Datura Metel L. Ameliorates Imiquimod-Induced Psoriasis-Like Dermatitis and Inhibits Inflammatory Cytokines Production through TLR7/8–MyD88–NF-κB–NLRP3 Inflammasome Pathway

Background: Psoriasis is a chronic, immune-mediated inflammatory skin disease, and the inflammatory response plays an important role in its development and progression. Datura metel L. is a traditional Chinese medicine that exhibited a significant therapeutic effect on psoriasis in our previous study due to its remarkable anti-inflammatory effect. Meanwhile, the mechanism underlying its effects on psoriasis is still unclear. Methods: An imiquimod-induced psoriasis-like dermatitis mouse model was constructed to evaluate the protective effect of the effective part of Datura metel L. (EPD), which was verified by evaluations of the Psoriasis Area and Severity Index (PASI) score. Hematoxylin and eosin (H&E) staining, immunohistochemical examination, enzyme-linked immunosorbent assay (ELISA), and Western blot were used to measure the inflammatory cytokines and the protein expression associated with the Toll-like receptor 7– myeloid differentiation primary response gene 88–nuclear Factor-κB–nucleotide-binding oligomerization domain (Nod)-like receptor family pyrin domain-containing 3 (TLR7/8–MyD88–NF-κB–NLRP3) inflammasome pathway. Results: EPD significantly decreased the PASI, reduced epidermal thickness, and decreased the proliferation and differentiation of epidermal cells in psoriasis-like dermatitis C57BL/6 mice induced by imiquimod (IMQ). Furthermore, EPD reduced the infiltration of CD3+ cells to psoriatic lesions, as well as ameliorated the elevations of intercellular adhesion molecule 1 (ICAM-1) and inhibited the production of imiquimod-induced inflammatory cytokines, including IL-1β, IL-2, IL-6, IL-10, IL-12, IL-17, IL-22, IL-23, tumor necrosis factor-α (TNF-α), monocyte chemotactic protein 1 (MCP-1), and interferon-γ (IFN-γ). Besides, EPD decreased the imiquimod-induced expression levels of TLR7, TLR8, TRAF6, MyD88, p-IKKα, p-IKBα, p-NF-κB, NLRP3, apoptosis-associated speck-like protein contained a caspase recruitment domain (ASC), cysteinyl aspartate specific proteinase 1 (caspase-1), and IL-1β. Conclusion: This study demonstrated that EPD exhibited a protective effect on an imiquimod-induced psoriasis mice model by inhibiting the inflammatory response, which might be ascribed to the inhibition of the TLR7/8–MyD88–NF-κb–NLRP3 inflammasome pathway.


Introduction
Psoriasis, a chronic inflammatory skin condition, is commonly considered to be induced by multiple environmental and genetic factors. It is characterized by scaly reddish plaques because of the hyperproliferation and aberrant differentiation of keratinocytes [1]. Psoriasis has been estimated to globally affect approximately 2% to 4% of the population [2]. Meanwhile, besides affliction by a skin disorder, psoriatic patients might have a higher incidence of cardiovascular disease, diabetes, arthritis, depression, and even cancer [3][4][5][6][7]. These conditions had a significant impact on the physical and psychological health of psoriatic patients, and have also caused tremendous socioeconomic and psychological burden [8,9].
The exact pathogenesis of psoriasis is unclear, but it is commonly believed that the inflammatory reaction and abnormal activation of immune cells functions played the key roles in the onset and development of psoriasis. Excessive inflammatory factors activated multiple intracellular signaling pathways and stimulated transcription factors; thus, the cytokines released by immune cells dramatically increased, and the epidermal symptoms proliferated, finally leading to the aggravation of psoriasis. Toll-like receptors (TLRs) are pattern recognition receptors that play important roles in the development of psoriasis. Generally, the main function of TLRs was to mediate the inflammatory response [10,11]. TLR signaling involved the recruitment of adaptor proteins; TLR7/8 were the main TLRs which induced the recruitment of myeloid differentiation primary response gene 88 (MyD88); then, the MyD88-dependent pathway activated nuclear Factor-κB (NF-κB), and this caused various inflammatory cytokines to extensively express, as well as provided the positive feedback effect on the TLR7/8-MyD88-NF-κB signaling, thus resulting in the persistent inflammation [2,12]. Additionally, as one of the downstream factors of TLRs, the nucleotide-binding oligomerization domain (Nod)-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is a cytoplasmic macromolecular complex and consists of NLRP3, cysteinyl aspartate specific proteinase 1 (caspase-1), and apoptosis-associated speck-like protein contained a caspase recruitment domain (ASC). The activated NLRP3 inflammasome could result in the activation of ASC, caspase-1, and the release of mature IL-1β, and eventually promoted the development of inflammation [13]. Accordingly, the pathogenesis and progress of psoriasis were closely related to TLR7/8-MyD88-NF-κB and NLRP3 pathways [14,15].
Nowadays, the treatment of psoriasis mainly included topical treatment, systemic medications, and phototherapy [16,17]. However, these methods induced numerous side effects, such as relapse and adverse drug effects [17]. Since the pathological mechanism behind psoriasis was still not well understood, there was still a lack of safe, effective, and commonly accepted therapeutics [18]. Hence, it was urgent to find an agent with obvious therapeutic effect and low side effects in the treatment of psoriasis.
Numerous natural products with anti-inflammatory effects derived from medicinal plants, especially those used in traditional Chinese medicine, have been extensively used in clinical settings to prevent and treat many diseases with the advantages of minimal side effects and significant effectiveness [19,20]. The flowers of Datura metel L., namely Flos daturae, have been confirmed with obvious effects in the treatment of psoriasis because of their remarkable anti-inflammatory activity in our previous studies, and have been used in clinical settings [21][22][23][24][25][26]. Anolides and flavonoids were the main active ingredients of Flos daturae for psoriasis [22][23][24][25][26]. However, the precise mechanism by which the intragastric administration of Flos daturae exerted its anti-psoriatic effect remained unknown, which has restricted its widespread use.
Hence, this research was aimed at further proving and evaluating the anti-psoriasis effect of Flos daturae as well as exploring the roles of the TLR7/8-MyD88-NF-κB and NLRP3 pathways in its underlying mechanisms.

The Effective Part of Datura metel L. (EPD) Treatment Ameliorated IMQ-Induced Psoriasis-Like Skin Lesions
For investigating the protective effects of EPD on psoriasis mice induced by imiquimod (IMQ), EPD was orally administrated once daily 4 h after modeling for seven consecutive days, and the severity of lesions (thickening, scaling, erythema) on the back skin were scored on the basis of PASI. As shown in Figure 1A, the dorsal skin of the mice in the control group remained smooth and showed no morphological changes; however, compared to the control, typical symptoms of scaling, thickness, and erythema were observed after two to three days of modeling with IMQ, and presented a rough lesion resembling human plaque psoriasis. As expected, skin lesions in the EPD treatment groups were obviously alleviated in a dose-dependent manner, and displayed a significant improvement in scaling, thickening, and erythema as well as the reduced cumulative score compared to the model group ( Figure 1B). The above results indicated that EPD significantly improved the clinical symptoms of the psoriasiform mice induced by IMQ.
The histopathological changes of EPD on the skin lesions induced by IMQ were evaluated used hematoxylin and eosin (H&E) staining ( Figure 1C). The mice induced by IMQ showed pathological psoriatic lesions, including loss of the granular layer, epidermal hyperplasia, thickening of the acanthosis cell layer, and parakeratosis, as well as inflammatory cell infiltration. Compared to 13.01 ± 1.20 µm in the control ( Figure 1D), the thickness of epidermis in the model was 64.36 ± 4.80 µm. Oral administration of a high dose and low dose of EPD could weaken most of these changes mentioned above; the thickness of epidermis were 32.18 ± 4.09 and 38.52 ± 2.06 µm, respectively.

EPD Inhibited the Aberrant Expression of the Proliferation and Differentiation Markers of Keratinocytes in Mice Dorsal Skin Induced by IMQ
Studies have revealed that proliferating cell nuclear antigen (PCNA) was an important indicator of cell mitosis and expressed in proliferative cells, especially basal cells, while Ki-67 was the hallmark protein involved in cell proliferation [2]. To investigate the relevance of the therapeutic effects of EPD on psoriasis and the inhibition of the hyperproliferation of keratinocyte, the expression of PCNA and Ki-67 in the lesional skin of mice were detected by immunohistochemistry. The quantification of positive immunostaining cells showed that abundant amounts of activated PCNA and Ki-67 were significantly observed in the model compared to the control group (P < 0.01); however, the expression of PCNA and Ki-67 in the skin lesions were reduced after treating with EPD, and these findings indicated that EPD reduced the IMQ-induced abnormal proliferation and differentiation of keratinocytes (Figure 2A-C).
Involucrin was a marker of epidermal differentiation and played a key role in the skin barrier function of the epidermis, which was mainly expressed in the cytoplasm [27,28]. Western blot analyses reveled that the expression of involucrin was significantly higher in the model compared to the control (P < 0.01). However, EPD treatment (both for group with a high dose of EPD, or EPD-H, and low dose of EPD, or EPD-L) remarkably decreased involucrin expression (P < 0.01, Figure 2D,E).

EPD Inhibited Inflammatory Cell Infiltration in IMQ-Induced Psoriasis-Like Mouse Mode
The infiltration of leukocytes into the skin could be regarded as one of characteristics of inflammatory immune responses in the psoriasis, and the increment of intercellular adhesion molecule 1 (ICAM-1) expression in the psoriatic lesions could improve the infiltration of leukocytes [29]. In our study, the expression of ICAM-1 was remarkably increased in the model group, and this trend was reversed through EPD treatment (P < 0.01, Figure 3A,B). Moreover, CD3+ T cells play an important role in the skin lesion in psoriasis [30]. Our immunohistochemical results revealed that CD3 expression in the skin of the model group mice was significantly higher than that of the control group. Importantly, a significant decrease in CD3 expression was observed in mice treated with EPD (P < 0.01, Figure 3A-C).

EPD Administration Downregulated the Expression of Inflammatory Cytokines
The expressions of crucial inflammatory mediators (including IL-1β, IL-2, IL-6, IL-10, IL-17, IL-22, IL-23, IFN-γ, TNF-α) in skin tissues were measured by ELISA due to the import role of inflammatory cytokines in psoriasis. Our results showed that the levels of IL-1β, IL-2, IL-6, IL-10, IL-12, IL-17, IL-22, IL-23, IFN-γ, monocyte chemotactic protein 1 (MCP-1), and tumor necrosis factor-α (TNF-α) in the skin of mice were all significantly increased after IMQ treatment, whereas the expressions of these pro-inflammatory cytokines were dramatically reduced in a dose-dependent manner after oral administration with EPD ( Figure 4). Our experimental data displayed that regulating inflammatory cytokines might contribute to the treatment effect of EPD for psoriasis.

EPD Suppressed the TLR7/8-MyD88-NF-κB Signaling Pathways and Accordingly Exerted the Therapeutic Effect for Psoriasis
Various studies presented evidence that the transduction pathway of the TLR7/8-MyD88-NF-κB signal was considered as an early event that was essential for inflammatory responses [28,31]. Thus, the protein expression levels of TLR7 and TLR8 in IMQ-induced psoriasis mice were detected. As shown in Figure 5, the TLR7 and TLR8 expression were increased by IMQ stimulation, which were not shown in the control group, and the increase was resisted by EPD treatment (P < 0.05). TLRs' signaling involved the recruitment of the MyD88 adapter protein and eventually activated NF-κB; in our study, Western blot analysis was used to monitor the changes of downstream-signaling molecules related to TLR7/8 in skin tissues. The expressions of both TRAF6 and MyD88 were obviously elevated by IMQ treatment, as well as the phosphorylation level of IKKα, IKBα, and NF-κB in the model compared to control (P < 0.01), while the administration of EPD significantly reversed those (P < 0.01, Figure 5). The results indicated that the inhibition of the TLR7/8-MyD88-NF-κB pathway was relevant to the protective effects of EPD on IMQ-induced psoriasis.

EPD Inhibited the IMQ-Activated NLRP3 Inflammasome Signaling Pathway
The expression of inflammatory mediators was closely associated with the activation of the NLRP3 inflammasome [32]. Here, the proteins' expression of the NLRP3 inflammasome pathway was determined by immunohistochemical and Western blot analysis. The immunohistochemical results showed that the NLRP3 inflammasome signals were observed to be significantly increased in the psoriasis mice; furthermore, the expressions of NLRP3, ASC, caspase-1, and IL-1β were upregulated, which were all inhibited through EPD treatment ( Figure 6A-E). Similar results were also found using Western blotting ( Figure 6F-J).

Discussion
Psoriasis is a skin disease that has been characterized as common, chronic, and recurring. The mechanisms that participate in the genesis of psoriasis are complex, and previous research studies demonstrated that the typical form of psoriasis exaggerated the infiltration of specific immune and inflammatory cells in the epidermis and dermis. These effects in turn sustained the inflammatory extent of the skin layer, and subsequently functionally increased the proliferation and aberrant differentiation of keratinocytes and the destruction of the skin [33,34]. Previously, we found that Datura metel L. had an obvious effect on psoriasis, and withanolides and flavonoids were the main constituents of the EPD [22][23][24]. A growing body of research also proved that withanolides and flavonoids exhibited excellent anti-inflammatory activity [35].
IMQ was both a potent immune activator and Toll-like receptor agonist. A high-dose topical application of IMQ repeatedly on mouse ear or dorsal skin could induce erythema, scaling, keratinocyte proliferation with acanthosis, and also increase the expressions of immune cells and their related cytokines; all of these features are consistent with psoriatic conditions [36,37]. Therefore, the IMQ-induced mice model was widely applied in the research studies on psoriasis [38]. Additionally, Swindell WR's research demonstrated that the IMQ-induced dermatitis was strain-dependent, and C57BL/6 mice were more suitable for the psoriasis modeling and pathogenesis research because of their better genetic background than other strains [39]. In our study, the symptom of psoriasis-like lesions in animal C57BL/6 mice, which were induced by the topical administration of IMQ, was similar to that found in the literature [40,41]. The administration of EPD could lower scores of PASI and significantly attenuate the thickened epidermis in a psoriasis-like mice model. In addition, histological analyses showed that the mice treated with EPD exhibited smoother epidermis, less epidermal thickening, and parakeratosis. These findings suggested that EPD had remarkable treatment effects on a psoriasis-like mice model induced by IMQ. In the development of psoriasis, activated keratinocytes were considered to play a crucial role [41,42]. The results suggested that EPD obviously inhibited the expression of PCNA, Ki-67, and involucrin, which were the important markers of the proliferation or differentiation of keratinocytes [43]. Several studies indicated that the excessive proliferation of keratinocytes could lead to the activation of cytokine production, which was closely related to the psoriatic pathogenesis in the inflammatory regulation, and the levels of these cytokines were positively correlated with the severity of disease [44][45][46]. More importantly, the activated keratinocytes could produce large amounts of inflammatory cytokines, which induced proliferation and survival and in turn elevated keratinocyte proliferation and epidermal thickening, and then formed a positive loop to perpetuate the psoriatic lesions [36]. The data in our study results showed that the expression of inflammatory factors was markedly inhibited after the treatment of EPD in psoriasiform mice induced by IMQ.
Generally, imiquimod was an agonist for TLR7/8, and TLR7/8 signaling was essential for innate immune response inflammatory signaling pathways, and considered to be involved in the pathology of imiquimod-induced psoriasis-like dermatitis [41]. Once activated by their ligands, TLR7/8 played a regulatory role in the inflammation by interacting with MyD88 and the Toll receptor-associated activator of interferon, which were two distinct adaptor proteins. Activated TLR7/8 promoted the expression of MyD88 and influenced the expression of its downstream protein TRAF6, as well as increased the level of inflammatory factors with the upregulation of MyD88 and TRAF6. In turn, these inflammatory factors played a vital role in the TLR-mediated MyD88-dependent pathway [47]. Moreover, activated TRAF6 degraded the inhibitor of NF-κB kinases (IKKs), led to phosphorylation, and also degraded the IκB-α, which was the inhibitory protein of NF-κB. Then, the NF-κB translocated into the nucleus in order to trigger NF-κB signaling pathways after separating from IκB-α, which had been reported to be involved in the pathological process of IMQ-induced psoriasis lesions [48,49]. Our present study showed that the protein expression levels of TLR7, TLR8, MyD88, and p-NF-κB in the skin of mice induced by IMQ were upregulated significantly; consequently, TLR7/8-MyD88-NF-κB signaling was considered to play a critical role in IMQ-induced psoriasis. It was worth noting that all of these changes were obviously reversed by EPD treatment, which provided evidence that TLR7/8-MyD88-NF-κB signaling was involved in the therapeutic action of EPD on psoriasis.
The NLRP3 inflammasome was an important intracellular multiprotein inflammatory pathway and governed the productions of pro-inflammatory cytokines [14,50]. The NLRP3 inflammasome consisted of three sections, including NLRP3, ASC, and caspase-1. Ample evidence had shown that NLRP3 was particularly activated in psoriasis, and it was expressed both in the inflammatory cellular infiltrate in the dermis and in the epithelial cells of the psoriatic epidermis. The activation of NLRP3 triggered a cascade process, including ASC and pro-caspase-1 recruitment, caspase-1 activation, and pro-IL-1β being cleavaged into its mature form. Here, the activated NLRP3 inflammasome as an upstream trigger could govern the pathogenesis of tissular inflammatory injuries in psoriasis [51]. Thus, the targeted inhibition of the NLRP3 pathway, and subsequent reducing inflammatory injury might be an effective method of psoriasis treatment. The results from our current experiments suggested that the oral administration of EPD effectively downregulated the expressions of IL-1β and ASC caspase-1 as well as NLRP3.

The Effective Part of Datura metel L. (EPD) Preparation
The EPD was prepared according to our previous study [52]. Briefly, dried Flos daturae were refluxed with 70% alcohol (1:30, w/v, 120 min × 3). Then, the extract was acidified with 0.1% hydrochloric acid. After filtration, the filtrate was subjected to 732 cation exchange resin; then, the effluent was chromatographed on AB-8 macroporous resin and eluted by H 2 O, 50% ethanol, and 95% ethanol, respectively. Then, 50% ethanol eluate was collected and vacuum evaporated to obtain dried powder, which was regarded as the EPD for psoriasis. Kaempferol-3-O-β-d-glucopyranosyl-(1→2)-β-d-galactopyranosyl-7-O-α-l-rhamnopyranoside and daturataturin A were used as control standards; the contents of the total flavonoids and total withanolides of EPD were determined by ultraviolet-visible spectrophotometry, with contents of 55.25% and 23.26%, respectively [53,54]. EPD was stored at 4 • C before use and dissolved homogeneously by using saline when administrated.

Experimental Animal
Male C57BL/6 mice (No. SCXK(JING) 2016-0006; Beijing Vital River Laboratory Animal Technology Co., Ltd., China) 7 weeks to 8 weeks old, weighing between 20-24 g, were used in the current study. In addition, all the mice were allowed to acclimatize to drink and eat at will and housed under controlled conditions (maintained at 23 ± 2 • C, 40% to 50% relative humidity) for 7 days before the start of the experiment.

Establishment of Psoriasis-Like Dermatitis Mice Model and the Administration with EPD
The experimental process was strictly approved by the Animal Ethics Committee of Heilongjiang University of Traditional Chinese Medicine (Heilongjiang, China) on 14 September 2017 (number 2017091403). Healthy male C57BL/6 mice (n = 32) were assigned into four groups after shaved to dorsal skin randomly: control, model, high dose of EPD (EPD-H), and low dose of EPD (EPD-L). According to the previous study, 62.5 mg of commercially available IMQ cream (Sichuan Mingxin Pharmaceutical Co., Ltd., Chengdu, China) was applied topically once per day to a 2 cm × 3 cm shaved area dorsal skin of mice in the model, EPD-H, and EPD-L groups for 7 days in a row to induce a psoriasis-like mice model [39]. Mice in the control were treated similarly with petroleum jelly to the same skin sites according to the same schedule. The treatment and experimental design are shown in Figure 7. The mice in the EPD-H and EPD-L groups received an intragastric administration of EPD 104 mg/kg/day and 52 mg/kg/day after 4 h of each modeling [23]. Furthermore, the mice in the control and model received oral administration with the same volume of saline.

Psoriasis Area and Severity Index (PASI) Assessment
Based on PASI, the severity of the skin inflammation was evaluated once daily, including the measurements for skin erythema, scaling, thickening and scored separately using a five-point scale from 0 to 4, namely: 0, none; 1, slight; 2, moderate; 3, marked; and 4, very marked [36].

Histological Analysis
After 24 h of final modeling, the dorsal skin tissues were harvested from mice and excised on ice. Then, part of the excised skin samples were washed with saline and wiped. Then, after liquid nitrogen freezing, the skin samples were stored under −80 • C and used for the further experiments. The other part of the skin samples was fixed in 10% neutral formalin, embedded in paraffin, and sectioned into 5-µm thick sections and stained with hematoxylin and eosin (H&E) under a light microscope for pathological observation (OLYMPUS BX60, 200× magnification). The average optical density (AOD) of related indicators and acanthosis (epidermal thickness) of skin were measured by image analysis software (OLYMPUS DP72).

Detection of Inflammatory Cytokines in Skin Tissues Lysate
Skin samples were ground with liquid nitrogen, and the Tissue Protein Extraction Reagent (78510, Thermo Fisher Scientific, MA, USA) was then used to extract the skin protein. The extracted proteins were centrifuged for 20 min (12,000 rpm). Subsequently, the supernatant was collected, and the concentrations of protein were measured using the bicinchoninic acid (BCA) protein assay kit (23227, Thermo Fisher Scientific). Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used to determine the expression of cytokines, including IL-1β, IL-2, IL-6, IL-10, IL-12, IL-17, IL-22, IL-23, tumor necrosis factor-α (TNF-α), and monocyte chemotactic protein 1 (MCP-1) as well as interferon-γ (IFN-γ) (CUSABIO BIOTECH, Ltd. Wuhan, China); all the procedures were strictly according to the instructions of manufacturer.

Statistical Analysis
The data were all expressed as mean ± SD. All the standard statistical analysis was performed using GraphPad Prism Version 7.0 (GraphPad Software, La Jolla, CA, USA) software, including one-way Analysis of Variance (ANOVA) followed by multiple comparisons between groups using Tukey's post-hoc test. Statistically significant differences were identified as either P less than 0.05 or P less than 0.01.

Conclusions
The mouse model of psoriasis-like dermatitis induced by IMQ was first used for evaluating the therapeutic effect of EPD for psoriasis, and exploring the mechanism of the inflammation-signaling pathway. Our study indicated that EPD could alleviate psoriasis in the IMQ-induced mice model and that the potential mechanisms that were involved were, at least in part, due to inhibiting the inflammation responses via the TLR7/8-MyD88-NF-κb-NLRP3 inflammasome signaling pathway (Figure 8). Our findings provided the more scientific information in vivo about a clinical value of Datura metel L. for psoriasis.

Conflicts of Interest:
The authors declare that there are no conflicts of interest.