Therapeutic effects of a purified chitin-binding protein from Moringa oleifera seeds on irinotecan-induced intestinal mucositis in mice

Abstract

Intestinal mucositis is a common side effect of irinotecan-based anticancer regimens, affecting approximately 85% of colorectal cancer (CRC) patients undergoing chemotherapy. Current treatment options are mainly palliative. Moringa oleifera Lamarck, native to Northeast India, is known for its nutritional and therapeutic properties. Our research group has demonstrated that MoCBP4 (11.78 kDa), a thermostable chitin-binding protein isolated from Moringa oleifera seeds, possesses potent antinociceptive, antifungal, wound-healing, and anti-inflammatory activity, both orally and intraperitoneally. Therefore, this study aimed to evaluate the protective effect of Mo-CBP4 in a model of irinotecan (CPT-11)-induced intestinal mucositis. This study was approved by the Animal Research Ethics Committee of UFC – CEPA (9892300120) and (7796300120). Male Swiss mice (25-30 g) were divided into 3 groups: Group 1 received saline solution (0.9%, i.p.) once a day for seven days; Group 2 received irinotecan (75 mg/kg, i.p.) once a day for four days; Group 3 was treated for 7 days with Mo-CBP4 at a dose of 10 mg/kg e.v., respectively, 30 minutes before CPT-11, which was administered for 4 days. During the seven days, weight loss, diarrhea scores, and survival were evaluated. On the seventh day, blood was collected for leukocyte count, followed by euthanasia for duodenum collection and evaluation of the following parameters: small intestine length, intestinal contractility, histopathological and morphometric alterations, MPO, GSH, MDA, NO, cytokines (IL-1β, IL-6, KC, TGF-β, and IL-10), and PCR (IL-33, IL-17, Claudin-2, Occludin, and ZO-1). We evaluated the cytotoxicity of MoCBP4 in normal and tumor cells for 24h and 72h using the Sulforhodamine B (SRB) assay and its interference with the effect of SN38 [2.5μM] (active metabolite of CPT-11) for 48h in murine colon cancer (MC38). We injected MC38 cells into male C57BL/6 mice (n=10; 25±2g) to induce a tumor amenable to treatment with CPT-11. We analyzed daily weight and palpable tumor growth with a digital caliper, and after sacrifice, we measured final tumor growth and MPO. Irinotecan (CPT-11) induced intestinal mucositis in mice, characterized by weight loss, diarrhea, increased mortality, leukopenia, decreased intestinal length, increased intestinal contractility, histopathological alterations (villous blunting, loss of crypt architecture, vacuolation, inflammatory cell infiltrate), and increased levels of MPO, IL-1β, IL-6, KC, TGF-β, IL-33, and Claudin 2. Increased MDA and decreased GSH levels were also observed. Treatment with Mo-CBP4 (10 mg/kg) significantly attenuated CPT-11-induced intestinal mucositis, improving diarrhea, increasing survival, reducing intestinal damage, and attenuating histopathological alterations. MoCBP4 was also able to decrease levels of MPO (35%), NO (48%), IL-1β (52%), IL-6 (98%), KC (88%), TGF-β (62%), IL-33, decrease MDA levels, and increase GSH. MoCBP4 did not exhibit cytotoxic activity in MC38, L929, SK-MEL, and B16F10 cell lines, and did not interfere with the cytotoxic effect of SN38. It also did not interfere with the antitumor effect of CPT-11 in a tumor transplant model. Therefore, Mo-CBP4 demonstrates important antidiarrheal, anti-inflammatory, and antioxidant activities that make it a promising therapeutic option for preventing and attenuating the severity of intestinal mucositis during CPT-11 chemotherapy treatment without interfering with the antitumor effect of irinotecan.