Topical astilbin ameliorates imiquimod‐induced psoriasis‐like skin lesions in SKH‐1 mice via suppression dendritic cell‐Th17 inflammation axis

Abstract Astilbin, an essential component of Rhizoma smilacis glabrae, exerts significant antioxidant and anti‐inflammatory effects against various autoimmune diseases. We have previously reported that astilbin decreases proliferation and improves differentiation of HaCaT keratinocytes in a psoriatic model. The present study was designed to evaluate the potential therapeutic effects of topical administration of astilbin on an imiquimod (IMQ)‐induced psoriasis‐like murine model and to reveal their underlying mechanisms. Topical administration of astilbin at a lower dose alleviated IMQ‐induced psoriasis‐like skin lesions by inducing the differentiation of epidermal keratinocytes in mice, and the therapeutic effect was even better than that of calcipotriol. Moreover, the inflammatory skin disorder was relieved by astilbin treatment characterized by a reduction in both IL‐17‐producing T cell accumulation and psoriasis‐specific cytokine expression in skin lesions. Furthermore, we found that astilbin inhibited R837‐induced maturation and activation of bone marrow‐derived dendritic cells and decreased the expression of pro‐inflammatory cytokines by downregulating myeloid differentiation factor 88. Our findings provide the convincing evidence that lower doses of astilbin might attenuate psoriasis by interfering with the abnormal activation and differentiation of keratinocytes and accumulation of IL‐17‐producing T cells in skin lesions. Our results strongly support the pre‐clinical application of astilbin for psoriasis treatment.

Astilbin, an important bioactive ingredient derived from Rhizoma smilacis glabrae, is widely used in traditional Chinese medicine for the treatment of autoimmune diseases and possesses antiinflammatory activity and immunoregulatory effects. [9][10][11][12] Oral astilbin (50 or 25 mg/kg) is effective in the treatment of psoriasis by inhibiting Th17 cell differentiation. 13 Moreover, astilbin regulated in vitro keratinocyte differentiation in our study. 14 Previous study showed that astilbin at 50 μg/ml began to play an inhibitory role on HaCaT cell proliferation, but 50 μg/ml is a very high concentration for most compounds. Given that the DC-Th17 inflammation axis plays a central role in the pathology of psoriasis, we detected the effect of astilbin on DCs. A lower dose of astilbin at a concentration of 10-20 μg/ml can inhibit the maturation and activation of bone marrowderived dendritic cells (BMDCs). Therefore, in this study, we aimed to evaluate the potential therapeutic effects of topical administration of astilbin on an imiquimod (IMQ)-induced psoriasis-like murine model and reveal their underlying mechanisms. IMQ is a ligand of TLR7/8 and acts as a potent immune activator of DCs and causes them to mature, leading to psoriasis-like skin lesions when applied to the skin. 15,16 IMQ-induced skin lesions are a classic model of psoriasis. This study used the hairless, but immunocompetent SKH-1 mice.
This murine strain lacks active hair follicles and a thicker surface epidermis that closely mimics human skin. SKH-1 mice are suitable for experimental dermatology and have been widely used in research on wound healing, atopic dermatitis and ultraviolet radiation-induced skin photoaging. [17][18][19] The results showed that topical administration of astilbin at a lower dose level (4 or 10 mg/kg daily) alleviated the IMQ-induced psoriasis-like skin lesions by inducing the differentiation of epidermal keratinocytes in mice. Moreover, the inflammatory skin disorder was restored by astilbin treatment characterized by reduced accumulation of both IL-17-producing T cells and expression of psoriasisspecific cytokines in skin lesions via downregulation of myeloid differentiation factor 88 (MyD88). Therefore, use of astilbin to control the maturation and activation of DCs by reducing MyD88 in TLR7/8 signals may be an effective strategy to treat psoriasis.

| Animals
SKH-1 hairless mice were purchased from Charles River Laboratories (Wilmington) and housed in a pathogen-free facility at a temperature of 25 ± 5°C and 55 ± 5% humidity. The mice were given access to a standard laboratory diet and water. All animal experiments were

| Scoring severity of skin inflammation
To evaluate the severity of psoriasis-like skin lesions, an objective scoring system was adopted based on the clinical Psoriasis Area and Severity Index (PASI). Erythema, scaling and infiltration were scored independently on a scale from 0 to 4 as follows: 0, none; 1, slight; 2, moderate; 3, marked and 4, very marked. Erythema was scored according to the red tints. The total score (erythema plus scaling plus infiltration) served as a measure of the severity of inflammation (scale from 0 to 12).

| Skin cell preparation and flow cytometry assay
Mouse back skin was incubated in 1.

| Skin histology and immunochemical staining
The mice were sacrificed, and the skin samples were fixed in paraformaldehyde and embedded in paraffin. Skin sections (5 μm) were stained with haematoxylin and eosin and Ki67 antibodies (Abcam), and staining was assessed using light microscopy (Olympus). Epidermal thickness was determined by measuring the average interfollicular distance under a microscope in a blinded manner.

| Immunofluorescence staining
Skin samples from the back lesions of mice embedded and frozen in OCT medium for immunofluorescence staining. Skin sections (10 μm) were stained for KRT5 (Abcam), and staining was assessed in a blinded manner using a NanoZoomer S60 fluorescent microscope (Hamamatsu Photonics K.K.) to determine the thickness of the undifferentiated epidermis.

| ELISA
Skin tissue samples from the back lesions of mice were obtained and snap-frozen in liquid nitrogen. Then, the tissues were weighed and homogenized in ice-cold PBS with a protease inhibitor cocktail, followed by centrifugation at 12,000 g for 20 min at 4°C. The supernatant was stored at −80°C and used for ELISA assays within a month.
Supernatants from cultured BMDCs stimulated with astilbin and/or R837 were collected and stored at −80°C for ELISA assays within a month. Protein levels of IL-1β, IL-6, IL-17A and IL-23 in the skin tissue samples, and culture samples were measured using ELISA kits from R&D Systems, following the manufacturer's protocol.

| Real-time polymerase chain reaction (RT-PCR)
Total RNA was extracted from skin lesions using an RNAiso Plus kit The primer sequences are listed in Table 1.

| Western blot assay
After treatment with chemicals, the cells or skin were lysed for Western blotting as described previously. 14 The blots were incu-

| Statistical analysis
Data were expressed as the mean ± SD or the mean ± SEM. For comparison of the significant differences between more than two groups, one-way ANOVA tests were used, while other comparisons were performed with Student's unpaired t test. All the statistical analyses were conducted using the GraphPad Prism software.

| Topical astilbin significantly attenuated IMQinduced psoriasis-like skin lesions in mice in a dosedependent manner
We first investigated the effect of astilbin on an IMQ-induced pso-

| Topical astilbin reduced proliferation and promoted differentiation of keratinocytes in IMQinduced psoriasis-like skin lesions
The epidermis is mainly composed of keratinocytes that undergo a gradual process of differentiation from basal to supra-basal layers.
The balance between proliferation and differentiation of keratino-

| Topical astilbin decreased IMQ-induced skin inflammation in mice
The IL-23/Th17 axis plays a role in the pathology of psoriasis. 6 To investigate the effect of topical astilbin on IL-17-producing T cells, CD4 + IL-17A + T cells in CD3 + cells were investigated by flow cytometry. As shown in Figure 3A,B, in CD3 + cells of IMQ-induced psoriasis-like skin lesions, CD4 + IL-17A + T cells were significantly increased (12.760 ± 3.12% vs. 0.250 ± 0.082% in control skin lesions); while in astilbin-treated mice, IL-17A + cells were significantly reduced in a dose-dependent manner (5.410 ± 2.260% in the AL group and 1.575 ± 0.410% in the AH group). In addition, the mRNA level of the transcription factor RORγt, which specifically induces Th17 cell differentiation was reduced ( Figure 3C).
The mRNA levels of psoriasis-related inflammatory factors such as TNFα, IL-23, IL-17A, IL-1β and IL-6 were measured by RT-PCR.

| Astilbin inhibited the maturation and activation of BMDCs and decreased the expression of pro-inflammatory cytokines
It is well known that mature dendritic cells (mDCs) affect the mat- In IMQ-induced psoriasis-like skin lesions, CD11c + MHCII + DCs were significantly increased (1.19 ± 0.11% vs. 0.38 ± 0.04% in control skin lesions); while in astilbin-treated mice, CD11c + MHCⅡ + DCs were significantly reduced in a dose-dependent manner (0.96 ± 0.078% in the AL group and 0.492 ± 0.08% in the AH group).
We further explored whether astilbin inhibits the activation of TLR7/8 signalling by detecting MyD88. The results ( Figure 5C) showed that astilbin significantly prevented the overexpression

F I G U R E 4 Astilbin inhibits R837-induced maturation and activation of bone marrow-derived dendritic cells (BMDCs). (A) Gating strategy
for CD11c+ BMDCs (gate on the right). BMDCs were stained with CD11c and isotype control mAbs to reveal CD11c + cells. (B) Flow cytometry was performed to analyse the expression change of MHCⅡ/CD86/CD80 in BMDCs that were exposed to R837 and/or different concentration of astilbin. (C-E) The rate of BMDCs expressing MHCⅡ/CD86/CD80 stimulated by R837 and/or different concentration of astilbin was statistically analysed. Data are expressed as the mean ± SD (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001. (F-I) ELISA was performed to assess the concentration of TNFα, IL-1β, IL-6 and IL-23 in the supernatant of R837-induced BMDCs pretreated or not with astilbin (10 or 20 μg/ml, 6 h). The results are shown as the mean ± SD of three separate experiments (*p < 0.05; **p < 0.01; ***p < 0.001) of MyD88 stimulated by R837 in DCs. In addition, lower protein expression levels of MyD88 were observed in skin lesions of the astilbin-treated group as compared to those levels in the IMQinduced model ( Figure 5D).

| DISCUSS ION
Astilbin is the predominant bioactive component extracted from the Chinese medicinal herb-Rhizome smilacis glabrae, which is traditionally used to treat inflammatory diseases. 21 In recent years, astilbin has attracted considerable attention for its antioxidant and anti-inflammatory effects, 22,23 with no hepatotoxicity, renal toxicity or genotoxicity. 24 Our previous study 14 and the study by Li et al. 13   In conclusion, previous studies have reported that oral astilbin could inhibit Th17 cell differentiation and ameliorate IMQ-induced psoriasis-like skin lesions by inhibiting the Jak3/Stat3 signalling pathway in BALB/c mice. 13 Furthermore, astilbin possesses antiproliferative and differentiation-modulating effects on keratinocytes in vitro. This study revealed that topical administration of astilbin at a lower dose targeted the TLR7/8 signalling pathway, subsequently inhibiting Th17/IL-17A-induced immune responses and excessive keratinocyte proliferation to ameliorate IMQinduced psoriasis-like skin lesions in SKH-1 mice. Therefore, topical administration of astilbin may be a promising agent for psoriasis treatment, and appropriate dosage forms for external use are under development.

ACK N OWLED G EM ENTS
We would like to thank Dr. Xiaochen Wang for providing support in the preparation of drugs and writing in the early stages of the article. This work was supported by grants from the National Natural

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.