Abstract
Methotrexate (MTX) is an efficient chemotherapeutic and immunosuppressant drug, but the hepatotoxicity of MTX limits its clinical use. Naringin (Nar) is a flavonoid derived from Citrus paradise, and has been shown to possess several pharmacological activities, including free-radical scavenging and antioxidant properties. In the present study, we first tested the possible protective effects of multiple doses of Nar against MTX-induced acute hepatotoxicity in rats, and then we investigated the growth inhibition and apoptotic effects of MTX and/or Nar against the HepG2 hepatocarcinoma cell line. Our in vivo results showed that Nar significantly reduced MTX-induced increases in serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and total bilirubin levels. Nar also reduced MTX-induced oxidative stress by significantly reducing liver malondialdehyde (MDA) and nitric oxide (NO) content and increasing superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione (GSH). In addition, Nar significantly counteracted MTX-induced increases in hepatic interleukin-6 and tumor necrosis factor-α (TNF-α). Further, Nar greatly protected hepatocyte ultrastructure against MTX-induced injury. In contrast, in vitro MTX and/or Nar treatment of HepG2 cells for 48 h exhibited a cytotoxic effect and induced apoptosis in a dose-dependent manner mediated by a significant increase in the Bax/Bcl-2 protein expression ratio. Noticeably, Nar potentiated the MTX effect on the Bax/Bcl-2 ratio. In conclusion, Nar decreased MTX-induced functional and ultrastructural liver damage in a tumor-free animal model. Also, our data introduce MTX and Nar as promising antiproliferative agents with a distinctive mode of action, inducing apoptosis in HepG2 tumor cells through activation of Bax and down-regulation of Bcl-2 protein expression.
Highlights
Methotrexate (MTX), a folic acid antagonist, is widely used in the treatment and prophylaxis of several types of malignant tumors [1,2]
Nitric oxide (NO) is a free radical molecule with a multitude of physiological functions. This highly reactive molecule is synthesized from l-arginine by a group of isoenzymes collectively termed as NO synthases (NOSs)
We found a significant reduction in superoxide dismutase (SOD), CAT, glutathione peroxidase (GPx), glutathione reductase (GR), and GSH in rats treated with MTX
Summary
Methotrexate (MTX), a folic acid antagonist, is widely used in the treatment and prophylaxis of several types of malignant tumors [1,2]. Several studies have shown that MTX toxicity is mediated by oxidative stress and dysregulation of antioxidant defenses, thereby increasing the generation of reactive oxygen species (ROS) as superoxide anions, hydrogen peroxide and hydroxyl radicals [4]. ROS formation can initiate lipid peroxidation (LPO) and the release of inflammatory mediators, including tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) [5]. Increased expression of TNF-α has been reported in a model of MTX-induced hepatic, renal and intestinal damage [8]. Nitric oxide (NO) is a free radical molecule with a multitude of physiological functions. This highly reactive molecule is synthesized from l-arginine by a group of isoenzymes collectively termed as NO synthases (NOSs). Attenuation of oxidative stress can represent an effective strategy to protect against MTX-induced hepatotoxicity
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