Rutin (3,3’,4’,5,7-pentahydroxyflavone-3rhamnoglucoside), a flavonoid glycoside, consists of flavonol quercetin and disaccharide rutinose. Many plant species such as passion flower, buckwheat, tea and apple contain rutin ¹⁵. Rutin consumed in the daily diet is an important flavonoid ¹⁶. Rutin has many pharmacologic activities with potent antioxidant and anti-inflammatory effects. Ameliorative effects of rutin on hepatic, renal, reproductive, neurologic, and cardiac diseases have been demonstrated in many studies ^17-22.

Few studies evaluated the protective effects of rutin against cisplatin-induced nephrotoxicity. However, there is no studies on anti-apoptotic protein Bcl-3 levels in cisplatin nephrotoxicity to the best of the author’s knowledge. Thus, this study investigated the effects of rutin on oxidative stress, the levels of apoptotic protein caspase-3 and anti-apoptotic protein Bcl-3 in nephrotoxicity caused by cisplatin in rats.

Animals: The study consisted of 21 male Sprague-Dawley rats, 230-250 g weight, and eight weeks old. The animals were obtained from Atatürk University, Experimental Animal Unit, Erzurum, Turkey, and kept under standard conditions (at 24±1 °C temperature and 45±5% humidity and in 12 h light and dark cycle). The rats were fed ad libitum. They were acclimatized for 1 week prior to the experimental process. This study carried out on laboratory animals was approved by Local Ethics Committee of Atatürk University.

The rats were randomly separated into three groups, including 7 rats in each group. Physiological saline was administered to Group I for 14 days. Group II was treated with cisplatin on the tenth day of the study. Group III was treated with rutin 150 mg/kg orally for 14 days ¹⁷ plus cisplatin 10 mg/kg intraperitoneally on the tenth day of the study.

Blood and Kidney Samples Collection: Blood samples taken to the tubes without any anticoagulant were allowed to coagulate for 15 min at room temperature and centrifuged at 800 g for 5 min at 4 °C. The serum samples were allocated to the Eppendorf tubes and kept at -20 °C.

The rat kidneys were removed after euthanasia under sevoflurane anesthesia. They were washed with physiological saline and stored at -20 °C for further analyses.

Biochemical Analyses: The commercial kits were used for the analyses of the serum urea and creatinine concentrations (Diasis Diagnostic Systems, Istanbul, Turkey). In the, kidney, malondialdehyde (MDA) levels ^23, catalase activity ^24, protein concentration in the supernatant ^25, superoxide dismutase (SOD) activity ^26, glutathione (GSH) content ^27, and glutathione peroxidase activity (GSH-Px) ²⁸ were analyzed. In addition, caspase 3 and B-cell lymphoma protein 3 (Bcl-3) levels were determined according to the ELISA kit procedures.

Statistical Analysis: The variables of this study had normal distribution according to Kolmogorov-Smirnov test. Accordingly, the data were analyzed with one-way ANOVA and Tukey HSD tests using SPSS package program. The data were presented as the mean ± standard error of means (SEM). Statistical significance level was determined as P<0.05 ²⁹.

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Table 1: Effects of rutin on serum renal function markers, and on oxidants, antioxidants, apoptotic and anti-apoptotic parameters in rats with cisplatin nephrotoxicity

MDA Levels: The kidney MDA levels were significantly elevated in cisplatin group than control group. The kidney MDA levels were significantly reduced in cisplatin plus rutin treated group than cisplatin group. However, the decrease in MDA levels in cisplatin plus rutin treated group was still significantly higher than control group (Table 1).

Evaluation of Antioxidants: The kidney GSH level, catalase, GSH-Px, and SOD activities were significantly reduced in cisplatin group than control group. The kidney GSH level, catalase, GSH-Px, and SOD activities were significantly increased in cisplatin plus rutin group than cisplatin group, but were still lower than control group (Table 1).

Evaluation of Apoptosis-Related Parameters: The kidney caspase-3 and Bcl-3 levels were significantly elevated in cisplatin group than control group. The kidney caspase-3 and Bcl-3 levels were significantly reduced in cisplatin-plus-rutin-treated group than cisplatin group. However, these levels were still higher in cisplatin-plus-rutin-treated group than control group (Table 1).

In the pathogenesis of cisplatin-induced kidney injury, the mitochondrial dysfunction caused by reactive oxygen species (ROS) with increase in oxidants such as superoxide anion, hydrogen peroxide and decrease in SOD activities were noted ^31,32. Another study also confirmed the role of increased ROS, and apoptotic pathway proteins such as caspase-3, NF-κB, and TNF-α in cisplatin related kidney injury ³³. Similarly, another study showed that cisplatin induced the release of proinflammatory cytokines such as NF-κB, TNF-α, IL-1β, IL-6, IL-33, iNOS and COX-2 and decreased the kidney SOD, catalase and GSH-Px activities and GSH levels ³⁴.

In this study, the increased kidney MDA levels revealed that cisplatin could cause lipid peroxidation in the kidney epithelial cells via decrease in the kidney SOD, catalase and GSH-Px activities and GSH levels, and could activate apoptotic protein pathways as revealed by increased caspase-3 levels. This suggests the activation of caspases and the induction of apoptosis. Caspases are activated by the release of cytochrome C to cytosol ³⁵. Malik et al. ³⁵ have showed that cisplatin causes increase in caspase expression and apoptosis in the kidney cells.

In this study, rutin pretreatment prevented significantly apoptotic protein caspase-3 increase in cisplatin plus rutin group. Thus, rutin pretreatment provided decrease in anti-apoptotic protein Bcl-3 due to alleviation of apoptosis in the kidney of cisplatin plus rutin group. This suggests that rutin has significant anti-apoptotic effects against cisplatin nephrotoxicity in the rat kidney tissue.

Several studies have demonstrated that the antioxidant compounds such as rutin ^33, tangeretin ^36, embelin ^30, melatonin (367) prevent oxidative stress and inflammation on cisplatin-induced kidney injury.

Rutin, an active citrus flavonoid compound, exerts its effects via potent antioxidant properties. Rutin can scavenge radicals and activate intrinsic antioxidant defense systems ^17,19,38.

Cisplatin can lead to the depletion of GSH and other antioxidants ³⁴. In this study, rutin pretreatment reversed the kidney GSH depletion due to cisplatin treatment by increasing significantly GSH contents in the kidney. In addition, rutin pretreatment reversed oxidative stress induced by cisplatin as demonstrated by increased GSH levels, SOD, catalase, and GSH-Px activities, and decreased MDA levels.

B-cell lymphoma protein 3 (Bcl-3) is described as a negative regulator of transcription factor NF-ĸB ^39,40. Poveda et al. ⁴¹ have revealed that Bcl-3 is upregulated in acute kidney injury and supports to block inflammatory and lethal responses related to inflammatory stimuli in tubular cells. In addition, cleaved caspase-3 is increased by Bcl-3 silencing in inflammatory condition. Accordingly, rutin pretreatment in cisplatin nephrotoxicity provided the reduction of kidney Bcl-3 levels due to alleviation of oxidative stress, the reduction of the apoptotic protein caspase-3, and increasing of GSH levels and other antioxidants such as SOD, catalase and GSH-Px activities in the rat kidneys.

This study demonstrated that rutin pretreatment given at a dose of 150 mg/kg alleviated cisplatin nephrotoxicity in the rats by decreasing serum urea and creatinine concentrations, the kidney MDA and the kidney caspase-3 levels, and by increasing the kidney SOD, catalase and GSH-Px activities, and GSH levels.

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