Fasolino, Ines (2013) Plant-derived compounds in experimental inflammatory bowel disease and colon carcinogenesis. [Tesi di dottorato]

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Abstract

Abstract Background Inflammatory bowel disease (IBD) and colorectal cancer (CRC) are widespread intestinal diseases. The link between these two diseases is highlighted by the observation that patients with IBD are at increased risk for CRC. Plants have been traditionally used in folk medicine and are actually practiced in industrialized countries where their use is often integrated into conventional medicine. Most survey agree that digestive tract ailments cure/prevention, including IBD and CRC, is one of the most frequent reasons for trying plant medicines. Aim To evaluate the effect of a number of plant-derived compounds [i.e. cannabigerol (CBG), a non-psychotropic cannabinoid from Cannabis sativa, diallyl sulfide (DAS) and diallyl disulfide (DADS), two organosulfur compounds from Allium sativum, bromelain, a cysteine protease from Ananas comosus and boeravinone G, a rotenoid isolated from the Ayurvedic plant Boerhaavia diffusa) in experimental models of IBD and colon cancer. Methods. Experimental colitis was induced in mice by intracolonic administration of dinitrobenzene sulfonic acid (DNBS). Inflammation was assessed by evaluating inflammatory markers/parameters (e.g. colon weight/colon length ratio and myeloperoxidase activity), by histological analysis and by immunohistochemistry; interleukin-1β, interleukin-10, interferon-γ (IFN-γ), interferon-γ-induced protein 10 (IP-10) levels were measured by ELISA, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) by western blot and RT-PCR; CuZn-superoxide dismutase (SOD) activity were evaluated by a colorimetric assay. Murine macrophages and intestinal epithelial cells were used to evaluate the in vitro antinflammatory action (nitric oxide production, oxidative stress, interferon-γ, interleukin-6 (IL-6) and IP-10 levels or mRNA expression). Motility changes were investigated in vivo in the model of intestinal inflammation induced by croton oil. Colon carcinogenesis was induced by the administration of the carcinogenic agent azoxymethane (AOM) in mice, which induces the formation of preneoplastic lesions (aberrant crypt foci), polyps and tumours. In colorectal carcinoma (Caco-2) cells, proliferation was evaluated using the 3H-thymidine incorporation assay; ERK, Akt and phospho-NF-kB p65 expression was evaluated by western blot analysis, ROS production by fluorescent probe, DNA damage by Comet assay. Results Main results indicate that 1) CBG exerts therapeutic actions in the DNBS model of colitis in mice. The effect of CBG was associated to modulation of intestinal cytokine levels and down-regulation of intestinal iNOS expression. In macrophages, CBG inhibited iNOS-derived nitric oxide production. Also, CBG protected intestinal epithelial cells exposed to oxidative stress. 2) DAS and DADS exert beneficial actions in the DNBS model of IBD. Both compounds reduced intestinal inflammation and damage, as revealed by gross evaluation, histology and immunohistochemistry. In intestinal epithelial cells, DADS reduced IP-10 and IL-6 levels, while DAS inhibited IFN-γ-stimulated nitrite production. 3) Bromelain normalises intestinal inflammation-induced hypermotility and exerts chemopreventive actions in the AOM model of colon carcinogenesis. In Caco-2 cells, bromelain stimulated apoptotic processes via blockade of MAP kinase and (PI3K)/Akt signalling. 4). Boeravinone G exerts potent antioxidant and genoprotective actions in Caco-2 cells. The genoprotective effect of boeravinone G was associated to up-regulation of pERK1 and NF-kB expression. Conclusions. The plant ingredients CBG, DAS, DADS, bromelain and boeravinone G exerts promising intestinal antiinflammatory and antitumoural actions. In view of their safety records, these plant-derived compounds might be worthy of consideration in future clinical trials for the evaluation of new therapeutics in IBD and CRC prevention/treatment

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