The effects of apocynin against chloroquine and gamma radiation induced intestinal injury

Today, radiotherapy maintains its importance as one of the most effective cancer treatment methods. As in healthy cells, in some cancer cells, an autophagic process is activated for the survival of the cell in nutrient and oxygen deficiency. Autophagy, which develops in response to radiotherapy, limits this treatment modality. Chloroquine (CQ) has been used as an antimalarial drug for over eighty years. In recent years, chloroquine has been shown as an autophagy inhibitor and has begun to attract great attention. Various studies have shown that some tumor cells treated with CQ cannot autophagy and die by consuming their energy. It has been shown that when autophagy is suppressed in response to radiotherapy, there is a significant increase in the rate of apoptosis of cells, resulting in a significant reduction in tumor size. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) is a membrane-bound enzymatic complex responsible for the production of reactive oxygen species (ROT). Studies have shown that NOX-induced ROT initiates autophagy. Aposine (APO) is a substance with antioxidant and antiapoptotic properties, known as an effective NOX inhibitor. In our study, it was aimed to determine how the use of CQ as a support to radiotherapy in cancer treatment will create a cellular response on healthy tissues and to reveal the protective effects of APO with its antioxidant role in this combined treatment response for the first time. In this study, randomly selected animals were divided into eight groups with 7 Wistar albino rats each. Physiological saline to the first and second groups, CQ to the third and fifth groups, APO to the fourth and sixth groups, CQ and APO to the seventh and eighth groups, were injected. All injections were administered intraperitoneally (i.p.) five times, every 24 hours for 5 days. One hour after the fifth injection, 8 Gray (Gy) total body irradiation was applied to the second, fifth, sixth and seventh groups. Small intestine tissues were obtained from dissected rats one day after radiation administration. Histological changes in the small intestine tissue were applied by hematoxylin-eosin (HE), Masson's triple dye (Masson) and Periodic Acid Schiff (PAS) reaction, inhibition of autophagy with the specific cargo protein (p62) marker of autophagosome, cell proliferation, proliferation of cell nuclear antigen (PCNA) was determined by immunohistochemistry. Biochemically, microtubule-associated protein 1A/1B-light chain 3 (LC3) as autophagosomal biomarker for autophagy and lysosome membrane-associated protein 2 (LAMP 2) for lysosomal inhibition, cysteine aspartate-specific protease 3 (caspase-3) and B-cell lymphoma 2 (BCL-2) for apoptosis, NOX, myeloperoxidase (MPO) and malondialdehyde (MDA) markers for the presence and change of oxidative stress, Tumor necrosis factor -α (TNF-α) markers for the presence and change of inflammation were determined using enzyme-linked immunosorbent method (ELISA). In addition, lipid hydroperoxide (LPO) markers for the presence and change of oxidative stress, total antioxidant capacity (TAK), superoxide dismutase (SOD), catalase (KAT) and glutathione (GSH) markers for the presence and change of antioxidant effect were determined with routine biochemical methods. As a result of our study, we determined that radiation caused significant histological damage in the rat small intestine, caused a significant increase in oxidative damage parameters, weakened the antioxidant system, had antiproliferative, inflammatory and apoptotic effects, and also stimulated autophagy. We found that CQ given together with RAD increased oxidative stress, weakened antioxidant capacity, had antiproliferative and anti-inflammatory effects, and did not cause a significant change on apoptosis by itself. Due to the inhibition effect of CQ on the lysosomal step of autophagy, we observed a significant increase in p62, LC3 II and LAMP 2 levels in this group. The fact that APO significantly reverses these changes caused by RAD and CQ shows its protective effect. We anticipate that our results may shed light on clinical applications.