Modulation of the KEAP1-NRF2 pathway by Erianin: A novel approach to reduce psoriasiform inflammation and inflammatory signaling

2.1 Cell culture

The human differentiated keratinocytes (HaCaT; Procell, Wuhan, China) were cultivated in a moist incubator at 37°C and 5% CO₂ using DMEM supplied with 10% FBS and 1% penicillin-streptomycin. At 37°C and 5% CO₂, an in vitro cell model for psoriasis was created by inducing 10 ng/mL TNF-α in a logarithmic growth phase of HaCaT cells [36], for 48 h.

2.2 CCK8 assay

To ascertain the antiproliferative effect of ERN on HaCaT, the CCK-8 reagent was used. For a brief duration, HaCaT cells were seeded at a rate of 5 × 10³ cells/well in 96-well dishes, administered ERN at various doses (0, 5, 10, 20, 40, and 80 μM) for 24 h, and then maintained with CCK-8 reagent for 1 h at 37°C in the dark. ERN was administered to HaCaT cells at doses ranging from 5 to 20 μM following a 48 h TNF-α treatment at 37°C and 5% CO₂. Utilizing a Microplate Reader, the absorption coefficient of the assessments was measured at an apparent wavelength of 450 nm.

2.3 EdU assessment

The HaCaT cells were cultivated in 24-well plates and exposed to varying doses of combined TNF-α and ERN. As directed by the manufacturer, the cell proliferation EdU (5-ethynyl-2′-deoxyuridine) assay kit (Thermo Fisher Scientific, Catalog No. C10337) was utilized to measure the proliferative growth rate of HaCaT cells. Immunofluorescence was used to evaluate EdU accumulation.

2.4 ELISA

ERN (5–10 μM) was applied to the TNF-α-persuaded HaCaT cells for 24 h. The ELISA kit (BlueGene Biotech, Catalog No. E03P0661) was then used to identify the various inflammatory cytokines (IL-6, IL-8, IL-22, and IL-1β) in the supernatant following the directions provided by the maker.

2.5 Reverse transcription-polymerase chain reaction (RT-PCR) analysis

After seeding HaCaT keratinocytes in 12-well plates, they were either triggered using TNF-α (10 ng/mL) or without it for 2 h after being treated with ERN (5, 10, and 20 μM) or not for 24 h. RNA was collected from cells and used for qRT-PCR investigation of Keratin 1 (KRT1) and Keratin 6 (KRT6) mRNA levels. As directed by the supplier, the TRIzol^TM reagent was used for extracting total RNA from mouse skin tissue or HaCaT keratinocytes. 2 µg of total RNA was used to create the first strand of cDNA using the cDNA Synthesis Kit (Servicebio, Catalog No. G3331). Using the SYBR Green PCR Master Mix kit (Thermo Fisher Scientific, Catalog No. 4309155), GAPDH was used as an internal control gene, and the relative mRNA expression levels of the target genes were calculated using the 2^−ΔΔCt method. This method allowed for the comparison of gene expression across different experimental groups. All reactions were performed in triplicate, and data were analyzed using the CFX96 Touch™ Real-Time PCR Detection System software. To target the genes in HaCaT keratinocytes, the following primer sequences were used (Table 1).

2.6 Animal studies

All experiments on animals were conducted on male BALB/c mice in optimal condition, evaluating between 24 and 28 g at 5 weeks of age. The mice were housed in the animal house facility of the Department of Dermatology, The Second Children and Healthcare of Jinan, China, for approximately 7 days to allow them to become used to the laboratory environment (CPCSEA No: LL2024250001). A regular pellet meal and unlimited access to clean water were provided to the animals. For the duration of the investigative protocol, animals were kept in typical laboratory settings with a 12 h light/dark cycle, a temperature of 22 ± 2°C, and a moisture content of 40–70%. All that was possible has been taken to minimize the discomfort that the animals went through and to use only a few in totals for the assessment.

1. Ethical approval: The research related to animal use has been complied with all the relevant national regulations and institutional policies for the care and use of animals, and has been approved by the Animal Care and Use Committee of the Department of Dermatology, The Second Children and Healthcare of Jinan, China (Approval No: CPCSEA No: LL2024250001).

2.7 Fabrication of an experimental psoriasis system and therapy strategy in mice activated by IMQ

An animal model of IMQ-induced testing psoriasis was created [37], by topically treating mice with IMQ 5% (w/w) for 6 days in a row (days 1–6). A 2.5 cm × 2.5 cm section of every mouse’s dorsal skin was shaved to remove mouse hairs. IMQ and ERN were not applied topically to the control group. Based on day 1 to day 6, all the animals aside from the control group, were topically administered with commercially accessible IMQ, at an administration of 62.5 mg lotion on their smooth-shaven rear skin (50 mg of IMQ lotion), every morning. This equates to a daily dose of 3.125 mg of the effective IMQ. For the conventional clobetasol group of controls, 40 mg of commercially accessible clobetasol cream (0.05% w/w) was administered topically on all the animals’ trimmed back skin (equivalent to 32 mg of clobetasol cream) every day, resulting in a daily supply of 20 μg of the influential substance.

External administration of ERN was performed in an anionic suspension base containing 100 mg at three distinct concentrations of 0.5, 1, and 2% w/w per animal. Each animal's shaved dorsal skin was topically treated with 100 mg of the produced anionic emulsifying cream containing ERN (0.5, 1, and 2% w/w) for six days in a row. This amounts to 80 mg of ERN cream each application. This equates to a daily allowance of 0.5, 1, and 2 mg of the ERN dose for each animal, Accordingly, every aspect concentration was directed at 12 h intervals after the use of IMQ for 6 successive days, except for control and IMQ control. Six groups of six mice were chosen at random from among the mice (n = 6).

2.8 Experimental groups

Group I: control (without IMQ and ERN)

Group II: IMQ control (IMQ alone; without ERN)

Group III: IMQ + 0.05% w/w Clobetasol (standard drug)

Group IV: IMQ + 0.5% w/w ERN (IMQ + ERN 0.5% w/w)

Group V: IMQ + 1% w/w ERN (IMQ + ERN 1% w/w)

Group VI: IMQ + 2% w/w ERN (IMQ + ERN 2% w/w)

The psoriasis area and severity index (PASI), which is rated using successive days’ aggregate scores for scaling, skin redness, and thickness, was used to track the infection’s progression. Day 7 saw the aseptic collection of the animals’ blood, spleen, skin, and ears, as well as their CO₂ asphyxia death. The sections of the epidermis and ear tissues that were extracted were preserved in 10% formalin, and for later analysis, the remaining tissues were stored at −80°C. Blood was extracted to determine the hematological and biochemical features. The skin tissue in the IMQ-treated region provided the expected parameters for the entire investigation.?A3B2 tpb -13pt?>

2.9 Evaluation of the PASI

Animals were examined from day 1 to day 6 to calculate the PASI (for instance, following IMQ administration). The PASI was rated by the days that followed. On a scale of 0–4, erythema, skin thickness, and scales were rated distinctly as follows: Erythema: 0 (None), 1 (Weak), 2 (Moderate), 3 (Severe), 4 (Very severe), Skin thickness: 0 (None), 1 (Slightly thickened), 2 (Moderately thickened), 3 (Markedly thickened), 4 (Very markedly thickened), and Scales: 0 (None), 1 (Few scales), 2 (Moderate scales), 3 (Numerous scales), and 4 (Very numerous scales). The overall PASI score, which ranged from 0 (no inflammation) to 3 (severe inflammation), was computed by adding the distinct scores for erythema, skin thickness, and scales for every animal. This score was used to measure the level of inflammation involved with the psoriasis-like erythema on each day of the examination [38]. An aerospace digimatic micrometer Screw gauge was used to measure the thickness of the ear folds. On different days, the dorsal skin photos were captured to investigate the complete macroscopic structure in IMQ-induced animals.

2.10 Spleen to the mass ratio of the body

The animals’ weights were recorded before their killing and continued throughout the conclusion of the research, till the moment when the spleens were removed, cleansed, and evaluated. The spleen masses were regulated using body weight to compute the organ index. The results that were obtained were expressed in grams per gram.

2.11 Determining parameters for hematology

On the seventh day, blood from every testing animal was collected via the retroorbital plexus and placed into containers containing the anticoagulant substance EDTA. To ascertain the hematological features, a blood cell counter was employed. Hematological cell counts, measured in WBC, have been defined as (×10³ cell/μL).

2.12 Histopathological evaluation

Before being embedded in paraffin, the rear skin of the assassinated animals had been eliminated and treated in 4% paraformaldehyde. H & E staining was applied to the paraffin-embedded portions after they had been cut to a thickness of 5 μm for histological assessment.

2.13 Measuring oxidative stress markers and antioxidant defenses

Commercial kits for malondialdehyde (MDA) (Catalog No. A003-1), NO (Catalog No. A012-1-1), glutathione (GSH) (Catalog No. A006-2), glutathione s-transferases (GST) (Catalog No. A004), total superoxide dismutase (SOD; A001-1), and catalase (CAT; A007-1), Vitamin C (Catalog No. A009), and thiobarbituric acid reactive substances (TBARS) (Catalog No. A003-1) were purchased from Nanjing Jiancheng Bio Ins (Nanjing, China). Protein samples from skin tissues or cell supernatants were isolated and utilized to evaluate the contents of the aforementioned oxidative stress components using commercially available kits following the manufacturer’s instructions.

2.14 Myeloperoxidase (MPO) activity assay

MPO activity was measured using Bradley et al.’s [39] methodology. MPO activity is an indication of neutrophils’ infiltration of inflammatory cells in the dermis layer of psoriasis. Skin tissues were dissolved in ice-cold 0.1 M potassium phosphate buffer (pH 6.5) with 0.5% hexadecyltrimethylammonium bromide and 10 mM EDTA. Tissue homogenates have been centrifuged at 13,100 × g for 20 min at 4°C. An aliquot of the supernatant (0.1 mL) was mixed with 2.9 mL of 50 mM phosphate buffer containing 0.167 mg o-dianisidine hydrochloride and 0.0005% hydrogen peroxide. The absorbance change over 5 min was measured at 460 nm. The data were presented as U/g of tissue.

2.15 Western blot analysis

BCA kit (Sigma-Aldrich, Catalog No. 71285-M) was used to homogenize and measure the cellular and tissue homogenates that are used to measure the protein fractions. Polyvinylidene fluoride membrane were equally loaded with the protein samples after they had been separated on 10% SDS polyacrylamide gels. The membranes were treated with primary antibodies overnight at 4°C following a 1 h incubation period with 5% bovine serum albumin. All primary (Rabbit IgG) and secondary antibodies were purchased from Cell Signaling Technology, USA, and were employed at the specified dilutions. The expressions of Nf-κB p65 (1:500), NRF2 (1:1,000), COX (1:1,000), iNOS (1:1,000) p62 (1:1,000), and KEAP1 (1:1,000) were studied. β-actin (1:1,000) was used as a housekeeping protein for whole-tissue extracts, and Lamin B (1:1,000) was used as a housekeeping protein for nuclear extracts. Horseradish-peroxidase (HRP)-conjugated anti-mouse or anti-rabbit antibodies (1:5,000) were used as secondary antibodies. Digital imaging devices used ECL to identify protein expression on blots after they were treated for 1 h with secondary antibodies coupled with HRP. The western blot findings’ intensity was examined using ImageJ software.

2.16 Examining data statistically

All statistical analyses were performed using GraphPad Prism software. Data were expressed as mean value ± standard error of the mean (SEM). To assess statistical significance, one-way analysis of variance was used, followed by Tukey’s post hoc test to compare differences between groups. A p-value of less than 0.05 was considered statistically significant.

3.1 Effect of ERN on keratinocyte viability and TNF-α-induced proliferation

The molecular structure of ERN is shown in Figure 1a. The effects of ERN on human keratinocyte viability were assessed using the CCK8 assay. Following ERN therapy, no discernible toxicity was seen at dosages lower than 40 μM (Figure 1b). The inhibitory effect was particularly pronounced at higher concentrations (above 40 µM), suggesting that ERN exerts cytotoxic effects on keratinocytes at elevated doses. Thus, for the following investigations, keratinocytes were exposed to 5, 10, and 20 μM ERN.

Figure 1

The impact of ERN on TNF-α-stimulated HaCaT cell proliferation. (a) ERN molecular structure. (b) and (c) Cell viability was assessed using the CCK-8 assay for 24 h. (d) The EdU assay was utilized to quantify the proliferation rate. (e) Quantification of EdU-positive cells in HaCaT cells treated with TNF-α (10 ng/mL) and increasing concentrations of ERN (5, 10, and 20 μM) for 24 h. The bar graph represents the percentage of proliferating (EdU-positive) cells. Data are expressed as mean value ± SD (n = 3). (f) and (g) KRT6 and KRT1 mRNA levels were analyzed by qRT-PCR. The internal reference is GAPDH. **P < 0.01 vs control. ^## P < 0.01 vs TNF-α group. Data are expressed as mean value ± SD.

To confirm the impact of ERN on TNF-α-induced proliferation of HaCaT cells, the growth of HaCaT cells was measured using the CCK-8 and EdU assays. Proliferating cells were triggered by TNF-α, but this effect was mitigated by ERN administration at 5, 10, and 20 μM (Figure 1c). The findings of EdU staining verified that the proliferation of HaCaT cells was significantly increased by TNF-α therapy. Intriguingly, ERN reduced the rate of proliferation brought on by TNF-α induction in a manner that was dose-related (Figure 1d).

KRT6 is recognized as a marker of hyperproliferation in psoriatic keratinocytes. qRT-PCR analysis demonstrated that TNF-α exposure significantly upregulated KRT6 mRNA expression (Figure 1e), indicating increased keratinocyte proliferation. Conversely, KRT1, a marker of keratinocyte differentiation, was significantly downregulated following TNF-α treatment (Figure 1f). Cotreatment with ERN effectively reduced KRT6 expression in a dose reliant on the way, suggesting that ERN inhibits TNF-α-persuaded keratinocyte hyperproliferation. However, KRT1 expression was also increased upon ERN treatment, which implies that ERN restores keratinocyte differentiation. These findings suggest that ERN exerts an anti-proliferative effect on TNF-α-stimulated keratinocytes, supporting the successful establishment of an in vitro psoriasis model.

3.2 ERN reduces inflammatory cytokine stimulation

Measurement of various inflammatory cytokines linked to the emergence of inflammation, including IL-6, IL-8, IL-22, and IL-1β, was done using ELISA to ascertain the effect of ERN (5, 10, and 20 μM) on TNF-α-induced inflammation of HaCaT cells (Figure 2). According to the ELISA results, the TNF-α monotherapy group’s expressions of inflammatory factors were considerably higher than those of the untreated group. However, in a dose-dependent manner, ERN significantly decreased them. In a dose-responsive way, the data showed that ERN reduced the inflammation of HaCaT cells caused by TNF-α.

Figure 2

ERN reduced the inflammation of HaCaT cells caused by TNF-α. (a–d) Interleukin-8 (IL-8), IL-1β, IL-6, and IL-22 levels are all reduced by ERN. **P < 0.01 vs control. ^## P < 0.01 vs TNF-α group. Data are expressed as mean value ± SD.

To confirm its promise as a treatment, we expanded our research to include an in vivo psoriasis model. We used an IMQ-induced psoriasis approach, which particularly resembles human psoriasiform inflammatory processes, to assess ERN’s effectiveness in a physiologically appropriate environment. The implications of ERN on oxidative stress, inflammatory release of cytokines, and keratin hyperplasia can be evaluated in vivo using this paradigm.

3.3 Therapeutic impact of ERN on psoriasis severity, body weight, and spleen index

To determine the degree of psoriasis lesions in the IMQ-induced psoriasis model, the PASI was measured regularly from day 1 to day 6 following IMQ administration. Erythema, skin thickness, and scaling were individually recorded on a scale from 0 to 4: 0, none; 1, weak; 2, modest; 3, noticeable; and 4, very noticeable. The scores for each parameter were then summed to obtain a composite PASI score, which could range from 0 (no inflammation) to 12 (severe inflammation). Here are the specific findings based on the PASI and other measures such as erythema, skin thickness, scaling, composite PASI score, and macroscopic morphology (Figure 3).

Figure 3

Effect of ERN on IMQ-induced psoriasiform severity in mice. ERN treatment significantly alleviated psoriatic skin changes in mice after 6 days, resembling human psoriasis. The severity of psoriasiform lesions was evaluated using the PASI based on erythema (redness), scaling, and skin thickness, scored from 0 to 4. The following parameters were assessed: (a) Erythema, (b) scaling, (c) thickness, (d) cumulative PASI score, (e) ear thickness, measured using a screw gauge on days 1, 3, 5, and 6, (f) changes in body weight induced by IMQ and its modulation by ERN treatment, (g) spleen mass, calculated as the spleen-to-body weight ratio, comparing ERN-treated and IMQ control groups. Results are expressed as mean value ± SEM (n = 8). ^### p < 0.001; ^## p < 0.01, ^# p < 0.05 compared with the sham control group; *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the IMQ alone treated group; ns, non-significant.

IMQ administration induced a progressive increase in erythema from day 1, peaking around day 4 and remaining at a high level through day 6. Animals treated with ERN showed an important drop in erythema related to the IMQ-only group, with erythema scores in the ERN-treated group remaining 1–2 points lower throughout the treatment period (Figure 3a).

IMQ-induced scaling became evident by day 2 and intensified through day 6 (Figure 3b). In the IMQ group, scales were thick and widespread, with scores reaching 3–4 by day 4. ERN-treated animals showed a marked reduction in scaling, with scores approximately 1–1.5 points lower than the IMQ-only group. On Day 6, the 1% ERN group had marginally higher PASI scaling scores than the 0.5% group. These data point to a potential plateau or nonlinear connection in ERN’s therapeutic impact at increasing dosages. By the end of the observation period, scaling was significantly reduced in ERN-treated animals, suggesting ERN’s effectiveness in controlling hyperkeratosis.

Measurement of skin thickness using an Aerospace digimatic micrometer screw gauge demonstrated that the IMQ-treated animals developed substantial epidermal thickening by day 2, which continued to increase until day 6. ERN treatment significantly attenuated this increase, with skin thickness measurements consistently showing a lower degree of thickening compared to the IMQ-only group. By day 6, ERN-treated animals displayed skin thickness scores that were approximately 30–40% lower than untreated IMQ animals (Figure 3c).

The overall PASI score, obtained by summing the erythema, skin thickness, and scaling scores, provided a cumulative measure of psoriasis severity. In IMQ-only animals, the PASI score increased from an average of 2 on day 1 to around 10 by day 6, indicating severe psoriasiform inflammation. ERN treatment consistently reduced the PASI score across all days, with a final average PASI score of approximately 5 by day 6. This reduction in PASI score indicates a substantial alleviation of psoriasis-like symptoms with ERN treatment (Figure 3d). ERN considerably decreased and maintained the ear’s IMQ-induced epidermal thickness, as seen in Figure 3e. Also, it was observed that the animals in the common drug group demonstrated a reduction in their psoriatic symptoms.

Macroscopic morphology of the dorsal skin on different days revealed visible differences between IMQ-only and ERN-treated animals (Figure S1). IMQ-treated animals exhibited progressively more pronounced erythema, thickening, and scaling over time. In contrast, the ERN-treated animals maintained relatively smooth and less inflamed skin, with a reduced severity of scales and erythema, supporting the quantitative PASI findings.

Body weight measurements were made daily to analyze the consequences of ERN therapy (0.5, 1, and 2% w/w) and IMQ treatment. The IMQ-treated category’s body weight steadily dropped for 6 days, which was probably due to systemic inflammation and stress caused by psoriasis-like skin lesions. At all dosages (0.5, 1, and 2%), ERN treatment reduced IMQ-induced weight loss, with higher doses showing a more pronounced prophylactic effect (Figure 3f). There existed no notable distinctions between the ERN-treated category and the untreated group, indicating that ERN therapy had no adverse systemic effects.

The weight of every animal’s spleen was divided by its total weight to determine the spleen-to-bodyweight ratio, often known as the organ’s index. In the IMQ-only group, the organ index was notably elevated, with a mean increase of approximately 40–50% compared to control animals. This elevated organ index reflects the systemic inflammatory response triggered by IMQ treatment.

In contrast, ERN treatment led to a substantial decline in the organ index associated with the IMQ-only group. ERN-treated animals displayed an organ index that was approximately 20–30% lower than the IMQ group, indicating mitigation of spleen enlargement and suggesting an anti-inflammatory effect of ERN on systemic immune responses (Figure 3g). Statistical comparisons between groups revealed that the organ index in the IMQ-only group was significantly higher than in the control group (p < 0.05). ERN treatment significantly condensed the organ index associated with the IMQ-only group (p < 0.05), indicating that ERN treatment attenuated the systemic inflammatory response. The enhanced immunological activity brought on by psoriasiform inflammation is consistent with the higher organ index in the IMQ-only group. The spleen-to-bodyweight ratio decreased in the group that received ERN treatment, indicating that ERN has an immune-regulating impact that lowers the level of systemic inflammation. These results indicate that ERN may help to control not only local skin inflammation but also the broader immune activation associated with psoriasis.

3.4 Effect of ERN on oxidative stress markers in IMQ-induced skin inflammation in animal tissue

Figure 4 shows that treatment with IMQ alone substantially exacerbated oxidative stress, as demonstrated by elevated levels of MDA and nitric oxide (NO), as well as a substantial decline in antioxidant defense markers such as GSH, SOD, CAT, and vitamin C when compared to the control group. These data demonstrate that IMQ weakens the antioxidant barrier, resulting in increased oxidative damage.

Figure 4

Effects of ERN treatment on antioxidant and oxidative stress markers in IMQ-induced psoriasis- skin tissue. (a)–(h) Biochemical analysis of antioxidant enzyme activity and oxidative stress markers in all experimental groups. Results are presented as mean values ± SEM (n = 6). ^### p < 0.001, ^## p < 0.01, ^# p < 0.05 compared with the sham control group; *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the IMQ alone treated group; ns, non-significant.

On the other hand, ERN therapy dramatically reduced the oxidative damage caused by IMQ. This was demonstrated by reduced MDA and NO levels, as well as the restoration of antioxidant indices such as GSH, SOD, CAT, and vitamin C activities (Figure 4a–h). These findings imply that ERN improves the endogenous antioxidant defence system and might defend against IMQ-induced oxidative damage. The antioxidant enzyme levels (SOD and CAT) in the 2% ERN group were not substantially higher than those in the 1% group (Figure 4c and d). The results indicate an expected threshold or nonlinear connection in ERN’s therapeutic impact as doses increase. More research is needed to better understand the molecular processes behind ERN’s antioxidant capability.

3.5 Histopathological and clinical assessment of skin inflammation

Skin tissues were histologically examined with hematoxylin and eosin (H&E) staining to determine the therapeutic potential of ERN in IMQ-induced psoriatic mice (Figure 5a and b). Histological depiction of the IMQ-only group exhibited classic psoriatic characteristics such as extensive acanthosis, hyperkeratosis, parakeratosis, rete ridge elongation, and extensive dermal inflammatory infiltrates. In comparison, the control group’s skin had normal epidermal thickness and was free of inflammation.

Figure 5

Impact of ERN treatment on histopathological alterations in IMQ tempted psoriasis. Representative H&E-stained photomicrographs of (a) skin tissue for all experimental groups. Histopathological assessments include blue arrows indicating hyperkeratosis, red arrows denoting acanthosis, and green arrows highlighting the infiltration of neutrophils. (b) Quantification of epidermal thickness from H&E-stained dorsal skin sections. Epidermal thickness was measured at five randomly selected sites per section using ImageJ software. IMQ-induced epidermal thickening was significantly reduced following ERN treatment in a dose-dependent manner. (c) Inflammatory score of the skin and (d) MPO activity in skin tissue. Blue arrows indicate hyperkeratosis, red arrows indicate acanthosis, and green arrows indicate infiltration of neutrophils in histopathology images. Data are expressed as mean values ± SD. n = 6 animals per group, experiments were repeated three times. ^### p < 0.001; ^## p < 0.01, ^# p < 0.05 compared with the sham control group; *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the IMQ alone treated group; ns, non-significant.

ERN-treated groups (0.5, 1, and 2% w/w) showed dose-dependent enhancement in histopathological characteristics. The 2% ERN group had the greatest loss in epidermal thickness and immune cell infiltration, closely mirroring the skin architecture seen in the clobetasol-treated standard group. ERN treatment significantly reduced inflammatory severity, epidermal hyperplasia, and keratinocyte proliferation compared to IMQ alone (p < 0.001; Figure 5b). These refined histological data highlight the capacity of ERN to dramatically restore close to typical skin architecture in psoriatic mice. The combined visual and quantitative evaluations show strong evidence for ERN’s anti-psoriatic effectiveness.

3.6 Effect of ERN on skin tissue MPO

The skin tissue data showed that the IMQ monotherapy recipients had significantly greater MPO activity, a marker of neutrophil infiltration, than those who were not treated. As opposed to the IMQ monotherapy group of cures, MPO activities significantly decreased MPO levels during exposure to increased ERN doses (Figure 5c).

3.7 Determining parameters for hematology

The blood cell count data revealed differential responses between the experiments, IMQ and control groups (Figure 6). Outcomes exhibited that the levels of WBC in the blood were noticeably greater in the IMQ individually treated group than those of the control group, indicating an inflammatory or immunological response to the therapy. Compared to the group that was obtaining IMQ alone, the treatment groups (IMQ + ERN) showed a substantial drop in WBC count (Figure 6a), which may indicate immunological suppression. The RBC count, hemoglobin, and hematocrit values did not significantly differ across groups, indicating that the experimental treatments did not affect these parameters. Consequently, our results demonstrate that ERN therapy effectively declined the quantity of WBCs in the distribution of whole blood, this has thus decreased the blood’s production of inflammatory chemicals, resulting in an anti-inflammatory impact.

Figure 6

Effect of ERN treatment on hematological parameters in IMQ-exposure mice. Hematological analysis of whole blood from all experimental groups on day 7 post-treatment. (a) WBC count, (b) neutrophil count, (c) lymphocyte count, and (d) monocyte count. Results are expressed as mean value ± SEM (n = 6). ^### p < 0.001, ^## p < 0.01, and ^# p < 0.05 compared with the sham control group; *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the IMQ alone treated group; ns, non-significant.

The IMQ-only group showed a significant increase in neutrophils, monocytes, and lymphocyte count indicating an inflammatory response (Figure 6b–d). ERN treatment (0.5, 1, and 2% w/w) significantly modulated these changes in a dose-dependent manner. Neutrophil and monocyte levels were notably decreased, while lymphocyte counts were restored toward normal levels, suggesting an immunomodulatory and anti-inflammatory effect of ERN.

3.8 ERN-mediated modulation of pro-inflammatory and anti-inflammatory cytokines

The proportions of pro-inflammatory (TNF-α, IL-6, IL-17, IL-23, and IL-1β) and anti-inflammatory (IL-10) cytokines were assessed in the serum and cutaneous tissue of psoriatic animals produced by IMQ to assess the immunomodulatory impact of ERN in psoriasis-like inflammation. Pro-inflammatory cytokines were significantly reduced in the skin tissue and serum in dissimilarity to the subjects treated with IMQ (Figure 7a–g), suggesting that ERN effectively modulates systemic and localized inflammation in psoriasis.

Figure 7

Proinflammatory and anti-inflammatory cytokine levels in IMQ-stimulated psoriatic skin tissue and serum. (a) TNF-α, (b) IL-6 levels, (c) TNF-α, (d) IL-6, (e) IL-17, (f) IL-23, and (g) IL-1β levels in mice skin tissue at 7 days post-ERN treatment. (h) IL-10 level in mouse skin tissue with ERN treatment, representing anti-inflammatory response. Cytokine levels were quantified using ELISA kits. The mean value ± SEM is used to present the data (n = 6). ^### p < 0.001, ^## p < 0.01, and ^# p < 0.05 compared with the sham control group; *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the IMQ alone treated group; ns, non-significant.

The levels of anti-inflammatory cytokines elevated after ERN treatment, especially IL-10, and these had elevated levels in the treated animals’ blood and epidermis (Figure 7h). This supports ERN’s promise as a treatment for psoriasis by indicating that it encourages the skin to respond in a way that is less inflammatory.

3.9 Impact of ERN on NF-κB signaling

The present study assessed the expression profiles of key proteins involved in the NF-κB signaling pathway to evaluate the effect of ERN in IMQ-induced psoriatic skin. IMQ treatment markedly increased the total expression and nuclear localization of NF-κB p65, as demonstrated by Western blot (Figure 8a), indicating enhanced activation of the NF-κB pathway. In parallel, expression of downstream pro-inflammatory mediators iNOS and COX-2 was also significantly upregulated in IMQ-treated mice (Figure 8b). Treatment with ERN at concentrations of 0.5, 1, and 2% resulted in a dose-dependent reduction in nuclear NF-κB p65 expression (Figure 8c). Furthermore, this finding supported ERN’s function in preventing NF-κB-mediated inflammation by confirming lower nuclear translocation of NF-κB p65 in ERN-treated groups. This effect was accompanied by a substantial decrease in the expression of iNOS and COX-2 (Figure 8d and e), suggesting attenuation of the NF-κB-mediated inflammatory response. Taken together, these findings suggest that ERN mitigates IMQ-induced psoriasis-like inflammation by suppressing NF-κB signaling and its downstream inflammatory mediators.

Figure 8

Impact of ERN therapy on the NF-κB signaling pathway in skin tissue produced by IMQ. The appearance levels of p65, COX-2, and iNOS in the skin tissue of IMQ compared to ERN are shown in (a) and (b), an illustrative immunological blot analysis. β-actin and Lamin B served as the internal reference. (c)–(e) shows the band intensities measured concerning protein expression using Image J software analyses. The findings were expressed using the mean value ± SEM (n = 3). ^### p < 0.001 compared with the sham control group; **p < 0.01 and ***p < 0.001 compared with the IMQ alone treated group.

3.10 Activation of KEAP1-NRF2 pathway in animal models

The impact of ERN was then assessed by using immunoblotting techniques to influence the localization of HO-1 in the IMQ-induced skin and Nrf2 in the nuclear region. When comparing the IMQ-only treated group to the untreated control group, we discovered a substantial p < 0.001 drop in KEAP1 and nuclear deposition of NRF2 (Figure 9). On the contrary, western blot analysis demonstrated that ERN treatment expressively improved the levels of KEAP1, p62, HO-1, and NRF2 in the skin tissue (Figure 9a–e). Altogether, ERN progresses the antioxidant enzyme status in IMQ-induced psoriatic mice. These results propose that ERN modulates the KEAP1-NRF2 pathway in vivo, enhancing antioxidant defence mechanisms and reducing oxidative stress in psoriasis-like skin inflammation.

Figure 9

Impact of ERN therapy on the KEAP1-Nrf-2 signaling cascade in skin tissue that could be stimulated by IMQ. (a) The protein expression levels of Nrf2, p62, Keap1, and HO-1 in IMQ + ERN skin tissues are demonstrated by exemplary immunoblot studies. The internal control in this case was β-actin. (b)–(e) Shows various band intensities assessed by Image J software evaluation to protein expression levels graphically. Results are expressed as mean values ± SEM (n = 3). ^### p < 0.001, ^## p < 0.01, and ^# p < 0.05 compared with the sham control group; *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the IMQ alone treated group; ns, non-significant.