MINISTRY OF EDUCATION AND TRAININGNONG LAM UNIVERSITY- HO CHI MINH CITYFACULTY OF BIOLOGICAL SCIENCES UNDERGRADUATE THESIS EVALUATION OF THE EFFECTS OF CORTICOSTEROIDS ON IL17A AND IL23
Purpose of this thesis 6
Purpose 1: Creating a standard psoriasis mouse model using imiquimod
Purpose 2: Evaluating the effects of topical corticosteroids on IL17A and IL23 expression in imiquimod psoriasis mouse model.
Content 1: Creating imiquimod-induced mouse model of psoriasis.
Content 2: Applying then evaluating the effect of corticosteroid on the imiquimod- induced psoriasis mouse model
LITERATURE REN LE W ssunncunmmannnennmnanamriianan qummammacanciis 2
Epidermis — outer layer Of the skii Sen“ EHE440E8000 0000744 2
The epidermis, the outermost layer of skin, serves as a protective barrier against external threats and consists of five distinct layers: stratum basale, stratum spinosum, stratum granulosum, stratum lucidum, and stratum corneum, with the stratum corneum playing a crucial role in its protective function (Wickett and Visscher, 2006) This layer undergoes a natural turnover every two weeks, facilitating wound healing, a process supported by progenitor basal cells located in the stratum basale (Lechler, 2019) These basal cells are the origin of keratinocytes, essential for maintaining skin integrity and health (Yousef et al.).
Keratinocytes are the primary cell type in the epidermis, essential for producing keratin, which contributes to the mechanical stability and integrity of epithelial cells and tissues They play crucial roles in regulatory functions and intracellular signaling pathways, aiding in stress protection, wound healing, and apoptosis (Moll et al., 2008) Additionally, keratinocytes are vital for regulating calcium absorption through the activation of cholesterol precursors by UVB light to synthesize vitamin D (Yousef et al., 2022) These cells are organized in layers within the skin, transitioning to ghost cells that serve primarily as a barrier with minimal energy consumption (Garza, 2019).
2.1.3 Dermis - middle layer of the skin
The dermis, a connective tissue layer situated between the epidermis and hypodermis, provides the primary structure of the skin through its collagen complex This complex consists of collagen and elastic fibers that interact to form a fibrous composition Additionally, the dermis contains various extracellular components, including vasculature, nerve endings, hair follicles, and glands, which work together to support skin structures, facilitate thermoregulation, and enhance sensation.
2.1.4 Hypodermis — bottom layer of the skin
The hypodermis is final part of skin construction and is characterized by subcutaneous adipose tissue It is attached the dermis to the underlying muscle and fascia.
The hypodermis is primarily composed of lipid-rich adipocytes, interspersed with neurovascular structures that support the skin and the deeper regions of sweat glands (Avram et al., 2005) This layer plays a crucial role in providing insulation, storing energy, and protecting the body from environmental forces (Harbour and Song, 2019).
Psoriasis is a prevalent skin condition that impacts approximately 125 million individuals globally, affecting both men and women across all age groups Research indicates that the average age of onset is around 33 years, with two significant peaks: the first occurring between the ages of 16 and 22, and the second between 57 and 60 years.
Psoriasis is a chronic, immune-mediated inflammatory disease that leads to skin inflammation and epidermal hyperplasia throughout the body This condition is associated with a heightened risk of painful arthritis, cardiovascular issues, and various psychosocial challenges Additionally, psoriasis often coexists with other health disorders, including obesity, hypertension, hyperlipidemia, diabetes, metabolic syndrome, cardiovascular disease—collectively referred to as the "psoriatic march" or "inflammatory march"—as well as chronic kidney disease.
Psoriasis can be classified into three main types based on its clinical presentation: psoriasis vulgaris, psoriatic arthritis, and generalized pustular psoriasis (Tokuyama and Mabuchi, 2020) Among these, psoriasis vulgaris, also known as plaque-like psoriasis, accounts for 90% of diagnosed cases (Boehncke and Schön, 2015).
Psoriasis has been recognized for over 2000 years, with Hippocrates referring to it as psora and /Jepra (Gudjonsson and Elder, 2019) Despite this long history, the exact cause of psoriasis remains unknown, though it is believed to stem from the excessive activation of certain components of the adaptive immune system Key players in the early stages of psoriasis pathogenesis include plasmacytoid dendritic cells, keratinocytes, natural killer T cells, and macrophages, which release cytokines that activate myeloid dendritic cells (Nestle et al., 2009) A significant discovery in understanding psoriasis mechanisms is the role of interleukin 17 (IL17) and interleukin 23 (IL23), which has advanced research towards more effective treatments over the past two decades (Di Cesare et al., 2009).
Plaque-like psoriasis affects 90% of patients and is characterized by well-demarcated, raised red plaques with a white scaly surface Lesions vary in size, ranging from small papules to large plaques that can cover extensive areas of the body Underneath the scales, the skin appears glossy and homogeneous with erythema, and pinpoint bleeding may occur when the scales are lifted, known as the Auspitz sign This condition typically presents as symmetric eruptions across the body, particularly localized on the extensor surfaces of the elbows and knees, as well as the scalp, lower back, buttocks, and genital areas Patients often experience significant itching, especially during flare-ups of moderate to severe psoriasis.
2.2.3 Roles of 1L17A/IL23 axis in Psoriasis
IL23 is a protein made up of two subunits, p40 and p19, belonging to the IL12 family, and it plays a crucial role in activating naive T cells through its shared p40 subunit As a pro-cytokine, IL23 is primarily responsible for the differentiation and proliferation of T-helper 17 cells (Th17 cells), making it a significant cytokine in the context of psoriasis disease (Rendon and Schọkel, 2019).
IL17 is a cytokine family consisting of six members, with IL17A and IL17F being the most significant, particularly IL17A, which plays a crucial role in inflammatory diseases like psoriasis Th17 cells produce IL17 and IL21 through IL23 signaling, with IL17 being the predominant cytokine involved in neutrophil recruitment and psoriasis lesion formation Elevated levels of IL17 are found in psoriasis lesions, especially in CD8+Th17 cells, where it triggers keratinocytes to release chemo-attractant cytokines and chemokines, facilitating leukocyte infiltration The presence of IL17-messenger RNA in different psoriasis subtypes compared to normal tissues highlights its role in endothelial and immunological functions When IL17A binds to its trimeric receptor complex, it recruits the ACTI adaptor protein, activating intracellular kinases that lead to the production of pro-inflammatory cytokines, chemokines, and antimicrobial peptides through NFkB, AP-1, and C/EBP pathways.
2.2.4 Psoriasis Area Severity Index (PASI
The Psoriasis Area and Severity Index (PASI), introduced in 1978 by Fredriksson and Pettersson, is the most widely used method for assessing the severity of skin symptoms in psoriasis It serves as a crucial diagnostic tool in selecting appropriate treatments for psoriasis patients (Pathirana et al., 2009) In a 2009 study by van der Fits et al., the PASI score in an imiquimod-induced mouse model was determined by evaluating three factors: erythema, scaling, and thickening, each rated on a scale from 0 (none) to 4 (very marked).
2.3 Imiquimod-induced psoriasis mouse model.
The Imiquimod-induced psoriasis mouse model is a widely used method for studying psoriasis due to its speed, convenience, and the significant symptoms it replicates, including psoriatic plaques (Bochenska et al., 2017) Imiquimod (IMQ) cream, commonly used for treating external genital warts and certain skin cancers, has been shown to exacerbate psoriasis in some patients (Beutner and Tyring, 1997; Geisse et al., 2002; Szeimies et al., 2004; Wu et al., 2004; Gilliet et al., 2004) In mice, daily IMQ application leads to skin lesions that mirror human psoriatic plaques, characterized by increased epidermal proliferation, erythema, altered vascularity, impaired keratinocyte differentiation, and an influx of immune cells such as CD4+ T cells and dendritic cells (Bochenska et al., 2017).
Figure 2.3 Structure of imiquimod (NCBI).
IMQ, a nucleoside analogue from the imidazoquinoline family, effectively activates anti-tumoral cellular immune responses by targeting TLR7 and TLR8, which recognize viral single-stranded RNA Research by van der Fits et al (2009) demonstrated that applying IMQ to mouse skin resulted in lesions resembling human psoriasis, with increased levels of IL23 and IL17, indicating its potential as a robust in-vivo model for studying psoriasis Additionally, a 2019 study by Singh et al corroborated these findings, showing that IMQ combined with IL23 injection led to elevated IL17A levels and inflammatory cell accumulation, mirroring the conditions found in human psoriasis.
Corticosteroids are versatile medications used across various medical settings for both administration and therapeutic purposes This broad category includes natural steroid hormones, specifically glucocorticoids and mineralocorticoids Glucocorticoids primarily influence metabolism and exhibit immunosuppressive, anti-inflammatory, and vasoconstrictive properties, while mineralocorticoids regulate ion transport and maintain electrolyte and water balance in epithelial cells of the tubules.
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3.1 Time and place of experiment
The undergraduate thesis has been performed since middle December 2021 to the middle of August 2022 at Stem Cell Institute, University of Science - Vietnam National University Ho Chi Minh City.
In this study, Balc/c mice, aged 8 to 10 weeks and weighing between 18g and 25g, were used as experimental subjects The mice were sourced from the Laboratory for Animal Care and Use at the Stem Cell Institute, University of Science — VNU, and included both male and female specimens.
The experiment involving mice has received approval from the head of the Laboratory for Animal Care and Use, ensuring compliance with biomedical ethics standards for animal research.
4 Easy-BLUETM Total RNA Extraction Kit iNtRON
II Potasstum Phosphate Monobasic China
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Methods 1
Effects of protection Effects of prevention
Group 2: Appling IMQ Group 4: Appling IMQ (day tay O-) palicmige Group 1: Healthy mice LG ung
Group 3: Appling IMQ Group 5: Appling IMQ (day
: (Control) (day 0 -6) with cort (day 0 — 12) + cort (day 7 — 12)
: - 4 - qRT PCR £ : : PASI scoring H&E q OF :
- Images of wound Indirect TP quantification of 17A and _
3.2.2.2 IMQ-induced psoriasis mouse modeling and treatment
Imiquimod: Apply once every 24 hours, 62.5 mg each time for each mouse
Corticosteroids: Apply twice every 24 hours, 0.015g each time and at least 2 hours apart for each mouse.
After a one-day health observation period, Balc/c mice will be evaluated for their well-being Once confirmed healthy, the fur on the backs of the mice will be shaved to an area of approximately 9 cm, followed by the division of the mice into five distinct groups for further study.
Group 1: Control group: Not using any topical drugs, including 10 mice.
Group 2: The group of mice applied topical IMQ for 6 days, including 10 mice. Group 3: The group of mice applying topical IMQ for 6 days and applying corticosteroids for the next 6 days starting from day 7, including 30 mice Groups 2 and 3 were proposed to evaluate the therapeutic effects of corticosteroids on some acute symptoms of psoriasis.
Group 4: The group of mice applied topical IMQ for 12 days, including 10 mice. Group 5: The group of mice applied IMQ for 12 days and applied corticosteroids for
6 days starting from day 7, including 30 mice Groups 4 and 5 were included to evaluate the therapeutic effects of corticosteroids on some chronic symptoms of psoriasis.
All groups of mice were observed and evaluated based on the PASI scale (mentioned in the literature review) daily for up to 24 days.
Table 3.4 PASI scoring on mouse model
2: 4: Very Intensity 0: None 1: Slight 3: Marked
- dh Medium red , fe) redding surface
Erythema across ; Dark red change across with dark surface surface red pathches
= Moderate spots sll flaking Moderate
Minor dry ơ acrossa flaking across a majority if Seali spots large large surface
Xung the skin, without across a area; severe flaking flaking large flaking along along surface crevices crevices area
Biopsies were conducted on three random mice from both the topical IMQ group and the control group on days 3 and 6 From day 6 onward, all five groups underwent biopsies on days 8, 12, 18, and 24, with each sample consisting of three mice Prior to sample collection, mice were anesthetized with zoletin and xylazine Tissue samples intended for PCR were placed in 1.5 ml tubes, frozen in liquid nitrogen, and stored at -20°C, while samples for histological staining were preserved in a 4% Paraformaldehyde (PFA) solution in 2 ml tubes at 4°C Each mouse was sampled twice before being sacrificed.
Mouse skin samples preserved in 4% PFA solution were sent to Apatha company,
No 85 Tan Khai, Ward 04, District 11 for slicing and H&E staining services.
3.2.2.4 Indirect immunofluorescence staining of [L23 and IL17A antigen
Mouse skin samples preserved in a 4% PFA solution were sent to Apatha Company in Ho Chi Minh City for tissue fragmentation and fixation using paraffin Upon receipt of the samples, indirect immunofluorescence staining was carried out following a specific protocol.
Step 1: Deparaffinization, In the following order:
Chemicals Amount of time Times
Double Distilled Water Slight washing 1
Step 2: Add 1 - 2 drops of antiserum: rabbit primary antibody IL23p19, rabbit primary antibody IL17A to the samples fixed on the respective slide and leave for 30 min in a closed, moistened container at 37 °C.
Step 3: Discard excess serum and rinse thoroughly and gently with pH 7,2 phosphate buffer.
Step 4: Soak with pH 7,2 phosphate buffer for 30 minutes twice.
Step 5: Drain at room temperature.
Step 6: While the cuttings are still slightly damp, add one drop of Mouse IgGk light chain binding protein (secondary antibody) and leave for 30 minutes in a humidified container at 37°C.
Step 7: Discard excess fluorescent antibodies and rinse thoroughly.
Step 8: Soak with pH 7,2 phosphate buffer for 10 minutes twice.
Step 9: Dye with 1-2 drops of Hoechst for 15-30 minutes
Step 10: Drain at room temperature, while the cuttings are still slightly damp, add a drop of mounting Cover the glass and observe the dyed sample under the microscope. 3.2.2.5 qRT-PCR of IL23 and IL17A mRNA
To begin the RNA extraction process, add 200 µl of Easyblue - Total RNA Extraction Kit to a sample tube and use a glass rod to thoroughly crush the tissue Next, incorporate an additional 300 µl of Easyblue into the tube and vortex the mixture for 3 minutes Subsequently, incubate the mixture at 4°C for a minimum of 15 minutes After incubation, centrifuge the mixture at 6000 RPM for 5 minutes, then carefully extract about 500 µl and add Chloroform in a specified ratio of Chloroform to Easyblue.
To prepare the solution, mix the components in a 1:5 ratio and vortex for 40-60 seconds, followed by a 5-minute incubation at 4°C Centrifuge the incubation solution at 13,000 RPM for 10 minutes, then transfer 200 µl of the supernatant to a new 1.5 ml tube and add isopropanol in a 1:1 ratio Stir the mixture thoroughly and incubate it at -20°C for at least 15 minutes After incubation, centrifuge again at 13,000 RPM for 10 minutes, discard the supernatant, and collect the residue Finally, add 700 µl of cold 75% ethanol and centrifuge the mixture.
To extract RNA, centrifuge the sample at 13,000 RPM for 5 minutes, repeating this step twice Carefully pour out the alcohol, using a pipette tip to remove any remaining liquid Allow the alcohol to evaporate for 5 minutes, then add 50 µL of DEPC to the sample Vortex the mixture and store the extracted RNA at -20°C for preservation.
The kit utilized for the quantitative reaction of IL23 and IL17A includes reverse transcriptase activity, enabling the conversion of total RNA into cDNA Commercially available primer pairs specific to the IL23 and IL17A genes, along with the internal control gene GAPDH, were employed Prior to application, the annealing temperatures were optimized in the laboratory.
Table 3.6 Sequence of primers participating in RT-qPCR for IL23 and IL17A
Gene Primer sequence Annealing name 6 = 3") temperature Manufacturer
Table 3.7 Components participating in RT-qPCR
Component 20 ul reaction Final concentration Luna Universal One-Step Reaction Mix 10 1X
Luna WarmStart RT Enzyme Mix 1 wl 1X
Template RNA variable < lpg (total RNA) Nuclease-free Water To 20 pl
Table 3.8 RT-qPCR temperature cycles
This thesis utilizes Graphpad Prism software, version 8.0.1, for statistical analysis, employing both two-way and one-way ANOVA methods All results are considered significant with a p-value of less than 0.05, maintaining a confidence level of 95%, and are presented as Mean ± SD.
4.1 Results IMQ-induced psoriasis mouse model
4.1.1 Results of the PASI assessment
The mouse model induced by IMQ demonstrates distinct clinical characteristics over time, as illustrated in Figure 4.1 On day 0, there are no visible signs of psoriasis By day 3, the model exhibits a scaling degree of 1 to 2 and an erythema degree of 3 By day 6, the severity increases, with a scaling degree of 4 and an erythema degree of 4, highlighting the progression of three typical psoriasis symptoms.
Daily assessments of psoriasis symptoms, including thickening, scaling, and erythema, are conducted and evaluated using the PASI scale This index is updated every day for six consecutive days during the modeling period to accurately assess the severity of the condition.
Figure 4.2 PASI scale from day 0 to day 6 between control group and IMQ application group p < 0.05.
The PASI index analysis indicates that day 6 marks the peak of symptom severity during treatment, with the IMQ application group showing the highest value of 9.222 ± 1.331 One-Way ANOVA results confirm significant differences among the groups, with p-values consistently below 0.001.
The study revealed that the clinical progression of psoriasis varied daily, consistently trending upward until the conclusion Additionally, all mathematical analyses indicated a statistically significant difference across the observed days.
Treatment effects of corticosteroids on model resuÌts 7.7111 31 1 Evaluation of treatment effects based on clinical manifestations
4.2.1 Evaluation of treatment effects based on clinical manifestations
Figure 4.7 illustrates the clinical manifestations across four distinct groups: (a) the day 8 group, (b) the day 12 group, (c) the day 18 group, (d) the day 24 group, and (e) the control group The groups are categorized based on treatment duration and type: (1) the group treated with IMQ for 12 days, (2) the group receiving IMQ for 12 days alongside corticosteroids from day 7 to day 12, (3) the group treated for 6 days, and (4) the group receiving IMQ for 6 days with corticosteroids from day 7 to day 12.
Figure 4.7 evaluates cort based on its therapeutic and preventive effects In the therapeutic aspect, a comparison was made between a group treated with IMQ for 6 days and another group that received both IMQ and cort from days 7 to 12 Results showed that by day 12, external manifestations were nearly normal in both groups; however, the cort group exhibited a quicker recovery, with significant reduction in inflammation symptoms by day 8, unlike the group that did not receive cort For the preventive effect, the study involved a 12-day IMQ treatment alongside cort administration from day 7.
Research indicated that on day 18, mice exposed to IMQ continued to exhibit symptoms of psoriasis, although the severity showed a tendency to decrease In contrast, the group treated with corticosteroids demonstrated a more rapid reduction in symptom expression These findings suggest that corticosteroids are effective in alleviating clinical psoriasis symptoms.
DAY8 DAY12 DAY18 DAY 24 Figure 4.8 PASI scale at day 8, day 12, day 18 and day 24 between 2 main groups: Therapeutic efficacy assessment group and tpreventive effectiveness assessment group p < 0.05.
The PASI index highlights notable clinical differences, particularly on days 8 and 12 when IMQ was still active During these days, the 12IMQ and 12IMQ/Cort groups, which focus on preventive effects, demonstrated a significant reduction in psoriasis symptoms.
The study revealed a statistically significant difference between two groups, while no significant difference was observed between the 6IMQ and 6IMQ/Cort groups, despite the cort-treated group showing a reduction in psoriasis symptoms compared to the 6IMQ group By day 18, most groups exhibited nearly normal conditions, except for the 12IMQ group, and by day 24, all groups had returned to or were near normal levels.
Day 12 was the day after the last day of treatment, so it was collected skin samples
Figure 4.9 Comparison of histological alterations following of 5 groups: 6IMQ, 6IMQ/Cort, 12IMQ, 12IMQ/Cort groups and Control group.
Figure 4.9 Comparison of histological alterations following of 5 groups: 6IMQ, 6IMQ/Cort,12IMQ, 12IMQ/Cort groups and Control group (Continued).
Figure 4.10 Epidermal thickness between control group and 4 groups: 6IMQ, 6IMQ/Cort, 12IMQ, 12IMQ/Cort groups at day 12 p $0.05.
Day 12 was the day after the last day of treatment, so I collected skin samples of this day to check the results of the treatment in histological analysis In histological side, in
The study observed distinct differences in skin texture between the IMQ group treated for 6 days and the IMQ group treated with cort for the same duration Notably, the stratum corneum in both groups lacked living cells, indicating normal skin, as evidenced by imaging findings The IMQ/Cort group exhibited minimal inflammatory cell presence, resembling the histological characteristics of the control group, with no significant difference in thickness In the preventive effect analysis, the 12-day IMQ group displayed pronounced changes, including elongated rete ridges, new pore formation, hyperplasia, and hyperkeratosis, while the cort-treated group showed none of these symptoms Additionally, the epidermal thickness in the cort-treated group was significantly thinner compared to the control group.
4.2.3 Changes in gene expression of IL23 and IL17A during and after treatment
6IMQ-IL23 6IMQ/Cort-IL17A 6IMQ/Cort-IL23 12IMQ-IL17A 12IMQ-IL23 12IMQ/Cort-IL17A 12IMQ/Cort-IL23 Control-IL17A Control-IL23
The expression levels of IL23 and IL17A were analyzed across different time points—day 8, day 12, day 18, and day 24—in various treatment groups, including 6IMQ, 6IMQ/Cort, 12IMQ, 12IM/Cort, and a control group The data was transformed using log10 to facilitate a clearer representation on the decreasing x-axis, with results showing statistical significance at p < 0.05.
RT-qPCR experiments were conducted on day 8, day 12, day 18, and day 24 to assess transcription levels across all groups Notably, day 8 and day 12 marked key points in the treatment process In the therapeutic effect group, specifically in the 6IMQ and 6IMQ/Cort groups, a significant decrease in the gene expression of IL23 and IL17A was observed during these landmarks.
At day 12, the treated group exhibited higher IL23 gene expression, though the difference was not statistically significant IL17A levels nearly returned to normal compared to the control group, showing higher expression than the IMQ-only group, but this was also not statistically significant In the preventive groups (12IMQ and 12IMQ/Cort), IL23 expression was stronger in the IMQ-only group compared to both the cort treatment and control groups Similarly, IL17A expression was lower in the treatment group than in the IMQ-only group By days 18 and 24, gene expression for both IL23 and IL17A gradually decreased and stabilized, with day 24 showing nearly identical expression levels across the groups.
4.2.4 Protein expression of IL17A and IL23 gene in skin structure
IL23 Nucleus Skin texture Merge
Figure 4.12 Tissue staining by indirect immunofluorescence technique with IL23 assessment of day 12 skin tissue samples of control and experiment groups.
In the study of IL23 expression, the cort-treated group exhibited a notable decrease in protein expression compared to the negative control group and the IMQ-only group In the therapeutic survey group, the cort treatment resulted in sparse, small orange spots that did not cover the cell nuclei, indicating weak expression Conversely, the 6IMQ and control groups displayed prominent orange spots that fully enveloped the nuclei, demonstrating clear expression In the chronic survey group, the 12IMQ group showed dense, round orange spots surrounding the nuclei, while the 12IMQ/Cort group revealed only a few small spots that failed to cover the nuclei, indicating a significant loss of IL23 expression.
ILI7A Nucleus Skin texture Merge
Figure 4.13 Tissue staining by indirect immunofluorescence technique with ILI7A assessment of day 12 skin tissue samples of control and experiment groups.
In the acute effect group, IL17A expression was similar in both the 6IMQ and 6IMQ/Cort groups, showing no significant differences in the density of orange circles Conversely, in the preventive effect group, the 12IMQ group displayed numerous, round orange spots fully surrounding the nuclear region, indicating higher IL17A expression In contrast, the 12IMQ/Cort group exhibited very small orange spots that failed to cover the nuclear sites, suggesting a marked reduction in IL17A expression.
4.3.1 Discussion in model creating results
Psoriasis accelerates the skin cell lifecycle, with cells regenerating every week compared to the normal four-week cycle In a mouse model of psoriasis induced by IMQ, the experiment was conducted over a span of 7 days, from day 0 to day 6, to observe the effects of this rapid cell turnover.
Psoriasis characteristics are most pronounced in patients with severe clinical features, as indicated by the PASI scale, which reflects the actual progression of the disease This finding aligns with the 2009 study by van der Fits et al., although their results showed no significant differences between days 2 and 3, likely due to the limited sample size Increasing the sample size could potentially yield different outcomes Additionally, the research conducted by Singh et al further supports these observations.
2019, and Riol-Blanco et al., 2014, also clearly showing clinical characteristics when creating model by IMQ, the severity reached the highest day 6.
On day 6 of the modeling group, H&E images revealed significant epidermal changes characterized by keratinocyte hyperproliferation and altered differentiation, along with papillomatosis, an influx of inflammatory cells, and modified vascularity, all of which are hallmark features of the psoriatic model (Roark et al.).