Colonic Tissue Levels Research Articles

Anti-inflammatory and antioxidant effect of methanol extract of latex of Calotropis procera in rat model of colorectal cancer

Ethnopharmacological relevancePreparations derived from the plant Calotropis procera, have been used for medicinal purpose though the plant is known for its toxic effects. The aerial parts of the plant contain latex in plenty and have been found effective in treating disorders of gastrointestinal system and cancer. Aim of the studyThis study evaluated the efficacy of C. procera dried latex extract prepared in methanol (MeDL) against inflammation and oxidative stress in experimental model of colorectal carcinoma (CRC). Materials and methodsTwo subcutaneous injections of chemical carcinogen, 1,2-dimethylhydrazine (DMH; 150 mg/kg) were given at an interval of one week to induce CRC in rats. The MeDL (50 and 150 mg/kg) and aspirin (60 mg/kg) were given daily and their effect was evaluated on markers of oxidative stress and inflammation after completion of 8 weeks following second injection of carcinogen. A comparison was made with normal and experimental control groups. The colon tissue levels of glutathione (GSH), thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD), nitrite and myeloperoxidase (MPO) were determined. Enzyme-linked immunosorbent assay was performed to determine the levels of prostaglandin E2 (PGE2) and tumor necrosis factor-alpha (TNF-α) and immunohistochemical analysis was performed for IL-1β. ResultsInduction of cancerous changes in the colon resulted in altered oxidative homeostasis as evident from a reduction in GSH level and SOD activity and rise in TBARS level when compared with normal rats. Elevated levels of nitrite, MPO, TNF-α, PGE2 and immunoreactivity of IL-1β were also observed in these rats. The levels of these markers were normalized when the rats were treated with MeDL or anti-inflammatory drug, aspirin. ConclusionThis study demonstrates that suppression of oxidative stress and inflammation contributes to the beneficial effect of MeDL in rat model of colon carcinogenesis.

AIMP2-DX2 provides therapeutic interface to control KRAS-driven tumorigenesis

Recent development of the chemical inhibitors specific to oncogenic KRAS (Kirsten Rat Sarcoma 2 Viral Oncogene Homolog) mutants revives much interest to control KRAS-driven cancers. Here, we report that AIMP2-DX2, a variant of the tumor suppressor AIMP2 (aminoacyl-tRNA synthetase-interacting multi-functional protein 2), acts as a cancer-specific regulator of KRAS stability, augmenting KRAS-driven tumorigenesis. AIMP2-DX2 specifically binds to the hypervariable region and G-domain of KRAS in the cytosol prior to farnesylation. Then, AIMP2-DX2 competitively blocks the access of Smurf2 (SMAD Ubiquitination Regulatory Factor 2) to KRAS, thus preventing ubiquitin-mediated degradation. Moreover, AIMP2-DX2 levels are positively correlated with KRAS levels in colon and lung cancer cell lines and tissues. We also identified a small molecule that specifically bound to the KRAS-binding region of AIMP2-DX2 and inhibited the interaction between these two factors. Treatment with this compound reduces the cellular levels of KRAS, leading to the suppression of KRAS-dependent cancer cell growth in vitro and in vivo. These results suggest the interface of AIMP2-DX2 and KRAS as a route to control KRAS-driven cancers.

Protective Effects of Grape Seed Proanthocyanidin Extract in Preventing DSS Induced Ulcerative Colitis Based on Pharmacodynamic, Pharmacokinetic and Tissue Distribution.

Based on pharmacodynamic, pharmacokinetic and tissue distribution studies, we explored the potential effect of grape seed proanthocyanidin extract (GSPE) on dextran sodium sulfate (DSS) -induced ulcerative colitis in mice and its underlying mechanism. A liquid chromatography-mass spectrometry method was developed to measure the content of five components of GSPE in rat plasma and tissue. After oral administration of GSPE, correlative index levels of interleukin- 1β (IL-1β), interleukin-6 (IL-6), factor-α (TNF-α), Nitric Oxide (NO), malonaldehyde (MDA), and superoxide dismutase (SOD) were detected in the serum and colon tissues. The protein expression levels of HO-1, Nrf2 and NF-κB in the mouse colonic mucosa were analysed using immunohistochemistry. Pharmacodynamic tests showed substantially reduced mice body weight, diarrhea, and bloody stool in the model group. The pathological damage to the colonic mucosa of mice in the GSPE groups was remarkably reduced in a dose-dependent manner. The histopathological score of the colon in the model group was significantly higher than that of the control group (P <0.05), suggesting that DSS caused severe damage to the colon. After oral administration of GSPE, the serum and colonic tissue levels of IL-1β, IL-6, TNF-α, NO, and MDA decreased, whereas SOD content increased. Moreover, the protein levels of NF-κB and Keap-1 were significantly decreased, whereas the expression levels of Nrf2 and HO-1 proteins increased (P<0.01) based on the results of the microwaveimmunohistochemical assay. The pharmacokinetic results showed that catechin, epicatechin, and procyanidins B1, B2, and B4 are widely distributed in the tissues and blood of rats and may accumulate in some tissues. Catechin and epicatechin peaked at 0.25 and 1.5 h for the first and second time, respectively. Procyanidin B1, B2, and B4 peaked at 0.5 and 1.5 h for the first and second time, respectively, owing to the effect of the hepato-enteric circulation. The active components of GSPE can reach the colon of the lesion site, and hepatoenteric circulation can increase the residence time of the active components in the body, further increasing the anti-ulcer activity. Our findings suggest that GSPE has a potential protective effect against DSS-induced ulcerative colitis in mice.

Transdermal Administration of Volatile Oil from Citrus aurantium-Rhizoma Atractylodis Macrocephalae Alleviates Constipation in Rats by Altering Host Metabolome and Intestinal Microbiota Composition.

Background The Citrus aurantium- (ZhiShi, ZS-) Rhizoma Atractylodis Macrocephalae (BaiZhu, BZ) pairs are often found in herbal formulas for constipation. The volatile oils of ZS and BZ (ZBVO) have good pharmacological activity against constipation, but the mechanism for treatment of slow transit constipation (STC) remains unclear. Method A rat model using diphenoxylate tablets was constructed to investigate if transdermal administration of ZBVO would mediate intestinal microorganisms and fecal metabolites and improve STC symptoms. The regulatory effects of ZBVO at 0.15, 0.30, and 0.60 mL kg−1 d−1 on STC rats were assessed by measuring fecal water content, intestinal propulsion rate, histopathology, expression of gastrointestinal hormones, brain and intestinal peptides, and inflammatory factors. The changes in intestinal flora of STC rats were analyzed by 16S rRNA gene sequencing. Moreover, the untargeted fecal metabolomics analysis was performed by ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometer (UPLC-Q-TOF-MS) technology. Results The results showed that ZBVO had a modulating effect on STC by increasing the fecal water content and intestinal propulsion rate. Transdermal administration of ZBVO decreased serum levels of interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) and increased the levels of gastrin (GAS) and substance P (SP). In addition, ZBVO increased 5-hydroxytryptamine (5-HT) levels and decreased vasoactive intestinal peptide (VIP) levels in colon and hippocampus tissues. The results of intestinal microbiota showed that ZBVO improved the diversity and abundance of intestinal microbiota and changed the community composition by decreasing Romboutsia and increasing Proteobacteria, Allobaculum, and Ruminococcaceae. And the feces metabolomics found that nicotinate and nicotinamide metabolism, purine metabolism, citrate cycle (TCA cycle), pyruvate metabolism, arachidonic acid metabolism, pyrimidine metabolism, and primary bile acid biosynthesis were modulated. Conclusion These findings suggest that ZBVO can alleviate STC symptoms by promoting intestinal peristalsis, increasing fecal water content, regulating gastrointestinal hormone level, reducing the inflammatory response, and regulating brain and intestinal peptides after transdermal administration. And structural changes in the intestinal microbiota are closely related to host metabolism and intestinal microbiota destroyed in STC modeling could be significantly improved by the ZBVO, which provides a reference for the development of aromatic drug macrohealth products.

The protective effect of Boswellic acid and Ellagic acid loaded, colon targeted, and pH-sensitive N-succinyl chitosan in ulcerative colitis rat model

Targeted drug delivery systems that provide therapeutic activity with lower concentrations of both herbal and other substances, while reducing access to non-target sites and undesirable effects; have an important potential for future projections in terms of pharmacological and toxicological studies. The present study aimed to investigate the protective effects of Acetyl 11-keto-β-boswellic acid (AKBA) and Ellagic acid (EA) loaded, colon targeted, and pH-Sensitive N-succinyl chitosans (NsCh) in ulcerative colitis (UC) model induced by acetic acid (AA) in rats. The physicochemical characterization (elemental analysis, FT-IR, DSC, XRD, SEM, NMR) analyses, swelling, and releasing tests of NsCh's suggested that AKBA and EA were successfully loaded to pH-sensitive polymer and targeted to the colon. The targeted AKBA and EA were administered to Wistar rats (n:30, 5 groups) and damage, inflammatory cytokine, protein expression, oxidative stress, and antioxidant parameter levels in colon tissues of the groups were determined. Increase in the levels of CAT, GPx activities, and decrease in necrosis, inflammation, and levels of MDA, TNF-α, COX-2, NF-kB were observed in the targeted EA and AKBA groups compared to the UC group. The potential protective effect of EA and AKBA loaded, colon-targeted, pH-sensitive controlled releasing system against UC and the potential to be licensed preparations by completing the next stages of in vitro and in vivo studies were demonstrated.

Abstract 3089: Study of HOX genes in the stem cell origin of CRC

Abstract Our goal is to ascertain how HOX-regulatory mechanisms play a role in the SC origin of colorectal cancer (CRC). While HOX genes encode master regulatory transcription factors that regulate stem cells (SCs), their role in cancer etiology is under-studied. We and others find that HOX genes are aberrantly expressed in CRC. We previously reported that HOXA4 and HOXD10 along with aldehyde dehydrogenase (ALDH) and the other components of the retinoic acid (RA) signaling pathway are selectively expressed in human colon SCs. That RA signaling regulates HOX expression indicates that RA regulation of HOX gene expression mainly occurs through ALDH+ SCs in the colon. We hypothesize that specific HOX genes regulate growth and differentiation of SCs in human colon and HOX gene dysregulation leads to decreased differentiation and SC overpopulation. Indeed, our bioinformatics analysis shows that many HOX genes are overexpressed in CRCs, which parallels the overpopulation of ALDH+ SC that occurs during CRC tumorigenesis. We are using immunohistochemical mapping to measure proportions of SCs and HOX-positive cells in normal and malignant human colon tissues. We find that specific HOX proteins have increased expression levels in malignant colon tissues. In addition, immunofluorescence co-staining of human colon tissues is used to validate HOX gene expression in ALDH+ SCs. We are also evaluating the effects of all-trans-RA (ATRA) on HOX gene expression and on cellular proliferation and differentiation using CRC cell lines. Our results show that ATRA treatment of HT29 CRC cells leads to reduced proliferation and decreased expression of select HOX genes. These results indicate that HOX genes like HOXA4, HOXA9, and HOXD10, are expressed colonic SCs, and are regulated by RA signaling. Overall, the overexpression of HOX genes in cancer SCs appears to contribute to the SC origin of CRC. Citation Format: Brian Osmond, Caroline O. Facey, Lynn M. Opdenaker, Bruce M. Boman. Study of HOX genes in the stem cell origin of CRC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3089.

Lactobacillus paracasei DTA81, a cholesterol-lowering strain having immunomodulatory activity, reveals gut microbiota regulation capability in BALB/c mice receiving high-fat diet.

Aims In‐vitro/In‐vivo evaluation of cholesterol‐lowering probiotic strain Lactobacillus paracasei DTA81 and the possible connection with the gut microbiota modulation.Methods and ResultsIn the present study, strain DTA81 has been evaluated for the possible influence on blood lipid and glucose concentrations, modulation of the immune system, gastrointestinal survivability and modulation of gut microbiota in BALB/c mice receiving a high‐fat diet. After 6 weeks of treatment, a significant reduction of total cholesterol and fasting blood sugar (FBS) among animals treated with L. paracasei DTA81 has been recorded. Comparison of colon tissue levels of different cytokines revealed a significant reduction of the inflammatory cytokine interleukin‐6. The comparison of gut microbiota using the 16S rRNA approach indicated that the treatment with L. paracasei DTA81 significantly increased the taxa Bacteroidetes and Coprococcus. Moreover, the genome of DTA81 was sequenced for the in‐silico assessment, and the analysis indicated the presence of cholesterol assimilation‐related genes as well as the absence of negative traits such as transmissible antibiotic resistance genes, plasmids and prophage regions.ConclusionThe outcome of this study revealed the in‐vitro and in‐vivo properties of L. paracasei DTA81 and the possible mechanism between consumption of this strain, the abundance of Bacteriodetes/Coprococcus taxa, immunomodulatory activity and the subsequent reduction of cholesterol/FBS in BALB/c mice.Significance and Impact of the Study Lactobacillus paracasei DTA81 as a non‐pharmacological potential probiotic supplement can influence metabolic homeostasis in individuals, particularly those adopting high‐fat diets, and it can contribute to reduce coronary heart disease.

Novel functional variants in the Notch pathway and survival of Chinese colorectal cancer.

Notch signaling pathway plays crucial roles in progression of colorectal cancer (CRC), likely affecting overall survival (OS). In a two-stage survival analysis of 1116 CRC patients in East China, we found that one locus at MINAR1 out of 133 genes in the Notch signaling pathway was significantly associated with OS (P < 1 × 10-6 , false discovery rate < 0.01). This locus containing seven single-nucleotide polymorphisms (SNPs) in high linkage disequilibrium (R2 = 1) is located on chromosome 15, of which the MINAR1 rs72430409 G allele was associated with a greater death risk (HR = 1.98, 95% CI = 1.55-2.54, P = 6.8 × 10-8 ). Further analysis of ChIP-sequencing data from the encyclopedia of DNA Elements showed that rs72430409 and rs72630408 were potential cis-regulatory elements for the MINAR1 promoter. Additional expression quantitative trait loci analysis revealed that rs72430409 G>A and rs72630408 A>G were correlated with increased MINAR1 expression levels in both blood cells and colon tissues. Dual luciferase assays revealed that the rs72430409 A allele increased MINAR1 promoter activity. The Cancer Genome Atlas data showed that expression levels of MINAR1 in CRC samples were significantly higher than that in normal colorectal tissue and that high expression of MINAR1 was associated with a shortened OS, likely via activating the epithelial mesenchymal transition (EMT) pathway as shown in the gene-set enrichment analysis. In vitro, RNAi-mediated silencing of MINAR1 led to decreased migration and proliferation in CRC cancer cells, and MINAR1 silencing could downregulate the expression of key effector genes in EMT and glycolysis. Larger cohort studies and further experiments are needed to validate our findings.

Olmesartan modulates proliferating cell nuclear antigen expression and improves dextran sulfate: induced ulcerative colitis in rats

Background: Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by sudden attacks of remissions and exacerbations with increased incidence of cancer colon. The present study aims to determine the possible ameliorative mechanisms of Olmesartan in UC induced experimentally in rat.Methods: Adult albino rats were randomly grouped into control, UC model non treated group: Rats received dextran sodium (DSS) orally for 21 days with intra-colic administration of acetic acid (AA) for 3 consecutive days for induction of UC model, Olmesartan (1, 5, 10mg/kg/orally) and UC + Olmesartan in different doses (1 mg, 5 mg and 10 mg/kg/day orally).Results: DSS orally and AA intra-rectal produced sever colitis manifested by significant weight loss, watery and bloody diarrhea. Significant increase in serum and colonic tissue levels of tumor necrosis factor alpha and interleukine-1β. Pro-apoptotic Bax protein, myeloperoxidase (MPO) and expression of PCNA significantly increased in colonic tissue. Lipid peroxidation (MDA) significantly elevated while reduced glutathione (GSH) was depleted in UC non-treated group compared with normal control group. Treatment with Olmesartan (5 mg, 10 mg/kg/day, orally) ameliorated mucosal ulceration and improved inflammatory signs as confirmed by immunohistochemical and histopathological examination. Also, Olmesartan significantly attenuates overexpression of PCNA in colonic mucosa.Conclusions: Our results point out that Olmesartan had ameliorative effects on UC by its anti-inflammatory, antioxidant and anti-apoptotic effects and attenuates PCNA expression which is the main cause of dysplasia and colorectal cancer. Olmesartan may be a promising therapeutic drug for treating UC and protection of colorectal carcinoma.

ANXA3 is upregulated by hypoxia-inducible factor 1-alpha and promotes colon cancer growth.

BackgroundAnnexin A3 (ANXA3) is overexpressed in various cancers and is a potential target for cancer treatment. However, clinical implication and biological function of ANXA3 in colon cancer remain unknown. This study aimed to investigate the relationship between hypoxia-inducible factor 1-alpha (HIF-1α) and ANXA3, and explore the function of ANXA3 in colon carcinoma.MethodsExpression levels of HIF-1α and ANXA3 in human colon carcinoma specimens and colon cancer cell lines were detected by immunohistochemistry, real-time PCR and Western blot analysis. The proliferation of colon cancer cells was examined. Nude mice were used for xenograft tumor model, and HIF-1α siRNA or control adenovirus was injected into the tumor.ResultsHIF-1α and ANXA3 expression levels were higher in colon cancer tissues than their expression levels in normal colon tissues. In addition, HIF-1α and ANXA3 expression increased in colon cancer cells under hypoxic condition. Knockdown of HIF-1α decreased HIF-1α and ANXA3 expression, and inhibited the proliferation and growth of colon cancer cells. In nude mouse model, silencing HIF-1α decreased volume of xenograft tumor and ANXA3 expression.ConclusionsANXA3 expression is upregulated by HIF-1α in colon cancer in response to hypoxic stress and contributes to colon tumor growth. ANXA3 may represent a new therapeutic target for colon carcinoma.

Suppression of Inflammatory Cytokines Expression with Bitter Melon (Momordica Charantia) in TNBS-instigated Ulcerative Colitis.

Background and ObjectiveThis study was aimed to elucidate the molecular mechanism of Momordica charantia (MCh), along with a standard drug prednisolone, in a rat model of colitis induced by trinitrobenzene sulfonic acid (TNBS).MethodsAfter the induction of the experimental colitis, the animals were treated with MCh (4 g/kg/day) for 14 consecutive days by intragastric gavage. The colonic tissue expression levels of C-C motif chemokine ligand 17 (CCL-17), interleukin (IL)-1β, IL-6, IL-23, interferon-γ (IFN-γ), nuclear factor kappa B (NF-kB), and tumor necrosis factor-α (TNF-α), were determined at both mRNA and protein levels to estimate the effect of MCh. Besides, colonic specimens were analyzed histopathologically after staining with hematoxylin and eosin.ResultsThe body weights from TNBS-instigated colitis rats were found to be significantly lower than untreated animals. Also, the IFN-γ, IL-1β, IL-6, Il-23, TNF-α, CCL-17, and NF-kB mRNA and protein levels were increased significantly from 1.86-4.91-fold and 1.46-5.50-fold, respectively, in the TNBS-instigated colitis group as compared to the control. Both the MCh and prednisolone treatment significantly reduced the bodyweight loss. It also restored the induced colonic tissue levels of IL-1β, IL-6, IFN-γ, and TNF-α to normal levels seen in untreated animals. These results were also supported with the histochemical staining of the colonic tissues from both control and treated animals.ConclusionThe presented data strongly suggests that MCh has the anti-inflammatory effect that might be modulated through vitamin D metabolism. It is the right candidate for the treatment of UC as an alternative and complementary therapeutics.

Effects of Huoxiang Zhengqi Oral Liquid on intestinal barrier function in rats with dampness obstructing spleen-stomach syndrome

The aim of this paper was to investigate the effect of Huoxiang Zhengqi Oral Liquid on intestinal barrier functions in rats with dampness obstructing spleen-stomach syndrome and primarily explore the mechanism. The rat model of dampness obstructing spleen-stomach syndrome was established, and then the modeled rats were randomly divided into the model control group, Huoxiang Zhengqi Oral Liquid high and low dose groups, and natural recovery group according to gender and body weight, with 10 rats in each group. Another 10 rats were taken as blank control group. After each group received the corresponding treatment for 7 days, rat serum was isolated. D-lactic acid content was detected by the MTT method, and diamine oxidase(DAO) activity was detected by the rate method. Colon tissues of the rats were isolated to detect Na~+-K~+-ATPase activity and Ca~(2+)-Mg~(2+)-ATPase activity by phosphate determination method, glutathione peroxidase(GSH-Px) activity was detected by spectrophotometry, catalase(CAT) activity was detected by ammonium molybdate, superoxide dismutase(SOD) activity was detected by hydroxylamine, the expression of occludin protein and ZO-1 protein was detected by immunofluorescence, and the expression levels of occludin protein and ZO-1 protein were detected by Western blot. RESULTS:: showed that low dose Huoxiang Zhengqi Oral Liquid could improve the body weight, diet, stool and urine state of rats with dampness obstructing spleen-stomach syndrome obviously. The D-lactic acid content and the DAO activity in the serum of rats with dampness obstructing spleen-stomach syndrome were reduced obviously. The activities of Na~+-K~+-ATPase, Ca~(2+)-Mg~(2+)-ATPase, GSH-Px, CAT and SOD in rat colon tissues were increased obviously. The occludin proteins and ZO-1 protein expression levels in rat colon tissues were raised obviously. The differences in the above indexes between Huoxiang Zhengqi Oral Liquid group and the model control group were statistically significant(P&lt;0.05). Huoxiang Zhengqi Oral Liquid could effectively restore the intestinal barrier function in rats with dampness obstructing spleen-stomach syndrome and its mechanism may be related to the repair of intestinal mechanical barrier function.

Tussilagone Reduces Tumorigenesis by Diminishing Inflammation in Experimental Colitis-Associated Colon Cancer.

Background: Tussilagone, a major component of Tussilago farfara L., has anti-angiogenic and anti-inflammatory effects. However, the therapeutic and preventive activity of tussilagone in colitis-associated colon carcinogenesis is unknown. Methods: We intended to investigate the therapeutic effects and the potential mechanism of action underlying the pharmacological activity of tussilagone on colitis-associated colon cancer induced in mice using azoxymethane (AOM)/dextran sulfate sodium (DSS). We injected BALB/c mice with AOM and administered 2% DSS in drinking water. The mice were given tussilagone (2.5 and 5 mg/kg body weight) and colon tissues was collected at 72 days. We used Western blotting, immunohistochemistry and real-time RT-PCR analyses to examine the tumorigenesis and inflammatory status of the colon. Results: Tussilagone administration significantly reduced the formation of colonic tumors. In addition, tussilagone treatment markedly reduced the inflammatory mediators and increased heme oxygease-1 in protein and mRNA levels in colon tissues. Meanwhile, nuclear NF-κB-positive cells were elevated and nuclear Nrf2-positive cells were demised by tussilagone treatment in colon tissues. Tussilagone also reduced cell proliferation, induced apoptosis and decreased the β-catenin expression. Conclusions: Tussilagone administration decreases the inflammation and proliferation induced by AOM/DSS and induced apoptosis in colon tissue. Overall, this study indicates the potential value of tussilagone in suppressing colon tumorigenesis.

P157 FBXO3-FBXL2 AXIS MODULATORS AS A NOVEL CLASS OF ORAL SMALL MOLECULE COMPOUNDS FOR THE TREATMENT OF CROHN’S DISEASE

Abstract Background Ubiquitination is a common post-translational modification, tagging proteins for degradation. The ubiquitin proteasome system is activated in Crohn’s Disease (CD), and modulation of its components might be a novel strategy for therapeutic intervention to control inflammation. The conjugation of ubiquitin to a target protein is orchestrated by a series of enzymatic reactions, the last step being catalyzed by a selective ubiquitin E3 ligase. Among ubiquitin E3 ligases, Fbxl2 serves as a sentinel gatekeeper to limit inflammation by targeting and enhancing the degradation of tumor necrosis factor receptor associated factors (TRAF) proteins, which link cell surface signals (through NFkB signaling) to cytokine secretion. In addition, Fbxl2 controls the ubiquitination of the inflammasome, NOD-like receptor protein 3 (NLRP3), which mediates the release of interleukin (IL)1β and IL18 (Fig.1). Fbxl2 protein itself is ubiquitinated and degraded by another protein, called Fbxo3. Inhibition of Fbxo3 results in increased Fbxl2 levels and decreased TRAF and NLRP3. Individuals with a natural occurring polymorphism within Fbxo3V221Ihave decreased lipopolysaccharide(LPS)-induced cytokine (TNFα, IL1β &amp; IL6) production, Fbxo3 and TRAF levels and increased Fbxl2, providing human genetic target validation for Fbxo3-Fbxl2 axis modulation in inflammatory conditions. BC1261 is a first-in-class, selective, orally available Fbxo3 inhibitor in clinical development for the treatment of CD. Methods and Results In vitro data demonstrated that BC1261 binds to Fbxo3, prevents Fbxo3/Fbxl2 association and produces a concentration-dependent inhibition of LPS-induced release of TNFα and IL1β from human peripheral blood mononuclear cells. In an acute DSS-mouse model, administration of BC-1261 via drinking water ad libitum (30 μg/ml) or by daily intraperitoneal (IP) injection (150 μg) for 5 days resulted in the attenuation of the shortening of colonic length, tissue damage (Fig2.A), and TNFα and IL6 tissue levels (Fig.2B). In a repeated DSS-mouse model, BC-1261 (10 mg/kg bid) given orally for 19 days was comparable to the positive control (anti-p40) in the DAI composite (Fig2.C), stool consistency (Fig2.D), and histopathology score in the proximal colon (Fig2.E). BC-1261 also lead to clinically relevant reductions on endoscopy score (Fig2.F). Conclusion BC1261, a Fbxo3 inhibitor, was efficacious in both acute and chronic preclinical models of colitis. Taken together, these results suggest the potential utility of selective Fbxo3-Fbxl2 modulation in the treatment of CD.

Protective effects of myristicin against ulcerative colitis induced by acetic acid in male mice

ABSTRACT The present investigation aims to evaluate the prophylactic effects and mechanisms of Myr on experimental colitis model induced by acetic acid (AA) in mice. The obtained results showed that Myr treatment prior to induction of colitis significantly modulated the colonic oxidative stress by increasing the antioxidant enzymatic activities of catalse, superoxide dismutase, glutathione peroxidase and glutathione reductase, as well as by lowering the levels of lipid peroxidation. The treatment also significantly decreased the colonic tissue levels of inflammatory markers, including myeloperoxidase activity, tumour necrosis factor-alpha, interleukin-1 and interleukin-17. Besides, Myr significantly inhibited nuclear factor-kappa B (NF-κB) levels in AA-induced ulcerative colitis tissues. In the histological evaluation, the tissue damage scores in the mice colon were significantly suppressed with the administration of Myr + AA. Taken together, our findings suggested that Myr exerts marked protective effect against colonic inflammation through regulating antioxidative responses, NF-κB and subsequent pro-inflammatory cytokines production.

Buteelch-5, A Traditional Formula Ameliorates Dextran Sulfate Sodium-Induced Colitis in Mice

Objectives: To examine the therapeutic effects of buteelch-5, a traditional formula on DSS-induced colitis in C57/BL6 mice and its possible mechanisms. Methods: Colitis in mice was induced by oral administration of 5% dextran sulfate sodium (DSS) for seven days. On the eighth day after administration of DSS, buteelch-5 (500 mg/kg, twice a day) was given orally to mice for five consecutive days. Ciprofioxacin (50 mg/kg, once a day for 5 days) was given to mice as a comparison. Two hours after the last administration of buteelch-5 and ciprofioxacin, mice were euthanized, and colon tissues were removed. Protein and mRNA levels of occludin, claudin-1 and zonula occludens (ZO)-1 in colon tissues were determined by western blot and real time-qPCR respectively. Histopathological analysis of colon tissues was performed. Results: Histological analysis revealed successful establishments of colitis models. Treatment with buteelch-5 markedly inhibited DSS-induced colon injury. Furthermore, buteelch-5 increased (2.14-2.67 fold) the occludin, claudin and ZO-1 protein and mRNA levels in colon tissues of mice administered with DSS. Significant increase was observed in occludin mRNA levels after buteelch-5 treatment (p&lt;.05). Conclusion: Buteelch-5 improves microscopic inflammation and increases tight junction protein expressions such as occludin, claudin, and ZO-1 in mice with DSS-induced colitis.

Huang-Lian-Jie-Du Decoction Ameliorates Acute Ulcerative Colitis in Mice via Regulating NF-κB and Nrf2 Signaling Pathways and Enhancing Intestinal Barrier Function.

Evidence shows that intestinal inflammation, oxidative stress, and injury of mucosal barrier are closely related to the pathogenesis of ulcerative colitis (UC). Huang-lian-Jie-du Decoction (HLJDD) is a well-known prescription of traditional Chinese medicine with anti-inflammatory and antioxidative activities, which may be used to treat UC. However, its therapeutic effect and mechanism are still unclear. In this study, the UC model of BABL/c mice were established by DSS [3.5% (w/v)], and HLJDD was given orally for treatment at the same time. During the experiment, the clinical symptoms of mice were scored by disease activity index (DAI). Besides, the effects of HLJDD on immune function, oxidative stress, colon NF-κB and Nrf2 signaling pathway, and intestinal mucosal barrier function in UC mice were also investigated. The results showed that HLJDD could alleviate body weight loss and DAI score of UC mice, inhibit colonic shortening and relieve colonic pathological damage, and reduce plasma and colon MPO levels. In addition, HLJDD treatment significantly up-regulated plasma IL-10, down-regulated TNF-α and IL-1β levels, and inhibited the expression of NF-κB p65, p-IκKα/β, and p-IκBα proteins in the colon. Moreover, NO and MDA levels in colon tissues were significantly reduced after HLJDD treatment, while GSH, SOD levels and Nrf2, Keap1 protein expression levels were remarkably elevated. Additionally, HLJDD also protected intestinal mucosa by increasing the secretion of mucin and the expression of ZO-1 and occludin in colonic mucosa. These results indicate that HLJDD could effectively alleviate DSS-induced mice UC by suppressing NF-κB signaling pathway, activating Nrf2 signaling pathway, and enhancing intestinal barrier function.

Beneficial Effects of Oat Beta-Glucan Dietary Supplementation in Colitis Depend on its Molecular Weight.

Background: Inflammatory bowel diseases are an important health problem. Therefore, the aim of the present study was to compare the impact of isolated oat beta-glucan fractions of low and high molecular weight, taken as dietary supplementation, on inflammatory markers in the colitis model. Methods: Two groups of Sprague–Dawley rats—control and with experimentally induced colitis—were subsequently divided into three subgroups and fed over 21 days feed supplemented with 1% of low (βGl) or high (βGh) molecular weight oat beta-glucan fraction or feed without supplementation. The level of colon inflammatory markers, cytokines, and their receptors’ genes expressions and immune cells numbers were measured by ELISA, RT-PCR, and by flow cytometry methods, respectively. Results: The results showed moderate inflammation affecting the colon mucosa and submucosa, with significant changes in the number of lymphocytes in the colon tissue, elevated cytokines and eicosanoid levels, as well as disruption of the main cytokine and chemokine cell signaling pathways in colitis rats. Beta-glucans supplementation caused a reverse in the percentage of lymphocytes with stronger effects of βGh and reduction of the levels of the inflammatory markers, and improvement of cytokine and chemokine signaling pathways with stronger effects of βGl supplementation. Conclusions: The results indicate the therapeutic effect of dietary oat beta-glucan supplementation in the colitis in evident relation to the molecular weight of polymer.

Elevated expression of the leptin receptor ob‑R may contribute to inflammation in patients with ulcerative colitis.

The effect of leptin on ulcerative colitis (UC) has been controversial. The present study aimed to investigate the role of leptin and its receptor ob‑R in UC and the underlying mechanism of this role. The level of serum leptin and the protein expression of the leptin receptor ob‑R in the colonic mucosa were determined in patients with UC. Experimental colitis was induced through intrarectal administration of 2,4,6‑trinitrobenzene sulfonic acid (TNBS) in leptin receptor‑deficient Zucker rats (LR‑D). The body weight, disease activity index, colon length, and macroscopic and histopathological appearance were evaluated. Furthermore, the myeloperoxidase (MPO) enzyme activity and cytokine levels in colon tissues were also determined. The expression of the signal transducer and activator of transcription 3 (STAT3), phosphorylated STAT3 (p‑STAT3), nuclear factor (NF)‑κB‑p65, and Ras homolog gene family member A (RhoA) proteins in colon tissues was assessed. The results revealed that the expression of the leptin receptor ob‑R was increased in the colonic mucosa but the serum leptin level was not altered in patients with UC compared with healthy volunteers. The severity of experimental colitis, represented by body weight loss, disease activity index, colon length, and macroscopic and histological changes, was ameliorated in LR‑D rats compared with the wild‑type (WT) rats. Moreover, the MPO activity; levels of cytokines including interleukin (IL)‑1β, IL‑6, and tumor necrosis factor‑α; and expression of p‑STAT3, NF‑κB, and RhoA proteins were reduced in colon tissues of LR‑D rats compared with WT rats. In conclusion, activation of the leptin receptor ob‑R is an important pathogenic mechanism of UC, and leptin receptor deficiency may provide resistance against TNBS‑induced colitis by inhibiting the NF‑κB and RhoA signaling pathways.

Expression Levels of 4 Genes in Colon Tissue Might Be Used to Predict Which Patients Will Enter Endoscopic Remission After Vedolizumab Therapy for Inflammatory Bowel Diseases

Background & AimsWe aimed to identify biomarkers that might be used to predict responses of patients with inflammatory bowel diseases (IBD) to vedolizumab therapy.MethodsWe obtained biopsies from inflamed colon of patients with IBD who began treatment with vedolizumab (n = 31) or tumor necrosis factor (TNF) antagonists (n = 20) and performed RNA-sequencing analyses. We compared gene expression patterns between patients who did and did not enter endoscopic remission (absence of ulcerations at month 6 for patients with Crohn’s disease or Mayo endoscopic subscore ≤1 at week 14 for patients with ulcerative colitis) and performed pathway analysis and cell deconvolution for training (n = 20) and validation (n = 11) datasets. Colon biopsies were also analyzed by immunohistochemistry. We validated a baseline gene expression pattern associated with endoscopic remission after vedolizumab therapy using 3 independent datasets (n = 66).ResultsWe identified significant differences in expression levels of 44 genes between patients who entered remission after vedolizumab and those who did not; we found significant increases in leukocyte migration in colon tissues from patients who did not enter remission (P < .006). Deconvolution methods identified a significant enrichment of monocytes (P = .005), M1-macrophages (P = .05), and CD4+ T cells (P = .008) in colon tissues from patients who did not enter remission, whereas colon tissues from patients in remission had higher numbers of naïve B cells before treatment (P = .05). Baseline expression levels of PIWIL1, MAATS1, RGS13, and DCHS2 identified patients who did vs did not enter remission with 80% accuracy in the training set and 100% accuracy in validation dataset 1. We validated these findings in the 3 independent datasets by microarray, RNA sequencing and quantitative PCR analysis (P = .003). Expression levels of these 4 genes did not associate with response to anti-TNF agents. We confirmed the presence of proteins encoded by mRNAs using immunohistochemistry.ConclusionsWe identified 4 genes whose baseline expression levels in colon tissues of patients with IBD associate with endoscopic remission after vedolizumab, but not anti-TNF, treatment. We validated this signature in 4 independent datasets and also at the protein level. Studies of these genes might provide insights into the mechanisms of action of vedolizumab.