Chrysin alleviates imiquimod-induced psoriasis-like skin inflammation and reduces the release of CCL20 and antimicrobial peptides.

Psoriasis is a common non-contagious chronic inflammatory skin lesion, with frequent recurrence. It mainly occurs due to aberrant regulation of the immune system leading to abnormal proliferation of skin cells. However, the pathogenic mechanisms of psoriasis are not fully understood. Although most of the current therapies are mostly efficient, the side effects can result in therapy stop, which makes the effectiveness of treatment strategies limited. Therefore, it is urgent and necessary to develop novel therapeutics. Here, we investigated the efficacy of chrysin, a plant flavonoid, which we previously reported to possess strong antioxidant and anti-inflammatory effects, against psoriasis-like inflammation. Our results revealed that chrysin significantly attenuated imiquimod-induced psoriasis-like skin lesions in mice, and improved imiquimod-induced disruption of skin barrier. Moreover, the TNF-α, IL-17A, and IL-22-induced phosphorylation of MAPK and JAK-STAT pathways, and activation of the NF-κB pathway were also attenuated by chrysin pretreatment of epidermal keratinocytes. Most importantly, chrysin reduced TNF-α-, IL-17A-, and IL-22-induced CCL20 and antimicrobial peptide release from epidermal keratinocytes. Thus, our findings indicate that chrysin may have therapeutic potential against inflammatory skin diseases. Our study provides a basis for further investigating chrysin as a novel pharmacologic agent and contributes to the academic advancement in the field of Chinese herbal medicine.

www.nature.com/scientificreports www.nature.com/scientificreports/ by IMQ treatment was improved by chrysin pretreatment. The histology of chrysin-pretreated skin exhibited significant improvement with respect to inflammatory symptoms. The total scoring of Psoriasis Area and Severity Index (PASI) showed that chrysin-pretreated groups showed significantly reduced PASI score compared with that of the IMQ group (Fig. 2D).
After IMQ stimulation, trans-epidermal water loss and values of physiological parameters, such as erythema, blood flow, and ear thickness, increased significantly. In addition, the surface skin hydration (corneometer) was greatly reduced. These results showed that IMQ induced an inflammatory state in the skin. The pretreatment by topical application of chrysin significantly reduced the trans-epidermal water loss, erythema, blood flow, and Quantification of mouse skin histology examined using the PASI score. In each group, mice were stimulated with or without IMQ after pretreatment with chrysin. We performed in vivo experiments in at least six mice per treatment group, and data represent the mean ± SEM from at least six independent experiments. *p < 0.05 was considered to be statistically significant.  MTT assay, (B) crystal violet assay, and (C) trypan-blue exclusion method are the most common methods used to determine cell viability. We treated NHEKs with different concentrations of chrysin (1-50 μM), and the results showed that chrysin exhibited no cytotoxicity in the concentration range of 1-30 μM. However, at a concentration of 50 μM, the cell viability was decreased, it was shown that chrysin has cytotoxicity at this concentration. Therefore, in subsequent experiments, the concentrations of chrysin 3, 10, and 30 μM were selected. Results are expressed as a percentage of control value and are represented by mean ± SEM from at least three independent experiments. *p < 0.05 was considered to be statistically significant. www.nature.com/scientificreports www.nature.com/scientificreports/ chrysin was not cytotoxic to NHEK at concentrations between 0-30 μM, we chose three concentrations of 3, 10, and 30 μM for further experiments.
TNF-α-stimulated NHEK cells showed a significant increase in levels of the mitogen-activated protein kinase (MAPK) pathway components, including p38 kinase, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), when compared with those in the controls. Pretreatment with chrysin at various concentrations (3, 10, and 30 μM) significantly attenuated the TNF-α-stimulated increase in phosphorylation of p38 kinase, ERK, and JNK proteins (Fig. 5A,B). We also investigated the effect of chrysin on IL-17A-induced MAPK pathway activation using western blot analysis. The results showed that the phosphorylation of p38 kinase, ERK, and JNK activated only after IL-17A treatment. Pretreatment with chrysin at the working concentrations (3, 10, and 30 μM) inhibited the IL-17A-induced MAPK phosphorylation in a dose-dependent manner in NHEK Figure 5. Chrysin inhibits phosphorylation of MAPK and JAK/STAT pathway components induced by TNF-α, IL-17A, and IL-22 in normal human epidermal keratinocytes (NHEK). NHEK were pretreated with different doses of chrysin, and stimulated with either (A) TNF-α, (C) IL-17A, or (E) IL-22, and the quantification data are shown in the right panel (B,D,F). The total protein was extracted from the cells and associated protein expression was determined via western blotting. The results are presented as a percentage of the control. *p < 0.05 was considered to be statistically significant. (2020) 10:2932 | https://doi.org/10.1038/s41598-020-60050-1 www.nature.com/scientificreports www.nature.com/scientificreports/ ( Fig. 5C,D). In addition, we examined the effects of chrysin on IL-22-induced phosphorylation of MAPK protein in NHEK using western blot analysis. The results showed that IL-22 treatment upregulated the phosphorylation of p38 kinase, ERK, and JNK, and chrysin pretreatment downregulated the activation of MAPK proteins following IL-22 stimulation (Fig. 5E,F).
Previous studies showed that the JAK-STAT pathway is a classical signal transduction pathway and is triggered by IL-22 stimulation. Therefore, we investigated the effects of chrysin on the activation of these related signalling molecules. After chrysin treatment, western blot was performed to identify the protein expression of phosphorylated JAK2 and STAT3. As shown in Fig. 5E,F, p-JAK2 and p-STAT3 levels were decreased by chrysin pretreatment compared with the IL-22 only group. These results suggested that TNF-α, IL-17A, and IL-22 indeed elicit psoriatic inflammation, and chrysin can significantly inhibit the production of these proinflammatory mediators. chrysin suppressed tnf-α-or IL-17A-induced NF-κB pathway activation in NHEKs. NF-κB pathway is the key regulatory pathway during inflammation, and is considered to be a crucial mediator in the pathogenesis of psoriasis 42 . Therefore, we investigated the effects of chrysin on TNF-α-or IL-17A-mediated induction of α isoform of IκB (IκBα) in the NF-κB signalling pathway using western blot analysis. TNF-α-or IL-17A-stimulated NHEK cells showed a significant increase in IκBα protein levels when compared to the controls. In addition, pretreatment with chrysin downregulated the phosphorylation of IκBα protein. These results indicated that TNF-α and IL-17A treatments promote the NF-κB pathway, and this effect was reduced by chrysin pretreatment (Fig. 6A-D).
Chrysin downregulated the mRNA expression of CCL20 and AMPs in NHEKs induced by TNF-α, IL-17A, or IL-22 recombination proteins. In addition to providing a basic physical barrier, the skin is also an outpost of the immune response. These immune reactions are crucial responses to injury or infection, and can also regulate the skin symbiotic microbiota 43 . While the skin is infected with harmful pathogens (mostly microorganisms), keratinocytes are activated to secrete antimicrobial peptides and proteins (AMPs), cytokines, and chemokines to trigger an immune response. Chemokines, like CCL20, and AMPs, including cathelicidin, β-defensins, and S100 proteins, are speculated to intensify psoriasis lesions 44 , and excessive production of AMPs is an important characteristic of psoriasis lesions [43][44][45] . Previous studies have reported that TNFα, IL-17A, and IL-22 upregulate the expression of CCL20 and AMPs [46][47][48] . Therefore, in this study, we investigated whether chrysin can downregulate the expression of CCL20 and AMPs via TNFα, IL-17A, and IL-22 stimulation. Our results showed that the expression of CCL20, S100A7, S100A8, S100A9, hBD2, and LL-37 were significantly increased by TNFα, IL-17A, and IL-22 stimulation. In the group pretreated with chrysin, the expression of CCL20 and AMPs were significantly suppressed ( Fig. 7A-C).

Discussion
This study showed that chrysin can ameliorate the inflammation in psoriasis, and the underlying mechanism involves the regulation of three signalling pathways, namely MAPK, JAK-STAT, and IKK/NF-κB pathways. This is consistent with the results of our previous study, wherein chrysin was found to protect skin cells against the photodamage caused by UVA and UVB via the regulation of MAPK pathway and inhibition of ROS production 33 . In this study, we explored several factors that contributed to psoriasis, such as TNF-α, IL-17A, and IL-22 and found that chrysin could ameliorate the inflammatory reactions induced by these factors. www.nature.com/scientificreports www.nature.com/scientificreports/ The most important pathological characteristics of psoriasis include hyperinflammation, epidermal keratinocyte proliferation and abnormal differentiation, and angiogenesis. The pathogenesis of psoriasis is a highly complex molecular mechanism, involving a variety of immune and inflammatory mediators. Several innate immune , and RT-qPCR was used to detect the effects of chrysin on the mRNA expression of CCL20, S100A7, S100A8, S100A9, hBD2, and LL-37. Data are expressed as fold induction of relevant mRNA sequences compared to untreated controls. Data represent mean ± SEM from at least three independent experiments performed in triplicates; *p < 0.05 was considered to be statistically significant. www.nature.com/scientificreports www.nature.com/scientificreports/ cells, adaptive immune cells, and epidermal keratinocytes are stimulated by various cytokines, which is the main cause for the sustained development of psoriatic lesions. However, based on previous research and clinical experiences, the immune and inflammatory cells release a variety of cytokines, especially TNF-α, IL-17A, and IL-22, which are believed to affect epidermal keratinocytes directly, and are the most important cytokines responsible for a variety of pathological processes.
Conventional treatments of psoriasis include topical and systemic medication 49,50 . Systemic steroids are widely used as anti-inflammatory drugs in the treatment of several skin diseases; however, they exhibit potential side effects, such as skin atrophy, striae, and telangiectasias 51 , or systemic response, including inhibition of the hypothalamic-pituitary-adrenal axis and other endocrinological complications, such as diabetes and osteoporosis 52 . In addition, the side effects of steroids in the cardiovascular, gastrointestinal, and neurological systems are well known. Besides these common side effects, some reports have indicated that psoriasis leads to a deterioration of the rebound phenomenon after systemic steroid withdrawal 53 . Moreover, morphological changes from non-pustular to pustular psoriasis can become difficult to control after discontinuation of steroid therapy, making it difficult to perform a risk-benefit analysis 54,55 . The use of steroids may also cause erythroderma in patients with psoriasis 56 . Although steroids are very effective as first-line treatments in many cases, the efficacy of the drug weakens over time in most cases, and higher doses are then required to maintain the treatment response. Therefore, steroids are not conducive to long-term use 57 . In contrast biological agents, which are proteins produced by organisms, have advantages, such as lower side effects, less hepatotoxicity and nephrotoxicity, and selective towards immune system. However, their disadvantages are that they are expensive and less popular. At present, the main biological agents are antibodies and agents against TNF, IL-12/23, IL-17, JAK, and IκB kinase 58 . TNF therapy has long been used to treat various inflammatory diseases, including psoriasis 59 , and mainly neutralizes TNF secretion by inflammatory cells. IL-12/23 Inhibitors interfere with IL-12 and IL-23-mediated cell signal transduction 60 , while IL-17 inhibitor neutralizes the effects of IL-17A 61 and the downstream signalling pathways. In psoriasis, JAK inhibitors show good therapeutic effects. In a mouse model of contact dermatitis, topical application of JAK inhibitors could effectively inhibit the infiltration of lymphocyte, the phosphorylation of STAT3, and the proliferation of keratinocyte 62 . Recently, many studies have speculated that NF-κB pathway may be a new target for the treatment of psoriasis, and IκB kinase inhibitor can improve the severity of psoriasis 63 . However, compared with steroids and biological agents, Chinese herbal medicine ingredients are more advantageous, as they can be isolated from natural plants, can be conveniently obtained, have no usage restrictions, and show only few or no side effects. In modern medicine, there are more and more Chinese medicine treatments for psoriasis are extensively investigated 64 . Indigo naturalis has been used as a traditional Chinese medicine for the treatment of skin diseases. In recent years, many reports indicated that topical application of indigo naturalis and indirubin were effective and safe for the treatment of psoriasis, especially plaque psoriasis and nail psoriasis, and it also has the inhibitory effect on the inflammatory response of human neutrophils. It has also been found that indigo naturalis up-regulated the expression of claudin-1 and the function of tight junction in human epidermal cells [65][66][67] . However, although Chinese herbal medicine can be an alternative, safety, adverse effects and efficacy of long-term medication are still important issues that need to be addressed in the future. It is important to identify novel herbal ingredients that can be efficiently and safety used in abovementioned therapeutic approaches/strategies against psoriasis and other skin inflammatory diseases.
In this study, the topical application of chrysin suppressed the IMQ-induced psoriasis-like skin inflammation in the murine model, ameliorated the skin-related physiology, including trans-epidermal water loss (TEWL), erythema, blood flow speed, and ear thickness, and increased surface skin hydration (corneometer). Furthermore, pretreatment with chrysin reduced the IMQ-induced inflammation and infiltration of inflammatory cells, as observed in the histopathological analysis. We also found that chrysin effectively suppressed the downstream responses of these cytokines in epidermal keratinocytes, including phosphorylation of the MAPK pathway, activation of IκBα in the NF-κB pathway, and triggering of the JAK-STAT pathway at protein levels. Furthermore, chrysin treatment downregulated the expression of CCL20 and antimicrobial peptides (AMPs), consisting of S100A7, S100A8, S100A9, hBD2, and LL-37, at mRNA levels.
Many skin diseases, such as atopic dermatitis, contact dermatitis, psoriasis, and even ultraviolet radiation are related to skin inflammation. Inflammation of the skin is often accompanied by an increase in vascular permeability, together with the release of NO and prostaglandins, which trigger accumulation of tissue fluid in the lesion and occurrence of oedema. At the same time, leukocytes (especially neutrophils) also migrate to the skin lesions. The production of matrix metalloproteinase, in particular MMP-9, degrades the main structural component of connective tissue, collagen, in the epidermis and dermis 68 , which contributes to such migration of leukocytes 69 . Other cytokines released from epidermal cells or antigen-specific cells play a crucial role in inflammation of the skin, such as TNF-α, IL-1, and IL-6. Therefore, the above mentioned mediators can be used as indicators for exploring skin inflammation 4 . TNF-α plays a key role in inflammatory and barrier-impaired skin diseases, and also affects the regulation of many downstream molecules, such as cytokines, chemokines, enzymes, and proteins. Therefore, by elucidating the effects of Chinese herbal medicines on the cellular signalling pathways induced by TNF-α, we could reduce the effects of these inflammatory molecules, and identify the potential and feasibility of Chinese herbal medicines for the treatment of inflammatory skin diseases.
Cytokines involved in both Th1 and Th17 pathways are found in the skin of psoriasis patients, including IL-17A, IL-17F, IL-19, IL-20, IL-22, IL-23, IL-24, IL-26, and TNF-α, which can be detected in serum and lesions. The synergistic effect of IL-17 and IL-22 promotes the expression of antimicrobial peptides in keratinocytes, such as β-defensin-2 (BD-2), S100A7 (psoriasin), cathelicidin (LL37), and S100A8/9 (calprotectin), all of which may lead to the development of psoriasis in individuals with a higher resistance to skin infections 70,71 . In this study, the results showed that three cytokines, TNF-α, IL-17A, and IL-22, induced mRNA expression of CCL20 and antimicrobial peptides, and chrysin pretreatment downregulated their mRNA levels significantly. In recent years, an increasing number of studies have reported that interleukin-36 (IL-36), which belongs to the IL-1 superfamily, Scientific RepoRtS | (2020) 10:2932 | https://doi.org/10.1038/s41598-020-60050-1 www.nature.com/scientificreports www.nature.com/scientificreports/ affects the balance between pro-inflammatory and anti-inflammatory branches, and easily leads to tissue inflammation. IL-36 cytokines are mainly expressed at the barrier sites in the body, such as the skin epithelium, and the spectrum of psoriasis is one of the famous examples 72 . Therefore, IL-36 is likely to be an important diagnostic tool for dermatitis, and additional keratinocyte-linked cytokines like the inflammasome-related IL-1β, IL-36α, and IL-36γ should be investigated in the future. Previous studies have indicated that IL-17 exhibits the ability to activate transcription factors, such as NF-κB, in many cell types, including fibroblasts, macrophages, chondrocytes, intestinal epithelial cells, and myofibroblasts of the colon and pancreas 73 . When cells are stimulated by IL-17, the IL-17 receptor (IL-17R) is triggered to activate ERK1 and ERK2 and the stress-induced JNK-1 and JNK-2, as well as the p38 MAPK pathway; these signal transduction pathways contribute to the upregulation of IL-6, IL-1, and NF-κB 74 . Our results showed that the addition of TNF-α, IL-17A, and IL-22 induced the phosphorylation of p38, ERK, and JNK, and TNF-α and IL-17A activated the expression of IκBα in the NF-κB pathway. Previous studies have reported that IL-22 induces the phosphorylation of IKK and IκB in HaCaT cells 75 . However, we have not explored the effects of IL-22 on the NF-κB pathway in this study. If we can further explore the effects of this pathway, we can better understand the mechanism of chrysin on the pathogenesis of psoriasis. The phosphorylation of these related proteins was suppressed by the pretreatment of chrysin in a dose-dependent manner. These results demonstrated that chrysin effectively improved psoriasis-associated inflammation and exhibited anti-psoriatic potential.
With the advent of cellular molecular biotechnology, it has become increasingly clear that many molecules related to the skin barrier, as well as molecules that maintain skin hydration, such as filaggrin and junction protein, play a decisive role in maintaining normal physiological functions of the skin. Therefore, exploring and reducing the external factors that affect the above skin properties is an important research area in pathology and future drug development. Cornification (or keratinization) is the process of terminal differentiation of epidermal keratinocytes and plays an important role in the formation of the skin barrier 76,77 . In psoriatic lesions, abnormal differentiation of keratinocytes is observed. In particular, the expression of some crucial differentiation markers, such as keratin 10, loricrin, and filaggrin, is suppressed, leading to hypogranulosis in the psoriatic lesion epithelium. The three cytokines, TNF-α, IL-17A, and IL-22, affect the differentiation of keratinocytes and result in downregulation of the abovementioned differentiated proteins. In addition, proliferation and thickening of the skin in psoriatic lesions can also be observed, which is related to the hyperplasia of keratinocytes. Previous evidence showed that among the several cytokines that are pathogenic to psoriasis, IL-22 most likely promotes the proliferation of keratinocytes 78,79 . In the inflammatory pathway, we found that chrysin can effectively inhibit the expression of inflammation-related proteins and mRNAs induced by TNF-α, IL-17A, and IL-22. In future, we hope to address whether chrysin can reverse the inhibition of TNF-α, IL-17A, or IL-22-induced differentiation in keratinocytes in psoriasis.
In conclusion, the current study demonstrates the potential of chrysin in the treatment of inflammatory skin diseases, such as psoriasis, via its ability to relieve their symptoms. The findings also suggest that it may be useful as a daily health care supplement for prevention of inflammation.

Materials and Methods
Ethics statement. All animal experiments in this study were approved by the Institutional Animal Care and Use Committee of Fu Jen Catholic University (approval #A10367). The principles of the 3Rs (Replacement, Reduction, and Refinement) were followed to optimize the experimental design. The human primary epidermal keratinocytes were cells from human foreskins. The foreskins were provided by the Mackay Memorial Hospital, after obtaining the consent for use (#13MMHIS022) from the Institutional Review Board. There was no interaction between the researcher and the foreskin donors, and the foreskins do not have any relevant information to identify the donors. All experiments were performed in accordance with relevant guidelines and regulations. www.nature.com/scientificreports www.nature.com/scientificreports/ changes in the appearance of the skin and ears. At the end of the experiment, mice were sacrificed and tissues were collected and stored at −80 °C for subsequent homogenization or fixation in formalin.

Histopathological analysis.
Mouse tissues were fixed with 4% paraformaldehyde at 4 °C overnight.
Routine methods were used to prepare formalin-fixed paraffin-embedded blocks of the mice's skin tissues. These tissues were then cut into 5-μm sections, and stained with haematoxylin and eosin (H&E). ZEISS Axioskop 40 Inverted System microscope (NY, United States) and SPOT Cam software (Sterling Heights, MI) were used to visualize the Images from H&E staining. Psoriasis Area and Severity Index (PASI) is an objective scoring system. The erythema, scaling, and thickness were scored independently from 0 to 4 (0 no infection,1 mild, 2 intermediate, 3 severe, 4 very severe), and the total score was used as an index of psoriasis severity (scores 0-12).
Cell culture. The primary keratinocytes were isolated from human foreskin tissue, and were grown in Keratinocyte-SFM (Gibco BRL/Invitrogen, Carlsbad, CA). The primary keratinocytes were used between passages 2 to 4 in this study. Normal human keratinocytes were plated in 35-mm culture dishes, and 24 h prior the stimulus, keratinocyte medium was switched and chrysin (3, 10, or 30 μM) was added. The control medium contained an equal volume of DMSO. Finally, cells were stimulated with either TNF-α (50 ng ml −1 ), IL-17A (50 ng ml −1 ), or IL-22 (50 ng ml −1 ) from PeproTech (Rocky Hill, NJ).
Cell viability assays (MTT, trypan blue assay, and crystal violet assay). Cell viability was determined as previously described 32,34 by MTT, trypan blue, and crystal violet assays. In MTT assay, cells were pretreated with DMSO or chrysin for 24 h. After a brief wash, MTT (0.5 mg/mL in Keratinocyte-SFM) was used for the quantification of metabolically-active live cells and were analysed photometrically at 550 nm. In crystal violet assay, cells were treated as described, fixed with methanol, and then stained with 0.1% crystal violet solution for 1 h staining. Then, the cells were washed thrice with double-distilled water, followed by acetic acid to dissolve the cells. The optical density (OD) was then read using a Tecan Sunrise spectrophotometer (Tecan, Crailsheim, Germany) at 550 nm. The Trypan Blue exclusion method was performed according to the manufacturer's protocols. The normal human keratinocytes were pretreated with chrysin, and then suspended and stained with equal volume of trypan blue dye. The cells were counted using a dual-chamber haemocytometer and a light microscope.
Western blot analysis. After 5 and 15 min of stimulation using TNF-α, IL-17A, or IL-22, the total proteins were extracted by applying RIPA lysis buffer. Protein concentration was measured with the Pierce protein assay kit (Pierce, Rockford, IL). The proteins were separated by electrophoresis on 10% SDS-polyacrylamide gels, and then transferred onto a PVDF membrane (Millipore, Darmstadt, Hesse, Germany). After blocking with 5% non-fat dry milk for 1 h at room temperature, the membranes were probed using the indicated specific antibodies and visualized with ECL solution.
Real-time quantitative RT-PCR. After 6 and 8 h of stimulation with TNF-α, IL-17A, or IL-22, total RNA was isolated using the total RNA isolation kit (GeneDireX ® , Vegas, NV) according to manufacturer's instructions and reverse-transcribed into cDNA using SuperScript ™ III First-Strand Synthesis System kit (Invitrogen, Carlsbad, CA). The qPCR was performed using the CFX96 ™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA), following the previously described conditions 34 with SYBR green (Kapa Biosystems, Wilmington, MA). Primer sequences used in the PCR reactions are listed in Table 1