ABSTRACT
Although methotrexate is widely used in clinics as an anticancer agent, it's utility is limited by its gastrointestinal toxicity, the mechanism of which is unclear. The role of NFκB inflammatory pathway in MTX induced mucositis was investigated in the present study. GI injury was induced in adult Wistar rats by the administration of 3 consecutive i.p . injections of MTX. On the fourth day, the rats were sacrificed and the small intestine was removed; A piece was used for light microscopy, immunohistochemistry, immunofluorescence studies . The mucosa was collected and used for the analysis of protein and mRNA expressions of NFκB and its target genes by the western blot, RT-PCR respectively. MTX treatment resulted in NFκB activation and nuclear translocation as evidenced by immunofluorescence, immunohistochemistry , and western blot. NFκB mRNA was also increased. There was increased protein and mRNA expressions of NFκB target genes, TNF-α, iNOS, COX-2, PLA2, HO-1, HSP70, MMPs 2 and 9 . Aminoguanidine pretreatment (30mg/ 50mg /kg body wt.) attenuated MTX induced activation of NFκB and its proinflammatory target genes and improved MTX induced morphological changes. Aminoguanidine has protective effects against MTX induced gastrointestinal mucositis in rats.
Subject(s)
Antimetabolites, Antineoplastic/toxicity , Cyclooxygenase 2/metabolism , Inflammation/metabolism , Intestine, Small/drug effects , Methotrexate/toxicity , NF-kappa B/metabolism , Nitric Oxide Synthase Type II/metabolism , Signal Transduction , Tumor Necrosis Factor-alpha/metabolism , Animals , Guanidines/pharmacology , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , Heme Oxygenase-1/genetics , Heme Oxygenase-1/metabolism , Inflammation Mediators/metabolism , Intestine, Small/injuries , Male , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Phospholipases A2/metabolism , RNA, Messenger/genetics , Rats, Wistar , Up-Regulation/drug effectsABSTRACT
The efficacy of methotrexate (MTX), a commonly used chemotherapeutic drug, is limited by intestinal injury. As the mechanism of MTX-induced small intestinal injury is not clear, there is no definitive treatment for MTX-induced gastrointestinal injury. The present study investigates the role of mitochondrial apoptotic pathway in MTX-induced small intestinal injury and examines whether aminoguanidine is effective in preventing the damage. Eight Wistar rats were administered 3 consecutive i.p. injections of 7 mg/kg body wt. MTX. Some rats were pretreated with 30 mg or 50 mg/kg body wt. of aminoguanidine (n = 6 in each group). Protein expressions of cytochrome c, caspases 3 and 9, and PARP-1 were determined in the small intestines by immunohistochemistry and western blot. Mitochondrial pathway of apoptosis was activated in the small intestines of MTX-treated rats as evidenced by intense immunostaining for cyt c, caspases 9 and 3, and PARP-1 and mitochondrial release of cyt c, activation of caspases, and PARP-1 cleavage by Western blot. Immunofluorescence revealed increased nuclear localization of PARP-1. Aminoguanidine pretreatment ameliorated MTX-induced small intestinal injury in dose-dependent manner and inactivated the mitochondrial apoptotic pathway. Aminoguanidine may possess beneficial intestinal protective effects as an adjuvant co-drug against MTX intestinal toxicity during cancer chemotherapy. As the mechanism of MTX-induced small intestinal injury is not clear, there is no definitive treatment for MTX-induced gastrointestinal injury. The results of the present study show that the mitochondrial pathway of apoptosis plays a role in MTX-induced small intestinal injury as evidenced by cytochrome c release, activation of caspases 9 and 3, PARP-1 cleavage, and DNA fragmentation. Aminoguanidine (AG) pretreatment attenuates the severity of small-intestinal injury induced in rats by MTX treatment. The mechanisms of action of AG involve inhibition of iNOS, and mitochondrial pathway of apoptosis. It is suggested that aminoguanidine may possess beneficial intestinal protective effects as an adjuvant co-drug against MTX intestinal toxicity during cancer chemotherapy.
Subject(s)
Apoptosis/drug effects , Intestine, Small/drug effects , Methotrexate/toxicity , Mitochondria/drug effects , Animals , Caspase 3/metabolism , Caspase 9/metabolism , Cytochromes c/metabolism , DNA Fragmentation/drug effects , Guanidines/pharmacology , Intestine, Small/metabolism , Intestine, Small/pathology , Male , Microscopy, Fluorescence , Mitochondria/metabolism , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II/antagonists & inhibitors , Nitric Oxide Synthase Type II/metabolism , Poly (ADP-Ribose) Polymerase-1/metabolism , Protective Agents/pharmacology , Rats , Rats, WistarABSTRACT
BACKGROUND: One of the major toxic side effects of methotrexate (MTX) is enterocolitis, for which there is no efficient standard treatment. Nitric oxide overproduction has been reported to play an important role in MTX-induced mucositis. This study was designed to investigate whether pretreatment with aminoguanidine (AG) - a selective iNOS inhibitor - prevents MTX-induced mucositis in rats. METHODS: Rats were pretreated with AG (30 and 50 mg/kg body weight) i.p. daily 1 h before MTX (7 mg/kg body weight) administration for 3 consecutive days. After the final dose of MTX, the rats were killed, and the small intestines were used for analysis. RESULTS: The small intestines of MTX-treated rats showed moderate to severe injury. Pretreatment with AG had a dose-dependent protective effect on MTX-induced mucositis. AG pretreatment reduced iNOS protein levels, mucosal nitric oxide levels, and protein tyrosine nitration. AG pretreatment also restored the activities of electron transport chain (ETC) complexes, vital tricarboxylic acid (TCA cycle) enzymes, and mitochondrial antioxidant enzymes. CONCLUSIONS: These findings suggest that AG is beneficial in ameliorating MTX-induced enteritis in rats.
