Alleviative effects of combined topical melatonin and rutin on imiquimod-induced psoriasis mouse model

Background: Psoriasis is a longstanding autoimmune dermatosis with thickened, reddish-brown, and flaking skin lesions. Melatonin is an indolamine hormone exhibiting antioxidant, anti-inflammatory, and anti-proliferative actions. Rutin is a nutritional flavonoid possessing antioxidative, anti-inflammatory, and immunomodulatory properties. Aim: To explore the potential anti-psoriatic activity of combined topical melatonin and rutin. Methods: 70 albino mice were divided into 7 groups of 10 each. The impacts of clinical observation, histopathological examination, and biomarker analysis were estimated. Results: Combined topical melatonin and rutin effectively diminished imiquimod-induced elevated PASI and Baker’s scores and corrected histopathological aberrations. Diminished inflammatory indicators like TNF-α and IL-17A, angiogenic elements like VEGF, oxidative elements like MDA, and proliferative elements like Ki-67 were also noted. Conclusion: Combined topical melatonin and rutin have profound anti-psoriatic effects.


Introduction
Psoriasis is a chronic immune-mediated dermatological disorder distinguished by reddened, thickened, and flaky skin plaques.Multiple immunological, genetic, and environmental factors seem to contribute to the development of psoriasis (Petkova et al. 2014;Takeshita et al. 2017;Schön 2019).The tumor necrosis factor (TNF)-α and interleukin (IL)-17/IL-23 axis play a key role in psoriasis progression (Pandey et al. 2021).Overproduction of reactive oxygen species (ROS) is correlated with risk factors for the occurrence of autoimmune diseases, including psoriasis (Kadam et al. 2010;Jawad et al. 2022;Blagov et al. 2023).Malondialdehyde (MDA) is a helpful predictive marker for determining the severity of psoriasis (Dobrică et al. 2022;Cordiano et al. 2023).Vascular endothelial growth factor (VEGF) is a prospective target for psoriasis therapy due to its pathogenic involvement in controlling neovascularization during the disease's onset and progression (Fareed and Al-Qrimli 2024;Mahdi et al. 2024).The antigen Kiel 67 (Ki-67) serves as a prominent marker of cellular proliferation and is extensively expressed in psoriatic skin cells (Khairutdinov et al. 2017).Psoriasis treatment modalities involve topical therapies, systemic therapies, and phototherapy.Unfortunately, the adverse effects of these medicines often render patients intolerant to longterm therapy, limiting their clinical adoption (Xie et al. 2021).Local adverse effects of topical steroids include skin atrophy and infection, while systemic absorption can elicit Cushing syndrome, osteonecrosis, child growth retardation, and adrenal suppression (Del Rosso 2020;Al-Jabr et al. 2024).Tazarotene is labeled with a pregnancy category X warning (Sami and Feld 2021).Oral mucositis, hepatotoxicity, and myelosuppression are notable side effects of methotrexate (Zhu et al. 2022;Attarbashee et al. 2023).Hypertension and nephrotoxicity restrict cyclosporine's long-term use (Krueger et al. 2022).Mucocutaneous dryness is the common negative effect of acitretin observed in most patients (Kakarala et al. 2021).Apremilast may cause nausea and metabolic effects (Langley and Beecker 2018).With the debut of biological medicines, the efficacy and tolerability of psoriasis therapies have jumped significantly in the past twenty years.Drugs that block TNF-α, IL-17, IL-12/23, and IL-23 are authorized for managing psoriasis.Comorbidity factors of psoriasis can be further impacted by persistent inflammatory modifications induced by anti-psoriatic drugs (Salman et al. 2024a).Investigative studies on the safety of biologic products aid in tailoring anti-psoriasis therapies.Specifically, IL-17 antagonists are associated with ulcerative colitis.TNF-α blockers need to be administered cautiously to people with psoriasis accompanied by cardiac problems, multiple sclerosis, or malignancy.Thus, uncovering innovative anti-psoriatic medicines remains difficult (Jiang et al. 2023).
Melatonin is an indoleamine, a complex hormone synthesized primarily by the pineal gland, possessing antioxidative, anti-inflammatory, anti-angiogenic properties, and antiapoptotic characteristics (Amaral and Cipolla-Neto 2018;Ma et al. 2020;Muñoz-Jurado et al. 2022).Treatments with melatonin were extensively investigated across an assortment of experimental studies of autoimmune and autoinflammatory diseases (Zhao et al. 2019;Ahmed et al. 2022;Ahmad et al. 2023;Obaid 2024).Rutin is a polyphenolic natural glycosylated flavonol-type flavonoid that is derived from many plants and fruits (De Jesus et al. 2024).Rutin's actions might be mediated via modifications of gonadotropins, reproductive steroidal hormones, prostaglandin eicosanoids, and cytokines, along with biomarkers of oxidation, inflammation, hyperproliferation, and apoptotic and angiogenic processes (Jahan et al. 2016;Sirotkin and Kolesarova 2022;Sirotkin 2024).
However, the most effective method of treating psoriasis was shown to involve a combination of several different drugs (Gustafson et al. 2013;Elmets et al. 2021;Sreya et al. 2023).To the best of our knowledge, melatonin and rutin have not been examined in combination to treat psoriasis, but they have been used to alleviate lead acetate-induced gastrointestinal and hepatic oxidative damage disturbances (Akinrinde et al. 2023).Supplementation with either flavonoid or melatonin could dampen inflammatory responses (Negi et al. 2011;Abu-Raghif et al. 2015).This may predict the synergistic protective impact of the combination of melatonin and flavonoids against immune-mediated conditions (Al-Rasheed et al. 2017).Rutin is composed of the flavonoids quercetin and rutinose (Amaretti et al. 2015;Yang et al. 2019).The combined melatonin and quercetin flavonoid could have improved hypoxia-induced kidney impairment (Alshanwani et al. 2020).Additionally, the flavonoids, when combined with melatonin, produced significant down-regulation of TNF-α and IFN-γ levels (Al-Rasheed et al. 2016).Another study showed that combining melatonin with the flavonoid quercetin significantly inhibited diabetic oxidative damage in various organs by decreasing MDA levels (Elbe et al. 2015).

Aim of the study
This research aimed to assess the probable alleviative actions of combined topical melatonin and rutin treatments on imiquimod-induced psoriasis in mice while comparing their efficacy to that of the traditional medication clobetasol propionate.

Study design
The study was carried out using a randomized, controlled experimental model.The study was conducted at the Pharmacology Department of the College of Medicine, AL-Nahrain University, between October 2023 and May 2024.The study was carefully reviewed for ethical and scientific care and received approval from the Institutional Review Board (IRB) of the College of Medicine at AL-Nahrain University (No. 20230786).

Drugs and reagents
The petroleum 15% jelly was supplied by the local market (Iraqi Federation of Industries, Baghdad, Iraq).Clobetasol propionate ointment was donated by Dermo-vate®, GlaxoSmithKline, Brentford, UK.Imiquimod was provided by Meda Pharmaceuticals, Solna, Sweden, under the brand name Aldara® 5% Cream.Jinlan Pharma-Drugs Technology, located in Hangzhou, China, delivered powdered melatonin and rutin drugs.

Pharmaceutical preparation of melatonin (2.5%) ointment
The ointment was prepared using the levigation method, which involved dissolving (2.5 g) of melatonin in a levigation solution of 2 ml castor oil, which was then mixed with (97.5 g) of Vaseline petroleum jelly to yield a homogenous, grit-free formulation.A pilot study was conducted to establish the optimal and effective dosage of the melatonin medication (Hameed et al. 2020).

Pharmaceutical preparation of rutin (2%) ointment
The levigation method was employed to prepare the ointment by dissolving (2 g) of rutin in a levigation solution of (2 ml) caster oil, which was then blended with (98 g) of Vaseline petroleum jelly to get a uniform and grit-free mixture.A pilot study was conducted to determine the appropriate and effective dosage of the drug (Kadhim et al. 2022).

Animal model and experimental design
Experimental mice were purchased from the Animal Facility of the Iraqi Center for Cancer and Medical Genetics Research.They were housed in polypropylene cages and maintained in a controlled environment at 25 °C.The environment was regulated by an inverted light-dark cycle lasting 12/12 hours.The mice were acclimated for a period of seven days at the same facility from which they were obtained.The animals were provided with a standard pellet diet and free access to water.This study involved 50 Swiss albino mice, weighing 24-30 g and aged 11-15 weeks.All mice underwent dorsal shaving with an electric razor, exposing a 6 cm 2 (2 × 3 cm) area of back skin.During the 14-day experiment, mice were haphazardly allocated to 7 distinct groups, each consisting of 10 mice, as follows: Control group: consisted of normal, healthy mice that had not received any sort of treatment.Induction group: Mice were administered topical imiquimod cream (5%) at a dosage of 62.5 mg once daily for up to 6 days.Vehicle group: Mice underwent topical imiquimod application (as in the induction group), and then topical vehicle ointment (petrolatum jelly) was applied two hours later for an additional 8 days.Clobetasol group: Imiquimod was administered to the mice (as in the induction group), and two hours thereafter, 0.25 g/kg of topical clobetasol ointment (0.05%) was administered twice a day over the next eight days.Melatonin group: Mice were given imiquimod (as in the induction group), followed by topical melatonin ointment (2.5%) two hours post-induction, twice a day for an extra 8 days.Rutin group: After receiving imiquimod (as in the induction group), mice were administered topical rutin ointment (2%) twice daily for an additional eight days, starting two hours after induction.Melatonin + Rutin combination group: Mice were given imiquimod (as in the induction group), followed by a combination of topical melatonin (2.5%) and rutin (2%) ointments applied two hours post-induction, twice a day for 8 days.Drug applications occurred between 8 a.m. and 8 p.m. for the four treatment groups (Clobetasol, rutin, melatonin, and rutin+melatonin combination groups).

The psoriasis area and severity index (PASI)
The severity of skin psoriasis was assessed based on measures of skin erythema, scaling, and thickness to evaluate the amount of skin inflammation and lesion severity.Psoriasis area and severity index (PASI) was assessed on a scale ranging from 0 to 4 for each category.The values correspond as follows: "0" = none, "1" = mild, "2" = moderate, "3" = marked, and "4" = severe.The lesion's severity was assessed by summing the additive values for erythema, scaling, and thickness to calculate the cumulative PASI scores (Van Der Fits et al. 2009;Abbas et al. 2023;Hassan et al. 2023).

Preparation and sampling of animals
On day 14, all animals were given intraperitoneal (IP) anesthesia with 80 mg/kg of ketamine and 10 mg/kg of xylazine.All mice were terminated by exsanguination, which is a technique appropriate for tissue harvesting and preservation, after being completely anesthetized (Pierozan et al. 2017;ALzubaidy et al. 2020;Hassan and Kadhim 2022b).Skin samples were collected and processed to prepare tissue homogenate for analyzing biomarkers and conducting histopathological examinations (Mekkey et al. 2020;Hassan et al. 2022;Oubaid et al. 2023).

Preparation of tissue homogenates
One gram of recently collected skin from the back has been preserved in a 9-ml buffer solution saturated with phosphate at a pH of 7.2.The tissues were homogenized using a pestle and mortar, then centrifuged for 10 minutes at 5000 rpm at a cold degree.The supernatants were stored at -80 °C for further examination (Alzubaidy et al. 2016).

Histological examination
Skin samples collected from several mouse groups were preserved in 10% neutralized buffered formalin following established guidelines (Ali et al. 2021;Kamal and Khadhim 2021;Shihab et al. 2024).Tissues were then fixed in paraffin, followed by xylol processing and heating to 55-60 °C before being immersed in liquid paraffin.The tissue was then dipped in paraffin and set on a cold dish (Salman et al. 2013;Khaleq et al. 2015;Naji et al. 2022).The paraffin-embedded specimens were cut into sections and stained with hematoxylin and eosin (H&E) (Atarbashe and Abu-Raghif 2020; AL-Bairmani and Kadhim 2023; Shihab and Kadhim 2023).The samples were subsequently examined using a microscope and evaluated using Barker's scoring system, which assigns a numerical value between 0 and 10 to determine the presence of any pathological changes (Baker and Fry 1992).

Measurement of inflammatory, oxidative, and proliferative biomarkers
A sandwich enzyme-linked immunosorbent assay (ELI-SA) kit was used to measure TNF-α, IL-17, VEGF, MDA, and KI67 concentrations in mouse skin tissues, according to the manufacturer's instructions (Cloud-Clone Corp).Anti-marker antibodies were added to the pre-coated micro-ELISA strip plate in this kit.In the late wells of the micro-ELISA strip, the sample or principle was added before the appropriate antibody (Hassan and Kadhim 2022a).Next, a horseradish peroxidase (HRP)-conjugated antibody was added to each micro-ELISA well and incubated.The optical density (OD) was measured spectrophotometrically at a wavelength of 450 nm (Abed-Mansoor and Abu-Raghif 2022).

Statistical analysis
The data sources were put into the most recent edition of SPSS 24, a statistical tool developed for social scientists.The descriptive statistical analysis includes the mean and standard deviation (SD).The results were presented in graphical form and submitted to appropriate statistical analysis.For more than two means, a one-way analysis of variance (ANOVA) was employed, and two means were compared using Tukey's Honestly Significant Difference (HSD) Test.The significance level in all statistical analyses was p < 0.05.

Results
The study found that the induction and vehicle groups experienced significantly higher cumulative PASI and pathological Baker's scores than the presumably healthy control group (p < 0.05).On the other hand, mice within the clobetasol, melatonin, rutin, and melatonin+rutin combination groups revealed substantial reductions in the degree of cumulative PASI and pathological Baker's scores compared to the induction and vehicle groups (p < 0.05).There was no significant difference seen in the levels of PASI and Baker's scores between the groups receiving clobetasol, melatonin, or rutin treatments.Meanwhile, the melatonin+rutin combination group revealed statistically noteworthy decrements in both total PASI and histological Baker's scores as opposed to the clobetasol group, as indicated in Fig. 1 (p < 0.05).
Importantly, the induction and vehicle groups reported significantly increased skin levels of inflammatory mediators, including TNF-α, IL-17, and VEGF (p < 0.05), compared with the control group.Besides, the clobetasol, melatonin, rutin, and melatonin+rutin combination groups had significantly fewer levels of TNF-α, IL-17, and VEGF compared with the induction and vehicle groups (p < 0.05).Furthermore, the levels of VEGF, TNF-α, and Il-17 did not significantly differ amongst the clobetasol, melatonin, and rutin groups (p > 0.05).On the other hand, the mel-atonin+rutin combination group showed noticeable decreases in IL-17 levels compared to the clobetasol group, while there were no significant differences between the clobetasol and melatonin+rutin combination groups in terms of TNF-α or VEGF levels (p < 0.05), as illustrated in Fig. 2.
Additionally, the induction and vehicle groups revealed markedly elevated concentrations of the oxidative marker MDA and the proliferative marker Ki-67 (p < 0.05) in comparison with the control group.In addition, there was a considerable decline in MDA and Ki-67 concentrations in the clobetasol, melatonin, rutin, and melatonin+rutin combination groups compared with the induction and vehicle groups (p < 0.05).Moreover, MDA and Ki-67 scores were not substantially different across the clobetasol, melatonin, and rutin groups (p > 0.05).Of particular importance, the melatonin+rutin combination group demonstrated major declines in MDA levels in comparison with the clobetasol group; nevertheless, there had been statistically insignificant changes among the clobetasol and melatonin+rutin combination groups in Ki-67 levels (p < 0.05), as shown in Fig. 3.
The control group had normal keratinization, epidermal thickness, and dermal layer appearance, containing hair roots and sebaceous glands.The induction and vehicle groups exhibited substantial histopathological alterations versus the control group, including increased cutaneous thickness, hyperkeratosis, Munro's microabscess, elongated rete ridges, and invasion of inflammatory cells.However, the clobetasol group showed important improvements in the skin histological features compared to the induction group, encompassing mild hyperkeratosis, thinned epidermal layers, and a minor influx of inflammatory cells into the skin.Similar improvements in psoriatic histologic characteristics were seen in both the melatonin and rutin separate groups, involving decreased skin thickness, moderate rete ridge elongation, and moderate inflammatory cell penetration, as opposed to the induction group.Still, the melatonin+rutin combination group demonstrated significant ameliorations in psoriatic histopathological details compared to the induction group, comprising reduced skin thickness and a mild inflammatory cell infiltration (H&E 10X) (Fig. 4).

Discussion
Despite management of psoriasis advancing, there are still challenges in treating the condition due to possible safety concerns regarding systemic medicines, phototherapy, topical therapies, and biological therapies used in prolonged administration (Mao and Ma 2022).The present research evaluated the effects of topical rutin and melatonin, either separately or in combination, on a mouse model of imiquimod-induced psoriasis.After being applied to the mouse dorsal skin for 6-8 days, imiquimod elicited psoriasis-like skin lesions, including erythema, desquamation, and acanthosis.This is because imiquimod acts as an agonist on the toll-like receptor-7/8 (TLR-7/8), which, when stimulated, causes unfavorable increases in oxidative, angiogenic, and inflammatory parameters (Almudaris and Gatea 2024;Ridha-Salman et al. 2024).Additionally, imiquimod boosts the number of neutrophils, T lymphocytes, and dendritic cells in the epidermis (Chen et al. 2023).This approach is recoverable and only lasts for a limited period after ceasing imiquimod treatment since the mouse cannot be transgenic and may reverse the inflammatory response (Sandra et al. 2017).However, clobetasol, the standard medication used in this trial, markedly alleviated the symptoms of psoriasis.This may be due to its anti-proliferative, anti-inflammatory, and immune-suppressive effects, which limit inflammatory cell invasion and suppress keratinocytic proliferation (Salman et al. 2024b).Clobetasol inhibits the generation of inflammatory cytokines by interacting with cytosolic glucocorticoid receptors and influencing gene transcription (Feldman and Yentzer 2009;Khafaji et al. 2024).Additionally, clobetasol may regulate T-cell responses and promote the expression of anti-inflammatory genes (Uva et al. 2012;Hasan and Gatea 2024).It also has the potential to attenuate psoriatic inflammation by suppressing the gene production of numerous cytokines, which may account for the diminution of keratosis and skin thickness (Abidi et al. 2010;Heim et al. 2022).In this work, the findings from imiquimod-aggravated psoriasis displayed a substantial decline in the overall PASI and Baker's scores following treatment with topical rutin, melatonin, or their combination, which was accomplished by improving epidermal thickening, silver-white scales, and erythema.The PASI and Baker's scores, depending on the aforementioned lesions, are the most regularly employed severity evaluation methods for psoriasis (Luo et al. 2016).Meanwhile, this experiment found that melatonin, rutin, and their combination improved imiquimod-induced histopathological alterations and significantly suppressed levels of the inflammatory, angiogenic, proliferative, and oxidative biomarkers, notably TNF-α, IL-17, VEGF, Ki-67, and MDA.
Melatonin was selected for this investigation owing to its robust anti-inflammatory, immunomodulatory, and antioxidant characteristics (Muñoz-Jurado et al. 2022).Melatonin supplementations were demonstrated before to mitigate skin inflammation in imiquimod-provoked psoriasis-like mice and reverse Th17/Treg cell imbalances (Shen et al. 2024).Another study found that melatonin ameliorated imiquimod-exacerbated pruritus and cutaneous hyperplasia (Zhang et al. 2022).Besides, topical ramelteon, a melatonin agonist, has significantly attenuated the PASI and Baker's scores in a mouse model of imiquimod-induced psoriasis (Khafaji et al. 2024).In a similar way, local melatonin actions were shown to ameliorate the severity scores of inflammatory eczematous lesions in atopic dermatitis patients (Jaworek et al. 2021).Also, people with psoriasis were observed to have inadequate levels of melatonin, and restoring melatonin levels was linked to amelioration of depressive symptoms and resolution of psoriatic plaques (Sahi et al. 2020).Meanwhile, psoriasis is mainly triggered by angiogenic and inflammatory signaling systems (Singh et al. 2013).Melatonin's anti-inflammatory and anti-angiogenic effects involve blocking the synthesis of profibrogenic and proinflammatory mediators comprising VEGF, transforming growth factor (TGF)-β, IL-17A, and fibroblast growth factors (Hosseinzadeh et al. 2018).Melatonin also dramatically reduced VEGF production and angiogenesis in bone marrow-originating endothelial progenitor cells (Lin et al. 2023).Other observations indicate that melatonin dramatically decreases the mRNA expression of the inflammatory cytokines TNF-α, IL-1β, and IL-6 (Escames et al. 2007;Jung et al. 2009;Veneroso et al. 2009).Further support for our hypothesis comes from melatonin's capacity to suppress Th1 and Th17 cells while inhibiting dendritic cell stimulation and hindering the synthesis of IL-1β, IL-6, IL-17, and IFN-γ proteins in a mouse model of encapsulating peritoneal sclerosis (Huang et al. 2024).Biologics that target the majority of the aforementioned inflammatory components have become the backbone of psoriasis management (Mateu-Arrom and Puig 2024).Furthermore, melatonin functions as a potent antioxidant and scavenger of free radicals (Aysun and Burcu 2018).MDA is a substance formed through lipid peroxidation and is linked to the worsening of psoriasis (Wroński et al. 2023).Melatonin is capable of mitigating oxidative parameters like MDA and myeloperoxidase (MPO) and promoting the activity of intrinsic antioxidants in numerous tissues (Janjetovic et al. 2017;Morvaridzadeh et al. 2020;Monteiro et al. 2024).Melatonin is also an effective antitumor agent, exhibiting proapoptotic and antiproliferative effects in melanoma and breast cancerous cells (Gatti et al. 2017).Subsequent research discovered that melatonin hampers proliferative Ki-67 scores and interrupts the cell cycle of malignant breast cells (Sonehara et al. 2019).However, psoriasis seems to be linked with higher levels of Ki-67, which is suggestive of keratinocyte proliferation (Yazici et al. 2005;Huang et al. 2019).
On the other hand, rutin's efficacy in alleviating psoriasis lesions could be attributed to its anti-inflammatory properties and capacity to inhibit T cell proliferation (Satari et al. 2021;Muvhulawa et al. 2022).A previous study suggests that rutin may reduce imiquimod-induced psoriatic inflammation in mice, and regulating the AGE-RAGE activation pathways could represent one of the possible anti-inflammatory approaches.Rutin also prevented TNF-α and IL-6 transcription in HaCaT keratinocytes, suggesting it might be an alternative therapy for psoriasis (Wang et al. 2023).In the same way, this flavonol can alleviate lupus-like manifestations in mice by modifying peroxisome proliferator-activated receptor-(PPAR)-γ activities, thus diminishing the expression of nuclear factor-kappa B (NF-κB) and signal transducer and activator of transcription 3 (STAT3) and inhibiting the release of cytokines including TNF-α, IL-17, and IL-6 (Yi et al. 2024), which are key factors in the development of psoriasis (Guo et al. 2023).A further investigation observed that rutin is one of the constituents of different types of tea extracts that dampen imiquimod-induced PASI-score while restoring histopathological abnormalities (Zhang et al. 2024).Additionally, rutin exhibits multiple anti-inflammatory activities, such as hindering the experimental models of colitis via controlling the NF-κB signaling cascade and intestinal flora (Liu et al. 2022), as well as attenuating colonic production of proinflammatory proteins, notably interferon (IFN)-γ, TNFα, and IL-1β (Mascaraque et al. 2014).Another study found that this flavonoid ameliorated skin thickness, lymphocyte proliferation, and the generation of cytokines, namely, IFN-γ, IL-4, IL-5, IL-10, IL-17, and TNF-α, in a murine model of atopic dermatitis (Choi and Kim 2013).Moreover, flavonoids markedly decreased the synthesis of pro-inflammatory mediators TNF-α, IL-1β, and IL-6 and restricted leukocyte migration to the injury site (Leyva-López et al. 2016;Bash and Abd 2020;Obaidi et al. 2024).Furthermore, oxidative stress appears to be implicated in the onset and progression of psoriasis and its accompanied complications (Dobrică et al. 2022).Emerging data suggests that rutin exerts antioxidative characteristics by preventing lipid peroxidation and combating oxidative stress (Yang et al. 2008;Enogieru et al. 2018).Similarly, topical rutin has improved wound healing by downregulating levels of oxidative MDA and upregulating levels of endogenous anti-oxidants like superoxide dismutase and catalase (Iova et al. 2021).In addition, pro-angiogenic factors play a critical role in psoriasis advancement, and angiogenesis represents an attractive target for new medicines (Heidenreich et al. 2009).However, rutin possesses potent anti-angiogenic potential and has been shown to ameliorate a rat model of diabetic retinopathy via decreasing VEGF and TNF-α expression (Gupta et al. 2020).Rutin flavonoid also has antiproliferative and apoptotic properties, which may strengthen the anticancer actions of 5-fluorouracil and oxaliplatin on colonic malignant cells (Nasiri et al. 2016).Meanwhile, it was shown that rutin significantly reduced proliferative cell nuclear antigen (PCNA) in lung tissues (Li et al. 2014).Still, PCNA may be an indicator of keratinocytic hypergrowth in psoriasis (Yazici et al. 2005;Huang et al. 2019).
Interestingly, mounting evidence suggests that combining two antipsoriatic drugs may result in higher efficacy, better safety profiles, lower doses, and fewer adverse effects than any one therapy used alone (Snehasis et al. 2024).The combined administration of etanercept, adalimumab, and infliximab with methotrexate is extensively applied for optimal management of psoriatic plaques (Philipp et al. 2012;Farhangian and Feldman 2015).Additionally, steroids are combined with steroid-sparing medicines like calcipotriene and tazarotene to improve effectiveness while minimizing side effects (Diotallevi et al. 2022).What's more, combining multiple nutraceutical supplements improves imiquimod-exacerbated psoriasis in mice and offers potential as game-changer substitutes for the use of steroids as the only antipsoriatic treatment (Vijayapoopathi et al. 2023).Therefore, rutin and melatonin were combined in the current study, which was found to be considerably more effective in reducing psoriatic inflammation than those demonstrated when rutin and melatonin were used individually, supporting the benefits of combination treatment.Despite experiments on mice yielding encouraging findings, further research is still required to assess the safety and efficacy of melatonin, rutin, and their combination in humans.Another drawback of our study is that we were unable to identify whether the medications we examined had any inhibitory effects on the NF-κB and STAT3 pathways.These pathways are crucial to the development and longevity of Th17 cells as well as their production of cytokines.

Conclusion
The combined topical treatment of melatonin (2.5%) and rutin (2%) had significant antipsoriatic benefits.Their potential to treat psoriasis might be attributed to their anti-inflammatory, antioxidant, anti-proliferative, and immune-modulating activities.

Figure 1 .
Figure1.Effects of the drugs under study on cumulative PASI and Baker's scores.PASI: psoriasis area and severity index.The data are indicated as mean ± SD; * denotes significant differences (p < 0.05) compared to the healthy controls; ** denotes significant differences (p < 0.05) compared to the vehicle and induction groups; # denotes significant differences (p < 0.05) compared to the clobetasol group.

Figure 2 .
Figure2.Effects of the drugs under study on inflammatory markers (TNF-α, IL-17, and VEGF).The data are indicated as mean ± SD; * denotes significant differences (p < 0.05) compared to the healthy controls; ** denotes significant differences (p < 0.05) compared to the vehicle and induction groups; # denotes significant differences (p < 0.05) compared to the clobetasol group.The data for TNF-α, IL-17, and VEGF levels were expressed as (pg/g).

Figure 3 .
Figure3.Effects of the drugs under study on the oxidative marker MDA and the proliferative marker Ki-67.The data are indicated as mean ± SD; * denotes significant differences (p < 0.05) compared to the healthy controls; ** denotes significant differences (p < 0.05) compared to the vehicle and induction groups; # denotes significant differences (p < 0.05) compared to the clobetasol group.The data for MDA amounts were expressed as (nmol/g), whereas the data for Ki-67 amounts were expressed as (ng/g).

Figure 4 .
Figure 4. Effects of the drugs under study on psoriatic histopathological changes.A. The skin segment of the control group demonstrated typical normal keratin layers (blue arrow), normal skin thickness (green arrow), normal dermis (red arrow), and the presence of hair roots and sebaceous glands (yellow arrow) (H&E 10X); B. The skin segment of the induction group showed prominent Munro's microabscess (yellow arrow), marked hyperkeratosis (blue arrow), severe elongation of rete ridges (black arrow), increased skin thickness (green arrow), and marked inflammatory infiltrate (red arrow) (H&E 10X); C. The skin segment of the vehicle group revealed a significant Munro's microabscess (yellow arrow), marked hyperkeratotic changes (blue arrow), severe rete ridge (black arrow), increased skin thickness (green arrow), and marked invasion of inflammatory cells (red arrow) (H&E 10X); D. The skin segment from the clobetasol group indicated mild hyperkeratosis (blue arrow), thinned skin layers (green arrow), and a minor influx of inflammatory cells (red arrow) (H&E 10X); E. The skin segment of the melatonin group presented decreased skin thickness (green arrow), moderate rete ridge (black arrow), and moderate inflammatory cell influx (red arrow) (H&E, 10X); F. The skin segment of the rutin group exhibited reduced skin thickness (green arrow), moderate rete ridge (black arrow), and moderate invasion of inflammatory cells (red arrow) (H&E, 10X); G The skin segment of the melatonin + rutin combination group exerted diminished skin thickness and a mild inflammatory cell infiltration group (H&E, 10X).