A Probiotic Combination of Limosilactobacillus reuteri and Clostridium butyricum Ameliorates 5-Fluorouracil-Induced Intestinal Mucositis in Rats by Strengthening Barrier Integrity and Modulating Immuno-Microbial Homeostasis.

Recent evidence suggests that transforming growth factor-alpha (TGF-alpha) enhances enterocyte proliferation and exerts a gut trophic effect. The purpose of the present study was to evaluate the effect of TGF-alpha on enterocyte proliferation and intestinal recovery following methotrexate (MTX)-induced intestinal mucositis in rats and in Caco-2 cells. Nonpretreated Caco-2 cells and those pretreated with MTX were incubated with increasing concentrations of TGF-alpha. Cell proliferation was determined by FACS cytometry. Adult rats were divided into three groups: control rats treated with vehicle, MTX rats treated with one dose (20 microg/kg) of MTX given intraperitoneally, and MTX-TGF-alpha rats treated with one dose of MTX followed by two doses of TGF-alpha (75 microg/kg a day). Three days after MTX injection, rats were sacrificed. Intestinal mucosal damage (Park's score), mucosal structural changes, and enterocyte proliferation were measured at sacrifice. Western blotting was used to determine the level of extracellular signal-related kinase (ERK) protein, a marker of cell proliferation. A nonparametric Kruskal-Wallis ANOVA test was used for statistical analysis with P value less than 0.05 considered statistically significant. The in vitro experiment demonstrated that treatment with TGF-alpha of Caco-2 cells resulted in a significant stimulation of cell proliferation in a dose-dependent manner. The in vivo experiment showed that treatment with TGF-alpha resulted in a significant increase in bowel and mucosal weight, DNA and protein content in jejunum and ileum, villus height in jejunum and ileum, crypt depth in ileum, and increased cell proliferation in jejunum and ileum compared to the MTX group. MTX-TGF-alpha rats also had a significantly lower intestinal injury score in ileum when compared to MTX animals. The increase in levels of cell proliferation in MTX-TGF-alpha rats corresponded with the increase in ERK protein levels in intestinal mucosa. Treatment with TGF-alpha prevents mucosal injury, enhances ERK-induced enterocyte proliferation, and improves intestinal recovery following MTX-induced intestinal mucositis in rats. These findings correlated with the observation that TGF-alpha also caused a significant stimulation of cell proliferation in a Caco-2 cell culture model treated with MTX. These observations may have significant implications for the treatment of patients on chemotherapy who develop severe mucositis.

Patchouli oil ameliorates 5-fluorouracil-induced intestinal mucositis in rats via protecting intestinal barrier and regulating water transport.

Worldwide, colorectal cancer (CRC) is a deleterious disease causing millions of death annually. 5-Fluorouracil (5-FU) is a first-line chemotherapy for CRC, but chemoresistance and gastrointestinal mucositis limit its efficacy. Polyphenol-rich foods are increasingly popular due to their potential beneficial roles in preventing and treating cancer. Ellagitannins are a group of phenolic compounds commonly found in pomegranate, strawberries, raspberries, etc. The objective of this study was to explore whether ellagitannins from pomegranate (PETs) could ameliorate 5-FU-induced intestinal mucositis and enhance the drug's efficacy against CRC. The results showed that PETs (100 mg/kg) counteracted 5-FU-induced intestinal mucositis in rats. The number of apoptotic cells per crypt was reduced from 1.50 ± 0.21 to 0.85 ± 0.18 ( P < 0.05). Moreover, PETs induced HT-29 CRC cell death through intrinsic apoptosis, as demonstrated by dissipation of mitochondrial membrane potential, increased Bax-to-Bcl-2 ratio, and cleavage of caspase 9 and caspase 3. PETs and 5-FU combination treatments exhibited synergistic cytotoxicity against HT-29 cells with a weighted combination index of 0.3494. PETs (80 μg/mL) and 5-FU (40 μg/mL) treatments for 48 h induced 14.03 ± 0.76% and 16.42 ± 1.15% of HT-29 cells to undergo apoptosis, while the combination treatment further increased apoptosis of cells to 34.00 ± 1.54% ( P < 0.05). Combination treatment of the cells also enhanced S phase cell cycle arrest as compared with PETs or 5-FU monotherapy ( P < 0.05). These results suggest that dietary ellagitannins from pomegranate could alleviate intestinal mucositis in rats induced by 5-FU while enhancing its toxicity against HT-29 cells through potentiation of apoptosis and cell cycle arrest.

Intestinal mucositis is a common toxic side effect in cancer patients receiving high-dose chemotherapy. This study aimed to evaluate the beneficial effects of Bifidobacterium infantis in a rat model of intestinal mucositis induced by 5-fluorouracil (5-FU). Thirty male Sprague-Dawley rats were divided into three groups: control, 5-FU, and 5-FU + B. infantis. A single intraperitoneal injection of 5-FU (150 mg/kg) was used to induce intestinal mucositis. B. infantis (1×109 cfu) was administered for 11 days, starting from 7 days before 5-FU injection. Intestinal mucositis was evaluated based on body weight, villus height, immunohistological expression of proliferating cell nuclear antigen (PCNA) and nuclear factor kappa beta (NF-κB), levels of the pro-inflammatory factors interleukin 1 beta and tumour necrosis factor alpha, and myeloperoxidase (MPO) concentration. The results showed that the 5-FU + B. infantis group demonstrated a higher body weight and villus height, increased expression of PCNA, reduced expression of NF-κB and pro-inflammatory factors, and lower MPO concentration compared to the 5-FU group. These data suggest that probiotic B. infantis is effective in reducing chemotherapy-induced intestinal mucositis in rats.

Cisplatin-based chemotherapy causes intestinal mucositis, which causes patients immense suffering and hinders the process of cancer treatment. Dioscin is a natural steroid saponin that exhibits strong anti-inflammatory and immunomodulatory properties. Herein, we investigate the protective effect of dioscin on cisplatin induced mucositis in rats from the perspective of gut microbiota and intestinal barrier. We established a rat model of intestinal mucositis by tail vein injection of cisplatin, and concurrently treated with dioscin oral administration. Parameters, such as body weight, diarrheal incidence, and D-Lactate levels, were assessed in order to evaluate the effects of dioscin on intestinal mucositis in rats. Furthermore, biological samples were collected for microscopic gut microbiota, intestinal integrity, and immune inflammation analyses to elucidate the protective mechanisms of dioscin on intestinal mucositis. The results revealed that administration of dioscin significantly attenuated clinical manifestations, histological injury and inflammation in mucositis rats. Besides this, dioscin markedly inhibited the gut microbiota dysbiosis induced by cisplatin. Meanwhile, dioscin partially alleviated junctions between ileum epithelial cells and increased mucus secretion. Moreover, dioscin effectively inhibited the TLR4-MyD88-NF-κB signal transduction pathway and reduced the secretion of subsequent inflammatory mediators. These results suggested that dioscin effectively attenuated cisplatin-induced mucositis in part by modulating the gut microflora profile, maintaining ileum integrity and inhibiting the inflammatory response through the TLR4-MyD88-NF-κB pathway.

Pre-treatment with insulin-like growth factor-I partially ameliorates 5-fluorouracil-induced intestinal mucositis in rats

Patchouli alcohol attenuates 5-fluorouracil-induced intestinal mucositis via TLR2/MyD88/NF-kB pathway and regulation of microbiota

One of the main secondary toxic side effects of anti-mitotic agents used to treat cancer patients is intestinal mucositis. Previous data showed that cathepsin D activity, contributing to the proteolytic lysosomal pathway, is up-regulated during intestinal mucositis in rats. At the same time, cathepsin inhibition limits intestinal damage in animal models of inflammatory bowel diseases. The aim of this study was to evaluate the effects of cathepsin inhibition on methotrexate-induced mucositis in rats. Male Sprague-Dawley rats received saline solution subcutaneously as the control group or 2·5 mg/kg of methotrexate for 3 days (D0-D2). From D0 to D3 methotrexate-treated rats also received intraperitoneal injections of pepstatin A, a specific inhibitor of cathepsin D or E64, an inhibitor of cathepsins B, H and L, or vehicle. Rats were euthanized at D4 and jejunal samples were collected. Body weight and food intake were partially preserved in rats receiving E64 compared with rats receiving vehicle or pepstatin A. Cathepsin D activity, used as a marker of lysosomal pathway, was reduced both in E64 and pepstatin-treated rats. However, villus atrophy and intestinal damage observed in methotrexate-treated rats were restored in rats receiving E64 but not in rats receiving pepstatin A. The intramucosal concentration of proinflammatory cytokines, interleukin-1β and cytokine-induced neutrophil chemoattractant (CINC)-2, was markedly increased in methotrexate-treated rats receiving vehicle or pepstatin A but not after E64 treatment. In conclusion, a large broad inhibition of cathepsins could represent a new potential target to limit the severity of chemotherapy-induced mucositis as opposed to the inhibition of cathepsin D alone.

Chemotherapy-induced mucositis is characterized by inflammation and ulceration of the intestinal mucosa, compromising intestinal function. Exogenous nucleotides have been reported to repair the mucosa. The nucleotide preparation, Nucleoforce F0328 (Nucleoforce), was investigated for its potential to ameliorate intestinal mucositis in rats. Female Dark Agouti rats (n = 8/group) were gavaged once daily with Nucleoforce (175 mg/kg) or water from Days 0 to 8 and injected (i.p.) with 5-fluorouracil (5-FU; 150 mg/kg) or saline on Day 5. Histological parameters (disease severity, crypt depth, and villus height measurements) and myeloperoxidase activity were quantified. P < 0.05 was considered significant. Jejunal and ileal histological disease severity scores were significantly increased by 5-FU, compared to normal controls (P < 0.05). Nucleoforce treatment in 5-FU-injected rats significantly reduced jejunal and ileal disease severity compared to 5-FU controls (P < 0.05). In 5-FU-injected rats, jejunal and ileal villus heights and crypt depths were significantly decreased compared to 5-FU controls, with no additional Nucleoforce effect (P > 0.05). Intestinal myeloperoxidase activity was significantly elevated by 5-FU (8.8-fold), compared to normal controls (P < 0.05), which was not normalized by Nucleoforce treatment (P > 0.05). Nucleoforce only partially improved parameters associated with experimentally-induced mucositis. Future studies could investigate increased concentrations, more frequent administration, or protective microencapsulation delivery methods, to increase bioavailability.

Although probiotics are beginning to enter mainstream medicine for disorders of the colon, their effects on the small bowel remain largely unexplored. We investigated the recently identified probiotic, Lactobacillus fermentum (L. fermentum) BR11 (BR11) and the prebiotic, fructo-oligosaccharide (FOS), both individually and in synbiotic combination, for their potential to alleviate intestinal mucositis. From Days 0–9, rats consumed skim milk (SM; saline + SM), low dose (LD-BR11; 1 × 10 6 cfu/ml), high dose (HD-BR11; 1 × 10 9 cfu/ml), LD-FOS (3%), HD-FOS (6%), or synbiotic (HD-BR11/FOS). On Day 7, rats were injected with 5-fluorouracil (5-FU; 150 mg/kg). All rats were sacrificed on Day 10. Intestinal tissues were collected for quantitative histology, sucrase, and myeloperoxidase (MPO) determinations. 5-FU decreased sucrase activity, villus height, crypt depth, and crypt cell proliferation compared to controls. Compared to 5-FU + SM, histological damage severity scores were increased for all treatments, although all were effective at reducing jejunal inflammation, indicated by reduced MPO activity ( P < 0.05). The combination of BR11 and FOS did not provide additional protection. Moreover, HD-FOS and the synbiotic actually increased clinical mucositis severity ( P < 0.05). We conclude that L. fermentum BR11 has the potential to reduce inflammation of the upper small intestine. However, its combination with FOS does not appear to confer any further therapeutic benefit for the alleviation of mucositis.

5-Fluorouracil (5-FU) is an anticancer drug with intestinal mucositis (IM) as a deleterious side effect. Thymol is a monoterpene phenol which has been reported to possess an antioxidant and anti-inflammatory activity versus 5-FU-induced IM. The Notch pathway affects multiple cellular activities, such as cellular proliferation, in addition to inflammatory responses modulation. Accordingly, this work was carried out in order to elucidate the role of the Notch pathway in 5-FU-induced IM and to further elucidate the immunomodulatory protective mechanisms of thymol. Experimental rats were divided randomly into four groups: Control, 5-FU, 5-FU+thymol (60 mg/kg/day), and 5-FU+thymol (120 mg/kg/day). 5-FU was injected intraperitoneally at a dose of 150 mg/kg on days 6 and 7, while thymol was orally administered daily for 11 days. By the end of the study, intestinal tissues were collected for the determination of IL-17, CD4, CD8, Notch1, Hes-1, pSTAT3, and STAT-3 protein expressions. The effect of thymol on 5-FU cytotoxicity was also examined using WST1 assay. 5-FU induced a marked increase in IL-17 levels, along with a marked downregulation of CD4 and the upregulation of CD8, Notch1, Hes-1 protein expressions, and activation of STAT3 in the intestinal tissue when compared with the control group. Thymol ameliorated the changes that occurred in these parameters. Additionally, cytotoxicity testing revealed that thymol augmented the antiproliferative action of 5-FU against breast and colorectal human cancer cell lines. This study was the first to show that the IL-17/Notch1/STAT3 pathway is involved in the molecular mechanism of 5-FU-induced IM, as well as the immunomodulatory activity of thymol.

A Diet Containing Whey Protein, Free Glutamine, and Transforming Growth Factor-β Ameliorates Nutritional Outcome and Intestinal Mucositis during Repeated Chemotherapeutic Challenges in Rats1–

Objective:Intestinal mucositis is a significant problem haunting clinicians for decades. One of the major reasons for its occurrence is high-dose chemotherapy. The study is aimed at investigating effect of intestinal mucositis on pharmacokinetics, organ distribution, and bioavailability of azathioprine (AZA) (6-mercaptopurine).Materials and Methods:Intestinal mucositis was induced with methotrexate (MTX) (2.5 mg/kg). The oral absorption of AZA and 6-mercaptopurine (metabolite) levels were determined in control and MTX-treated rats: ex vivo (noneverted sac technique) and in vivo (pharmacokinetics and organ-distribution) using high-performance liquid chromatography. Immunohistochemistry was conducted to evaluate peptide transporter expression on luminal membrane of small intestine.Results:Intestinal permeation of AZA into systemic circulation of rats was lower after MTX administration, widely found in intestinal segments of mucositis-induced rats leading to decline in systemic bioavailability of AZA. Immunohistochemistry findings indicated diminution of peptide transporter expression representing hampered absorption of drugs absorbed via this transporter.Conclusion:Study outcome has thrown light on altered fate of AZA when administered to individuals with mucositis which suggests modified drug therapy. These findings can further be investigated in different drug classes which might be administered concomitantly in mucositis and study outcome can be further confirmed in mucositis patients in clinical practice also.

Forsythiaside A alleviates methotrexate-induced intestinal mucositis in rats by modulating the NLRP3 signaling pathways

Milk Growth Factors Enriched from Cheese Whey Ameliorate Intestinal Damage by Methotrexate When Administered Orally to Rats