention of adenoma formation in APCmin/+ mice. We found that knockdown of the adenomatous polyposis coli (APC) gene led to subsequent activation of Wnt signaling in colon epithelial cells (NCM460-APCsiRNA) and induction of β-catenin and its downstream target proteins c-MYC, cyclin D1, and survivin. When aspirin and DT3 were combined, cell growth and survival were inhibited and apoptosis was induced in colon epithelial cells and in CCSCs. However, DT3 and/or aspirin had little or no effect on control normal colon epithelial cells (NCM460-NCsiRNA). The induction of apoptosis was directly related to activation of caspase 8 and cleavage of BID to truncated BID. In addition, DT3 and/or aspirin-induced apoptosis was associated with cleaved PARP, elevated levels of cytosolic cytochrome c and BAX, and depletion of anti-apoptotic protein BCl-2 in CCSCs. The combination of aspirin and DT3 inhibited the self-renewal capacity, Wnt/β-catenin receptor activity, and expression of β-catenin and its downstream targets c-MYC, cyclin D1 and survivin in CCSCs. We also found that treatment with DT3 alone or combined with aspirin significantly inhibited intestinal adenoma formation and Wnt/ β-catenin signaling and induced apoptosis, compared to vehicle, in APCmin/+ mice. Our study demonstrated a rationale for further investigation of the combination of DT3 and aspirin for colorectal cancer prevention and therapy.

Circadian rhythm disruption-mediated downregulation of Bmal1 exacerbates DSS-induced colitis by impairing intestinal barrier.

Circadian rhythm disruption (CRD) is thought to increase the risk of inflammatory bowel disease. The deletion of Bmal1, a core transcription factor, leads to a complete loss of the circadian rhythm and exacerbates the severity of dextran sodium sulfate (DSS)-induced colitis in mice. However, the underlying mechanisms by which CRD and Bmal1 mediate IBD are still unclear. We used a CRD mouse model, a mouse colitis model, and an in vitro model of colonic epithelial cell monolayers. We also knocked down and overexpressed Bmal1 in Caco-2 cells by transfecting lentivirus in vitro. The collected colon tissue and treated cells were assessed and analyzed using immunohistochemistry, immunofluorescence staining, quantitative reverse transcription-polymerase chain reaction, western blot, flow cytometry, transmission electron microscopy, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling staining. We found that CRD mice with downregulated Bmal1 expression were more sensitive to DSS-induced colitis and had more severely impaired intestinal barrier function than wild-type mice. Bmal1-/- mice exhibited more severe colitis, accompanied by decreased tight junction protein levels and increased apoptosis of intestinal epithelial cells compared with wild-type mice, which were alleviated by using the autophagy agonist rapamycin. Bmal1 overexpression attenuated Lipopolysaccharide-induced apoptosis of intestinal epithelial cells and impaired intestinal epithelial cells barrier function in vitro, while inhibition of autophagy reversed this protective effect. This study suggests that CRD leads to the downregulation of Bmal1 expression in the colon, which may exacerbate DSS-induced colitis in mice, and that Bmal1 may serve as a novel target for treating inflammatory bowel disease.

Lectin microarray based glycan profiling of exosomes for dynamic monitoring of colorectal cancer progression

BackgroundExosomes, as emerging biomarkers in liquid biopsies in recent years, offer profound insights into cancer diagnostics due to their unique molecular signatures. The glycosylation profiles of exosomes have emerged as potential biomarkers, offering a novel and less invasive method for cancer diagnosis and monitoring. Colorectal cancer (CRC) represents a substantial global health challenge and burden. Thus there is a great need for the aberrant glycosylation patterns on the surface of CRC cell-derived exosomes, proposing them as potential biomarkers for tumor characterization. ResultsThe interactions of 27 lectins with exosomes from three CRC cell lines (SW480, SW620, HCT116) and one normal colon epithelial cell line (NCM460) have been analyzed by the lectin microarray. The result indicates that Ulex Europaeus Agglutinin I (UEA-I) exhibits high affinity and specificity towards exosomes derived from SW480 cells. The expression of glycosylation related genes within cells has been analyzed by high-throughput quantitative polymerase chain reaction (HT-qPCR). The experimental result of HT-qPCR is consistent with that of lectin microarray. Moreover, the limit of detection (LOD) of UEA-I microarray is calculated to be as low as 2.7 × 105 extracellular vehicles (EVs) mL−1 (three times standard deviation (3σ) of blank sample). The UEA-I microarray has been successfully utilized to dynamically monitor the progression of tumors in mice-bearing SW480 CRC subtype, applicable in tumor sizes ranging from 2 mm to 20 mm in diameter. SignificanceThe results reveal that glycan expression pattern of exosome is linked to specific CRC subtypes, and regulated by glycosyltransferase and glycosidase genes of mother cells. Our findings illuminate the potential of glycosylation molecules on the surface of exosomes as reliable biomarkers for diagnosis of tumor at early stage and monitoring of cancer progression.

Milk-derived extracellular vesicles functionalized with anti-tumour necrosis factor-α nanobody and anti-microbial peptide alleviate ulcerative colitis in mice.

Ulcerative colitis (UC) manifests clinically with chronic intestinal inflammation and microflora dysbiosis. Although biologics can effectively control inflammation, efficient delivery to the colon and colon epithelial cells remains challenging. Milk-derived extracellular vesicles (EV) show promise as an oral delivery tool, however, the ability to load biologics into EV presents challenges to therapeutic applications. Here, we demonstrate that fusing cell-penetrating peptide (TAT) to green fluorescent protein (GFP) enabled biologics loading into EV and protected against degradation in the gastrointestinal environment in vitro and in vivo after oral delivery. Oral administration of EV loaded with anti-tumour necrosis factor-α (TNF-α) nanobody (VHHm3F) (EVVHH) via TAT significantly reduced tissue TNF-α levels and alleviated pathologies in mice with acute UC, compared to VHH alone. In mice with chronic UC, simultaneously introducing VHH and an antimicrobial peptide LL37 into EV (EVLV), then administering orally improved intestinal barrier, inflammation and microbiota balance, resulted in relief of UC-induced depression and anxiety. Collectively, we demonstrated that oral delivery of EVLV effectively alleviated UC in mice and TAT efficiently loaded biologics into EV to confer protection from degradation in the gastrointestinal tract. This therapeutic strategy is promising for UC and is a simple and generalizable approach towards drug-loaded orally-administrable EV treatment for other diseases.

UPLC-ESI-TQD-MS/MS Identification and Antioxidant, Anti-Inflammatory, Anti-Diabetic, Anti-Obesity and Anticancer Properties of Polyphenolic Compounds of Hawthorn Seeds

Hawthorn seeds are a by-product of fruit processing and due to the scale of processing of this raw material, they can be an important source of bioactive compounds. This work is the first report on the phenolic composition of hawthorn seeds and their antioxidant, anti-inflammatory, antidiabetic, antiobesity and anticancer activities. In the isolated phenolic fraction of six seed species, 23 phenolic compounds were identified using the UPLC-ESI-TQD-MS/MS method, the key ones of which included the B-type procyanidin dimer. The seeds of the tested species showed high antioxidant activity (mainly by scavenging O2•− and OH• radicals), anti-inflammatory (mainly through LOX inhibition), anti-diabetic, anti-obesity and anti-cancer, with the highest activity against colon cancer cells (Dld-1 line), showing no activity against healthy colon epithelial cells (CCD841CoN). This activity was significantly dependent on the analyzed hawthorn species and, according to PCA analysis, on the content of flavan-3-ols. These discoveries provided the theoretical basis for the possibility of industrial use of hawthorn seeds.Graphical

Encapsulation of zingerone by self-assembling peptides derived from fish viscera: Characterization, interaction and effects on colon epithelial cells

The purpose of the present work was to encapsulate zingerone (a bioactive compound from ginger) by self-assembling peptides derived from fish viscera. The encapsulation conditions were investigated and the structure of fish peptides-zingerone complex was characterized. The interaction between zingerone and fish peptides was investigated using fluorescence spectroscopy. Further research was performed on the in vitro release of zingerone and fish peptide-zingerone as well as their antiproliferative effects on colon epithelial Caco-2 cells. The results demonstrated that zingerone can be successfully encapsulated by self-assembling peptides derived from fish viscera with high encapsulation efficiency and loading capacity. Furthermore, transmission electron microscope and confocal laser scanning microscope observations revealed the successful encapsulation of zingerone by fish viscera peptides. In addition, in vitro release and antiproliferative activity against Caco-2 cells can be significantly increased by encapsulating zingerone via peptide self-assembly. The current study advances knowledge of encapsulation of bioactive compounds through peptide self-assembly.

Faecalibacterium prausnitzii A2-165 metabolizes host- and media-derived chemicals and induces transcriptional changes in colonic epithelium in GuMI human gut microphysiological system.

Aim: Recently, a GuMI gut microphysiological system has been established to coculture oxygen-intolerant Faecalibacterium prausnitzii (F. prausnitzii) A2-165 with organoids-derived primary human colonic epithelium. This study aims to test if this GuMI system applies to different donors with different healthy states and uses metabolomics to reveal the role of gut microbes in modulating host- and diet-derived molecules in the gut lumen. Methods: Organoids-derived colonic monolayers were generated from an uninflamed region of diverticulitis, ulcerative colitis, and Crohn's disease patients and then integrated into the GuMI system to coculture with F. prausnitzii A2-165 for 2 to 4 days. Apical media was collected for metabolomic analysis. Targeted metabolomics was performed to profile 169 polar chemicals under three conditions: conventional static culture without bacteria, GuMI without bacteria, and GuMI with F. prausnitzii. The barrier function of monolayers was measured using transepithelial resistance. Results: GuMI successfully cocultured patient-derived monolayers and F. prausnitzii for up to 4 days, with active bacterial growth. Introducing flow and oxygen gradient significantly increases the barrier function, while exposure to F. prausnitzii slightly increases the barrier function. Targeted metabolomics screened 169 compounds and detected 76 metabolites, of which 70 significantly differed between at least two conditions. F. prausnitzii significantly modulates the levels of nucleosides, nucleobases, and amino acids on the apical side. Further analysis suggests that F. prausnitzii changes the mRNA level of 260 transcription factor genes in colonic epithelial cells. Conclusion: The GuMI physiomimetic system can maintain the coculture of F. prausnitzii and colonic epithelium from different donors. Together with metabolomics, we identified the modulation of F. prausnitzii in extracellular chemicals and colonic epithelial cell transcription in coculture with human colonic epithelium, which may reflect its function in gut lumen in vivo.

Tedizolid phosphate alleviates DSS-induced ulcerative colitis by inhibiting senescence of cell and colon tissue through activating AMPK signaling pathway

Ulcerative colitis (UC) is a subtype of inflammatory bowel disease. Previous studies have suggested a link between senescence process and the body’s inflammatory reaction, indicating that senescence may exacerbate UC, yet the relation between UC and senescence remains unclear. Tedizolid Phosphate (TED), a novel oxazolidinone antimicrobial, is indicated in acute bacterial skin infections, its impact on senescence is not known. Our research revealed that the UC inducer dextran sulfate sodium (DSS) triggers senescence in both colon epithelial NCM460 cells and colon tissues, and TED that screened from a compound library demonstrated a strong anti-senescence effect on DSS treated NCM460 cells. As an anti-senescence medication identified in this research, TED efficiently alleviated UC and colonic senescence in mice caused by DSS. By proteomic analysis and experimental validation, we found that DSS significantly inhibits the AMPK signaling pathway, while TED counteracts senescence by restoring AMPK activity. This research verified that the development of UC is accompanied with colon tissue senescence, and TED, an anti-senescence medication, can effectively treat UC caused by DSS and alleviate colon senescence. Our work suggests anti-senescence strategy is an effective approach for UC treatment.

High expression of fragile X mental retardation protein inhibits ferroptosis of colorectal tumor cells by activating the RAS/MAPK signaling pathway

To investigate the mechanism by which fragile X mental retardation protein (FMRP) regulates ferroptosis evasion in colorectal cancer (CRC) cells. We examined FMRP expression levels in CRC cell lines using RT-qPCR and Western blotting and analyzed the biological functions and signaling pathways involved in FMRP-mediated regulation of CRC progression using the TCGA database. A lentiviral FMRP overexpression vector (Lv-FMRP) and 3 knockdown vectors (siFMRP-1, siFMRP-2, and siFMRP-3) were constructed, and their effects on proliferation of HCT116 cells were examined using CCK8 assay and plate clone formation assay; the changes in cell ferroptosis level was determined using MDA/ROS/GSH/Fe2+ kits, mitochondrial membrane potential changes were detected using JC-1 fluorescence staining, and the expressions of proteins associated with ferroptosis and the RAS/MAPK signaling pathway were detected using Western blotting. The subcutaneous tumorigenic potential of the transfected cells was evaluated in nude mice. Compared with normal colonic mucosal epithelial NCM460 cells, the CRC cell lines had significantly higher FMRP expression level. Bioinformatics analysis suggested the involvement of FMRP in regulation of reactive oxygen, oxidative stress-induced cell death, mitochondrial respiration, and glutathione metabolism pathways. In the cell experiments, FMRP knockdown significantly inhibited proliferation of HCT116 cells, lowered cellular GSH content, increased MDA and ROS levels, Fe2+ fluorescence intensity, and mitochondrial membrane potential, and decreased SLC7A11/GPX4 protein expressions and the phosphorylation levels of ERK, MEK, MAPK, and RAS proteins; FMRP overexpression resulted in the opposite changes in the cells. In the tumor-bearing nude mice, HCT116 cells with FMRP knockdown showed attenuated tumorigenic potential with lowered xenograft growth rate and reduced SLC7A11 expression in the xenograft. The high expression of FMRP inhibits ferroptosis in CRC cells and promotes progression of CRC by activating the RAS/MAPK signaling pathway.

Testis-specific serine kinase 6 (TSSK6) is abnormally expressed in colorectal cancer and promotes oncogenic behaviors

Cancer testis antigens (CTAs) are a collection of proteins whose expression is normally restricted to the gamete, but abnormally activated in a wide variety of tumors. The CTA, Testis specific serine kinase 6 (TSSK6), is essential for male fertility in mice. Functional relevance of TSSK6 to cancer, if any, has not previously been investigated. Here we find that TSSK6 is frequently anomalously expressed in colorectal cancer and patients with elevated TSSK6 expression have reduced relapse free survival. Depletion of TSSK6 from colorectal cancer cells attenuates anchorage independent growth, invasion and growth in vivo. Conversely, overexpression of TSSK6 enhances anchorage independence and invasion in vitro as well as in vivo tumor growth. Notably, ectopic expression of TSSK6 in semi-transformed human colonic epithelial cells is sufficient to confer anchorage independence and enhance invasion. In somatic cells, TSSK6 co-localizes with and enhances the formation of paxillin and tensin positive foci at the cell periphery, suggesting a function in focal adhesion formation. Importantly, TSSK6 kinase activity is essential to induce these tumorigenic behaviors. Our findings establish that TSSK6 exhibits oncogenic activity when abnormally expressed in colorectal cancer cells. Thus, TSSK6 is a previously unrecognized intervention target for therapy, which could exhibit an exceptionally broad therapeutic window.

Influence of Vitamin D Receptor Signalling and Vitamin D on Colonic Epithelial Cell Fate Decisions in Ulcerative Colitis.

Epidemiological studies have shown that subnormal levels of vitamin D (25[OH]D) are associated with a more aggravated clinical course of ulcerative colitis [UC]. Despite an increased focus on the therapeutic importance of vitamin D and vitamin D receptor [VDR] signalling, the mechanisms underlying the effects of the vitamin D-VDR axis on UC remain elusive. Therefore, we aimed to investigate whether exposure to active vitamin D (1,25[OH]2D3/VDR) signalling in human organoids could influence the maintenance of the colonic epithelium. Intestinal VDR expression was studied by immunohistochemistry, RNA expression arrays, and single-cell RNA sequencing of colonic biopsy specimens obtained from patients with UC and healthy individuals. To characterise the functional and transcriptional effects of 1,25[OH]2D3, we used patient-derived colonic organoids. The dependency of VDR was assessed by knocking out the receptor with CRISPR/Cas9. Our results suggest that 1,25[OH]2D3/VDR stimulation supports differentiation of the colonic epithelium and that impaired 1,25[OH]2D3/VDR signalling thereby may compromise the structure of the intestinal epithelial barrier, leading to flares of UC. Furthermore, a transcriptional response to VDR activity was observed primarily in fully differentiated cells at the top of the colonic crypt, and this response was reduced during flares of UC. We identified an important role of vitamin D signalling in supporting differentiated cell states in the human colonic epithelium, and thereby maintenance of the intestinal barrier integrity. This makes the vitamin D-VDR signalling axis an interesting target for therapeutic efforts to achieve and maintain remission in patients with UC.

A dual model of normal vs isogenic Nrf2-depleted murine epithelial cells to explore oxidative stress involvement

Cancer-derived cell lines are useful tools for studying cellular metabolism and xenobiotic toxicity, but they are not suitable for modeling the biological effects of food contaminants or natural biomolecules on healthy colonic epithelial cells in a normal genetic context. The toxicological properties of such compounds may rely on their oxidative properties. Therefore, it appears to be necessary to develop a dual-cell model in a normal genetic context that allows to define the importance of oxidative stress in the observed toxicity. Given that the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is considered to be the master regulator of antioxidant defenses, our aim was to develop a cellular model comparing normal and Nrf2-depleted isogenic cells to qualify oxidative stress–related toxicity. We generated these cells by using the CRISPR/Cas9 technique. Whole-genome sequencing enabled us to confirm that our cell lines were free of cancer-related mutations. We used 4-hydroxy-2-nonenal (HNE), a lipid peroxidation product closely related to oxidative stress, as a model molecule. Here we report significant differences between the two cell lines in glutathione levels, gene regulation, and cell viability after HNE treatment. The results support the ability of our dual-cell model to study the role of oxidative stress in xenobiotic toxicity.

Taking SCFAs produced by Lactobacillus reuteri orally reshapes gut microbiota and elicits antitumor responses

BackgroundColorectal cancer (CRC) incidence is increasing in recent years due to intestinal flora imbalance, making oral probiotics a hotspot for research. However, numerous studies related to intestinal flora regulation ignore its internal mechanisms without in-depth research.ResultsHere, we developed a probiotic microgel delivery system (L.r@(SA-CS)2) through the layer-by-layer encapsulation technology of alginate (SA) and chitosan (CS) to improve gut microbiota dysbiosis and enhance anti-tumor therapeutic effect. Short chain fatty acids (SCFAs) produced by L.r have direct anti-tumor effects. Additionally, it reduces harmful bacteria such as Proteobacteria and Fusobacteriota, and through bacteria mutualophy increases beneficial bacteria such as Bacteroidota and Firmicutes which produce butyric acid. By binding to the G protein-coupled receptor 109A (GPR109A) on the surface of colonic epithelial cells, butyric acid can induce apoptosis in abnormal cells. Due to the low expression of GPR109A in colon cancer cells, MK-6892 (MK) can be used to stimulate GPR109A. With increased production of butyrate, activated GPR109A is able to bind more butyrate, which further promotes apoptosis of cancer cells and triggers an antitumor response.ConclusionIt appears that the oral administration of L.r@(SA-CS)2 microgels may provide a treatment option for CRC by modifying the gut microbiota.

Bacillus cereus CwpFM induces colonic tissue damage and inflammatory responses through oxidative stress and the NLRP3/NF-κB pathway

Bacillus cereus (B. cereus) from cow milk poses a threat to public health, causing food poisoning and gastrointestinal disorders in humans. We identified CwpFM, an enterotoxin from B. cereus, caused oxidative stress and inflammatory responses in mouse colon and colonic epithelial cells. Colon proteomics revealed that CwpFM elevated proteins associated with inflammation and oxidative stress. Notably, CwpFM induced activation of the NLRP3/NF-κB signaling, but suppressed antioxidant NFE2L2/HO-1 expression in the intestine and epithelial cells. Consistently, CwpFM exposure led to cytotoxicity and ROS accumulation in Caco-2 cells in a dose-dependent manner. Further, NAC (ROS inhibitor) treatment abolished NLRP3/NF-κB activation due to CwpFM. Moreover, overexpression of Nfe2l2 or activation of NFE2L2 by NK-252 reduced ROS production and inhibited activation of the NLRP3/NF-κB pathway. Inhibition of NF-κB by ADPC and/or suppression of NLRP3 by MCC950 attenuated CwpFM-induced inflammatory responses in Caco-2 cells. Collectively, CwpFM induced oxidative stress and NLRP3/NF-κB activation by inhibiting the NFE2L2/HO-1 signaling and ROS accumulation, leading to the development of intestinal inflammation. Our data elucidate the role of oxidative stress and innate immunity in CwpFM enterotoxicity and contribute to developing diagnostic and therapeutic products for B. cereus-related food safety issues.