Pregabalin mitigates methotrexate-induced pulmonary injury via suppression of inflammatory and endothelial activation mechanisms: a histopathological and immunohistochemical approach

Abstract

Background

Methotrexate (MTX) is a cornerstone chemotherapeutic and immunosuppressive agent widely used in the treatment of hematological malignancies, including acute lymphoblastic leukemia, Burkitt lymphoma, and primary central nervous system lymphoma. Despite its clinical efficacy, MTX use may be limited by pulmonary toxicity, a rare but potentially life-threatening adverse effect. Previous studies suggest that MTX-induced lung injury is associated with inflammatory responses, endothelial activation, and oxidative stress–related mechanisms reported in the literature. These processes may contribute to structural lung damage and impaired pulmonary integrity. Pregabalin (PREG), a structural analog of γ-aminobutyric acid, has been reported to exhibit anti-inflammatory, and cytoprotective properties in several experimental models. In addition, previous experimental studies suggest that PREG may attenuate endothelial activation and inflammatory mediator release. Therefore, the present study aimed to investigate the potential protective effects of PREG against MTX-induced pulmonary injury, focusing on histopathological and immunohistochemical alterations.

Methods

Adult male Wistar rats were randomly allocated into four experimental groups: Control, MTX, MTX + PREG, and PREG. Pulmonary injury was induced by a single intraperitoneal administration of MTX (20 mg/kg). PREG (30 mg/kg/day) was administered orally for seven consecutive days. At the end of the experimental period, lung tissues were collected for histopathological examination using hematoxylin and eosin staining. Immunohistochemical analyses were performed to evaluate the expression of cyclooxygenase-2 (COX-2), interleukin 6 (IL-6), and vascular cell adhesion molecule-1 (VCAM-1). Immunoreactivity was assessed semi-quantitatively across the experimental groups.

Results

MTX exposure resulted in marked pulmonary injury characterized by pronounced alveolar septal thickening, interstitial and alveolar edema, hemorrhage, and extensive inflammatory infiltration. These histopathological changes were accompanied by a significant increase in COX-2, IL-6, and VCAM-1 immunoreactivity, indicating enhanced inflammatory and endothelial activation within lung tissue. In contrast, PREG co-treatment significantly attenuated MTX-induced lung injury, as evidenced by lower histopathological injury scores and reduced numbers of immunopositive cells for all markers. The PREG-only group exhibited preserved pulmonary architecture without detectable pathological alterations.

Conclusion

PREG significantly attenuated MTX-induced pulmonary injury in this experimental model, as demonstrated by improved histopathological findings and reduced expression of inflammatory and endothelial activation markers, including COX-2, IL-6, and VCAM-1. These results provide tissue-level evidence supporting the potential protective effects of PREG against MTX-associated lung injury. However, oxidative stress parameters, functional pulmonary assessments, and intracellular signaling pathways were not directly evaluated in the present study. Therefore, the findings should be interpreted as morphological and hypothesis-generating evidence. Further investigations incorporating molecular, biochemical, and functional analyses are required to clarify the underlying mechanisms and to determine the translational relevance of these observations.

Introduction

Methotrexate (MTX) remains one of the most widely used chemotherapeutic and immunosuppressive agents in the management of hematologic malignancies and autoimmune disorders, including acute lymphoblastic leukemia, Burkitt’s lymphoma, and primary central nervous system lymphoma [1]. Despite its clinical efficacy, MTX use is associated with a spectrum of dose-independent toxicities, among which pulmonary toxicity is a rare but potentially severe complication. MTX-induced lung injury can present as acute or chronic interstitial pneumonitis, characterized histologically by inflammatory cell infiltration, alveolar septal thickening, pulmonary edema, hemorrhage, and progressive fibrosis [2, 3].

The pathogenesis of MTX-induced pulmonary toxicity is multifactorial and involves complex molecular and cellular processes, including inflammatory cytokine release, endothelial activation, oxidative stress, mitochondrial dysfunction [4, 5]. Experimental studies have shown that MTX exposure increase the expression of pro-inflammatory mediators, such as cyclooxygenase 2 (COX-2) and interleukin 6 (IL-6), and enhance endothelial adhesion molecule expression, including vascular cell adhesion molecule-1 (VCAM-1) [6, 7]. These changes are thought to contribute to alveolar and vascular alterations observed in MTX-induced pneumonitis [8].

Pregabalin (PREG), a structural analog of γ-aminobutyric acid (GABA), is clinically used for neuropathic pain and seizure disorders [9]. Preclinical evidence suggests that PREG may influence calcium influx, cytokine release, and tissue injury in various experimental models, and it has been associated with modulation of oxidative stress, apoptosis, and endothelial function [10–13]. However, its potential effects in MTX-induced pulmonary toxicity have not been fully explored.

Therefore, the present study aimed to investigate the protective effects of PREG against MTX-induced lung injury through histopathological evaluation and immunohistochemical assessment of COX-2, IL-6, and VCAM-1 expression. By integrating morphological findings with immunohistochemical evidence of inflammatory and endothelial activation, this study provides tissue-level evidence supporting the anti-inflammatory and endothelial-modulatory properties of PREG. These findings should be interpreted as supportive experimental observations rather than direct mechanistic proof. The proposed mechanism of PREG action in MTX-induced lung injury, based on the present findings is schematically illustrated in Fig. 1.

Fig. 1.

Schematic representation of the potential effects of PREG on MTX-induced ALI. MTX administration was associated with alveolar alterations, including atelectasis, emphysema, and edema. In the experimental model, co-treatment with PREG was associated with reduced histopathological changes and lower immunoexpression of IL-6, COX-2, and VCAM-1 compared with MTX alone. These observations are based on tissue-level histopathological and immunohistochemical assessments and are intended to illustrate potential effects rather than direct mechanistic pathways. ALI: Acute lung injury, MTX: Methotrexate, PREG: Pregabalin, VCAM-1: Vascular Cell Adhesion Molecule 1, COX-2: Cyclooxygenase-2, IL-6: Interleukin 6

Materials and methods

Animals and experimental design

This study utilized lung tissues obtained from rats originally included in the project entitled “Investigation of the Protective Effects of Pregabalin on Methotrexate-Induced Hepatotoxicity via the SIRT1-p53 and Mitochondrial Pathways” (Project No. TTU-2024-9577), conducted under the Scientific Research Projects Unit of Süleyman Demirel University. A total of thirty-two healthy adult male Wistar albino rats (250–350 g) were obtained from the Experimental Animal Research Center of the same university.

All experimental procedures were conducted in strict accordance with institutional and national guidelines for the care and use of laboratory animals and were approved by the Süleyman Demirel University Local Ethics Committee for Animal Experiments (Ethics Approval No: 01/250 Date:15.02.2024). The study was performed in compliance with internationally recognized ethical standards, including the principles outlined in the Basel Declaration.

In addition, the study adhered to the ARRIVE 2.0 (Animal Research: Reporting of In Vivo Experiments) and Basel Deceleration guidelines to ensure transparency and reproducibility. All efforts were made to minimize animal suffering and to reduce the number of animals used, in accordance with the principles of the 3Rs (Replacement, Reduction, and Refinement). The reuse of tissues from an existing ethically approved project contributed to the reduction principle. All procedures were performed by trained personnel, and appropriate measures were taken to minimize pain and distress, including the use of anesthesia and humane endpoints where necessary.

The animals were housed under controlled environmental conditions (temperature 21–22 °C, relative humidity 55–60%) with a 12-hour light/dark cycle and free access to standard laboratory chow and tap water. Following a seven-day adaptation period, the rats were randomly assigned to four experimental groups (n = 8 per group):

1. Control group: Animals received 1 mL of physiological saline (SF) orally by gavage once daily for seven consecutive days. On day 2, 1 mL of SF was also administered intraperitoneally (i.p.) in the contralateral inguinal region.

2. MTX group: A single i.p. dose of MTX (20 mg/kg; Methotrexate DBL 500 mg/20 mL vial, Koçak, Türkiye) was administered on day 2. Rats also received 1 mL of SF orally once daily for seven days [9, 14].

3. MTX + PREG group: Animals received MTX (20 mg/kg, i.p.) on day 2 and were treated with PREG (30 mg/kg; Lyrica, Pfizer, Türkiye) orally once daily for seven days [15].

4. PREG group: Rats received PREG (30 mg/kg, orally by gavage) once daily for seven days, with an additional i.p. administration of 1 mL SF on day 2.

Two hours after the final treatment, all rats were anesthetized with ketamine (90 mg/kg; Keta-Control, Doğa İlaç, Türkiye) and xylazine (8–10 mg/kg; XylazinBio 2%, Bioveta PLC, Czech Republic). Following anesthesia, laparotomy was performed, and blood samples were collected from the inferior vena cava. Euthanasia was carried out via surgical exsanguination. Lung tissues were harvested and fixed in 10% buffered formalin for histopathological examination (hematoxylin and eosin [HE] staining) and immunohistochemical analysis of COX-2, IL-6, and VCAM-1.

Histopathological analysis

Lung tissue specimens were collected during necropsy and immediately fixed in 10% neutral buffered formalin for 48 h to preserve tissue architecture. Following fixation, the samples were processed using a fully automated tissue processor (ASP300S, Leica Microsystems, Nussloch, Germany), embedded in paraffin wax, and serially sectioned at 5 μm thickness with an automated rotary microtome (RM2155, Leica Microsystems, Nussloch, Germany).

Sections were mounted on glass slides, stained with HE for general histopathological evaluation, and coverslipped with a synthetic mounting medium. Microscopic examinations were conducted under a standard light microscope at various magnifications (×100–×400), and representative microphotographs were captured using a digital imaging system for documentation and comparative analysis.

Histopathological changes were assessed semi-quantitatively using a severity-based grading system (0–3), where 0 = no lesion, 1 = mild, 2 = moderate, and 3 = severe. The parameters evaluated included hyperemia, interstitial or alveolar edema, hemorrhage, inflammatory cell infiltration, epithelial degeneration or loss and alveolar collapse. Scoring was performed in 10 randomly selected high-power fields (HPFs, ×400) per section by the same observed, blinded to group allocation, on two separate occasions. The mean scores for each animal was used for statistical analysis.

Scoring criteria were adapted with minor modifications from Ozmen et al. (2024) (Table 1) [8]. In addition to semi-quantitative assessment, qualitative morphological descriptions were provided to complement the histopathological evaluation of MTX-induced pulmonary injury.

Table 1.

Histopathological score criterion for lungs

Score	0	1	2	3
Hyperemia	Normal thin alveolar septae	Slight hyperemic alveolar septae (in less than 1/3 of the fields)	Moderate hyperemic alveolar septae (in 1/3 to 2/3 of the fields)	Severe hyperemic alveolar septae (in greater than 2/3 of the fields)
Edema	No edema	Slight edema (in less than 1/3 of the fields)	Moderate edema (in 1/3 to 2/3 of the fields)	Severe edema (in greater than 2/3 of the fields)
Hemorrhage	None	Slight and local	Moderate and multi focal	Severe and diffuse
Infiltrations	Less than 5 in the fields	5 to 10 in the fields	10 to 20 in the fields	More than 20 in the fields
Epithelial loss	None	Slight loss (in less than 1/3 of the fields)	Moderate loss (in 1/3 to 2/3 of the fields)	Severe loss (in greater than 2/3 of the fields)
Atelectasis	None	Local	Multifocal	Diffuse
Emphysema	None	Slight	Moderate	Marked

Examinations done high-power field (HPF) (10 fields of view at ×400 magnification)

Immunohistochemical analysis

Three serial sections were obtained from each paraffin-embedded lung tissue block and mounted on poly-L-lysine–coated glass slides for immunohistochemical analysis. Immunostaining was performed to evaluate cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), and vascular cell adhesion molecule-1 (VCAM-1) using specific recombinant antibodies: COX-2 (Anti-COX2/Cyclooxygenase-2 antibody [EPR12012], ab179800), IL-6 (Anti-IL-6 antibody [1.2-2B11-2G10], ab9324), and VCAM-1 (Anti-VCAM1 antibody [EPR5047], ab134047) (Abcam, Cambridge, UK).

Sections were deparaffinized in xylene and rehydrated through graded ethanol series, and subjected to heat-induced antigen retrieval in citrate buffer (pH 6.0) using a microwave oven for 15 min. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 min, followed by blocking of nonspecific binding with 5% normal goat serum for 20 min at room temperature.

Primary antibodies were diluted 1:100 in phosphate-buffered saline and applied overnight at 4 °C in a humidified chamber. Sections were then incubated with a biotinylated secondary antibody, followed by a streptavidin– horseradish peroxidase (HRP) conjugate according to the manufacturer’s instructions. Immunoreactivity was visualized using the EXPOSE Mouse and Rabbit Specific HRP/ 3,3′-diaminobenzidine (DAB) Detection IHC Kit (ab80436, Abcam, Cambridge, UK) with DAB serving as the chromogen. Nuclei were counterstained with Mayer’s hematoxylin and sections were dehydrated, cleared in xylene, and mounted with a permanent medium. Negative controls were prepared by replacing the primary antibody with the antibody diluent.

Immunohistochemical evaluation was performed by a single veterinary pathologist blinded to experimental groups and treatment allocation. For quantification, immunopositive cells were analyzed at ×400 magnification in 10 randomly selected HPFs per slide using a light microscope equipped with a digital camera (Olympus Co., Tokyo, Japan) and quantified using ImageJ software (version 1.48, NIH, Bethesda, MD, USA) and Database Manual Cell Sens Life Science Imaging Software System (Olympus Co., Tokyo, Japan). Digital images were standardized for cropping, color-channel separation, and artifact removal to ensure consistency across samples. Immunopositive cells were identified by distinct brown cytoplasmic DAB staining, and the percentage of positive cells was calculated relative to the total number of cells per field.

For semi-quantitative assessment, the percentage of positive cells was scored staining intensity were each scored on a 0–3 scale (0 = none, 1 = weak, 2 = moderate, 3 = strong) as detailed in Table 2. The total immunohistochemical (IHC) score for each specimen was obtained by summing the positivity and intensity scores, and mean total IHC scores were used for statistical comparisons among the experimental groups.

Table 2.

Immunohistochemical scoring system for expressions

Score	Staining intensity	Percentage of positive cells	Overall evaluation (interpretation)
0	No detectable staining	< 5% positive cells	Negative (no expression)
1	Weak cytoplasmic/membranous staining visible only at high magnification	5–25% positive cells	Mild (low expression)
2	Moderate, clearly visible brown staining in cytoplasm or membrane	26–50% positive cells	Moderate expression
3	Strong, dark brown staining easily observed at low magnification	> 50% positive cells	Strong (high expression)

Statistical analysis

All statistical evaluations were conducted using GraphPad Prism software (version 10.1; GraphPad Software, San Diego, CA, USA). Data normality was assessed with the Shapiro–Wilk test, and homogeneity of variances was evaluated using Levene’s test. As the datasets did not meet the assumptions of normality, non-parametric tests were applied. Group comparisons were performed using the Kruskal–Wallis test followed by Dunn’s multiple comparison test for pairwise analyses. Data are presented as mean ± standard deviation (SD), and p-values < 0.05 were considered statistically significant.

Results

Histopathological findings

Lung tissue sections from both the control and PREG-only groups exhibited normal pulmonary architecture, characterized by thin and intact alveolar septa, well-preserved alveolar spaces, and the absence of inflammatory infiltration. The bronchial and bronchiolar epithelium displayed normal morphology with intact ciliated columnar cells, without evidence of desquamation or necrosis. In contrast, the MTX-treated group demonstrated pronounced histopathological alterations consistent with drug-induced pulmonary toxicity. The lung parenchyma exhibited marked hyperemia of alveolar capillaries and parenchymal vessels, prominent interstitial and alveolar edema, and multifocal hemorrhagic areas ranging from mild to severe intensity. A substantial increase in alveolar septal thickness was observed, accompanied by dense inflammatory cell infiltration. Alveolar spaces appeared variably reduced or completely obliterated in some regions due to alveolar collapse and focal microatelectasis. In addition, type II pneumocyte hyperplasia, bronchiolar epithelial desquamation, and degenerative cellular changes were evident, indicating ongoing tissue injury. In the MTX+PREG group, the severity of these histopathological alterations was markedly reduced. Lung sections from this group exhibited only mild septal thickening and limited inflammatory cell infiltration, while the overall pulmonary architecture remained largely preserved. Hyperemia, edema and hemorrhagic changes were less prominent compared with the MTX group. These histopathological observations indicate that PREG co-treatment was associated with attenuation of MTX-induced pulmonary damage at the tissue level (Fig. 2).

Fig. 2.

Representative histopathological images of lung tissue sections from experimental groups. A Control group showing normal pulmonary architecture with thin alveolar septa and intact bronchiolar epithelium. B MTX-treated group displaying alveolar septal thickening (arrow), interstitial inflammatory cell accumulation, and hyperemia. C MTX+PREG group showing relatively preserved alveolar spaces and fewer inflammatory cells compared with the MTX group. D PREG-only group with tissue morphology similar to the control group. HE staining, scale bars = 50 μm

Quantitative histopathological evaluation demonstrated a marked increase in pulmonary injury parameters in the MTX group compared with the control group. MTX administration resulted in significant alveolar structural damage, reflected by higher scores for emphysema (p < 0.01), interstitial and alveolar edema (p < 0.001), inflammatory cell infiltration (p < 0.01), hemorrhage (p < 0.01), epithelial loss (p < 0.001), atelectasis (p < 0.001), and hyperemia (p < 0.001). These findings are consistent with diffuse alveolar injury, capillary hyperemia, and vascular leakage observed in the histopathological examination.

Co-administration of PREG (MTX + PREG) was associated with a marked reduction in the severity of MTX-induced histopathological alterations. Semi-quantitative scores for all evaluated parameters were significantly lower than those in the MTX group (p < 0.001 for edema, epithelial loss, and hyperemia; p = 0.003 for atelectasis; p = 0.035 for inflammatory infiltration; p = 0.043 for hemorrhage), whereas emphysema scores did not differ statistically between these groups (p = 0.486). Among the evaluated parameters, demonstrated the most pronounced reduction, approaching values observed in the control group, indicating no detectable histopathological alterations.

Overall, the semi-quantitative scoring analysis supports the histopathological observations observed in HE-stained sections, indicating that PREG co-treatment was associated with attenuation of MTX-induced pulmonary injury at the tissue level. These findings are consistent with reduced hyperemia, interstitial edema, and epithelial damage in lung tissue (Fig. 3; Table 3).

Fig. 3.

Semi-quantitative histopathological scores of lung tissue in experimental groups. Semi-quantitative analysis of emphysema, edema, inflammatory cell infiltration, hemorrhage, epithelial loss, atelectasis, and hyperemia parameters in the Control, MTX, MTX+PREG, and PREG groups. Each bar represents mean ± SD (n = 8). administration was associated with higher histopathological scores compared with the control group. In the MTX + PREG group, the scores for all parameters were lower than those observed in the MTX group. Statistical comparisons were performed using Kruskal–Wallis test followed by Dunn’s multiple comparison test (*p < 0.05; ** p < 0.01; *** p < 0.001). MTX: Methotrexate, PREG: Pregabalin

Table 3.

Statistical evaluation of histopathological expression levels across different groups

Individual P Value	Control vs. MTX	Control vs. MTX+PREG	Control vs. PREG	MTX vs. MTX+PREG	MTX vs. PREG	MTX+PREG vs. PREG
Hyperemia	< 0.001	= 0.015	> 0.999	< 0.001	< 0.001	= 0.015
Edema	< 0.001	= 0.096	> 0.999	< 0.001	< 0.001	= 0.096
Hemorrhage	= 0.006	= 0.402	= 0.674	= 0.043	= 0.002	= 0.213
Inflammation	= 0.002	= 0.192	> 0.999	= 0.035	= 0.002	= 0.192
Epithelial Loss	< 0.001	= 0.044	> 0.999	< 0.001	< 0.001	= 0.044
Atelectasis	< 0.001	= 0.355	= 0.642	= 0.003	< 0.001	= 0.642
Emphysema	= 0.009	= 0.044	> 0.999	= 0.486	= 0.009	= 0.044

Kruskal–Wallis test followed by Dunn’s multiple comparison test

*p < 0.05

**p < 0.01

***p < 0.001

Immunohistochemical findings

COX-2 analysis

MTX administration resulted in a significant increase in COX-2 immunoexpression compared with the control group, as demonstrated by higher staining intensity, an increased percentage of positive cells, and elevated total immunoreactivity scores (p < 0.001). These findings indicate enhanced COX-2 expression in lung tissue following MTX exposure.

PREG co-treatment was associated with a significant reduction in COX-2 immunoreactivity compared with the MTX group (p < 0.001 for all evaluated parameters). Although COX-2 expression in the MTX + PREG group remained slightly higher than in the control group, the difference was limited and did not correspond to marked histopathological alterations. No significant differences were observed between the control and PREG-only groups, indicating that PREG administration alone did not induce detectable changes in COX-2 expression in lung tissue (Fig. 4; Table 4).

Fig. 4.

COX-2 immunohistochemical expression in lung tissues from experimental groups.Representative immunohistochemical staining showing COX-2 expression in lung tissue sections. The control group (A) exhibited minimal COX-2 immunoreactivity. MTX administration (B) resulted in increased COX-2 staining, primarily observed in alveolar macrophages, type II pneumocytes, and interstitial and perivascular inflammatory cells. In the MTX + PREG group (C), COX-2 immunoreactivity appeared reduced compared with the MTX group. The PREG-only group (D) showed COX-2 expression similar to the control group. Quantitative analysis demonstrated a significant increase in staining intensity, percentage of positive cells, and total COX-2 scores in the MTX group compared with the control (p < 0.001). These parameters were significantly lower in the MTX + PREG group than in the MTX group (p < 0.001). Streptavidin-biotin peroxidase method; scale bars = 50 μm. Statistical analysis was performed using the Kruskal–Wallis test followed by Dunn’s multiple comparison test. ***p < 0.001. MTX: Methotrexate, PREG: Pregabalin, COX-2: Cyclooxygenase-2

Table 4.

Statistical evaluation of COX-2 immunohistochemical expression across different groups

Individual p value of COX-2	Control vs. MTX	Control vs. MTX+PREG	Control vs. PREG	MTX vs. MTX+PREG	MTX vs. PREG	MTX+PREG vs. PREG
Staining intensity	< 0.001	= 0.696	= 0.946	< 0.001	< 0.001	= 0.375
Percentage score	< 0.001	> 0.999	= 0.974	< 0.001	< 0.001	= 0.974
Total score	< 0.001	= 0.904	= 0.904	< 0.001	< 0.001	> 0.999

Kruskal–Wallis test followed by Dunn’s multiple comparison test

COX-2: Cyclooxygenase-2

***p < 0.001

IL-6 analysis

MTX exposure resulted in a significant increase in IL-6 immunoexpression compared with the control group, as evidenced by higher staining intensity, an increased percentage of positive cells, and elevated total immunoreactivity scores (p < 0.001). These findings indicate increased IL-6 expression in lung tissue following MTX administration, particularly within alveolar and interstitial compartments.

PREG co-treatment was associated with a significant reduction in IL-6 immunoreactivity compared with the MTX group (p < 0.001 for all evaluated parameters). Although IL-6 expression in the MTX + PREG group remained slightly higher than in the control group, the values were markedly lower than those observed in the MTX group. No significant differences were detected between the control and PREG-only groups, indicating that PREG administration alone did not produce detectable changes in IL-6 expression in lung tissue (Fig. 5; Table 5).

Fig. 5.

IL-6 expression in lung tissues from experimental groups. Representative immunohistochemical staining showing IL-6 expression in lung tissue sections. The control group (A) displayed minimal IL-6 immunoreactivity in alveolar and interstitial regions. MTX administration (B) resulted in increased IL-6 staining, primarily in alveolar macrophages, type II pneumocytes, and interstitial and perivascular inflammatory cells. In the MTX + PREG group (C), IL-6 immunoreactivity appeared reduced compared with the MTX group, with fewer positive cells and weaker staining intensity. The PREG-only group (D) exhibited IL-6 expression similar to the control group. Quantitative analysis revealed a significant increase in IL-6 staining intensity, percentage of positive cells, and total score in the MTX group compared with the control (p < 0.001). These values were significantly lower in the MTX + PREG group than in the MTX group (p < 0.001). Streptavidin–biotin peroxidase method; scale bar = 50 μm. Statistical analysis was performed using the Kruskal–Wallis test followed by Dunn’s multiple comparison test. ***p < 0.001. MTX: Methotrexate, PREG: Pregabalin, IL-6: Interleukin-6

Table 5.

Statistical evaluation of IL-6 immunohistochemical expression across different groups

Individual p value of IL-6	Control vs. MTX	Control vs. MTX+PREG	Control vs. PREG	MTX vs. MTX+PREG	MTX vs. PREG	MTX+PREG vs. PREG
Staining intensity	< 0.001	= 0.593	> 0.999	< 0.001	< 0.001	= 0.593
Percentage score	< 0.001	= 0.953	> 0.999	< 0.001	< 0.001	= 0.953
Total score	< 0.001	= 0.865	> 0.999	< 0.001	< 0.001	= 0.865

Kruskal–Wallis test followed by Dunn’s multiple comparison test

IL-6: Interleukin-6

***p < 0.001

VCAM-1 analysis

MTX administration resulted in a significant increase in VCAM-1 immunoexpression compared with the control group, as evidenced by higher staining intensity, an increased percentage of positive cells, and elevated total immunoreactivity scores (p < 0.001). VCAM-1 immunoreactivity was predominantly. VCAM-1 immunoreactivity was predominantly localized in vascular endothelial cells and perivascular inflammatory regions.

PREG co-treatment was associated with a significant reduction in VCAM-1 expression, immunoreactivity compared with the MTX group across all evaluated parameters (p < 0.001). Although VCAM-1 expression in the MTX + PREG group remained slightly higher than in the control group, the values were substantially lower than those observed in the MTX group. No significant differences were detected between the control and PREG-only groups, indicating that PREG administration alone did not produce detectable changes in VCAM-1 expression in lung tissue (Fig. 6; Table 6).

Fig. 6.

Representative immunohistochemical staining demonstrating VCAM-1 expression in lung tissue sections. The control group (A) showed minimal VCAM-1 immunoreactivity in alveolar and vascular structures. The MTX-treated group (B) exhibited increased VCAM-1 immunostaining, predominantly localized in vascular endothelial cells and perivascular regions. In the MTX + PREG group (C), VCAM-1 immunoreactivity appeared reduced compared with the MTX group, with weaker and more limited staining patterns. The PREG-only group (D) displayed minimal VCAM-1 expression similar to that observed in the control group. Quantitative analysis demonstrated significantly higher staining intensity, percentage of positive cells, and total VCAM-1 immunoreactivity score in the MTX group compared with the control group (p < 0.001). These values were significantly lower in the MTX + PREG group than in the MTX group (p < 0.001). Streptavidin–biotin peroxidase method; scale bar = 50 μm. Statistical analysis was performed by Kruskal–Wallis test followed by Dunn’s multiple comparison test. ***p < 0.001. MTX: Methotrexate, PREG: Pregabalin, VCAM-1: Vascular Cell Adhesion Molecule 1

Table 6.

Statistical evaluation of VCAM-1 immunohistochemical expression across different groups

Individual p value of VCAM-1	Control vs. MTX	Control vs. MTX+PREG		Control vs. PREG	MTX vs. MTX+PREG		MTX vs. PREG	MTX+PREG vs. PREG
Staining intensity	< 0.001		= 0.566	= 0.913		< 0.001	< 0.001	= 0.913
Percentage score	< 0.001		= 0.927	> 0.999		< 0.001	< 0.001	= 0.927
Total score	< 0.001		= 0.536	= 0.904		< 0.001	< 0.001	= 0.904

Kruskal–Wallis test followed by Dunn’s multiple comparison test

VCAM-1: Vascular Cell Adhesion Molecule 1

***p < 0.001

Overall, histopathological and immunohistochemical evaluations demonstrated that MTX administration was associated with severe pulmonary injury characterized by alveolar septal thickening, inflammatory cell infiltration, edema, hemorrhage, and epithelial degeneration. These morphological alterations were accompanied by significant increased immunoexpression of COX-2, IL-6, and VCAM-1 in lung tissue. In contrast, PREG co-treatment was associated with a marked reduction in the severity of these histopathological alterations and lower immunoreactivity for the evaluated markers compared with the MTX group. Lung architecture in the MTX + PREG group appeared largely preserved relative to the MTX-treated animals. Collectively, these findings provide tissue-level evidence suggesting that PREG may attenuate MTX- associated pulmonary injury in this experimental model. However, the present observations are based on histopathological and immunohistochemical assessments and should therefore be interpreted as supportive experimental evidence rather than direct mechanistic confirmation.

Discussion

MTX, although indispensable in the treatment of autoimmune disorders and hematological malignancies, is paradoxically associated with dose-dependent pulmonary toxicity that often limits its clinical use [5, 16]. The present study provides histopathological and immunohistochemical evidence that PREG, a structural analog of GABA [9], markedly attenuates MTX-induced lung injury. The protective efficacy of PREG was demonstrated by preservation of alveolar architecture together with reduced expression of inflammatory and endothelial activation markers, suggesting that PREG may influence key pathogenic processes involved in MTX-related pulmonary toxicity.

MTX exposure produced a characteristic pattern of pulmonary injury including alveolar edema, hyperemia, inflammatory infiltration, epithelial degeneration, hemorrhage, atelectasis, and emphysematous changes. These alterations are consistent with previous descriptions of MTX-induced pneumonitis, in which septal thickening, vascular congestion, and inflammatory cell accumulation represent common histopathological findings [17, 18]. In parallel with these structural alterations, MTX administration was associated with increased immunoexpression of COX-2, IL-6, and VCAM-1. Previous studies have suggested that MTX -related pulmonary injury may involve inflammatory amplification and endothelial activation processes that are frequently linked to oxidative stress–mediated cellular injury [19]. In the present study, the increased expression of these markers is consistent with such mechanisms, although oxidative stress parameters were not directly evaluated.

Co-administration of PREG significantly ameliorated MTX-induced histopathological damage, as evidenced by reduced scores for edema, hyperemia, inflammation, epithelial loss, and hemorrhage. These morphological improvements were accompanied by marked reductions in COX-2 and IL-6 immunoexpression [20, 21]. Because these mediators are commonly regulated through NF-κB –related inflammatory pathways, the observed reduction in their expression may indicate modulation of inflammatory signaling processes. However, as NF-κB activity was not directly assessed in this study, this interpretation should be considered a literature-based inference rather than direct experimental evidence. Previous experimental studies have reported that PREG may attenuate inflammatory responses and cytokine release in models of neuropathic and systemic inflammation, supporting the plausibility of such mechanism [22, 23].

In MTX-treated lungs, extensive inflammatory infiltration and epithelial desquamation were associated with alveolar collapse and structural disruption. Increased IL-6 expression has been reported to contribute to epithelial injury and mitochondrial dysfunction in inflammatory lung conditions [24]. The preservation of epithelial continuity and reduced inflammatory infiltration observed in PREG-treated groups therefore suggests that PREG may exert cytoprotective effects on alveolar structures. Nevertheless, the precise cellular mechanisms responsible for these protective effects cannot be determined from the present data.

Endothelial activation also appeared to be an important component of MTX-induced lung injury. VCAM-1 expression was markedly increased in MTX -exposed lungs, particularly in vascular endothelium and perivascular inflammatory regions. VCAM-1 facilitates leukocyte adhesion and transendothelial migration and is widely recognized as a marker of endothelial activation during inflammatory responses [25]. The reduction of VCAM-1 expression in PREG-treated animals suggests a potential stabilizing effect on the pulmonary vascular endothelium and a possible reduction in leukocyte recruitment. Similar endothelial-protective effects of PREG have been reported experimental inflammatory models, including lipopolysaccharide-induced cardiac and vascular injury [26].

The coexistence of atelectasis and emphysematous regions in MTX-treated lungs reflects severe disruption of alveolar structure, and characterized by regional collapse and compensatory hyperinflation [5, 27–31]. The reduction of these lesions in PREG-treated animals indicates preservation of alveolar integrity [32, 33]. Although mitochondrial function and type II pneumocyte activity were not directly assessed in the present study, previous reports have suggested that mitochondrial dysfunction may contribute to MTX-induced pulmonary injury [34]. In this context, it is conceivable that preservation of cellular integrity may contribute to maintenance of alveolar stability.

Oxidative stress has been widely implicated in the pathogenesis of MTX-induced tissue injury [34]. Experimental studies have suggested that PREG may enhance antioxidant defense systems, including glutathione and superoxide dismutase activity, thereby reducing ROS accumulation [35]. Reduced oxidative stress may, in turn, attenuate NF-κB–dependent transcription of COX-2 and IL-6 and disrupt prostaglandin-mediated endothelial activation loops involving VCAM-1 [36]. Because oxidative stress parameters were not measured in the present study, the potential contribution of antioxidant mechanisms should be considered speculative and based on previously published findings.

Preservation of epithelial integrity is essential for maintaining pulmonary barrier function. MTX has been reported to disrupts epithelial tight junctions and induce apoptosis through mitochondrial and caspase-dependent pathways [37, 38]. The reduced epithelial degeneration observed in the PREG -treated groups may therefore reflect improved structural stability of the alveolar epithelium. However, intracellular signaling pathways potentially involved in this protection, including Protein Kinase B/ Glycogen Synthase Kinase-3 β/ β-catenin signaling were not evaluated in this study and remain hypothetical [9, 39, 40].

Similarly, IL-6–mediated permeability has been reported to contribute to pulmonary edema through mechanisms involving STAT3-dependent phosphorylation of vascular endothelial cadherin [41]. The reduction of IL-6 expression by PREG therefore represents a plausible mechanism underlying the observed reductions in edema and hyperemia. This interpretation is supported by prior studies demonstrating that inhibition of IL-6–driven inflammation alleviates vascular permeability and tissue edema, and that PREG exerts similar effects in systemic inflammatory models [42, 43]. The reduction of IL-6 expression observed in the present study may therefore be associated with decreased vascular permeability. Nevertheless, the underlying signaling pathways were not experimentally investigated and cannot be directly inferred from the present data.

Although functional respiratory parameters were not assessed, the preservation of alveolar architecture together with reduced inflammatory marker suggests that structural protection of lung tissue occurred in the PREG-treated groups. Clinically, such protection could mitigate the declines in arterial oxygenation and diffusion capacity characteristic of MTX-associated pneumonitis [44]. However, whether these histological improvements translate into functional pulmonary benefit requires further investigation.

Oxidative stress parameters were not measured in the present study. However, previous studies have suggested that PREG may activate the Nuclear Factor Erythroid 2-Related Factor 2/Heme Oxygenase-1 (NRF2/HO-1) [45]. There for, it is possible that similar mechanisms may contribute to the protective effects observed here, although this remains to be experimentally confirmed. Overall, the present findings suggest that MTX-induced pulmonary injury is associated with inflammatory and endothelial activation processes characterized by increased expression of COX-2, IL-6, and VCAM-1. The reduced expression of these markers together with improved alveolar morphology in the PREG-treated groups indicates a protective tissue-level effect, likely related to attenuation of inflammatory responses and preservation of epithelial integrity.

Several limitations of this study should be acknowledged. First, pulmonary injury was evaluated mainly through histopathological and immunohistochemical analyses and functional respiratory parameters such as pulmonary function tests or arterial blood gas measurements were not assessed. Second, although key inflammatory and endothelial markers were examined, oxidative stress biomarkers and intracellular signaling pathways implicated in MTX-induced pulmonary toxicity (e.g., NF-κB, STAT3, and NRF2/HO-1) were not directly investigated. Therefore, mechanistic interpretations are based on marker expression patterns and existing literature rather than direct molecular evidence. Third, the study used an acute, single-dose MTX model and a single dose of pregabalin which may not fully reflect the subacute or chronic exposure patterns seen in clinical practice.

Taken together, these limitations indicate that the present results should be interpreted primarily as tissue-level and hypothesis-generating evidence. Future studies incorporating functional pulmonary assessments, oxidative stress analyses, and molecular pathway investigations are required to clarify the underlying mechanisms and the potential therapeutic role of pregabalin in MTX-induced pulmonary injury.

Conclusion

In summary, the present study shows that PREG co-treatment was associated with attenuation of MTX-induced pulmonary injury, as reflected by improved alveolar architecture and reduced immunoexpression of inflammatory and endothelial-associated markers, including COX-2, IL-6, and VCAM-1. These histopathological and immunohistochemical findings indicate provide tissue-level suggesting a potential protective role of PREG against MTX-associated lung injury in this experimental model. Although previous studies have suggested that PREG may influence oxidative stress–related and cytokine-mediated inflammatory processes, such mechanisms were not directly evaluated in the present study. Therefore, the current observations should be interpreted as supportive and hypothesis-generating rather than as direct mechanistic evidence. Future studies incorporating molecular analyses, including evaluation of oxidative stress pathways (e.g., the NRF2/HO-1 axis), together with functional pulmonary assessments, are warranted to clarify the underlying mechanisms and to determine the translational relevance of PREG as a potential adjunctive strategy for mitigating MTX-induced pulmonary toxicity.

Author contributions

S.K.H. and O.O. contributed to the literature search and manuscript editing. S.K.H., O.O., and H.A. contributed to the study design, data analysis, and interpretation. O.O. and H.A. contributed to data collection and/or assembly. All authors participated in data verification. All authors reviewed and approved the final version of the manuscript.

Funding

This study was supported by the Scientific Coordination Unit of the Suleyman Demirel University (Project No. TTU-2024-9577).

Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

This study was approved by the Süleyman Demirel University Local Ethics Committee for Animal Experiments (Approval No: 01/250; Date: 15.02.2024) and conducted in accordance with institutional and international guidelines for laboratory animal care and use, including the ARRIVE 2.0 and Basel Declaration principles. Lung tissues were obtained from rats previously included in an ethically approved project, thereby supporting the 3Rs principle, particularly reduction.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.