Therapeutic potential of β-sitosterol in methotrexate-induced liver injury: association with STING and ERK-1 pathways

Abstract

Background

Methotrexate (MTX) is a commonly used drug to treat various cancers and autoimmune disorders, but its clinical utility is often limited by hepatotoxicity. β-sitosterol is a bioactive phytosterol compound that is naturally present in various plant foods and exhibits multiple antioxidant, anticancer and immunomodulatory activities. It has not been fully explored for its potential protective effects against liver injury. This study investigated whether β-sitosterol is associated with reduced hepatic injury in a short-term MTX model.

Methods

The rats were arbitrarily assigned to three groups as control, MTX and MTX + β-sitosterol groups. A single intraperitoneal dose of MTX was used to induce liver toxicity, and animals subsequently received either β-sitosterol or vehicle by oral gavage once daily for ten days. Liver tissues were evaluated using semi-quantitative histopathological scoring. Plasma alanine transaminase (ALT) and malondialdehyde (MDA), as well as liver transforming growth factor-β (TGF-β), MDA, stimulator of interferon genes (STING) and extracellular signal-regulated kinase 1 (ERK-1) levels were measured.

Results

β-sitosterol treatment was associated with lower plasma ALT and MDA levels compared with the MTX group. Hepatic TGF-β, MDA, STING and ERK-1 levels were also reduced in the MTX + β-sitosterol group. Histopathology showed attenuated hepatocyte necrosis, inflammatory infiltration and early fibrotic changes.

Conclusions

β-sitosterol was associated with reduced oxidative stress markers and lower hepatic TGF-β, STING and ERK-1 levels in this short-term MTX injury model. These findings suggest that β-sitosterol may have potential therapeutic value in mitigating MTX-related hepatotoxicity.

Graphical Abstract

Introduction

Fibrogenic processes in the liver are characterized by excessive accumulation of extracellular matrix proteins, which may ultimately progress to liver fibrosis under chronic conditions [1–3]. Although primarily associated with chronic liver diseases, drug-induced hepatotoxicity is also an important cause of fibrosis in both humans and animals [4, 5]. Methotrexate (MTX), a widely used chemotherapeutic and immunosuppressive agent, is known to induce hepatocellular injury through mechanisms involving oxidative stress, inflammation, and fibrotic signaling pathways [6–9]. Previous studies have reported MTX-induced hepatocyte degeneration, necrosis, inflammatory cell infiltration, and collagen deposition, underscoring the need for effective hepatoprotective strategies [10–12].

Accumulating evidence suggests that MTX toxicity is closely linked to disruptions in redox homeostasis and activation of profibrotic mediators. In particular, oxidative stress plays a central role in MTX-induced liver injury, promoting cellular damage and enhancing fibrogenesis [9, 12]. At the molecular level, signaling pathways such as transforming growth factor-β (TGF-β), stimulator of interferon genes (STING), and extracellular signal-regulated kinase-1 (ERK-1) have been implicated in hepatic stellate cell activation and extracellular matrix deposition [13–17]. However, despite growing knowledge about MTX-related molecular alterations, therapeutic options capable of effectively modulating these pathways remain limited. This gap highlights the need for compounds with both antioxidant and antifibrotic potential.

β-Sitosterol, a naturally occurring phytosterol abundant in plant-derived foods, exhibits well-documented antioxidant, anti-inflammatory, and metabolic regulatory activities [18–20]. Several experimental studies indicate that β-sitosterol can improve liver histology, reduce lipid peroxidation, and suppress inflammation-related signaling [21–23]. However, its possible role in MTX-induced hepatic injury has not been adequately investigated.

Although β-sitosterol has been studied in several liver injury models [21–23], its effects in MTX-induced hepatic injury and its interaction with TGF-β, STING and ERK-1 pathways have not been fully clarified. Therefore, the present study was designed to evaluate the potential hepatoprotective effects of β-sitosterol in an MTX-induced liver injury model, with a specific focus on its influence on oxidative stress markers and key fibrogenic pathways, including STING and ERK-1. By addressing this gap, we aimed to determine whether β-sitosterol could serve as a promising candidate for mitigating MTX-related hepatic injury.

Materials and methods

Animals and experimental design

This investigation included thirty adult rats (female Wistar albino), aged 10 to 12 weeks and weighing 150 to 200 g. All animals were sourced from the Experimental Animals Laboratory at Demiroglu Science University. During the investigation, the rats were maintained in standard laboratory circumstances, residing in stainless steel cages at a controlled temperature of 22 ± 2 °C, subjected to a 12-hour light/dark cycle, and granted unrestricted access to food (35% lipids, 18% proteins, 47% carbohydrates) and water.

The animals were arbitrarily assigned into three groups (Group 1: Control; Group 2: MTX; Group 3: MTX + β-sitosterol), each consisting of 10 rats. The control group received only the vehicle. In the MTX group, liver injury was triggered by a single administration of MTX at a dose of 20 mg/kg (intraperitoneal), as described in previous studies [24, 25]. The MTX + β-sitosterol group was subjected to the same MTX injection protocol and subsequently received β-sitosterol at 80 mg/kg/day by oral administration for 10 days, a dose within the non-toxic and pharmacologically effective range reported in previous rodent studies [26, 27] (Fig. 1). β-sitosterol was prepared as a suspension in 0.5% carboxymethylcellulose (CMC) in tap water and administered via oral gavage in a volume of 1 mL/kg. To ensure consistency across groups, the control and MTX-only groups received the same CMC vehicle (1 mL/kg/day). All animals completed the experimental protocol, and no mortality occurred in any group (n = 10 per group). Animals were randomly allocated to experimental groups using simple randomization, and both the pathologist and the investigators conducting biochemical assays were blinded to group assignments to minimize bias, in accordance with ARRIVE guidelines.

Fig. 1.

Experimental Design

All operations adhered to the National Institutes of Health (USA) Guide for the Care and Use of Laboratory Animals. The Local Ethics Committee for Animal Experiments at Demiroglu Science University gave ethical permission for the experimental protocol (Permission No: 1525063304; Date: January 9, 2023). Unless otherwise noted, all chemicals and reagents were procured from Sigma‒Aldrich, Inc.

Sample collection and preparation

At the conclusion of the experimental protocol, anesthesia was administered intraperitoneally using 100 mg/kg ketamine (Ketasol, Richterpharma AG, Wels, Austria) in combination with 10 mg/kg xylazine (Rompun, Bayer, Leverkusen, Germany). Following anesthesia, blood samples were drawn through cardiac puncture to perform biochemical analyses and euthanasia was applied through cervical dislocation. Then, a midline incision was conducted in the abdominal wall and the liver was excised from the abdomen. After that, a part of the liver was fixed in a 10% buffered neutral formalin solution for 24 h. Following routine histological processing, the tissue samples were encased in paraffin and were sectioned at 4 μm thickness at 100 μm intervals for comprehensive histomorphological examination. In addition, the other part of the liver was kept at -20 °C for quantitative biochemical assays.

Histopathological evaluation

Tissue sections were subjected to hematoxylin and eosin staining for histopathological assessment. The slices were analyzed for hepatocyte necrosis, fibrogenic alterations, and cellular infiltration. In this short-term MTX injury model, liver sections were assessed semi-quantitatively, with severity scored from 0 to 4 (0 = absent, 1 = minimal < 5%, 2 = mild 5–25%, 3 = moderate 26–50%, 4 = marked > 50%) [28]. All assessments were conducted by a pathologist unaware of the experimental groups. Microscopic analysis and photography were conducted utilizing an Olympus BX51 light microscope paired with an Olympus C-5050 digital camera (Olympus Co., Tokyo, Japan).

Biochemical analysis

By using a glass homogenizer, the tissue samples were mixed with 5 mL of phosphate-buffered saline (PBS, pH 7.4) until completely homogenized. In order to collect the supernatants for biochemical examination, the tissue lysates were centrifuged at 5000× g for 15 min at 4 °C. Total protein concentration in homogenized liver samples was measured via Bradford’s method [29], using bovine serum albumin as the standard, and the obtained values were used solely for normalization of tissue biochemical data.

Determination of liver TGF-β, STING and ERK-1 levels

The concentrations of TGF-β, STING, and ERK-1 in samples of liver supernatant were ascertained using rat-specific ELISA kits (Elabscience, Houston, TX, USA). The manufacturer’s recommendations were followed for the analysis of each sample in duplicate. A MultiscanGo microplate spectrophotometer (Thermo Fisher Scientific, NH, USA) was used at spectral ranges specific to the experiment to get the optical density measurements.

Quantification of lipid peroxidation

To assess lipid peroxidation, malondialdehyde (MDA) levels in tissue and plasma were quantified by the thiobarbituric acid reactive substances (TBARS) assay, as previously outlined [30]. Tissue samples were subjected to treatment with trichloroacetic acid and TBARS reagent, thoroughly mixed and heated at 100 °C for 60 min. Subsequent to cooling on ice, the samples underwent centrifugation at 3000 rpm for 20 min, and the optical density was assessed at 535 nm. MDA concentrations were determined via a standard curve established with tetraethoxypropane and represented as nmol per gram of protein.

Assessment of plasma ALT level

Following the thorough instructions provided by the manufacturer, plasma ALT levels were determined using ELISA kits that are commercially available (USCN, Life Science Inc., Wuhan, China).

Statistical evaluation

Data analysis was conducted utilizing SPSS software version 15.0 for Windows (SPSS Inc., Chicago, IL, USA). Before comparing groups, data distribution was assessed using the Kolmogorov–Smirnov test, and the homogeneity of variances was verified using the Bartlett test. Parameters that did not meet normal distribution criteria (STING, ERK-1, liver MDA, and histological scores) were analyzed using the Kruskal–Wallis test followed by the Mann–Whitney U test. Normally distributed variables (ALT, plasma MDA, and TGF-β) were analyzed using one-way ANOVA followed by Tukey’s Honestly Significant Difference (HSD) post-hoc test, which controls for Type I error in multiple pairwise comparisons. Findings are expressed as mean ± SEM. A p-value below 0.05 was accepted as statistically significant, whereas values under 0.01 and 0.001 indicated a highly significant difference.

Results

Effects of β-sitosterol on biochemical parameters

The concentrations of liver TGF-β, MDA, STING, ERK-1, as well as plasma ALT and MDA, are presented in Table 1. Plasma levels of ALT and MDA were significantly increased in the MTX group compared with the control group (p < 0.01, p < 0.001), whereas these parameters were reduced in the MTX + β-sitosterol group relative to the MTX group (p < 0.05, p < 0.01). However, the improvement in ALT levels in the MTX + β-sitosterol group was relatively modest compared with the more pronounced reduction observed in MDA, indicating that ALT responded less markedly to MTX-induced injury and subsequent treatment. Moreover, hepatic levels of TGF-β, MDA, STING, and ERK-1 were substantially elevated in the MTX group (p < 0.001), while these parameters were significantly decreased in the MTX + β-sitosterol group compared with the MTX group (p < 0.05).

Table 1.

Biochemical parameters in plasma and liver tissues (n = 10)

Parameters	Control	MTX	MTX + β-sitosterol
Liver TGF-β level (pg/g)	0.19 ± 0.10	1.95 ± 0.40**	0.92 ± 0.20#
Liver MDA level (nmol/g)	26.40 ± 0.50	50.20 ± 1.60**	38.30 ± 2.90#
Liver STING level (pg/g)	9.10 ± 0.60	21.70 ± 2.20**	13.50 ± 4.80#
Liver ERK-1 level (pg/mg)	1.03 ± 0.80	9.80 ± 1.60**	3.30 ± 0.90#
Plasma MDA level (nM)	13.70 ± 1.60	89.40 ± 2.40**	51.03 ± 3.80##
Plasma ALT (IU/L)	39.80 ± 4.10	63.30 ± 3.70*	47.10 ± 5.50#

Data are expressed as mean ± SEM. Control group: only vehicle (1 mL/kg/day); MTX group: MTX (20 mg/kg, single dose, i.p.) and vehicle (1 mL/kg/day); MTX + β-sitosterol group: MTX (20 mg/kg, single dose, i.p.) and β-sitosterol (80 mg/kg/day)

Statistical analyses were performed using one-way ANOVA or Kruskal-Wallis, as appropriate. * p < 0.01, ** p < 0.001 relative to the control group

^#p < 0.05, ^##p < 0.01 distinct from MTX group

TGF-β Transforming Growth Factor-β, MDA Malondialdehyde, STING Stimulator of Interferon Genes, ERK-1 Extracellular Signal-Regulated Kinase 1, ALT Alanine Aminotransferase

Effects of β-sitosterol on histopathological parameters

Microscopic analysis of rat liver sections revealed significant morphological differences among the groups (Fig. 2). Hepatic sections from the control group exhibited well-preserved tissue architecture, with intact parenchymal cells arranged in typical cord-like patterns radiating from the centrally located vein. The hepatocytes maintained normal structural features, including distinct cellular borders, centrally positioned nuclei, and eosinophilic cytoplasm. Sinusoidal areas appeared unaltered, and there were no significant signs of necrosis, fibrotic changes, or inflammatory cell infiltration (Fig. 2A and B). Conversely, the MTX group revealed substantial histological alterations in the portal area, noted for bridging necrosis, fibrogenic changes and prominent cellular infiltration in comparison to the control group (p < 0.001) (Fig. 2C and D). Additionally, the MTX + β-sitosterol group had a marked reduction in bridging necrosis, fibrogenic changes, and cellular infiltration relative to the MTX group (p < 0.05) (Fig. 2E and F) (Table 2).

Fig. 2.

Liver histopathological images stained with hematoxylin and eosin (H&E) at ×10 and ×20 magnifications. (A-B); The control group shows preserved hepatic architecture with normal hepatocytes and central vein (CV). (C-D); MTX group displays severe histopathological alterations (arrows), including bridging necrosis, fibrosis-like changes (f) and cellular infiltration in the portal area (p.a.). (E-F); MTX + β-sitosterol group, arrows indicate reduced bridging necrosis, less fibrogenic changes and mild cellular infiltration

Table 2.

Histopathological findings across control and treatment groups (n = 10)

Parameters	Control	MTX	MTX + β-Sitosterol
Hepatocyte necrosis	0.1 ± 0.1	1.3 ± 0.1**	0.6 ± 0.2#
Fibrogenic alterations	0.2 ± 0.1	2.1 ± 0.3**	1.3 ± 0.3#
Cellular infiltration	0.2 ± 0.1	1.6 ± 0.2**	0.9 ± 0.2#

Data are shown as mean ± SEM. Control group: only vehicle (1 mL/kg/day); MTX group: MTX (20 mg/kg, single dose, i.p.) and vehicle (1 mL/kg/day); MTX + β-sitosterol group: MTX (20 mg/kg, single dose, i.p.) and β-sitosterol (80 mg/kg/day)

Statistical analyses were performed using Kruskal-Wallis test

^**p < 0.001 distinct from control group

^#p < 0.05 distinct from MTX group

Discussion

Fibrogenic alterations are characterized by the deterioration of liver functions [3], which occurs as a result of excessive accumulation of extracellular matrix proteins in the tissue due to various factors such as diseases and drug-related toxicities [31]. Methotrexate, a drug widely used in the treatment of psoriasis and rheumatoid arthritis, causes changes such as necrosis and fibrosis in the liver due to its side effects [14]. Although several pharmacological compounds have been explored the harmful effects of MTX, the protective effects of these treatment approaches on liver fibrosis are generally insufficient [32]. In this context, this study focuses to investigate the biochemical and histopathological changes occurred by β-sitosterol, known to have attenuation of fibrogenic responses, in an MTX-induced liver fibrogenic injury model through the regulation of STING and ERK-1 signals [23].

Alanine aminotransferase is an important enzyme that increases in the blood when liver cells are damaged or die, making it a primary biomarker for detecting liver injury [33]. Previous studies revealed a significant elevation in plasma ALT levels following MTX treatment, suggesting the presence of hepatotoxic effects [34, 35]. This finding is consistent with our study. Furthermore, it has been found that β-sitosterol reduced plasma ALT levels in rats fed a high-fat diet [22]. Similarly, the present study determined that β-sitosterol significantly decreased ALT levels in MTX-induced liver injury. Based on this information, it can be hypothesized that β-sitosterol may contribute to the amelioration of liver injury.

Lipid peroxidation refers to the oxidative degradation of unsaturated fatty acids, leading to irreversible damage to cellular membranes. This destructive process can ultimately result in cell death [36]. Malondialdehyde, a byproduct of the oxidation of polyunsaturated fatty acids, serves as a key indicator of oxidative stress [37]. Oxidative stress is a major contributor to liver injury, including that triggered by MTX toxicity [38]. Alfwuaires et al. [39] reported increased levels of MDA in the liver, accompanied by a reduction in antioxidant defenses following MTX treatment. Consistently, in our study, MTX administration led to a significant rise in MDA levels in both liver tissue and plasma. Importantly, β-sitosterol markedly reduced MDA concentrations in the MTX + β-sitosterol group, indicating its capacity to alleviate MTX-induced oxidative injury. Supporting this observation, prior research has shown that β-sitosterol restores intracellular antioxidant enzyme activities and decreases lipid peroxidation and fibrosis markers in carbon tetrachloride-induced liver damage, further confirming its antioxidant and antifibrotic potential [23]. Taken together, these results suggest that β-sitosterol counteracts MTX-induced oxidative stress by strengthening endogenous antioxidant defenses and limiting lipid peroxidation. In contrast to the marked reduction in MDA, the decrease in ALT levels was less pronounced, suggesting that β-sitosterol’s protective effect on hepatocellular leakage markers may be more limited compared with its impact on oxidative stress parameters.

Transforming growth factor-β is a critical mediator of liver fibrosis, promoting hepatic stellate cell activation and enhancing extracellular matrix deposition [40]. Consistent with previous studies reporting that MTX elevates plasma and hepatic TGF-β levels [31, 41], our results showed a marked increase in TGF-β in the MTX group, supporting its involvement in MTX-induced fibrogenesis. Furthermore, Wen et al. [42] demonstrated that β-sitosterol inhibited TGF-β1 activity in endometrial cells. Similarly, β-sitosterol administration markedly reduced TGF-β expression in our MTX model, suggesting a potential antifibrotic effect mediated, at least in part, through modulation of TGF-β signaling. The marked reduction of hepatic and plasma MDA levels in the β-sitosterol–treated group suggests attenuation of lipid peroxidation–driven oxidative injury. Therefore, the concomitant decline in TGF-β levels observed in our study may, in part, reflect β-sitosterol’s ability to modulate oxidative stress, thereby limiting downstream stellate cell activation and extracellular matrix accumulation.

It has been reported that the STING signaling pathway [43], activated after cell injury, increases fat accumulation in hepatocytes and promotes fibrogenic activation of hepatic stellate cells via macrophage-derived mediators [44]. STING expression has been shown to rise significantly in MTX-induced liver damage [45] and in liver fibrosis tissues from liver cancer patients [46]. Consistent with these reports, hepatic STING levels were significantly higher in the MTX group in our study, supporting a role for STING signaling in MTX-induced liver injury.

Previous studies have indicated that β-sitosterol can inhibit STING activation in pulmonary arterial smooth muscle cells under hypoxia [47] and in pulmonary vascular endothelial cells cultured in high glucose [48]. Similarly, in our study, β-sitosterol administration significantly reduced hepatic STING levels, suggesting suppression of excessive innate immune signaling and mitigation of early fibrogenic responses. These findings indicate that inhibition of the STING pathway by β-sitosterol may contribute to its protective effect against MTX-induced liver injury by limiting inflammatory infiltration and fibrogenic signaling.

Activation of the extracellular signal-regulated kinase 1 signaling pathway in the liver leads to the differentiation of hepatic stellate cells (HSCs) into myofibroblasts [49], stimulation of collagen gene expression and extracellular matrix deposition [50]. Thus, the fibrogenesis process is triggered in the liver [51]. In previous studies, it has been determined that ERK-1 levels increased in MTX-induced liver toxicity [52], in duct ligation-induced bile fibrotic liver tissue [53], and in the liver of chronic ethanol-consuming rats [54]. Consistent with the literature, it was noted in our study that liver ERK-1 level significantly elevated in the MTX group in comparison to the control group. Accordingly, ERK-1 signaling may play a role in the pathogenesis of HSC proliferation and hepatic fibrogenesis in MTX-induced liver toxicity. Furthermore, it has been reported that β-sitosterol can prevent inflammation in mouse microglia cells exposed to LPS by inhibiting the activation of the ERK-1 pathway [55]. In the presented study, it was found that β-sitosterol significantly reduced liver ERK-1 level in MTX-induced liver injury. ERK-1 is also a key downstream regulator of cellular stress responses and matrix remodeling [50]. The marked decrease in ERK-1 following β-sitosterol treatment suggests that β-sitosterol may attenuate ERK-dependent profibrotic signaling, thereby limiting stellate cell activation and collagen formation.

Methotrexate is used in the treatment of cancer and some autoimmune diseases [6, 7]. Studies have reported that MTX causes lobular degeneration in the liver, inflammatory cell infiltration [12], congestion in sinusoids [10] and fibrin deposition in periportal areas [11]. In our study, consistent with previous studies, it was observed that MTX administration caused liver necrosis, fibrosis-like changes, and inflammatory cell infiltration. Furthermore, studies have shown that β-sitosterol reduced hepatocyte ballooning degeneration and intrahepatic fat droplets in experimental models of liver injury, and also improved narrowed sinusoids and irregular hepatic cords [21, 22, 56]. Similarly, in the present study, β-sitosterol significantly reduced liver bridging necrosis, fibrogenic changes, and cellular infiltration in MTX-administered rats.

The biochemical improvements observed in the β-sitosterol–treated group were also consistent with these histopathological findings. The reductions in plasma ALT and MDA levels paralleled the attenuation of hepatocellular degeneration and inflammatory infiltration seen microscopically. Likewise, the decreases in hepatic TGF-β, STING, and ERK-1 levels corresponded with reduced fibrogenic changes, fewer inflammatory cells in the portal regions, and diminished bridging necrosis. Together, these findings indicate that the biochemical changes closely reflect the histologically verified improvement in liver architecture.

Limitations

This study has several limitations that should be considered when interpreting the findings. First, the MTX protocol employed represents an acute hepatotoxicity model that induces early fibrogenic alterations rather than fully established chronic liver fibrosis. Accordingly, the histopathological changes observed should be interpreted as indicative of early fibrogenic responses rather than advanced fibrotic remodeling. Second, a β-sitosterol–only treatment group was not included due to ethical constraints related to the allowable number of experimental animals. This limitation restricts the ability to determine whether β-sitosterol exerts intrinsic hepatic effects independent of MTX exposure. In addition, all experiments were conducted exclusively in female Wistar rats; therefore, potential sex-dependent differences in MTX-induced hepatotoxicity and phytosterol responsiveness cannot be excluded, and the findings should not be directly generalized to both sexes. Third, the use of a single β-sitosterol dose and a relatively short 10-day treatment period limits assessment of dose–response relationships as well as the evaluation of longer-term fibrogenic progression. Future studies employing multiple dosing regimens and extended observation periods will be necessary to better characterize the therapeutic window and durability of β-sitosterol–mediated hepatic protection. Furthermore, although this study provides integrated functional, biochemical, and histopathological evidence supporting a protective effect of β-sitosterol within this experimental model, advanced molecular analyses related to extracellular matrix remodeling, such as Western blotting or gene expression profiling (e.g., matrix metalloproteinases), were not performed. Specifically, collagen-targeted histological staining (e.g., Masson’s trichrome or Sirius Red), biochemical collagen quantification, and immunohistochemical markers of hepatic stellate cell activation (such as α-SMA or collagen I/III) were not included. The absence of these endpoints limits definitive classification of the observed structural alterations as established fibrosis. Finally, while β-sitosterol treatment was associated with reduced hepatic TGF-β, STING, and ERK-1 levels, these associations do not establish direct mechanistic causality. Downstream STING mediators, ERK phosphorylation status, and pathway-specific interventions were not assessed. Consequently, the involvement of these signaling pathways should be regarded as preliminary. Future investigations incorporating pathway-specific protein analyses, genetic markers of fibrosis, and targeted inhibition or cell-specific models are warranted to further substantiate the mechanistic interpretations proposed in this study.

Conclusions

The present study demonstrates that β-sitosterol treatment is associated with significant biochemical and histopathological improvements in an experimental model of MTX-induced hepatic injury. β-sitosterol markedly reduced plasma ALT and MDA levels, as well as hepatic TGF-β, STING, ERK-1, and oxidative stress markers, accompanied by attenuation of necrosis, inflammatory cell infiltration, and early fibrogenic alterations.

Collectively, these findings suggest that β-sitosterol may exert a protective effect against MTX-induced liver damage within the context of this experimental model. While modulation of fibrogenic and inflammatory signaling pathways was observed, further mechanistic investigations are warranted to clarify the molecular basis of these effects and to better define the therapeutic potential of β-sitosterol in MTX-associated liver injury.

Abbreviations

ALT

Alanine aminotransferase

ERK-1

Extracellular signal–regulated kinase 1

HSCs

Hepatic stellate cells

MDA

Malondialdehyde

PBS

Phosphate–buffered saline

STING

Stimulator of interferon genes

TBARS

Thiobarbituric acid reactive substances

TGF-β

Transforming growth factor–β

Authors’ contributions

Conceptualisation, M.U., M.A.E., and O.E.; data curation, M.U., M.A.E., and O.E.; formal analysis, M.U., M.A.E., and O.E.; investigation, M.U., M.A.E., and O.E.; methodology, M.U., M.A.E., O.S., and O.E.; writing—original draft preparation, M.U., M.Y., M.A.E., O.S., B.B.C., M.K., and O.E.; writing—review and editing, M.U., M.Y., M.A.E., O.S., B.B.C., M.K., and O.E. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data availability

The data are provided within the manuscript.

Declarations

Ethics approval and consent to participate

The animal study protocol was approved by the Institutional Animal Care and Ethical Committee of the Demiroglu Science University (protocol code: 1525063304; January 9, 2023). The authors confirm that all methods were performed in strict accordance with the guidelines for animal experiments.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.