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).