Colon Tissue Homogenates Research Articles

First report of isolation of Fusobacterium varium from liver abscesses and ruminal and colonic epithelial tissues of feedlot cattle*

ObjectiveOur objective was to isolate and determine prevalence of Fusobacterium varium in liver abscesses and the corresponding ruminal and colonic epithelial tissues and ruminal and colonic contents of feedlot cattle. Materials and MethodsA total of 96 intact liver abscess samples and matched ruminal and colonic tissues and contents from cattle, originating from feedlots that did not receive in-feed tylosin, were collected at slaughter. Liver abscesses and ruminal and colonic tissue were ho- mogenized and then plated, before and after enrichment in lactate or lysine medium with selective antibiotics, onto blood agar and selective lactate or lysine agar for isolation of Fusobacterium and to determine prevalence and concentration. Putative colonies were tested by a quantitative PCR assay targeting the hgdA gene for species confirma- tion. Results and DiscussionNone of the liver abscess samples yielded F. varium by direct plating; however, F. varium was isolated from 3 of 96 (3.1%) following enrich- ment of the homogenate in lactate or lysine medium. In contrast to liver abscesses, F. varium was isolated by direct plating from 27.1% (26/96) of ruminal epithelial and 3.1% (3/96) of colonic epithelial tissue homogenates. Overall, 10.1%, 77.1%, 44.8%, 86.5%, and 70.1% of liver abscess, ruminal, and colonic epithelial tissues and ruminal and colonic contents were positive for F. varium, respectively. Implications and ApplicationsThe increased fre- quency of isolation and high prevalence of F. varium in ruminal tissue and, to a lesser extent, in the colonic tissue confirms its ability to invade tissues and possibly cause bacterial ruminitis. However, the relatively low frequency of F. varium isolation and low prevalence in liver abscesses suggest that it is unlikely to be an etiologic agent. Inter- estingly, there is some evidence that ruminal strains of F. varium were resistant to tylosin; therefore, it would be of interest to determine the prevalence in cattle receiving in-feed tylosin.

Mangiferin (mango) attenuates AOM-induced colorectal cancer in rat’s colon by augmentation of apoptotic proteins and antioxidant mechanisms

Mangiferin (MF) is a natural C-glucosylxantone compound that has many substantial curative potentials against numerous illnesses including cancers. The present study's goal is to appraise the chemo preventive possessions of MF on azoxymethane (AOM)-mediated colonic aberrant crypt foci (ACF) in rats. Rats clustered into 5 groups, negative control (A), inoculated subcutaneously with normal saline twice and nourished on 0.5% CMC; groups B-E injected twice with 15 mg/kg azoxymethane followed by ingestion of 0.5% CMC (B, cancer control); intraperitoneal inoculation of 35 mg/kg 5-fluorouracil (C, reference rats) or nourished on 30 mg/kg (D) and 60 mg/kg (E) of MF. Results of gross morphology of colorectal specimens showed significantly lower total colonic ACF incidence in MF-treated rats than that of cancer controls. The colon tissue examination of cancer control rats showed increased ACF availability with bizarrely elongated nuclei, stratified cells, and higher depletion of the submucosal glands compared to MF-treated rats. Mangiferin treatment caused increased regulation of pro-apoptotic (increased Bax) proteins and reduced the β-catenin) proteins expression. Moreover, rats fed on MF had significantly higher glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT), and lower malondialdehyde (MDA) concentrations in their colonic tissue homogenates. Mangiferin supplementation significantly down-shifted pro-inflammatory cytokines (transforming growth factor-α and interleukine-6) and up-shifted anti-inflammatory cytokines (interleukine-10) based on serum analysis. The chemo-protective mechanistic of MF against AOM-induced ACF, shown by lower ACF values and colon tissue penetration, could be correlated with its positive modulation of apoptotic cascade, antioxidant enzymes, and inflammatory cytokines originating from AOM oxidative stress insults.

Effective enhancement of the immunomodulatory capacity of canine adipose-derived mesenchymal stromal cells on colitis by priming with colon tissue from mice with colitis.

The therapeutic efficacy of mesenchymal stromal cells (MSCs) in inflammatory bowel disease is not completely known and is not consistent. Priming with inflammatory cytokines has been proposed to adapt MSCs to an inflammatory environment to have them ready to counteract it, but may have undesirable effects on MSCs, such as increased immunogenicity. In this study, we hypothesized that priming MSCs with inflamed intestinal tissue would more effectively enhance their therapeutic effect on intestinal inflammation. The capacity of canine adipose-derived MSCs (cADSCs) primed with colon tissue homogenates from mice with experimentally induced colitis or a combination of tumor necrosis factor-α and interferon-γ to inhibit T-cell proliferation was analyzed, along with their own apoptosis, proliferation, cell surface marker expression, and transcriptome. In addition, colitis mice were treated with the primed cADSCs to assess colitis severity and immune cell profile. Priming with cytokines induced apoptosis, decreased cell proliferation, and major histocompatibility complex-II gene expression in cADSCs, but these adverse effects were mild or absent with colitis-tissue priming. cADSCs primed with colitis tissue reduced the severity of colitis via the induction of M2 macrophages and T-regulatory cells and suppression of T-helper (Th)1/Th17-cell responses, and their effects were comparable to those of cytokine-primed cells. Our results emphasize the importance of the activation of MSCs by the appropriate microenvironment to maximize their therapeutic effect.

Chemoprotective effect of arbutin on azoxymethane-induced aberrant crypt foci in rat colon via modulation of PCNA/Bax protein.

Arbutin is utilized in traditional remedies to cure numerous syndromes because of its anti-microbial, antioxidant, and anti-inflammatory properties. This study aimed to evaluate chemopreventive effects of arbutin on azoxymethane (AOM)-induced colon aberrant crypt foci (ACF) in rats. Five groups of rats were used: normal control group (rats injected hypodermically with sterile phosphate-buffered saline once per week for two weeks) and groups 2-5, which were subcutaneously inoculated with 15 mg/kg AOM once a week for two weeks. AOM control and 5-fluorouracil (5-FU) control groups were fed 10% Tween orally daily for 8 weeks using a feeding tube. The treated groups were fed 30 and 60 mg/kg arbutin every day for 2 months. ACF from the AOM control group had aberrant nuclei in addition to multilayered cells and an absence of goblet cells. The negative control group displayed spherical cells and nuclei in basal positions. Histological examination revealed a reduced number of AFC cells from colon tissues of the 5-FU reference group. Arbutin-fed animals showed down-regulation of proliferating cell nuclear antigen (PCNA) and up-regulation of Bax protein compared to AOM control. Rats fed with arbutin displayed a significant increase of superoxide dismutase (SOD) and catalase (CAT) activities in colon tissue homogenates compared to the AOM control group. In conclusion, arbutin showed therapeutic effects against colorectal cancer, explained by its ability to significantly decrease ACF, down-regulate PCNA protein, and up-regulate Bax protein. In addition, arbutin significantly increased SOD and CAT, and decreased malondialdehyde (MDA) levels, which might be due to its anti-proliferative and antioxidant properties.

Boric Acid (Boron) Attenuates AOM-Induced Colorectal Cancer in Rats by Augmentation of Apoptotic and Antioxidant Mechanisms.

Boric acid (BA) is a naturally occurring weak Lewis acid containing boron, oxygen, and hydrogen elements that can be found in water, soil, and plants. Because of its numerous biological potentials including anti-proliferation actions, the present investigates the chemopreventive possessions of BA on azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) in rats. Thirty laboratory rats were divided into 5 groups: negative control (A) received two subcutaneous inoculations of normal saline and nourished on 10% Tween 20; groups B-E had two injections of 15 mg/kg azoxymethane followed by ingestion of 10% Tween 20 (B, cancer control), inoculationwith intraperitoneal 35 mg/kg 5-fluorouracil injection (C, reference group), or ingested with boric acid 30 mg/kg (D) and 60 mg/kg (E). The gross morphology results showed significantly increased total colonic ACF in cancer controls, while BA treatment caused a significant reduction of ACF values. Histopathological evaluation of colons from cancer controls showed bizarrely elongated nuclei, stratified cells, and higher depletion of the submucosal glands than that of BA-treated groups. Boric acid treatment up-surged the pro-apoptotic (Bax) expression and reduced anti-apoptotic (Bcl-2) protein expressions. Moreover, BA ingestion caused upregulation of antioxidant enzymes (GPx, SOD, CAT), and lowered MDA contents in colon tissue homogenates. Boric acid-treated rats had significantly lower pro-inflammatory cytokines (TNF-α and IL-6) and higher anti-inflammatory cytokines (IL-10) based on serum analysis. The colorectal cancer attenuation by BA is shown by the reduced ACF numbers, anticipated by its regulatory potentials on the apoptotic proteins, antioxidants, and inflammatory cytokines originating from AOM-induced oxidative damage.

Investigation into the Potential Role of Propionibacterium freudenreichii in Prevention of Colorectal Cancer and Its Effects on the Diversity of Gut Microbiota in Rats.

Colorectal cancer (CRC) accounts for 10% of all cancer diagnoses and cancer-related deaths worldwide. Over the past two decades, several studies have demonstrated the clinical benefits of probiotic supplementation and some studies have shown that certain probiotics can modulate immunity and strengthen gut microbiota diversity. This study aims to assess the impact of the Propionibacterium freudenreichii (PF) probiotic against CRC induced by azoxymethane (AOM), and to investigate its effects on gut microbiota diversity in rats, as well as to evaluate the anti-proliferative activities of PF in HCT116 CRC cells. This experiment was performed using four groups of SD rats: normal control, AOM group, PF group (1 × 109 CFU/mL), and standard drug control (5-fluorouracil, 35 mg/kg). Methylene blue staining of colon tissues showed that the administration of PF significantly reduced the formation of colonic aberrant crypt foci (ACF) compared to the AOM control group. In addition, treated rats had lower levels of malondialdehyde in their colon tissue homogenates, indicating that lipid peroxidation was suppressed by PF supplementation. Furthermore, 16S rRNA gene analysis revealed that probiotic treatment enhanced the diversity of gut microbiota in rats. In vitro study showed that the viability of HCT116 cells was inhibited by the probiotic cell-free supernatant with an IC50 value of 13.3 ± 0.133. In conclusion, these results reveal that consuming PF as probiotic supplements modulates gut microbiota, inhibits the carcinogenic effects of AOM, and exerts anti-proliferative activity against CRC cells. Further studies are required to elucidate the role of PF on the immune response during the development and growth of CRC.

Pinostrobin attenuates azoxymethane-induced colorectal cytotoxicity in rats through augmentation of apoptotic Bax/Bcl-2 proteins and antioxidants

Objectives: Pinostrobin (5-hydroxy-7-methoxyflavanone; PN) is a natural active ingredient with numerous biological activities extensively utilized in tumour chemotherapy. The present study investigates the chemo-preventive potentials of PN on azoxymethane-mediated colonic aberrant crypt foci in rats. Methods: Sprague Dawley rats clustered into five groups, normal control (A) and cancer controls were subcutaneously injected with normal saline and 15 mg/kg azoxymethane, respectively, and nourished on 10% tween 20 and fed on 10% tween 20; reference control (C), injected with 15 mg/kg azoxymethane and injected (intraperitoneal) with 35 mg/kg 5-fluorouracil (5-FU); D and E rat groups received a subcutaneous injection of 15 mg/kg azoxymethane and nourished on 30 and 60 mg/kg of PN, respectively. Results: The acute toxicity trial showed a lack of any abnormal signs or mortality in rats ingested with 250 and 500 mg/kg of PN. The gross morphology of colon tissues revealed significantly lower total colonic aberrant crypt foci incidence in PN-treated rats than that of cancer controls. Histological examination of colon tissues showed increased aberrant crypt foci availability with bizarrely elongated nuclei, stratified cells and higher depletion of the submucosal glands in cancer controls. PN treatment caused positive modulation of apoptotic (Bax and Bcl-2) proteins and inflammatory cytokines (TNF-α, IL-6 and IL-10). Moreover, rats fed on PN had significantly higher antioxidants (superoxide dismutase) and lower malondialdehyde concentrations in their colon tissue homogenates. Conclusion: The chemoprotective efficiency of PN against azoxymethane-induced aberrant crypt foci is shown by lower aberrant crypt foci values and higher aberrant crypt foci inhibition percentage, possibly through augmentation of genes responsible for apoptotic cascade and inflammations originating from azoxymethane oxidative stress insults.

Curcumin alleviates experimental colitis via a potential mechanism involving memory B cells and Bcl-6-Syk-BLNK signaling

Immune dysfunction is the crucial cause in the pathogenesis of inflammatory bowel disease (IBD), which is mainly related to lymphocytes (T or B cells, incl-uding memory B cells), mast cells, activated neutrophils, and macrophages. As the precursor of B cells, the activation of memory B cells can trigger and differentiate B cells to produce a giant variety of inducible B cells and tolerant B cells, whose dysfunction can easily lead to autoimmune diseases, including IBD. To investigate whether or not curcumin (Cur) can alleviate experimental colitis by regulating memory B cells and Bcl-6-Syk-BLNK signaling. Colitis was induced in mice with a dextran sulphate sodium (DSS) solution in drinking water. Colitis mice were given Cur (100 mg/kg/d) orally for 14 con-secutive days. The colonic weight, colonic length, intestinal weight index, occult blood scores, and histological scores of mice were examined to evaluate the curative effect. The levels of memory B cells in peripheral blood of mice were measured by flow cytometry, and IL-1β, IL-6, IL-10, IL-7A, and TNF-α expression in colonic tissue homogenates were analyzed by enzyme-linked immunosorbent assay. Western blot was used to measure the expression of Bcl-6, BLNK, Syk, and other signaling pathway related proteins. After Cur treatment for 14 d, the body weight, colonic weight, colonic length, colonic weight index, and colonic pathological injury of mice with colitis were ameliorated. The secretion of IL-1β, IL-6, TNF-α, and IL-7A was statistically decreased, while the IL-35 and IL-10 levels were considerably increased. Activation of memory B cell subsets in colitis mice was confirmed by a remarkable reduction in the expression of IgM, IgG, IgA, FCRL5, CD103, FasL, PD-1, CD38, and CXCR3 on the surface of CD19+ CD27+ B cells, while the number of CD19+ CD27+ IL-10+ and CD19+ CD27+ Tim-3+ B cells increased significantly. In addition, Cur significantly inhibited the protein levels of Syk, p-Syk, Bcl-6, and CIN85, and increased BLNK and p-BLNK expression in colitis mice. Cur could effectively alleviate DSS-induced colitis in mice by regulating memory B cells and the Bcl-6-Syk-BLNK signaling pathway.

A6 THE ROLE OF TUFT CELLS IN INTESTINAL HOMEOSTASIS

Abstract Background Intestinal epithelial cells may actively regulate homeostasis by recognizing and responding to extracellular signals. One of these cell types, tuft cells, has been proposed to have a role in secretion, absorption, and reception. However, their role in the intestine has not been fully characterized. We have found that tuft cells express the SH2 domain-containing inositol 5’-phosphatase (SHIP), which was formerly thought to be restricted to hematopoietic cells. SHIP negatively regulates PI3K-mediated cell growth, proliferation, and activation. Tuft cells secrete IL-25, which activates group 2 innate lymphoid cells (ILC2s), leading to type 2 immune responses. Tuft cells may contribute to inflammation in the intestine by increasing ILC2 numbers and/or activation, leading to type II inflammation. Aims My hypothesis is that SHIP inhibits tuft cell responses to innate immune stimuli by limiting PI3K activation. Moreover, SHIP deficiency will increase tuft cell responses to commensal microbes, causing ILC2-mediated type II inflammation. To investigate the role of SHIP in tuft cell responses in vivo, I will use a tuft cell-specific SHIP deficient mouse in the dextran sodium sulfate (DSS)-induced colitis model. Methods We created a mouse deficient in SHIP only in intestinal tuft cells (Fabpcre x SHIPfl/fl) to investigate the impact of SHIP deficiency in tuft cells on responses to luminal microbes. Tuft cell-specific SHIP deficient mice (8-week-old) and their wild type littermates were subjected to DSS-induced colitis for 7 days. Clinical disease activity was monitored daily and gross pathology, including total colon length, was examined at the experimental endpoint. The concentrations of pro-inflammatory type I and type II cytokines were assessed in colonic tissue homogenates via ELISA. Results During DSS-induced colitis, mice with SHIP deficient tuft cells had increased disease activity compared to their wild type littermates, particularly evident in their weight loss. Mice with SHIP deficient tuft cells also had significantly shorter colons than their wild type littermates. IL-25 concentrations (produced by tuft cells) were increased in full thickness colon homogenates from mice with SHIP deficient tuft cells. In contrast, pro-inflammatory cytokines IL-1β, IL-6, and TNF did not differ between genotypes. Thus, increased tuft cell activity due to SHIP deficiency correlated with increased disease severity during DSS-induced colitis. Conclusions SHIP deficiency in intestinal tuft cells leads to increased tuft cell activity and exacerbated colitis during DSS treatment. Tuft cells may contribute to inflammation via IL-25 production, leading to increased type II inflammation by ILC2s. In future studies, we will target IL-25 in this model to determine whether increased tuft cell IL-25 production plays a causal role in disease exacerbation. Funding Agencies NSERC

In-Sample Calibration Curve Using Multiple Isotopologue Reaction Monitoring of a Stable Isotopically Labeled Analyte for Instant LC-MS/MS Bioanalysis and Quantitative Proteomics.

A novel methodology of in-sample calibration curves (ISCC) using multiple isotopologue reaction monitoring (MIRM) of multiple naturally occurring isotopologue transitions of a stable isotopically labeled (SIL) analyte for instant liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalysis of biomarkers, biotherapeutics, and small-molecule compounds is proposed and demonstrated for the first time. The theoretical isotopic abundances of the SIL analyte in its MIRM channels can be accurately calculated based on the isotopic distributions of its daughter ion and neutral loss. The isotopic abundances in these MIRM channels can also be accurately measured with a triple quadrupole mass spectrometer. By spiking a known amount of a SIL analyte into each study sample, an ISCC can be established based on the relationship between the calculated theoretical isotopic abundances (analyte concentration equivalents) in the selected MIRM channels of the SIL analyte and the measured MS/MS peak areas in the corresponding MIRM channels in each individual study sample. The analyte concentration of each study sample can then be calculated individually with the ISCC instantly without using an external calibration curve. The MIRM-ISCC-LC-MS/MS methodology was evaluated and demonstrated in this work with the examples of quantitation of a protein biomarker in human and monkey serum processed with immunocapture and trypsin digestion; three surrogate peptides in trypsin-digested human colon tissue homogenates; and a small-molecule drug in human and rat plasma extracted with liquid-liquid extraction. The potential applications of the MIRM-ISCC-LC-MS/MS methodology in quantitative proteomics, clinical laboratories, and other areas are also discussed in this paper. Without the need for using external calibration curves, this novel MIRM-ISCC-LC-MS/MS methodology can provide accurate and reliable bioanalysis in many potential applications, especially for cases where authentic matrices for external calibration curves are not available.

Tong-Xie-Yao-Fang Regulates 5-HT Level in Diarrhea Predominant Irritable Bowel Syndrome Through Gut Microbiota Modulation.

Tong-Xie-Yao-Fang (TXYF) has been widely used for the treatment of diarrhea-predominant irritable bowel syndrome (IBS-D) in traditional Chinese medicine. However, its mechanism of action in the treatment of IBS-D remains to be fully understood. Recent reports have shown that Clostridium species in the gut can induce 5-HT production in the colon, which then contributes to IBS-D. Due to the wide use of TXYF in the clinical treatment of IBS-D and the close relationship between gut microbiota and IBS-D, we hypothesize that TXYF treats IBS-D by modulating gut microbiota and regulating colonic 5-HT levels. In this study, variation analysis of 16S rRNA was conducted to evaluate changes in the distribution of gut microbiota in IBS-D model rats after TXYF treatment. Moreover, we investigated whether TXYF could affect colonic 5-HT levels in IBS-D model rats. We then performed fecal transplantation experiments to confirm the effects of TXYF on gut microbiota and 5-HT levels. We found that TXYF treatment can ameliorate IBS-D and regulate 5-HT levels in colon tissue homogenates. TXYF treatment also affected the diversity of gut microbiota and altered the relative abundance of Akkermansia and Clostridium sensu stricto 1 in gut flora populations. Finally, we showed that fecal transplantation from TXYF-treated rats could relieve IBS-D and regulate 5-HT levels in colon tissue homogenates. In conclusion, the present study demonstrates that TXYF treatment diminishes colonic 5-HT levels and alleviates the symptoms of IBS-D by favorably affecting microbiota levels in gut flora communities.

Comparison of anti-inflammatory activities of an anthocyanin-rich fraction from Portuguese blueberries (Vaccinium corymbosum L.) and 5-aminosalicylic acid in a TNBS-induced colitis rat model

Despite the actual therapeutic approaches for inflammatory bowel disease (IBD), efficient and secure alternative options remain a research focus. In this context, anthocyanins seem promising natural anti-inflammatory agents, but their action mechanisms and efficacy as compared with established drugs still require more clarification. The main aim of this study was to compare the anti-inflammatory action of a chemically characterized anthocyanin-rich fraction (ARF), obtained from Portuguese blueberries (Vaccinium corymbosum L.), with that of 5-aminosalicylic acid (5-ASA), a first-line drug in IBD, in a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis rat model. Such fraction showed a high content and great molecular diversity of anthocyanins, with malvidin-3-galactoside and petunidin-3-arabinoside in the highest concentrations. After daily administration by intragastric infusion for 8 days, ARF, at a molar anthocyanin concentration about 30 times lower than 5-ASA, showed a higher effectiveness in counteracting the intestinal inflammation, as assessed by i) body weight variation and colon damage score, ii) reduction in leukocyte infiltration, iii) increase in antioxidant defenses and iv) by downregulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in colon tissue homogenates. The strong inhibition of COX-2 expression seems to be a crucial anti-inflammatory mechanism common to both ARF and 5-ASA, but the additional higher abilities of anthocyanins to downregulate iNOS and to decrease leukocytes infiltration and to increase antioxidant defenses in colon may account for the much higher anti-inflammatory action of anthocyanins. These data may contribute to the development of a promising natural approach in IBD management.

Astragalus polysaccharide attenuates rat experimental colitis by inducing regulatory T cells in intestinal Peyer's patches.

To explore probable mechanism underlying the therapeutic effect of Astragalus polysaccharide (APS) against experimental colitis. Thirty-two Sprague-Dawley rats were randomly divided into four groups. Colitis was induced with 2, 4, 6-trinitrobenzene sulfonic acid (TNBS). The rats with colitis were treated with 400 mg/kg of APS for 7 d. The therapeutic effect was evaluated by colonic weight, weight index of the colon, colonic length, and macroscopic and histological scores. The levels of regulatory T (Treg) cells in Peyer's patches were measured by flow cytometry, and cytokines in colonic tissue homogenates were analyzed using enzyme-linked immunosorbent assay. The expression of related orphan receptor-γt (ROR-γt), IL-23 and STAT-5a was measured by Western blot. After 7-d treatment with APS, the weight index of the colon, colonic weight, macroscopical and histological scores were decreased, while the colonic length was increased compared with the model group. The expression of interleukin (IL)-2, IL-6, IL-17, IL-23 and ROR-γt in the colonic tissues was down-regulated, but Treg cells in Peyer's patches, TGF-β and STAT5a in the colonic tissues were up-regulated. APS effectively ameliorates TNBS-induced experimental colitis in rats, probably through restoring the number of Treg cells, and inhibiting IL-17 levels in Peyer's patches.

Vitamin D3 enhances the tumouricidal effects of 5-Fluorouracil through multipathway mechanisms in azoxymethane rat model of colon cancer.

BackgroundVitamin D3 and its analogues have recently been shown to enhance the anti-tumour effects of 5- Fluorouracil (5-FU) both in vitro and in xenograft mouse model of colon cancer. This study measured the potential mechanism(s) by which vitamin D3 could synergise the tumouricidal activities of 5-FU in azoxymethane (AOM) rat model of colon cancer.MethodsSeventy-five male Wistar rats were divided equally into 5 groups: Control, AOM, AOM-treated by 5-FU (5-FU), AOM-treated by vitamin D3 (VitD3), and AOM-treated by 5-FU + vitamin D3 (5-FU/D). The study duration was 15 weeks. AOM was injected subcutaneously for 2 weeks (15 mg/kg/week). 5-FU was injected intraperitoneally in the 9th and 10th weeks post AOM (8 total injections were given: 12 mg/kg/day for 4 successive days, then 6 mg/kg every other day for another 4 doses) and oral vitamin D3 (500 IU/rat/day; 3 days/week) was given from week 7 post AOM till the last week of the study. The colons were collected following euthanasia for gross and histopathological examination. The expression of β-catenin, transforming growth factor-β1 (TGF-β1), TGF-β type 2 receptor (TGF-βR2), smad4, inducible nitric oxide synthase (iNOS), and heat shock protein-90 (HSP-90) proteins was measured by immunohistochemistry. In colonic tissue homogenates, quantitative RT-PCR was used to measure the mRNA expression of Wnt, β-catenin, Dickkopf-1 (DKK-1) and cyclooxygenase-2 (COX-2) genes, while ELISA was used to measure the concentrations of TGF-β1, HSP-90 and COX-2 proteins.ResultsMonotherapy with 5-FU or vitamin D3 significantly decreased the number of grown tumours induced by AOM (P < 0.05); however, their combination resulted in more significant tumouricidal effects (P < 0.05) compared with monotherapy groups. Mechanistically, vitamin D3/5-FU co-therapy significantly decreased the expression of Wnt, β-catenin, iNOS, COX-2 and HSP-90 and significantly increased the expression of DKK-1, TGF-β1, TGF-βR2, smad4 (P < 0.05), in comparison with their corresponding monotherapy groups.ConclusionsVitamin D3 and 5-FU synergise together and exhibit better anticancer effects by modulating Wnt/β-catenin pathway, TGF-β1 signals, iNOS, COX-2 and HSP-90. Further studies are required to illustrate the clinical value of vitamin D supplementation during the treatment of colon cancer with 5-FU in human patients.

P0026 The chemopreventive potential of Curcuma purpurascens rhizome in reducing azoxymethane-induced aberrant crypt foci in rats

Background Curcuma purpurascens rhizome, a member of the Zingiberaceae family, is a popular spice in Indonesia and is traditionally used for assorted remedies. Dichloromethane extract of C. purpurascens rhizome (DECPR) has previously been shown to have apoptosis-inducing effect on colon cancer cells. Methods We examined the potential of DECPR to prevent colon cancer development in rats treated with azoxymethane (AOM, 15 ml/kg) by the incidence of aberrant crypt foci (ACF). Starting from the day immediately after AOM treatment, the rats were orally administered once a day for 2 months with 10% Tween-20 (5 ml/kg, cancer control), DECPR (250 mg/kg, low dose) and DECPR (500 mg/kg, high dose). Meanwhile, the control group was intra-peritoneally injected with fluorouracil (35 mg/kg) for 5 days consecutively. After euthanising the rats, the number of ACF was enumerated in colon tissues. Bax, Bcl-2, and PCNA protein levels were examined using immunohistochemical and western blot analyses. Antioxidant enzymatic activity was measured in colon tissue homogenates and associated with malondialdehyde level. Findings DECPR exposure at both doses significantly decreased AOM-induced ACF formation, which was accompanied with the reduced expression of PCNA. Up-regulation of Bax and down-regulation of Bcl-2 suggested the involvement of apoptosis in the chemopreventive effect of DECPR. In addition, the oxidative stress resulting from AOM treatment was significantly attenuated after administration of DECPR, which was shown by the elevated antioxidant enzymatic activity and reduced malondialdehyde level. Interpretation DECPR significantly inhibits ACF formation in AOM-treated rats and may offer protection against colon cancer development.

The chemopreventive potential of Curcuma purpurascens rhizome in reducing azoxymethane-induced aberrant crypt foci in rats

Curcuma purpurascens BI. rhizome, a member of the Zingiberaceae family, is a popular spice in Indonesia that is traditionally used in assorted remedies. Dichloromethane extract of C. purpurascens BI. rhizome (DECPR) has previously been shown to have an apoptosis-inducing effect on colon cancer cells. In the present study, we examined the potential of DECPR to prevent colon cancer development in rats treated with azoxymethane (AOM) (15 mg/kg) by determining the percentage inhibition in incidence of aberrant crypt foci (ACF). Starting from the day immediately after AOM treatment, three groups of rats were orally administered once a day for 2 months either 10% Tween 20 (5 mL/kg, cancer control), DECPR (250 mg/kg, low dose), or DECPR (500 mg/kg, high dose). Meanwhile, the control group was intraperitoneally injected with 5-fluorouracil (35 mg/kg) for 5 consecutive days. After euthanizing the rats, the number of ACF was enumerated in colon tissues. Bax, Bcl-2, and proliferating cell nuclear antigen (PCNA) protein expressions were examined using immunohistochemical and Western blot analyses. Antioxidant enzymatic activity was measured in colon tissue homogenates and associated with malondialdehyde level. The percentage inhibition of ACF was 56.04% and 68.68% in the low- and high-dose DECPR-treated groups, respectively. The ACF inhibition in the treatment control group was 74.17%. Results revealed that DECPR exposure at both doses significantly decreased AOM-induced ACF formation, which was accompanied by reduced expression of PCNA. Upregulation of Bax and downregulation of Bcl-2 suggested the involvement of apoptosis in the chemopreventive effect of DECPR. In addition, the oxidative stress resulting from AOM treatment was significantly attenuated after administration of DECPR, which was shown by the elevated antioxidant enzymatic activity and reduced malondialdehyde level. Taken together, the present data clearly indicate that DECPR significantly inhibits ACF formation in AOM-treated rats and may offer protection against colon cancer development.

The chemopotential effect of Annona muricata leaves against azoxymethane-induced colonic aberrant crypt foci in rats and the apoptotic effect of Acetogenin Annomuricin E in HT-29 cells: a bioassay-guided approach.

Annona muricata has been used in folk medicine for the treatment of cancer and tumors. This study evaluated the chemopreventive properties of an ethyl acetate extract of A. muricata leaves (EEAML) on azoxymethane-induced colonic aberrant crypt foci (ACF) in rats. Moreover, the cytotoxic compound of EEAML (Annomuricin E) was isolated, and its apoptosis-inducing effect was investigated against HT-29 colon cancer cell line using a bioassay-guided approach. This experiment was performed on five groups of rats: negative control, cancer control, EEAML (250 mg/kg), EEAML (500 mg/kg) and positive control (5-fluorouracil). Methylene blue staining of colorectal specimens showed that application of EEAML at both doses significantly reduced the colonic ACF formation compared with the cancer control group. Immunohistochemistry analysis showed the down-regulation of PCNA and Bcl-2 proteins and the up-regulation of Bax protein after administration of EEAML compared with the cancer control group. In addition, an increase in the levels of enzymatic antioxidants and a decrease in the malondialdehyde level of the colon tissue homogenates were observed, suggesting the suppression of lipid peroxidation. Annomuricin E inhibited the growth of HT-29 cells with an IC50 value of 1.62 ± 0.24 μg/ml after 48 h. The cytotoxic effect of annomuricin E was further substantiated by G1 cell cycle arrest and early apoptosis induction in HT-29 cells. Annomuricin E triggered mitochondria-initiated events, including the dissipation of the mitochondrial membrane potential and the leakage of cytochrome c from the mitochondria. Prior to these events, annomuricin E activated caspase 3/7 and caspase 9. Upstream, annomuricin E induced a time-dependent upregulation of Bax and downregulation of Bcl-2 at the mRNA and protein levels. In conclusion, these findings substantiate the usage of A. muricata leaves in ethnomedicine against cancer and highlight annomuricin E as one of the contributing compounds in the anticancer activity of A. muricata leaves.

Crotoxin from Crotalus durissus terrificus is able to down-modulate the acute intestinal inflammation in mice.

Inflammatory bowel diseases (IBD) is the result of dysregulation of mucosal innate and adaptive immune responses. Factors such as genetic, microbial and environmental are involved in the development of these disorders. Accordingly, animal models that mimic human diseases are tools for the understanding the immunological processes of the IBD as well as to evaluate new therapeutic strategies. Crotoxin (CTX) is the main component of Crotalus durissus terrificus snake venom and has an immunomodulatory effect. Thus, we aimed to evaluate the modulatory effect of CTX in a murine model of colitis induced by 2,4,6- trinitrobenzene sulfonic acid (TNBS). The CTX was administered intraperitoneally 18 hours after the TNBS intrarectal instillation in BALB/c mice. The CTX administration resulted in decreased weight loss, disease activity index (DAI), macroscopic tissue damage, histopathological score and myeloperoxidase (MPO) activity analyzed after 4 days of acute TNBS colitis. Furthermore, the levels of TNF-α, IL-1β and IL-6 were lower in colon tissue homogenates of TNBS-mice that received the CTX when compared with untreated TNBS mice. The analysis of distinct cell populations obtained from the intestinal lamina propria showed that CTX reduced the number of group 3 innate lymphoid cells (ILC3) and Th17 population; CTX decreased IL-17 secretion but did not alter the frequency of CD4+Tbet+ T cells induced by TNBS instillation in mice. In contrast, increased CD4+FoxP3+ cell population as well as secretion of TGF-β, prostaglandin E2 (PGE2) and lipoxin A4 (LXA4) was observed in TNBS-colitis mice treated with CTX compared with untreated TNBS-colitis mice. In conclusion, the CTX is able to modulate the intestinal acute inflammatory response induced by TNBS, resulting in the improvement of clinical status of the mice. This effect of CTX is complex and involves the suppression of the pro-inflammatory environment elicited by intrarectal instillation of TNBS due to the induction of a local anti-inflammatory profile in mice.

Probiotics increase T regulatory cells and reduce severity of experimental colitis in mice

To investigate the effect of probiotics on regulating T regulatory cells and reducing the severity of experimental colitis in mice. Forty C57/BL mice were randomly divided into four groups. Colitis was induced in the mice using 2,4,6-trinitrobenzene sulfonic acid (TNBS). After 10-d treatment with Bifico capsules (combined bifidobacterium, lactobacillus and enterococcus), body weight, colonic weight, colonic weight index, length of colon, and histological scores were evaluated. CD4+CD25+Foxp3+T cell in mesenteric lymph nodes were measured by flow cytometry, and cytokines in colonic tissue homogenates were analyzed by a cytometric bead array. The colonic weight index and the colonic weight of colitis mice treated with Bifico were lower than that of TNBS-induced mice without treatment. However, colonic length and percent of body weight amplification were higher than in TNBS-induced mice without treatment. Compared with TNBS-induced mice without treatment, the level of CD4+CD25+Foxp3+T cells in mesenteric lymph nodes, the expression of interleukin (IL)-2, IL-4 and IL-10 in colonic tissues from colitis mice treated with Bifico were upregulated, and tumor necrosis factor-α and interferon-γ were downregulated. Probiotics effectively treat experimental colitis by increasing CD4+CD25+Foxp3+T cell and regulating the balance of Th1 and Th2 cytokines in the colonic mucosa.

Pomegranate (Punica granatum) Peel Extract Efficacy as a Dietary Antioxidant against Azoxymethane-Induced Colon Cancer in Rat

Functional foods include antioxidant nutrients which may protect against many human chronic diseases by combating reactive oxygen species (ROS) generation. The purpose of the present study was to investigate the protective effect of pomegranate peel extract (PPE) on azoxymethane (AOM)-induced colon tumors in rats as an in vivo experimental model. Forty Sprague-Dawley rats (4 weeks old) were randomly divided into 4 groups containing 10 rats per group, and were treated with either AOM, PPE, or PPE plus AOM or injected with 0.9% physiological saline solution as a control. At 8 weeks of age, the rats in the AOM and PPE plus AOM groups were injected with 15 mg AOM/kg body weight, once a week for two weeks. After the last AOM injection, the rats were continuously fed ad-libitum their specific diets for another 6 weeks. At the end of the experiment (i.e. at the age of 4 months), all rats were killed and the colon tissues were examined microscopically for lesions suspected of being preneoplastic lesions or tumors as well as for biochemical measurement of oxidative stress indices. The results revealed a lower incidence of aberrant crypt foci in the PPE plus AOM administered group as compared to the AOM group. In addition, PPE blocked the AOM-induced impairment of biochemical indicators of oxidative stress in the examined colonic tissue homogenates. The results suggest that PPE can partially inhibit the development of colonic premalignant lesions in an AOM-induced colorectal carcinogenesis model, by abrogating oxidative stress and improving the redox status of colonic cells.