This study was conducted at the Center for Biotechnology, Al-Nahrain University. All experimental procedures followed standard laboratory practices for animal care and were conducted in accordance with internationally accepted guidelines (ARRIVE guidelines) to minimize animal suffering. All efforts were made to reduce the number of animals used and to ensure humane handling throughout the study.

A total of 48 male C57BL/6 mice (8–10 weeks old, 20–25 g) were randomly allocated into five groups using a computer-generated randomization table: negative control (no IMQ, n = 10), positive control (IMQ only, n = 10), IMQ + clobetasol 0.05% (n = 10), IMQ + tacrolimus 0.1% (n = 9), and IMQ + methotrexate 1% (n = 9). Investigators performing clinical scoring and histological assessment were blinded to group allocation. All experiments were performed at the Center for Biotechnology, Al-Nahrain University.

Psoriasis-like inflammation was induced by daily topical application of 5% imiquimod (IMQ) cream to the shaved dorsal skin for 7 consecutive days. Each mouse received 62.5 mg of 5% IMQ cream per day applied to a 2 × 3 cm area. After the 7-day induction period, mice received topical treatments twice daily for 14 days as follows:

The MTX ointment vehicle used in this study was prepared as follows: white soft paraffin 70% w/w, liquid paraffin 20% w/w, propylene glycol 10% w/w, with methotrexate incorporated at 1% w/w. Formulations were prepared under aseptic conditions, homogenized by mechanical mixing, and stored at 4 °C in amber containers. Short-term homogeneity was confirmed by visual inspection and sampling.

Clinical severity was assessed daily using a murine PASI adapted scoring system evaluating erythema, induration (thickness), and scaling on a 0–4 scale (0 = none; 4 = very marked). The three component scores were summed to give a daily total (maximum 12). Scoring was performed independently by two blinded investigators and the mean of the two scores was used for analysis. PASI was recorded throughout the 7-day induction and the 14-day treatment periods [4].

At study end (day 21 from first IMQ application), animals were euthanized by CO₂ inhalation followed by cervical dislocation. Full-thickness dorsal skin samples were excised, weighed, and divided: one portion was fixed in 10% neutral buffered formalin for histology; the remaining tissue was snap-frozen in liquid nitrogen and stored at −80 °C for biochemical assays.

Formalin-fixed tissues were processed, embedded in paraffin, and sectioned at 5 µm. Sections were stained with Hematoxylin and Eosin (H&E). A pathologist blinded to treatment scored histological features (inflammation, parakeratosis, epidermal thickness, vascular changes) using a standardized semiquantitative scale (0–3 for each feature). Epidermal thickness (basal layer to stratum granulosum) was measured morphometrically in ten random fields per section using ImageJ (NIH); the mean per animal was used for statistical analysis [5].

Approximately 50 mg of skin tissue was homogenized in 500 µL of ice-cold phosphate-buffered saline (PBS) containing protease inhibitors using a tissue homogenizer. The homogenates were centrifuged at 12,000× g for 15 min at 4 °C, and the supernatants were collected and stored at −80 °C until analysis [6].

Tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) concentrations were measured using commercially available ELISA kits (R&D Systems, Minneapolis, MN, USA), Mouse TNF-α Quantikine ELISA Kit (Catalog No.: MTA00B) and Mouse IL-6 Quantikine ELISA Kit (Catalog No.: M6000B), following the manufacturer’s instructions. Cytokine levels were expressed as pg/mg protein.

Target selection and preparation-selected protein targets and PDB IDs: DHFR (1U72), TYMS (1HVY), ATIC (1D3G), DHODH (6QU7), TNF-α (2AZ5), and IL-6 (1IL6). Protein structures were prepared by removing crystallographic waters, adding polar hydrogens, and assigning Kollman charges using AutoDock Tools. Ligands (methotrexate, clobetasol, tacrolimus) were energy-minimized using the MMFF94 force field in OpenBabel and converted to PDBQT format [7].

Molecular docking simulations were performed using AutoDock Vina v1.1.2. For each target protein, a grid box was centered on the reported active site or ligand-binding pocket; the grid box centers and dimensions are provided in Supplementary Table S1.

The docking parameters were set as follows: exhaustiveness = 8 and num_modes = 10. The lowest binding energy pose for each ligand–protein complex was selected for further analysis. The selected poses were visualized and analyzed using PyMOL to identify hydrogen bonds and hydrophobic interactions, while two-dimensional interaction diagrams were generated using LigPlot+ [8].

Interpretation docking results are reported as exploratory, hypothesis-generating data and do not constitute proof of in vivo binding or pharmacological targeting.

Data are presented as mean ± SD. Normality was assessed using the Shapiro–Wilk test. For normally distributed data, comparisons among groups were performed by one-way ANOVA followed by Tukey’s post hoc test; for non-normal data, Kruskal–Wallis with Dunn’s post hoc test was used. Repeated measures (PASI over time) were analyzed by two-way ANOVA with repeated measures and Sidak’s multiple comparisons test. Correlations were assessed by Pearson’s correlation for normally distributed variables. A two-tailed p < 0.05 was considered statistically significant. Analyses were performed using GraphPad Prism v9.0 [9].

Erythema, induration (thickness), and scaling became macroscopically evident 2–3 days after the first topical application of imiquimod (IMQ). All three component scores and the composite murine PASI showed a progressive increase during the induction phase, reaching maximal values around day 8 (Figure 1, top and bottom panels). The negative control group maintained scores close to zero throughout the study, confirming intact skin at baseline and no spontaneous lesion development. In contrast, IMQ-treated animals exhibited a clear and reproducible rise in PASI scores that validated successful establishment of the psoriasis-like model.

One animal died during the acclimation/adaptation week; consequently, data for that mouse were excluded and the control group was not available for the day-2 assessment. All remaining animals completed the induction and treatment phases and were included in the analyses described below.

PASI trajectories for the IMQ groups showed a rapid onset of inflammation (days 2–4), a peak around day 8, and a plateau or gradual change thereafter during the treatment period (Figure 1). The pattern was consistent across IMQ-only and IMQ + treatment groups, with differences in magnitude and temporal response apparent between treatment arms (see Figure 1 and Table 1 for group means ± SD and full statistical comparisons).

The divergence between the IMQ groups and the negative control in both component scores and total PASI confirms that the IMQ regimen produced a robust, reproducible inflammatory phenotype suitable for testing topical interventions. These clinical observations were corroborated by histopathological and biochemical endpoints (see Histology and Cytokine sections), which together support the validity of the model for evaluating topical anti-psoriatic effects.

The histopathological scoring system for all four groups was as follows: 0—normal skin; 1—mild inflammation, as evidenced by a sparse infiltrate of inflammatory cells, minimal tissue damage, and preservation of normal skin architecture; 2—moderate inflammation, as evidenced by a moderate infiltrate of inflammatory cells, moderate tissue damage, and partial disruption of normal skin architecture; 3—severe inflammation, as evidenced by a large infiltrate of inflammatory cells, significant tissue damage, and significant disruption of normal skin architecture; 4—most severe inflammation and tissue damage, as evidenced by extensive infiltrate of inflammatory cells, extensive disruption of normal skin architecture, and extensive tissue damage, as seen in the imiquimod-treated control group. The results were as follows: control negative—0; control positive—4; tacrolimus—3; clobetasol—2; methotrexate—1 (Figure 2: negative control; Figure 3: positive control; Figure 4 and Figure 5: tacrolimus group; Figure 6: clobetasol group; Figure 7 and Figure 8: MTX group) [10].

Cytokine concentrations (TNF-α and IL-6) measured in skin tissue homogenates across all treatment groups are presented in Table 2 and Figure 9. The positive control group showed significantly elevated TNF-α and IL-6 compared with negative controls, confirming successful model induction (Table 2). Treatment with clobetasol and topical MTX (1%) reduced both cytokines to near-baseline levels, while tacrolimus showed a significant reduction in TNF-α but not IL-6 (p = 0.060, ns).

Erythema, induration, and scaling were first evident 2–3 days after the initial topical IMQ application and increased progressively to a peak around day 8 (Figure 1). One mouse died during the acclimation week; data from that animal were excluded from all analyses. The negative control group maintained near-zero PASI scores throughout, confirming intact baseline skin, while IMQ treatment produced a reproducible psoriasis-like phenotype suitable for testing topical interventions.

For the Clobetasol-treated group, the cytokine levels of TNF-alpha and IL-6 are key findings.

Interpretation IMQ produced a clear pro-inflammatory cytokine profile in skin. Both clobetasol and topical MTX (1%) effectively reduced TNF-α and IL-6 to near-baseline levels, while tacrolimus showed a more modest effect, significantly lowering TNF-α but not IL-6. These biochemical results align with the clinical PASI trajectories and histopathological improvements observed in the corresponding treatment groups (Figure 9; see Figure 1 and histology results).

Molecular docking simulations were performed to study the interaction profiles of tacrolimus, methotrexate, and clobetasol propionate with TNF-α (PDB ID: 2AZ5) and IL-6 (PDB ID: 1IL6). The binding affinity values for top-ranked poses and key amino acid interactions are summarized in Table 3 and Table 4; representative binding interactions are visualized in Figure 10.

Topical MTX produced robust clinical improvement, reflected by significant reductions in PASI component scores (erythema, scaling, thickness) and by histological restoration toward normal epidermal architecture with decreased acanthosis and resolution of parakeratosis. These effects were comparable to clobetasol propionate (0.05%) and exceeded those observed with tacrolimus (0.1%) in this model. The histomorphological recovery observed with MTX—reduced epidermal proliferation and diminished inflammatory infiltrate—supports a direct antiproliferative effect on keratinocytes in addition to immunomodulation.

Although the biomarker panel was limited to TNF-α and IL-6, these cytokines represent key mediators in psoriasis inflammation. Future studies should include additional markers such as IL-17 and IL-23 to better characterize the immunological pathways.

Biochemical analyses showed that topical MTX significantly lowered TNF-α and IL-6 levels in skin homogenates to values similar to those achieved with clobetasol. Mechanistically, these findings align with MTX’s known actions as a folate antagonist that inhibits enzymes involved in nucleotide synthesis (e.g., DHFR, TYMS), thereby limiting proliferation of activated immune cells and hyperproliferative keratinocytes. MTX-mediated elevation of extracellular adenosine likely contributed to the observed suppression of proinflammatory cytokine production. The more modest effect of tacrolimus on IL-6—despite a significant reduction in TNF-α—suggests differential downstream modulation of cytokine networks by calcineurin inhibition versus antifolate mechanisms [11].

Docking simulations confirmed moderate predicted binding affinities of MTX, clobetasol, and tacrolimus to TNF-α and IL-6; these results should be interpreted cautiously as exploratory and not as evidence of direct cytokine antagonism in vivo. The combined in vivo and in silico data support a model in which topical MTX reduces cytokine levels primarily by acting on cellular sources (T cells, keratinocytes) and metabolic pathways rather than by direct blockade of soluble cytokines [12].

Previous reports on topical MTX efficacy have been inconsistent, likely reflecting variability in vehicle composition, drug concentration, and skin penetration. The efficacy demonstrated here suggests that the chosen ointment base and 1% concentration achieved sufficient local bioavailability to exert therapeutic effects no safety assessment was performed in this preclinical setting. These results underscore the importance of formulation optimization (vehicle selection, penetration enhancers, particle size, and occlusion) when developing topical MTX products [13,14].