DISCUSSION
Methotrexate (MTX) is commonly used for the treatment of cancer and autoimmune diseases (Pivovarov and Zipursky, 2019). However, its adverse effects on the liver, kidney, small intestine, and central nervous system remain a significant concern (Koroglu et al., 2021; Pannu, 2019; Samdanci et al., 2019; Vardi et al., 2012; Vardi et al., 2013). Various pathways, such as free oxygen radicals, intracellular ion imbalance, mitochondrial dysfunction, and apoptosis, play a crucial role in MTX-induced hepatotoxicity (El-Sheikh et al., 2015; Pivovarov and Zipursky, 2019). Suppression of these pathways and prevention of ROS formation are essential for reducing the severity of MTX-related organ damage (Jaeschke et al., 2002; Song et al., 2006). Recent studies have investigated the mechanism of action of ranitidine (RAN) in this regard (Naveena et al., 2018).
Our study demonstrated that increased levels of biochemical markers such as AST, ALT, ALP, and LDH in the blood of rats indicate hepatocyte damage caused by MTX (McGill, 2016). The elevation of malondialdehyde (MDA), an oxidant marker, suggests that oxidative stress plays a significant role in MTX-induced hepatotoxicity. In the RAN+MTX and MTX+RAN groups, we observed a decrease in liver function parameters and MDA levels, indicating that RAN’s mechanism of action in our hepatotoxicity model reduces oxidative stress. Our histopathological data also supports the potential of RAN as a treatment for MTX-induced hepatotoxicity.
In a study by Ghoneum et al. reported that ALT, AST, and ALP values significantly increased with MTX application, consistent with our findings (Ghoneum and El-Gerbed, 2021). We also observed an improvement in ALT, AST, ALP, and LDH values in RAN-treated groups. Additionally, Esenboğa et al. reported that RAN significantly reduced ALT and AST levels in rats with coronary artery disease and non-alcoholic fatty liver disease (Esenboga et al., 2022), further supporting the potential of RAN in preventing hepatocyte damage (Han et al., 2018; Imprialos et al., 2022).
MDA is a marker that increases in various conditions such as liver toxicity, cardiovascular diseases, diabetes, and cancers and is associated with oxidative stress (Aslankoç et al., 2019; Zhang et al., 2006). Our study revealed a significant increase in MDA levels in rats given MTX, suggesting that oxidative stress is one of the mechanisms by which MTX generates liver damage. However, the significant decrease in MDA levels in the MTX+RAN group indicates that RAN administration provides inhibition in oxidative stress and may be used in the treatment of hepatocyte damage. Notably, we observed a decrease in MDA levels in the RAN+MTX group compared to the MTX and MTX+RAN groups, suggesting that RAN may be more beneficial in prophylactic use than in treatment. Other studies in the literature corroborate our findings (Krishna et al., 2016; Matsumura et al., 1998; Ramakrishna et al., 2014; ullah Baig et al., 2020).
Jahovic and colleagues observed that MTX increased levels of MDA and myeloperoxidase in the blood, while decreasing tissue levels of GSH (Jahovic et al., 2003). In another study, Arinno et al. investigated the therapeutic effects of RAN, melatonin, and metformin on doxorubicin-induced cardiotoxicity. They demonstrated that RAN administration resulted in decreased MDA levels and improved left ventricular function (Arinno et al., 2021).
The balance between antioxidants and free oxygen radicals is crucial for maintaining cellular homeostasis. The disruption of this balance, favoring reactive oxygen species (ROS), can lead to various forms of cellular damage. Antioxidant mechanisms are characterized by markers such as superoxide dismutase (SOD), catalase (CAT), and GSH. MTX has been shown to activate the oxidative stress response (SOR) mechanism. Miyazono and colleagues investigated the role of oxidative stress in small intestine damage induced by MTX in rats. They found that MTX administration reduced levels of antioxidant markers such as SOD, CAT, and GSH, while increasing MDA levels as an indicator of oxidative stress. In another study, Had and colleagues evaluated the histological and biochemical effects of MTX on liver tissue in Wistar Albino rats. The biochemical parameters examined revealed increased MDA values, decreased SOD, CAT, and GSH levels, and elevated AST and ALT values by a factor of 7 (Vardi et al., 2012).
In our study, we observed a significant reduction in CAT levels in the MTX group compared to the control group, while levels increased in the RAN-treated groups. These results suggest that RAN acts through an antioxidant mechanism to prevent MTX-induced damage. Interestingly, there were no significant changes in GSH levels among the groups, and SOD levels decreased in the treatment and prophylaxis groups compared to the control and MTX groups. This may suggest that the antioxidant mechanism and the adaptation process against oxidative stress were not yet established, and the markers did not reach the desired level during the chronic phase. Alternatively, the study may be more effective by increasing the number of rats (Kisaoglu et al., 2013; Spolarics, 1998).
Our histopathological results showed that MTX caused damage to inflammatory lesions and hepatocytes, while RAN alleviated hepatocyte degeneration. The livers of rats in the control group showed normal morphological structures under microscopic observation, but the livers of rats in the MTX group exhibited statistically significant increases in mononuclear inflammation, vascular congestion, ductal profibrosis, and vacuolization findings consistent with hepatocyte damage. Upon examination of the Roening grade (Samdanci et al., 2019), a significant increase in fibrosis was observed in the MTX group as compared to the control group, providing evidence of the damaging effect of MTX on fibrosis. However, in the RAN+MTX and MTX+RAN groups, significant reductions in vascular congestion and ductal proliferation were noted as compared to the MTX group. These results suggest that RAN administration has the potential to prevent hepatocyte damage and facilitate liver tissue regeneration. Similar findings have been reported in other studies. For instance, Dalaklioglu et al. (2013) showed that the MTX group exhibited histopathological alterations such as degenerate hepatocytes, vascular occlusion in sinusoids, dilatation of sinusoids, and increased activation of Kupffer cells (Dalaklioglu et al., 2013).