Evaluation of Kynu, Defb2, Camp, and Penk Expression Levels as Psoriasis Marker in the Imiquimod-Induced Psoriasis Model

Background Psoriasis is a noncontagious auto-inflammatory chronic skin disease. So far, some of the inflammatory genes were upregulated in mouse model of psoriasis. This study examined changes in skin mRNA expression of L-kynureninase (Kynu), cathelicidin antimicrobial peptide (Camp), beta-defensin 2 (Defb2), and proenkephalin (Penk) in a mouse model of imiquimod-induced psoriasis. Materials and Methods Tree groups of C57BL/6 female mice were allocated. The imiquimod (IMQ) cream was administered to the mice dorsal skin of the two groups to induce psoriatic inflammation. In the treatment group, IMQ was administered 10 min after hydrogel-containing M7 anti-IL-17A aptamer treatment. Vaseline (Vas) was administered to the negative control group. The psoriatic skin lesions were evaluated based on the psoriasis area severity index (PASI) score, histopathology, and mRNA expression levels of Kynu, Camp, Defb2, and Penk using real-time PCR. In order to assess the systemic response, the spleen and lymph node indexes were also evaluated. Results The PASI and epidermal thickness scores were 6.01 and 1.96, respectively, in the IMQ group, and they significantly decreased after aptamer administration to 1.15 and 0.90, respectively (P < 0.05). Spleen and lymph node indexes showed an increase in the IMQ group, followed by a slight decrease after aptamer treatment (P > 0.05). Additionally, the mRNA expression levels of Kynu, Defb2, Camp, and Penk genes in the IMQ-treated region showed a significant 2.70, 4.56, 3.29, and 2.61-fold increase relative to the Vas mice, respectively (P < 0.05). The aptamer-treated region exhibited a significant decrease in these gene expression levels (P < 0.05). A positive correlation was found between Kynu, Penk, and Camp expression levels and erythema, as well as Camp expression with PASI, scaling, and thickness (P < 0.05). Conclusion According to our results, it seems that Kynu, Camp, and Penk can be considered appropriate markers for the evaluation of psoriasis in IMQ-induced psoriasis. Also, the anti-IL-17 aptamer downregulated these important genes in this mouse model.


Introduction
Psoriasis disease is a noncontagious chronic inflammatory skin condition that may be triggered by a trauma, infection, or drugs [1].The initiation and persistence of psoriatic inflammation contribute to the dysfunction of both adaptive and innate immune reactions in the skin.Therefore, psoriasis is an autoimmune and autoinflammatory disease [2,3].The prevalence of psoriasis is about 2%-4% of the worldwide population [4].Psoriasis includes a variety of types, with psoriasis vulgaris that is known by clinical manifestations, such as erythema, skin thickening, and scaling, being the most common type [5,6,7].
AMPs are first-line defense with a crucial role in the skin by killing pathogenic microorganisms [12,13].CAMP and DEFB2 are two types of AMPs secreted by keratinocytes in psoriasis [14].LL37, which interacts with deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), releases from apoptotic or necrotic cells and stimulates dendritic cells (DCs) to secrete inflammatory cytokines [15].On the other hand, DEFB2 has been shown to induce proliferative reactions as well as promote the secretion of inflammatory factors in keratinocytes [16].Therefore, it seems that the expression levels of both CAMP and DEFB2 elevate in lesional skin compared to healthy skin [14].
Enkephalin (ENK) is involved in nociception and pruritus [17,18], which is identified as a contributing factor to inflammation, which belongs to the family of endogenous opioidlike peptides.PENK is a protein precursor of ENKs [19,20], which has been found to facilitate an interaction between the neuroendocrine and immune systems [21].The PENK receptors are expressed on keratinocytes and regulate keratinocyte activities [22].They have direct antimicrobial activities and regulate cell proliferation and differentiation [23].The increase of PENK in psoriatic skin keratinocytes has been reported by studies [21,23,24].
Another factor involved in inflammation is KYNU, which is upregulated in psoriatic lesions [25].The pathway of tryptophan metabolism includes various enzymes, one of them being KYNU [26].Tryptophan metabolism, when mediated by the KYNU, causes inflammation.Kynurenine (KYN) is a metabolite of tryptophan that reduces the expression of some inflammatory genes in keratinocytes [25].It has been observed that the upregulation of KYNU leads to the destruction of KYN and induces excessive inflammation in psoriasis [27].
Skin problems caused by psoriasis can be extremely burdensome sometimes.So far, there are various treatments known for psoriasis, and one type of topical agents for modulating of psoriasis symptoms is aptamers [28].Aptamers are RNA and DNA (single-stranded form) molecules with 3D unique structures that specifically target biological molecules with a high affinity and specificity [29].One of the important cytokines with a main role in the development of psoriasis is IL-17A [30].M7 anti-IL-17A aptamer has been developed against IL-17A, which inhibits its cytokine effects and leads to a reduction in disease symptoms.So, it can be considered as a therapeutic agent that could prevent to engage of IL-17 with IL-17 receptor [31].One of the psoriasis animal models is the imiquimod (IMQ)-induced psoriasis mouse model, which induces psoriasis using IMQ cream [32].IMQ is an agonist of toll-like receptors (TLR) 7/8-ligand and induced lesions like psoriasis through this pathway [33].
It is very important to determine an appropriate marker to assess the induction of psoriasis caused by IMQ that can assess an adequate response after treatment with therapeutic agents.This study aimed to evaluate the psoriasis-correlated gene expression levels of Kynu, Defb2, Camp, and Penk in the IMQ-induced psoriasis mice skin and also in mice model treated with M7 anti-IL-17A aptamer for the purpose of determining a proper diagnostic marker for psoriasis and evaluating treatment response.

2
Mediators of Inflammation

Materials and Methods
Twelve female C57BL/6 mice with a weight range of 18.3-26 g, aged 7-8 weeks, were obtained from the Pasture Institute (Tehran, Iran) and maintained under standard situations at Bu-Ali Research Institute (Mashhad, Iran).Ethical Committee of Mashhad University of Medical Sciences (IR.MUMS.ACE.1401.096)approved the animal experiment procedure.
2.1.Hydrogel Preparation.In order to prepare the gel, 40 ml water was poured into a container and stirred with a stirrer, followed by slowly adding 0.4 g of Carbopol (gel-forming carbomer powder) to the water.Then, 400 µl of propylene glycol and 480 µl of triethanolamine were added dropwise.After preparation, the resulting gel was collected with a spoon and poured into a tube.
2.2.Animals' Procedure and Grouping.All of the animals were divided into the following three groups randomly, with four mice in each group.About 2 cm 2 of the mice dorsal skin was depilated using an electric shaver.In order to induce a psoriasis-like model, two experimental groups were received 40 mg (daily dose) of IMQ 5% cream (Aldara, UK).One group administered 40 mg/day of Vaseline (Vas) as the negative control group.This procedure was performed on the mice's dorsal skin topically, over a period of 5 consecutive days.The psoriasis area severity index (PASI) score and weight of the animals were recorded daily.One group of IMQ-administered mice received the discovered aptamer targeting IL-17A named M7 (mebep, China) as the treatment group.The aptamer utilized in this study was identified in our previous research with the sequence 5′-CGAC TAACTGTTTTCTTTTGTTTTTAGTCG-3′ (30-nt) [31].
All groups received 50 µl of carbomer-based hydrogel.About 4 µl of M7 anti-IL-17A aptamer (10 pmol) was topically applied along with the aforementioned hydrogel on the dorsal skin of the M7 anti-IL-17A aptamer group.IMQ or Vas is administered topically, 10 min after hydrogel application, once a day.Animals were sacrificed on the 6th day of the procedure, and their dorsal skin was isolated in order to histopathological and messenger RNA (mRNA) levels analyzing.Additionally, the mice popliteal lymph node and spleen were isolated for supplementary analysis [34].The experiment procedure is illustrated in Figure 2.
2.3.PASI Score.In our study, the mice dorsal skin lesions were evaluated with the PASI score, which represent the prominent scoring in clinical symptoms measurement of psoriasis [35].
The PASI score, as an indicative of an inflammatory condition in psoriatic lesions, was evaluated in all mice before the treatments daily by two individuals who were blinded to the experimental groups.A numerical score between 0 and 4 (0none, 1-light, 2-mild, 3-severe, and 4-very severe) was assigned for erythema, scaling, and thickness in mice's dorsal skin.The PASI cumulative score was presented as 0-12, determining the severity of psoriasis-related inflammation.

Popliteal Lymph
Node.We labeled the popliteal lymph node using an injection of Evans blue (1%) dye into the mouse left footpad.Following the sacrifice of animals, the lymph node was isolated, and the cells were collected in PBS with physiologic pH (7.4).Subsequently, a hemocytometer chamber was utilized to count the cells/1 ml PBS.
2.5.Spleen Size, Mass, and Total Cell Count.Following the sacrifice of the animals, the spleens of all mice were isolated and subsequently evaluated for their mass, size (length), and total cell count.The mass of the spleen (in milligrams), size (in millimeters), and spleen cell count were measured using a digital scale (A&D in Japan), a ruler, and a hemocytometer counting-chamber, respectively.In order to assess the spleen total cell count, RBCs were lysed by lysis solution (NH 4 Cl: 0.01 M, NaHCO 3 : 0.01 M, EDTA: 0.04 mM) and followed by washes with PBS, then the collected cells were adjusted up to a volume of 1 ml with PBS.The values were normalized according to the average of mice's last-day weight.

SYBR-Green Reverse Transcription Polymerase Chain
Reaction.About 10 mg of mice dorsal skin tissue was separated.Following the kit manufacture's instruction (Parstous Co., Iran), the cellular RNA was extracted, and then, the complementary DNA (cDNA) synthesis was accomplished utilizing reverse transcriptase enzyme and oligo-dT primers (Parstous Co., Iran).Specific primers (SinaClon, Iran) were designed for Kynu, Camp, Defb2, and Penk genes (Table 1).The evaluation of Kynu, Camp, Defb2, and Penk genes mRNA levels was performed according to the guidelines provided by the SYBR-Green Master Mix kit (Parstous Co., Iran).Hypoxanthine phosphoribosyltransferase (Hprt) primers, which were designed by Shobeiri et al. [36] utilized in this study as a housekeeping gene.The real-time PCR procedure was performed in duplicate by a real-time PCR (Four E's Scientific, Guangzhou, China) detection system.In the relative gene expression results, 2 −ΔCt was calculated for each mouse, and then the 2 −ΔCt mean results of each group were divided by the Vas group 2 −ΔCt results mean and reported as fold change.
2.7.Histopathology.In the context of histopathological analysis, a part of the mice dorsal lesional skin was separated and fixed.The process of fixation involved the use of 10% formaldehyde, and then the sample was embedded within a matrix of paraffin blocks, cut, subsequently placed upon slides, and stained with hematoxylin and eosin (H&E) stain.In order to assess the mice skin epidermal thickness, the tissue sections were analyzed utilizing an optical microscope (BEL Photonics, Italy).In this study, epidermis length and microscopic region diameter were measured by ImageJ software version 1.44 (NIH, Bethesda, MD, USA), and the epidermal thickness score was calculated.
2.8.Statistical Analysis.The Graph-Pad Prism software (Version 8.4.2,California, USA) was employed for the analysis of data.Weight, spleen, and lymph node data were presented in the form of mean AE standard error (SEM).The data of spleen normalized with the mouse weight.Statistical analysis, including Mann-Whitney U and two-way ANOVA, was used to assess group differences.Spearman's correlation coefficient analysis was conducted to evaluate the correlation between our research inflammatory genes expression levels and erythema, scaling, thickness, and PASI.The P-value < 0.05 was considered significant.

Evaluation of Weight
Changes in Mice Groups.The evaluation of weight mean AE SEM by two-way ANOVA analysis in mice revealed no statistically significant alterations in the IMQ-treated mice in comparison to the Vas and the M7 anti-IL-17A aptamer-treated groups (Figure 3(a)).) revealed a significant increase in the epidermal keratinocyte proliferation of the IMQ group versus the Vas group (P <0:001).The experimental group that received the M7 anti-IL-17A aptamer demonstrated a significant decrease in the epidermal layer thickness as compared to the IMQ group (P <0:001).

Discussion
We demonstrated that Kynu, Defb2, Camp, and Penk genes are upregulated by IMQ administration in a mouse model of psoriasis.Also, these gene expression levels were downregulated following mice treatment through inhibition of IL-17A with the anti-IL-17A aptamer.Additionally, these changes in Kynu, Camp, and Penk genes are related to the disease symptoms.
Psoriasis vulgaris is an inflammatory systemic disease with various manifestations [37].The keratinocytes proliferation in psoriasis vulgaris is correlated with the alterations of inflammatory gene expression levels [38].So far, studies have shown an association between psoriatic inflammation and changes in gene expression, such as SERPINB4, DEFB4, S100A, CCL20, KRT16, KYNU, IL-6, etc. [39].Additionally, the gene expression of IL-6, IL-1b, and IL-17 was investigated in the IMQ-induced psoriasis mice that were treated with anti-IL-17 aptamer [36].Therefore, it appears that changes in the gene expression of Kynu, Camp, Defb2, and Penk are Mediators of Inflammation more appropriate candidates for the analysis of IMQ-induced psoriasis and anti-IL-17A aptamer treatment in this model.As a result, we investigated the changes in the gene expression levels of Kynu, Defb2, Camp, and Penk to determine which genes are more appropriate factors for the evaluation of psoriasis and the aptamer treatment efficacy.Among the animal models used for studying psoriasis, direct induction with 5% IMQ has been widely utilized in various basic research [40].So, the IMQ was administered on the C57BL/6 mice skin in the IMQ group and in the aptamer-treated group following the M7 anti-IL-17A aptamer applied.
The evaluation of epidermal thickness and PASI scores on the 6th day of the experiment indicated a significant increase in the IMQ-treated mice and a decrease in the M7 anti-IL-17A aptamer-treated group.In this regard, it was observed that IMQ-induced psoriasis can be used as a mouse model for analyzing of psoriasis-like dermatitis pathogenesis and inflammatory cytokines [41].Also, Shobeiri et al. [36] demonstrated that the M7 anti-IL-17A aptamer administration on the IMQ-induced psoriasis C57BL/6 mice dorsal skin ameliorated severity manifestations of skin psoriasis and reduced the IL-17A, IL-1b, and S100a9 mRNA expression Results indicated a significant increase in the IMQ group versus the Vas group and a significant decrease in the M7 anti-IL-17A aptamer compared to the IMQ group.The mRNA expression levels of the animal's dorsal skin were evaluated by SYBR green real-time PCR.Data are expressed as the fold change AE SEM.RT-PCR procedures were carried out in duplicate and repeated 3 times.Spearman's correlation between inflammatory genes and PASI, erythema, scaling, and thickness of the IMQ group was conducted, "r" presented in the heat map (e).Vas, vaseline (negative control), IMQ, imiquimod, Kynu, kynureninase, Defb2, beta-defensin 2, Camp, cathelicidin antimicrobial peptide, Penk, proenkephalin.* P-value < 0.05.levels [36].We noticed that our findings on epidermal thickness and PASI scores, in line with the aforementioned studies results, demonstrated that the psoriasis was induced following IMQ application.Additionally, the anti-IL-17A aptamer was able to ameliorated psoriasis symptoms.CAMP and DEFB2 are two types of AMPs; they are synthesized in excessive quantities during psoriatic inflammation [42,43].In our research, Camp and Defb2 expression levels in the IMQ-applied area showed around three and five fold increase, respectively.Additionally, in the M7 anti-IL-17A aptamer-treated group, these genes expressions were downregulated near to their levels in the Vas group.Wolfram et al. [44] measured the expression of the Camp in overexpressed KC-Tie2 mice's back skin by RT-PCR.The results indicated a significant upregulation in the Camp expression, they were reported 40-fold upregulation of Camp relative to the control group [44].The local mouse CAMP levels of C57BL/6J mice dorsal skin exposed to IMQ, evaluated using immunoassay methods, revealed a significant 6.47fold increase [45].Kolbinger et al. [42] evaluated psoriatic patients before and after the administration of secukinumab and mentioned the expression of DEFB2 protein in the patient's lesional skin and sera increased, and after receiving secukinumab, along with the decrease in PASI score, the skin levels of DEFB2 were decreased.Also, the correlation was found between PASI disease activity and serum DEFB2 levels [42].In line with Kolbinger study, our research showed decrement in Defb2 expression levels after anti-IL-17 aptamer administration.Therefore, Defb2 decrement can be considered as a marker in the inhibition of IL-17 effects.Nevertheless, we observed a significant reduction in Camp gene expression in the anti-IL-17A aptamer-treated group, which contrasts with the results of the Peric study.Peric et al. [46] demonstrated vitamin D analogs (calcipotriol) therapy improves human skin inflammation in psoriasis, but it strongly induces the expression of CAMP in the skin [46].It seems that vitamin D analogs may ameliorate skin inflammation through pathways other than CAMP expression, and our treatment consequences may inhibit the effect of upregulated Camp in the development of the psoriatic manifestations.
It appears, another factor that demonstrates an upregulation in psoriatic inflammatory skin is KYNU.The KYNU enzyme is a part of the tryptophan metabolic pathway and is capable of eliciting inflammatory responses [47].We observed that the Kynu mRNA expression in the IMQ-applied area demonstrated approximately a three fold increase, whereas results related to the M7 anti-IL-17A aptamer-treated group showed decreased this gene mRNA levels near to the Vas group.A meta-analysis-derived transcriptomic study has indicated that the KYNU gene increases an excess of 20fold changes in psoriasis [48].Moreover, KYNU is upregulated by TNF-α and IL-17 synergistically in the psoriasis [49].Wang et al. [11] studied the BABL/c mice IMQ model, and their findings revealed that KYNU enzyme inhibition (by Carbidopa and Benserazide) reduced the inflammation of IMQ-applied mice dorsal skin [11].Harden et al. [25] demonstrated the upregulation of the KYNU gene in the inflammation of human psoriasis and its reduction following successful therapy with etanercept or ustekinumab.Additionally, they revealed a positive correlation between KYNU expression and PASI score.According to publicly available gene array data, indoleamine-2,3-dioxygenase (IDO) is overexpressed compared to the KYNU in cancers such as ovarian, lung, and cervical, while in inflammatory diseases, KYNU is overexpressed.Also, the KYNU/IDO-TDO ratio demonstrated a significant increase in inflammatory diseases [25].IDO promotes an anti-inflammatory environment in the skin by T-cell suppression and regulatory T-cell induction [50], which in turn reduces KYNU expression levels [25].Therefore, this appears that inhibiting the IL-17 in an aptamer-treated group helps the modulation of an inflammatory milieu in the skin, thereby may affecting tryptophan metabolism through the IDO.
The mRNA expression of the PENK increases in inflamed skin [23].PENK is a precursor of ENKs, which are endogenous analgesics [51].The PENK is a neurotransmitter involved in pruritus and nociception.It is also known as an AMP [20,21].Because ENKs are mediators of inflammation and induce pruritus [52], it is expected this mediator increased in psoriasis condition.Our results of Penk expression levels evaluating in the IMQ-applied skin indicated an approximately three fold increment.Also, it decreased in the M7 anti-IL-17A aptamer-treated group to near the Vas group level.Oishi et al. [53] performed a comprehensive analysis on the pruritus in IMQ-induced psoriasis BALB/c mice.The results revealed the upregulation of preproenkephalin (PPE) mRNA expression to an excess of 295-fold change in the IMQ-applied mice in comparison to the mice without IMQ [53].Loite et al. [54] indicated that the levels of the PENK mRNA expression in the human psoriatic lesional skin versus nonlesional skin were 1.6 times higher [54].Furthermore, another research accomplished by Slominski et al. [23] showed that the PENK was significantly expressed in human skin keratinocytes.Also, Met/Leu-ENK expression increased in psoriasis.Nissen et al. [22] applied calcipotriol and mometasone furoate on the psoriatic patient's skin and then measured ENK in the skin by using the radioimmunoassay method.They found that the levels of ENK were increased in psoriatic lesions and decreased after clinical improvement caused by treatment [22].Consequently, our findings are in line with the aforementioned studies showing that the Penk mRNA expression levels are an appropriate factor in assessing psoriasis.
Our findings indicated that in addition to increased expression levels of Camp, Defb2, Kynu, and Penk in the IMQ group, PASI and epidermal thickness scores also increased.Moreover, these measures decreased following treatment with the aptamer.Harden et al. [25] demonstrated that the upregulation of KYNU mRNA in psoriasis-affected skin is associated with an increase in PASI score [25].H&E staining analysis showed an increase in epidermal thickness of BALB/c mice treated with IMQ on the 4th day of the administration procedure, along with Kynu mRNA expression levels increment [11].Additionally, Salamah et al. [45], in agreement with our findings, indicated that CAMP skin levels and PASI scores increased in the C57/BL6J mice undertreated with IMQ.

Mediators of Inflammation
IMQ is an TLR7/8 agonist, which entry to the circulation after topical administration and leads to a systemic inflammation through the increased of inflammatory cytokines mRNA expression, especially TNF-α in the mice lungs and liver [55].Jabeen et al. [56] demonstrated an association between elevated TNF-α serum levels and spleen length.Furthermore, the enlargement and germinal center activity increment of the lymph node indicated the IMQ systemic effects.Spleen and lymph nodes are secondary lymphoid organs that can be associated with the psoriatic inflammation [56].It appears that IMQ-induced systemic psoriatic inflammation increases the spleen mass, splenocyte count, and size of the spleen and lymph node [56,57,58], which reverses after treatment [59].Our findings showed that spleen and lymph node indexes increased in the IMQ group, while the systemic effects of IMQ were not a notable improvement in comparison to the local effect of anti-IL-17 aptamer in the M7 aptamer group.In our study, the skin local inflammation induced by IMQ leads to skin Camp, Defb2, Kynu, and Penk mRNA expression increment along with effects on the spleen and lymph node.Also, anti-IL-17 local administration reduced the skin mRNA expression levels significantly, but the spleen and lymph node indexes slightly decreased.It seems acceptable because the targeting inhibition of immune pathways locally should not affect the systemic inflammation.

Conclusion
The results of our investigation in Camp, Defb2, Kynu, and Penk gene expression levels proved that changes in these genes occur in IMQ-induced psoriasis.Also, treatment with the anti-IL-17 aptamer showed a decrease in the expression of our studied genes.Interestingly, alterations in Camp, Kynu, and Penk are closely positively correlated with clinical symptoms, and Camp showing a stronger correlation.In conclusion, Camp, Kynu, and Penk can be used as markers to assess the induction of psoriasis vulgaris model and treatment effectiveness.
Score of Mice Groups.The mean AE SEM of erythema (Figure 3(c)), scaling (Figure 3(d)), thickness (Figure 3(e)), and PASI score (Figures 3(b) and 3(f )) in mice dorsal skin were assessed every day.The comparison of the aforementioned parameters 5th-day values average indicated a significant increase in the IMQ group versus the Vas group.The M7 anti-IL-17A aptamer-treated group findings demonstrated a significant decrease in erythema, scaling, thickening, and PASI score versus the IMQ group (P <0:05).

FIGURE 3 :FIGURE 4 : 3 . 5 .
FIGURE 3:  The evaluation of mice weight and PASI score.The measurements of mice weight on 5 consecutive days (a).Evaluation of dorsal skin manifestations following administered with Vas, IMQ, and M7 anti-IL-17A aptamer (b) indicated a significant increase in the IMQ group versus the Vas group, while a significant decrease was recorded in the treatments group versus the IMQ group with regards to the factors of erythema (c), scaling (d), thickness (e), and PASI score (f ).All data of aforementioned parameters are shown as mean AE SEM.Vas, Vaseline (negative control), IMQ, Imiquimod, PASI, psoriasis area and severity index, * P-value < 0.05, * * P-value < 0.01, ns, nonsignificant.

FIGURE 5 :
FIGURE 5: Histopathological analysis (H&E) of tissue sections on the mice dorsal skin (a).Statistical results of the epidermal thickness in the dorsal mice skin showed a significant elevation in the IMQ group and a significant reduction in the M7 anti-IL-17A aptamer (b).The value was displayed based on the epidermal length, and the microscopic region diameter was measured by ImageJ software.Data are displayed as relative to the Vas group.Vas, vaseline (negative control), IMQ, imiquimod, * * * P-value < 0.001.

ðeÞFIGURE 6 :
FIGURE 6:  Relative mRNA expression levels of Kynu (a), Defb2 (b), Camp (c), and Penk (d) analysis.Results indicated a significant increase in the IMQ group versus the Vas group and a significant decrease in the M7 anti-IL-17A aptamer compared to the IMQ group.The mRNA expression levels of the animal's dorsal skin were evaluated by SYBR green real-time PCR.Data are expressed as the fold change AE SEM.RT-PCR procedures were carried out in duplicate and repeated 3 times.Spearman's correlation between inflammatory genes and PASI, erythema, scaling, and thickness of the IMQ group was conducted, "r" presented in the heat map (e).Vas, vaseline (negative control), IMQ, imiquimod, Kynu, kynureninase, Defb2, beta-defensin 2, Camp, cathelicidin antimicrobial peptide, Penk, proenkephalin.* P-value < 0.05.
3.3.Evaluation of Spleen Size, Mass, and Total Cell Count in Mice Group.Evaluation of the spleen total cell count (Figure 4(b)) indicated a significant increase (P ¼ 0:021,) in the IMQ group ((1,030 AE 201.52) 3.4.Evaluation of Popliteal Lymph Node in Mice Group.However, the analysis of popliteal lymph nodes (Figure4(e)) revealed an increase in the count of lymph node cells in the IMQ group ((56 AE 9.94) × 10 4 cell/ml) as compared to the Vas ((33.33 AE 14.65) × 10 4 cell/ml) group and also, the results

TABLE 1 :
The primers were designed for the evaluation of Kynu, Camp, Defb2, and Penk mRNA expression levels.