Anti-Atopic Dermatitis Activity of Epi-Oxyzoanthamine Isolated from Zoanthid

Atopic dermatitis (AD, eczema) is a condition that causes dry, itchy, and inflamed skin and occurs most frequently in children but also affects adults. However, common clinical treatments provide limited relief and have some side effects. Therefore, there is a need to develop new effective therapies to treat AD. Epi-oxyzoanthamine is a small molecule alkaloid isolated from Formosan zoanthid. Relevant studies have shown that zoanthamine alkaloids have many pharmacological and biological activities, including anti-lymphangiogenic functions. However, there are no studies on the use of epi-oxyzoanthamine on the skin. In this paper, epi-oxyzoanthamine has been shown to have potential in the treatment of atopic dermatitis. Through in vitro studies, it was found that epi-oxyzoanthamine inhibited the expression of cytokines in TNF-α/IFN-γ-stimulated human keratinocyte (HaCaT) cells, and it reduced the phosphorylation of MAPK and the NF-κB signaling pathway. Atopic dermatitis-like skin inflammation was induced in a mouse model using 2,4-dinitrochlorobenzene (DNCB) in vivo. The results showed that epi-oxyzoanthamine significantly decreased skin barrier damage, scratching responses, and epidermal hyperplasia induced by DNCB. It significantly reduced transepidermal water loss (TEWL), erythema, ear thickness, and spleen weight, while also increasing surface skin hydration. These results indicate that epi-oxyzoanthamine from zoanthid has good potential as an alternative medicine for treating atopic dermatitis or other skin-related inflammatory diseases.


Introduction
Atopic dermatitis, also known as eczema, is a chronic inflammatory relapsing skin disease. It is one of the most common skin diseases affecting infants and young children [1]. About half of children with atopic dermatitis will develop allergic rhinitis, asthma, allergic

Effect of Epi-Oxyzoanthamine on HaCaT Cell Viability
At the beginning of the experiment, in order to prove the safety of the epi-oxyzoanthamine ( Figure 1A), whether the toxicity of the epi-oxyzoanthamine is proved by cell viability test is suitable for subsequent related in vitro or in vivo studies. Thiazolyl blue tetrazolium bromide (MTT) assay was used to evaluate the cell viability. After the cells were administered for 1 h from a low dose of 1 µM to a high dose of 50 µM, we still did not find any toxicity of epi-oxyzoanthamine to the cells ( Figure 1B). Therefore, experiments were conducted to investigate the mechanism of action of epi-oxyzoanthamine against atopic dermatitis in this concentration range.

The Anti-Inflammatory Effect of Epi-Oxyzoanthamine in Tumor Necrosis Factor-α (TNFα)/Interferon-γ (IFN-γ)-Induced Inflammation in HaCaT Cells
The increase of pro-inflammatory cytokines is closely related to the etiology of atopic dermatitis. Many studies have shown that IFN-γ and TNF-α stimulate keratinocytes to induce signaling pathways involved in pro-inflammatory responses. Therefore, this model is often used as an in vitro test method for skin anti-inflammatory effectiveness [24,25]. It is shown in Figure 2 that once these pro-inflammatory cytokines (TNF-α and IFN-γ) are added, the formation of intracellular cytokine mRNA is significantly increased. These cytokines include IL-1β, IL-6, and IL-8. Moreover, there was a direct relationship between the concentrations of epi-oxyzoanthamine (1-10 µM) and the inhibitory ability of the cytokines: as the drug concentration increased, so did the inhibitory ability (

The Anti-Inflammatory Effect of Epi-Oxyzoanthamine in Tumor Necrosis Factor-α (TNF-α)/Interferon-γ (IFN-γ)-Induced Inflammation in HaCaT Cells
The increase of pro-inflammatory cytokines is closely related to the etiology of atopic dermatitis. Many studies have shown that IFN-γ and TNF-α stimulate keratinocytes to induce signaling pathways involved in pro-inflammatory responses. Therefore, this model is often used as an in vitro test method for skin anti-inflammatory effectiveness [24,25]. It is shown in Figure 2 that once these pro-inflammatory cytokines (TNF-α and IFN-γ) are added, the formation of intracellular cytokine mRNA is significantly increased. These cytokines include IL-1β, IL-6, and IL-8. Moreover, there was a direct relationship between the concentrations of epi-oxyzoanthamine (1-10 µM) and the inhibitory ability of the cytokines: as the drug concentration increased, so did the inhibitory ability (

Effects of Epi-Oxyzoanthamine on Phosphrylation of MAPK Pathway in HaCaT Cells
Many studies have shown that cytokine stimulation of skin cells activates mitogenactivated protein kinase (MAPK) pathway. Therefore, the following experiments will elu- with different concentrations of epi-oxyzoanthamine for 1 h, and then the cells were treated with TNF-α/IFN-γ for 1 h. ## p < 0.01 compared with the no-treatment group; * p < 0.05 and ** p < 0.01 compared with the TNF-α/IFN-γ-induced group.

Effects of Epi-Oxyzoanthamine on Phosphrylation of MAPK Pathway in HaCaT Cells
Many studies have shown that cytokine stimulation of skin cells activates mitogenactivated protein kinase (MAPK) pathway. Therefore, the following experiments will elucidate whether or not epi-oxyzoanthamine affects these pathways. First, epi-oxyzoanthamine demonstrated no activation or effect on the MAPK pathway ( Figure 3). Next, cells were stimulated the cells with IFN-γ and TNF-α for one (or half) hour and found that the pathway to MAPK was activated and phosphorylated ( Figure 4). However, the study found that if the cells were pretreated with the epi-oxyzoanthamine, the increased phosphorylation was indeed significantly inhibited. This phenomenon was more effective at higher concentrations ( Figure 4A-C).

Effects of Epi-Oxyzoanthamine on Phosphrylation of MAPK Pathway in HaCaT Cells
Many studies have shown that cytokine stimulation of skin cells activates mitogenactivated protein kinase (MAPK) pathway. Therefore, the following experiments will elucidate whether or not epi-oxyzoanthamine affects these pathways. First, epi-oxyzoanthamine demonstrated no activation or effect on the MAPK pathway ( Figure 3). Next, cells were stimulated the cells with IFN-γ and TNF-α for one (or half) hour and found that the pathway to MAPK was activated and phosphorylated ( Figure 4). However, the study found that if the cells were pretreated with the epi-oxyzoanthamine, the increased phosphorylation was indeed significantly inhibited. This phenomenon was more effective at higher concentrations ( Figure 4A-C).

Epi-Oxyzoanthamine Reduced IκB and NF-κB Activation in TNF-α/IFN-γ-Stimulate Keratinocytes
Previous studies have cited MAP kinase as an upstream NF-κB regulatory [26]. Studies have also found that IFN-γ and TNF-α also increase the phosphoryla NF-κB and IκB. At the same time, it was found that an increase in the concentration oxyzoanthamine after pretreatment caused an increase in the inhibition of phosph tion ( Figure 5).

Epi-Oxyzoanthamine Reduced IκB and NF-κB Activation in TNF-α/IFN-γ-Stimulated Keratinocytes
Previous studies have cited MAP kinase as an upstream NF-κB regulatory kinase [26]. Studies have also found that IFN-γ and TNF-α also increase the phosphorylation of NF-κB and IκB. At the same time, it was found that an increase in the concentration of epi-

The Effect of Epi-Oxyzoanthamine on the Skin Appearance in DNCB-Induced BALB/c Mouse
After the previous in vitro studies, it was found that epi-oxyzoanthamine has the potential of anti-atopic dermatitis. Dinitrochlorobenzene (DNCB)-induced contact hypersensitivity (CHS) of the skin in mice is a commonly-used animal model for studying the pathogenesis of contact dermatitis [27]. Given the complexity of factors involved in the pathogenesis of AD, epidermal sensitization with stimuli such as DNCB is a common method used to identify testing drug candidates in AD [28]. When DNCB was applied to the animal's dorsal skin, it showed atopic dermatitis-like symptoms, such as desquamation and redness, epidermis thickening, skin inflammation, enlarged spleen, and the animal even had scratching behavior (Figures 6 and 7). The above pathological phenomena and symptoms after DNCB pretreatment will be alleviated with the increase of epi-oxyzoanthamine concentration. These same preventive effects are also commonly observed after pretreatment with dexamethasone ( Figures 6 and 7).

The Effect of Epi-Oxyzoanthamine on the Skin Appearance in DNCB-Induced BALB/c Mouse
After the previous in vitro studies, it was found that epi-oxyzoanthamine has the potential of anti-atopic dermatitis. Dinitrochlorobenzene (DNCB)-induced contact hypersensitivity (CHS) of the skin in mice is a commonly-used animal model for studying the pathogenesis of contact dermatitis [27]. Given the complexity of factors involved in the pathogenesis of AD, epidermal sensitization with stimuli such as DNCB is a common method used to identify testing drug candidates in AD [28]. When DNCB was applied to the animal's dorsal skin, it showed atopic dermatitis-like symptoms, such as desquamation and redness, epidermis thickening, skin inflammation, enlarged spleen, and the animal even had scratching behavior (Figures 6 and 7). The above pathological phenomena and symptoms after DNCB pretreatment will be alleviated with the increase of epi-oxyzoanthamine concentration. These same preventive effects are also commonly observed after pretreatment with dexamethasone ( Figures 6 and 7). the animal's dorsal skin, it showed atopic dermatitis-like symptoms, such as desqua tion and redness, epidermis thickening, skin inflammation, enlarged spleen, and the mal even had scratching behavior (Figures 6 and 7). The above pathological phenom and symptoms after DNCB pretreatment will be alleviated with the increase of epi-ox anthamine concentration. These same preventive effects are also commonly observed pretreatment with dexamethasone ( Figures 6 and 7).

Change in Physiological Functions of DNCB-Induced BALB/c Mouse Skin after Treatment with Epi-Oxyzoanthamine
From the previous pathological sections, epi-oxyzoanthamine does have an inhibitory effect on the inflammation caused by DNCB. The physiological values were measured to confirm the efficacy of these effects in improving physiological functions. As shown in Figure 8, improvements were found in physiological function, including transepidermal water loss, skin redness, and skin moisture content, after treatment of epi-oxyzoanthamine ( Figure 8A-C).
effect on the inflammation caused by DNCB. The physiological values were measured to confirm the efficacy of these effects in improving physiological functions. As shown in Figure 8, improvements were found in physiological function, including transepiderma water loss, skin redness, and skin moisture content, after treatment of epi-oxyzoanthamine ( Figure 8A

Discussion
Marine organisms are now a very large source of pharmaceutical resources. Scientists have also discovered many new structures of compounds here, and many structures have even been used to treat diseases. Some of them have even been used clinically or are al ready in clinical trials. Marine biological sources of these compounds typically include algae, bacteria, fungi, sponges, corals, and other marine animals [29]. Most of their dis covered physiological activities are anti-bacterial, anti-viral, anti-tumor, or anti-inflamma tory. Among them, the biological activities of sponges, algae, and bacteria have been stud ied the most, and have shown anti-tumor and anti-bacterial effects [29,30]. There are rela tively few drugs from corals [31]. Therefore, our study demonstrates that epi

Discussion
Marine organisms are now a very large source of pharmaceutical resources. Scientists have also discovered many new structures of compounds here, and many structures have even been used to treat diseases. Some of them have even been used clinically or are already in clinical trials. Marine biological sources of these compounds typically include algae, bacteria, fungi, sponges, corals, and other marine animals [29]. Most of their discovered physiological activities are anti-bacterial, anti-viral, anti-tumor, or antiinflammatory. Among them, the biological activities of sponges, algae, and bacteria have been studied the most, and have shown anti-tumor and anti-bacterial effects [29,30]. There are relatively few drugs from corals [31]. Therefore, our study demonstrates that epioxyzoanthamine isolated from zoanthid has good anti-dermatological potential, especially in the treatment of atopic dermatitis. These results will open up the application of related compounds produced by corals in the treatment of skin diseases, especially in the treatment of atopic dermatitis.
In previous studies on marine organisms against atopic dermatitis, some studies reported that the red algae-Pyropia yezoensis-extract inhibited the production of pro-inflammatory chemokines induced by IFN-γ and TNF-α in HaCaT cells by down-regulating NF-κB [32]. Down-regulation of NF-κB and STAT1 pathways by Polyopes affinis suppresses IFN-γ and TNF-α-induced inflammation in human keratinocytes [33]. Red algae-Sarcodia suiae sp. ethanol extract has anti-inflammatory effects, alleviates AD symptoms, suppresses inflammatory responses in skin tissue, and restores barrier function in DNCB-induced AD mice [34]. Polysaccharide of brown seaweed-laminarin-topical administration has a protective effect on oxazolone-induced atopic dermatitis-like lesions. Topical application of laminarin can alleviate the overproduction of IgE, mast cell infiltration, and expression of pro-inflammatory cytokines oxazolone-induced atopic dermatitis [35]. Trifuhalol A, a phlorotannin isolated from brown seaweed-Agarum cribrosum, inhibited the activations of immune cells and the biosynthesis of cytokines in differentiated B cells and keratinocytes, respectively. Trifuhalol A alleviated pruritus in the Compound 48/80-induced systemic anaphylaxis model and improved symptoms in house dust mite (HDM)-induced AD mice [36]. Sargassum polyphenol extract alleviates DNCB-induced atopic dermatitis in NC/Nga mice by restoring skin barrier function [37]. Based on these findings, the effects of marine drugs against atopic dermatitis are attributed to the immunomodulation and regulations of the skin barrier. As shown by the results of this study, epi-oxyzoanthamine reduced the cytokines-induced expression of pro-inflammatory cytokines. The mechanisms of action could regulate the map kinase and reduce the action of transcription factor (NF-κB). Therefore, epi-oxyzoanthamine could decrease the production of cytokines including IL1β, IL6, and IL8. The appearance of dysfunction of skin barrier is a major symptom in atopic dermatitis patients. Trans-epidermal water loss is an indicator of the skin barrier. Using in vivo studies, it was found that the increase of TEWL-induced DNCB was decreased after treatment of epi-oxyzoanthamine. On the other hand, the hydration of the epidermal is increased after the treatment of epi-oxyzoanthamine (Figure 8). The erythema and hyperplasia of dorsal skin are the appearance of inflammatory skin. The anti-inflammatory activity of epi-oxyzoanthamine was shown in Figures 7 and 8. It was also found that the infiltration of mast cells and the weight of the spleen after treatment of DNCB were decreased after pretreatment with epi-oxyzoanthamine. These effects indicate that dysregulation of the immune system could modulate by pretreatment with epi-oxyzoanthamine.
Soft corals are an indispensable source of metabolites with medicinal properties [38]. In the past, many studies have discovered many new compounds with anti-cancer, antibacterial, and anti-viral properties in soft corals [39][40][41][42]. Compounds found in soft corals have also been recently reported by many studies for their anti-inflammatory effects. Because inflammation plays an increasingly important role in many clinical diseases [43,44], these compounds are also of particular interest in the application of inflammatory diseases [45][46][47][48]. In recent years, the incidence of diverse chronic inflammatory skin diseases has been increasing, especially atopic dermatitis. Therefore, the discovery of a potent and potential chemical structure of zoanthenamine from zoanthid can be applied to inflammatory skin diseases. In the past, scholars only found that zoanthamines have anti-inflammatory, anti-bacterial, anti-platelet, anti-lymphangiogenesis neuroprotective effects [19,21,49,50]. However, no scholars have proposed the effect on inflammatory dermatitis. Therefore, this is the first to suggest that zoanthamines have anti-inflammatory dermatitis effects. Zoanthamine alkaloids are specific secondary metabolites of marine zoanthids. Importantly, these results show that its ability to resist atopic dermatitis is not inferior to that of components isolated from land plants. Moreover, their mechanism of action is very similar [24,25,51]. Future work will look to identify any unique mechanisms that are used by zoanthamine alkaloids to promote anti-inflammatory dermatitis effects.

Culture of Human Keratinocyte
The human epidermal keratinocyte line (HaCaT) was used for this study. The HaCaT cell line was provided by Dr. Nan-Lin Wu, Department of Dermatology, Mackay Memorial Hospital, Taiwan. HaCaT was cultured in a 75T flask containing 10% fetal bovine serum (fetal bovine serum, FBS) and Dulbecco's Modified Eagle Medium (DMEM) with 1% antibiotics (Grand Island, NY, USA). The cells were placed in an incubator at 37 • C and 5% CO 2 , and the cells were allowed to grow to about 80-90% saturation, and then the cells were subcultured.

Western Blotting
Western blots were used to analyze changes in various proteins in cells. Related methods are described in more depth in a previously published paper [24]. HaCaT cells were seeded in 3.5 cm culture dishes. HaCaT cells were seeded in 3.5 cm dishes. After cells reached 90% confluence and were starved for 24 h, they were pretreated with epioxyzoanthamine for 1 h and then stimulated with TNF-α/IFN-γ for 1 h, respectively. After scraping, cells were crushed by sonication and centrifuged (13,200 rpm, 10 min, 4 • C). After centrifugation, the supernatant was taken, and protein was quantified using the Pierce Protein Assay Kit (Pierce, Rockford, IL, USA). Approximately 20-40 µg of protein was electrophoresed on a 10% SDS-polyacrylamide gel, followed by electroporation with PVDF membranes. After the transfer, the PVDF membrane was placed in a TBS-T solution (Tris-buffered salt/0.05% tween 20) containing 5% nonfat dry milk for 1 h with continuous shaking to avoid nonspecific binding. Then, the PVDF membrane was washed 3 times with TBS-T (30 min in total). After that, the primary antibody was added (diluted 1:1000). PVDF membranes were left overnight at 4 • C and then washed 3 times with TBS-T for 10 min each. Finally, after adding the secondary antibody for 1 h (diluted to 1:1000), the PVDF membrane was washed 3 times with TBS-T, then the developing solution was added, and the membrane was put into the chemiluminescence extraction system (Biostep GmbH Chemiluminescence Imager CELVIN; Type CELVIN ® S420 FL, Calibre Scientific, LA, USA) for photography.

Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR)
HaCaT cells were seeded in 3.5 cm Petri dishes. Cells can reach 90% confluency after 24 h. Cells were pretreated with epi-oxyzoanthamine for 1 h and stimulated with TNFα/IFN-γ for 1 h. Cells were scraped and centrifuged (16,000× g, 10 min, 4 • C) and the supernatant was removed. RNA was purified using a total RNA isolation kit (GeneDireX ® , Vegas, NV, USA)according to the operating procedure of iScript™ cDNA Synthesis Kit (BIO-RAD, Hercules, CA, USA), reagents were added one by one and specified conditions were followed closely to convert RNA into cDNA. Additionally, PowerUp™ SYBR™ Green Master Mix (Applied Biosystems™, Waltham, MA, USA) was used. 7.5 µL ddH 2 O, 2 µL cDNA, 0.25 µL forward and reverse primers, and 10 µL SYBR GREEN were added and mixed well. The relevant primer sequences are listed in Table 1. Finally, RNA was quantified using the ABI StepOnePlus™ Real-Time PCR System (Applied Biosystems™, Waltham, MA, USA). Male mice (BALB/c, 8 weeks old) were purchased from Taiwan National Laboratory Animal Center. The mice were kept in a temperature-controlled and humidity-controlled animal room (controlled at 21 ± 2 • C and 50 ± 20%, respectively). Mice were housed at the Animal Center of Fu Jen Catholic University in Taiwan with controlled laminar filtered airflow and a 12-h light/dark cycle. This experiment was performed after review by the Institutional Animal Care and Use Committee of Fu Jen Catholic University, Taiwan (approval number A10964).
The mice were first divided into four groups: control group, DNCB group, epioxyzoanthamine (3 and 10 mg/kg) plus DNCB group, and 0.2 mg/kg dexamethasone plus DNCB group. For in vivo experiments, epi-oxyzoanthamine was dissolved in dimethylsulfoxide (DMSO), while DNCB was dissolved in 75% ethanol. The former was administered by intraperitoneal injection, while the latter was applied to the skin of the back and right ear in 100 µL and 20 µL, respectively. During the first three days, mice were anesthetized and had their dorsal hair removed. A small magnet (1 mm in diameter and 3 mm in length) was embedded in each mouse's hind paw. Three days later, the behavior of the mice was observed, and it was ensured that mice were in good physical condition and the skin in the depilated area on the back was normal before starting the experiment. Skin-related physiological values including transepidermal water loss (TEWL), erythema, skin moisture, ear thickness, and number of scratches were measured before the experiment. Photographs were also taken to document changes in the appearance of the skin and ears. The whole experimental process was carried out in a room with constant temperature and humidity. The first phase (days 1-4) is the allergic atopic dermatitis phase. After measuring the physiological value of the mice, 1% DNCB was evenly applied to the skin of the dorsal and right ear. On the fifth day, intraperitoneal injection of epi-oxyzoanthamine began. The second phase (days 5 to 14) involved reinduction of atopic dermatitis. We then evenly applied 0.5% DNCB on the back and right ear skin of mice in four experimental groups. The next day, the physiological values of the skin were tested and recorded and photographed. After completing all tests on day 15, mice were euthanized with carbon dioxide (CO 2 ) and dorsal skin tissue and spleens were removed for subsequent experimental analysis.

Data and Statistical Analysis
Sigma-Plot software (version 10.0) was used for all statistical analyses of the data. All data are presented as mean ± SEM. Statistical significance was assessed by unpaired two-tailed Student's t-test. p < 0.05 was considered significant (* p < 0.05, ## and ** p < 0.01).

Conclusions
The results showed that epi-oxyzoanthamine significantly decreased skin barrier damage, scratching responses, and epidermal hyperplasia induced by DNCB. It significantly reduced transepidermal water loss (TEWL), erythema, ear thickness, and spleen weight, while also increasing surface skin hydration. These results indicate that epi-oxyzoanthamine from zoanthid has good potential as an alternative medicine for treating atopic dermatitis or other skin-related inflammatory diseases.