Seungmagalgeun-Tang, a Traditional Herbal Formula, Alleviates Skin Inflammation and Depression-Like Behavior in Atopic Dermatitis Mice under Sleep Deprivation Conditions

Atopic dermatitis (AD) is a common inflammatory skin disease, which can be worsened under sleep deprivation (SD) conditions. This study investigated the efficacy and the mechanism of action of the traditional herbal formula Seungmagalgeun-tang (SMGGT) on the inflammation and behavioral changes in a mouse model of AD exposed to SD. SMGGT decreased levels of IgE, TNF-α, IL-4, IL-13, and mast cell infiltration and reduced the expression of CD3 in the mouse skin. SMGGT also reversed the SD-induced increase in corticosterone and decrease in melatonin level. Furthermore, SMGGT reduced the immobility time in the tail suspension test significantly. HaCaT cells and HMC-1 cells were used to investigate the effects of SMGGT on cell signaling pathways. In TNF-α/IFN-γ (TI) treated HaCaT cells, SMGGT reduced production of TARC/CCL17 and MDC/CCL22 and suppressed the p38 MAPK, STAT1, and NF-κB pathways. In substance P (SP)/CRH-stimulated HMC-1 cells, SMGGT decreased VEGF production and inhibited ERK phosphorylation. Network pharmacology and molecular docking analysis revealed that puerarin and paeoniflorin might contribute to the effects of SMGGT by targeting several AD-related molecules and pathways. Puerarin and paeoniflorin exerted anti-inflammatory effects by decreasing production of MDC/CCL22 and IL-6 in TI-treated HaCaT cells and VEGF production in SP/CRH-stimulated HMC-1 cells. This study suggests that SMGGT with puerarin and paeoniflorin as main bioactive components alleviates skin inflammation and depression-like behavior in a sleep-deprived mouse model of AD.


Introduction
Up to 87% of adults and 80% of children with atopic dermatitis (AD) suffer from sleep deprivation (SD). Nocturnal itching and scratching movements can severely disrupt the continuity and quality of sleep in AD patients [1]. Poor sleep at night can trigger an awareness of itching, leading to more scratching by these patients, indicating a vicious cycle between AD and SD [2]. A recent clinical trial reported the efficacy of melatonin supplementation on both sleep quality and skin symptoms in pediatric patients with AD [3]. Hence, the management of SD might be a promising approach for the treatment of AD. e cellular and molecular mechanisms underlying SD that could worsen AD have been suggested [1]. Acute and chronic SD can promote inflammation by increasing the production of various inflammatory cytokines, including TNF-α, IL-1β, IL-6, and IL-17 [4]. SD has also been reported to disrupt the normal functions of regulatory T cells and alter the 1/ 2 balance toward 2 dominance [5]. AD patients with sleep loss show a decrease in the ratio between the level of 1 cytokine IFN-c and 2 cytokine IL-4 than those without AD [6]. Moreover, SD could trigger psychological stress in AD patients, leading to a high risk of anxiety and depression [1]. SD-induced stress activates the hypothalamic-pituitary-adrenal (HPA) axis to stimulate the secretion of stress hormones, including corticotropin-releasing hormone (CRH) produced by the hypothalamus, adrenocorticotropic hormone (ACTH) from the pituitary gland, and glucocorticoids (cortisol in human or corticosterone in mouse/rat, CORT) from the adrenal gland [7]. Eight days of SD significantly increased CORT plasma level in a murine model of autoimmune disease [8]. CORT can disrupt skin barrier function and shift the immunity toward the 2 response [9,10]. CRH, another stress hormone, can activate mast cells in the skin and increase vascular permeability [11]. Activated mast cells can secret histamine and a range of inflammatory molecules, aggravating the itching symptoms in AD [12].
Seungmagalgeun-tang (SMGGT) is a traditional herbal prescription that consists of four medicinal herbs: Puerariae Radix, Glycyrrhizae Radix, Paeoniae Radix, and Cimicifugae Rhizoma. According to "Collection of Prescriptions with Notes (Yifang Jijie)," a classic book of traditional medicine, SMGGT has been used to treat both skin diseases, including purple rash, urticarial, and sleep disturbance. Previous studies reported that SMGGT had anti-inflammatory effects by reducing the expression of proinflammatory cytokines IL-4, IL-6, IL-8, and TNF-α in mast cells [13,14]. Paeoniae Radix, a herbal constituent of SMGGT, was reported to improve sleep quality in patients with restless leg syndrome [15]. However, the therapeutic capacity of SMGGT on AD exaggerated by SD has not been elucidated. Hence, the current study investigated the effects and mechanism of action of SMGGT extract on skin inflammation and behavioral changes in a mouse model of AD under SD conditions. In addition, network pharmacological analysis was performed to investigate the bioactive components of SMGGT and their possible targets for the treatment of AD.

Animal Model and Treatments.
Male BALB/c mice (five weeks old, n � 30) were procured from Koatech Laboratory Animal Inc. (Seoul, Korea). All animal experiments were conducted following the protocols from the Institutional Animal Care and Use Committee of Dongguk University (IACUC-2019-7). e animals were stabilized for seven days before starting the experiments. e mice were assigned randomly to six groups: normal control group (the NC group), DNCB treatment group (the AD group), DNCB + sleep deprivation (SD) group (the AD + SD group), DNCB + SD + 100 mg/kg of SMGGT (the SMGGT100 group), DNCB + SD + 500 mg/kg of SMGGT (the SMGGT500 group), and DNCB + SD + 0.1% dexamethasone (the DEX group). Figure 1(a) presents the experimental design diagram. DNCB and DEX for this experiment were obtained from Sigma-Aldrich (St. Louis, MO, USA). e dorsal skin area of each mouse was shaved, and then 200 μl of 1% DNCB (soluble in acetone/olive oil solution, 3 : 1 v/v) was treated three times for one week (sensitization). In the challenge period, 0.3% DNCB (200 μl) was treated to the mouse skins three times a week for six weeks. e mice were orally administered 100 or 500 mg/kg of SMGGTor topically treated with 0.1% DEX every day for six weeks. e SMGGT doses used in this experiment were the doses of other traditional herbal formulas used to treat AD-like symptoms in mouse models in previous studies [16,17]. ere were no side effects, abnormal behavior, or weight loss when the highest concentration of SMGGT was administered. Sleep deprivation was induced as described previously with modifications [18]. e mice were allowed to run on a treadmill at a slow speed (10 m/min) during the day (four cycles of 1 h running and 1 h resting, between 10 : 00 and 18 : 00). After running on the treadmill, the mice were put back in their normal cages. On the last day of the experiment, the skin severity score was recorded, including four different skin symptoms: redness, dryness, edema, and excoriation, with four intensity levels: none (0), mild (1), moderate (2), and severe (3). For sacrifice, the mice were anesthetized using isoflurane and the body weights were evaluated. e spleens were dissected, and the spleen weights were recorded. e skin and blood samples were collected for further analysis.

Tail Suspension Test (TST).
To assess depression-like behavior in mice, the TST was performed, as described previously [19,20]. e mice were acclimated to the test room for 2 h before testing. e mice were hung on a bar by a 15 cm-long adhesive tape attached approximately 1 cm from the tail tip. e distance from the head of the mouse to the floor was set at 50 cm. Each test session was conducted for 2 Evidence-Based Complementary and Alternative Medicine 6 min.
e immobility time was evaluated as the time without any limb movements.

Histological and Immunohistochemical Analysis.
e skin tissues were fixed using 4% paraformaldehyde and then embedded into paraffin. Skin sections were prepared at a thickness of 5 μm, and then hematoxylin and eosin (H&E) and toluidine blue (TB) staining of these sections were performed. For immunohistochemical staining, the sections were incubated with the anti-CD3 antibody (1 : 100, #ab5690, Abcam, Cambridge, MA) at 4°C overnight. e samples were then incubated with the anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. All stained samples were visualized using a Lionheart FX automated microscope and Gen5 software (BioTek Instruments, Winooski, VT, USA). e number of mast cells infiltrated in the dermis layer was counted. e CD3 intensity was evaluated by ImageJ Fiji software (WS Rasband, National Institutes of Health, Bethesda, MD, USA).

Cell Viability.
e effects of SMGGT on cell viability were determined using XTT assays. HaCaT and HMC-1 cells were treated with SMGGT (10, 100, 500, and 1000 μg/ml) or puerarin and paeoniflorin (1 and 10 μM) for 24 h and then incubated with 50 μl of XTT solution (Sigma-Aldrich, St. Louis, MO, USA) for 4 h. e absorbance was evaluated using a microplate reader (Tecan, Männedorf, Switzerland) at 450 nm wavelength (reference wavelength at 650 nm). e cell viability was presented as a percentage of the untreated controls.

Enzyme-Linked Immunosorbent Assay (ELISA).
e skin samples were homogenized and centrifuged as mentioned before. e supernatants were collected, and protein samples were prepared at a concentration of 0.5 mg/ml. e levels of cytokines and chemokines in skin lysates, serum, and cell culture media were assessed using commercial ELISA kits according to protocols from manufacturers. Human TARC/  Pharmacology (TCMSP) database [22] was used to predict target genes of the components of SMGGT. e compoundtarget network was visualized using Cytoscape 3.8.2 (Cytoscape Consortium, CA, USA). Cytoscape DisGeNET App was used to access atopic dermatitis-related genes [23]. Kyoto Encyclopedia Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis for biological processes were conducted using the Enrichr tool (https://maayanlab. cloud/Enrichr/) [

SMGGT Alleviated Skin Symptoms in AD Mice under SD
Conditions. SD exacerbated the AD symptoms significantly in the DNCB-treated mice. In contrast, treatment with SMGGT at 100 mg/kg reduced the SD-induced increases in the severity of skin symptoms (Figures 1(b) and 1(c)). e AD + SD group showed higher spleen weights compared with the AD group, and SMGGT at 100 and 500 mg/kg reduced these increases (Figure 1(d)). e body weights of SMGGT-treated groups were not significantly different from the NC group (Figure 1(e)).

SMGGT Reduced Inflammation in AD Mice under SD
Conditions. e AD + SD group showed higher skin levels of inflammatory cytokines, including IL-4, IL-13, TNF-α, and VEGF, compared with the NC group. Interestingly, TNF-α and VEGF production in the AD + SD group was significantly higher than that in the AD group. In contrast, treatment with SMGGT (100 and 500 mg/kg) significantly decreased the expression of TNF-α, IL-4, and VEGF in skin lysates, and the SMGGT100 group showed lower levels of IL-13 than the AD + SD group (Figure 2(a)). Moreover, serum IgE levels in the AD and AD + SD groups were significantly higher than those in the NC group, but these increases were reduced by treatment with SMGGT (100 mg/kg) (Figure 2(b)). e AD + SD mice had a thicker epidermis layer and a higher number of mast cell infiltrations than the AD mice (Figure 2(c)). However, SMGGT treatment (100 and 500 mg/ kg) significantly decreased the epidermal thickness and the number of mast cells compared with the AD + SD group (Figure 2(c)). In addition, the AD + SD group had a higher CD3 intensity than the AD group, which was reduced by treatment with SMGGT at 100 mg/kg (Figure 2(c)).

SMGGT Improved the Behavioral Change and Hormone
Levels in AD Mice under SD Conditions. SD was suggested to promote depression-like behavior in a mouse model [27]. In this study, the AD + SD mice increased the immobility time in the TST significantly, in comparison to the NC and AD groups. On the other hand, this increase was suppressed significantly by treatment with SMGGT (100 and 500 mg/kg) (Figure 3(a)). e levels of stress-related hormones, including CRH, ACTH, and CORT, were higher in the AD + SD group than in the NC and the AD group, which were decreased significantly in the SMGGT treatment groups (Figure 3(b)). e serum levels of melatonin were also lower in the AD + SD group than in the NC group, which recovered with SMGGT administration (Figure 3(c)). Moreover, the melatonin levels had negative correlations with the immobility time in the TST and the levels of inflammatory cytokines IL-4 and VEGF in skin lesions (Figure 3(d)).

SMGGT Reduced TI-Induced Inflammation in
HaCaT Cells. To understand the action mechanism of the SMGGT, this study examined its effects in HaCaT cells. e effects of SMGGT on the viability of HaCaT cells were measured by XTT assays, and following experiments were conducted at nontoxic concentrations (Figure 4(a)). e levels of 2-attracting chemokines (TARC/CCL17 and MDC/CCL22) were increased in HaCaT cells upon TI stimuli, but SMGGT (10 and 100 μg/ml) reduced these increases significantly (Figure 4(b)). Furthermore, SMGGT (100 μg/ml) significantly suppressed TI-induced phosphorylation of p38 and STAT1, as well as nuclear translocation of NF-κB p65 by downregulating phosphorylation of IκB-α (Figure 4(c)).

SMGGT Decreased SP/CRH-Induced Inflammation in
HMC-1 Cells. SP/CRH-treated HMC-1 cells were also used to evaluate the effects of SMGGT in vitro. SMGGT did not have cytotoxicity on HMC-1 cells (Figure 5(a)). SMGGT (10 and 100 μg/ml) reduced the level of VEGF and phosphorylation of ERK in SP/CRH-treated HMC-1 cells, which suggested that the effect of SMGGT might be through suppressing inflammatory response in mast cells (Figures 5(b) and 5(c)).

Network Pharmacological Analysis of SMGGT Compounds and Targets.
Network pharmacological analyses were conducted to determine potential targets of SMGGT  compounds and their mechanism of action in atopic dermatitis. We identified 84 targets of five compounds of SMGGT (paeoniflorin, puerarin, liquiritigenin, daidzin, and ferulic acid) using the TCMSP database (Figure 7(a)). Targets of albiflorin and glycyrrhizic acid have not been reported in this database. Figure 7(a) shows the relation between SMGGT compounds and their target genes. ADrelated targets were indicated according to the DisGeNET database. ese genes include AKT1, IFNA1, IFNB, IL-6, JAK3, JUN, MMP-9, NFKBIA, PTGS2, STAT3, TNF, VCAM-1, and VEGFA. Among SMGGT compounds, puerarin possesses the most AD-related targets with 12 targets, while the others possess only target 1-2 AD-related genes. KEGG enrichment analysis was performed to investigate the potential signaling pathways. Figure 7(b) shows that targets of SMGGT components are involved in several pathways related to inflammatory responses, such as TNF, VEGF, MAPK, NF-kappa B signaling pathways, or ADspecific pathways such as 1 and 2 differentiation. Top 10 GO Biological Process terms are shown in Figure 7(c), including cytokine-mediated signaling pathway, cellular response to cytokine stimulus, and positive regulation of intracellular signal transduction, which are the biological processes strongly related to AD.

Docking Analysis of SMGGT Compounds and Targets.
We conducted the molecular docking analysis of SMGGT compounds and their 13 AD-related targets. e docking scores indicating affinity between compounds and proteins are shown in Table 3. Most of the ligand-protein binding affinities are less than −6.0 kcal/mol, demonstrating potential binding between SMGGT compounds and targets. Figure 8 shows the binding modes of representative compounds and targets, including paeoniflorin-TNF-α, puerarin-IFN-α, daidzin-COX-2, liquiritigenin-COX-2, and ferulic acid-COX-2.

Anti-Inflammatory Effects of Puerarin and Paeoniflorin In
Vitro. TI-treated HaCaT cells and SP/CRH-treated HMC-1 cells were used to examine the anti-inflammatory effects of two main components of SMGGT, puerarin and paeoniflorin. Pretreatment with puerarin and paeoniflorin significantly reduced the production of MDC/CCL22 and IL-6 in TI-stimulated HaCaT cells as well as decreased VEGF production in SP/CRH-stimulated HMC-1 cells (Figures 9(b) and 9(d)). Puerarin and paeoniflorin did not show any cytotoxic effects in HaCaT and HMC-1 cells (Figure 9(a) and 9(c)).

Discussion
SD can exacerbate the severity of AD by causing immune dysregulation and inducing psychological stress, leading to significant impairment in the quality of life of patients with AD [1]. In this study, treadmill movement was utilized for the induction of SD in mice. A previous study demonstrated that running on a treadmill can disturb normal sleep homeostasis by reducing time spent in the rapid eye movement (REM) stage, non-rapid eye movement (NREM) stage, and total sleep in the mouse model [28]. Treadmill-induced SD can worsen the AD symptoms in NC/Nga mice by altering the expression of clock genes, in comparison with nontreadmill-treated mice [18]. In the current study, SD exacerbated skin inflammation and triggered behavior changes in DNCB-induced AD mice. SD increased the production of 2 cytokines (IL-4 and IL-13) significantly, compared to normal mice. ese results are in consonance with a previous study that SD favored a shift of the 1/ 2 balance toward 2 dominance [29]. Moreover, SD increased the immobility time in the TST in the AD mice.
SMGGT reduced the expression of CD3, a cell surface marker of T cells, and decreased the levels of 2 cytokines IL-4 and IL-13 in skin lesions in DNCB-treated mice under SD conditions. IL-4 and IL-13 are critical for the production of IgE, recruitment of immune cells, and skin barrier disruption in AD [30]. In addition to its inhibitory effects, SMGGTalso reduced the serum levels of IgE and the number of mast cells infiltrated in skin lesions. e chemokines, MDC and TARC, which are expressed strongly in the activated keratinocytes, play a crucial role in the recruitment of 2 lymphocytes in atopic lesions [31,32]. In vitro results suggested that SMGGT could suppress MDC/CCL22 and TARC/CCL17 production by suppressing p38 and STAT1 phosphorylation and the nuclear translocation of NF-κB in HaCaT cells.
ese results suggest that SMGGT could ameliorate the SD-worsened AD through anti-inflammatory activity. Interestingly, topical dexamethasone reduced inflammatory cytokines and cellular infiltrates in skin lesions, but they were unsuccessful in alleviating gross skin symptoms. is could be because, despite their anti-inflammatory qualities, topical corticosteroids interfere with epidermal barrier repair by affecting the formation of stratum corneum lipids [33].
Previous studies reported that the HPA axis is activated by SD [7,34], which is associated with the upregulation of 2 cytokines important in the pathogenesis of AD, such as IL-4 and IL-5 [35]. In this study, SD increased the serum levels of the hormones of the HPA axis, including CRH, ACTH, and CORT. CRH exerts its proinflammatory effects by binding to the CRH receptors (CRHR) on mast cells to enhance the production of VEGF; hence, AD can be aggravated [36]. e number of mast cells and VEGF levels in skin lesions from DNCB-treated mice under SD conditions were significantly higher than the only DNCB-treated mice. Treatment with SMGGT reduced the CRH levels as well as mast cell infiltration and skin level of VEGF in SD-exposed AD mice. Our previous study indicated that costimulation of   Evidence-Based Complementary and Alternative Medicine CRH and SP significantly increased the production of VEGF from mast cells in vitro [21]. In this study, SMGGT reduced VEGF production significantly by suppressing the phosphorylation of the ERK signaling pathway in SP/CRHtreated HMC-1 cells. e neuropeptide SP has been reported to induce sleep disturbances and itch behavior mediated by mast cells in mice [37,38]. In vitro studies suggested that SP induces the expression of CRHR1 on mast cells and a combination of SP and CRH could enhance the inflammatory responses in mast cells [39]. Overall, the effects of SMGGT on SD-worsened AD symptoms might be mediated by the regulation of CRH and inflammatory responses in mast cells. SD was reported to cause depression-like behavior in a mouse model [40].
e current study showed that SD increased the immobility time in the TST in AD mice, which was reduced by the SMGGT treatment. Melatonin, a hormone secreted by the pineal gland, plays a crucial role in the regulation of sleep and circadian rhythm [41]. Melatonin also has an antidepressant effect and alleviates AD-like symptoms by reducing levels of IgE in serum and IL-4 and IFN-c production [42,43]. In this study, under SD conditions, AD mice showed lower melatonin levels than normal mice. Moreover, melatonin levels had negative correlations with the levels of inflammatory cytokines VEGF and IL-4 in skin lesions and the immobility time in the TST; hence, the decrease in melatonin production might occur concomitantly with the increase in the inflammatory response and depression-like behavior in AD mice exposed to SD. ese results indicated that the SMGGT treatment recovered the reduction of melatonin level, suggesting that SMGGT might exert its effects on SD-exacerbated AD symptoms and behavior changes by increasing melatonin production.
HPLC analysis showed that puerarin and paeoniflorin are the major components in SMGGT with the highest amounts. Puerarin, the main isoflavonoid derived from the root of Pueraria montana var. lobata (Willd.) Sanjappa & Pradeep, has been reported to suppress the production of inflammatory mediators in AD models [44]. Paeoniflorin, an active ingredient of Paeonia lactiflora Pallas, has therapeutic effects on insomnia by promoting non-rapid eye movement sleep and reducing the wakefulness time in a mouse neuropathic pain model [45]. Moreover, both puerarin and paeoniflorin have antidepressant effects on chronic stressinduced depression in a rat model [46,47]. In addition, network pharmacological and molecular docking analysis also demonstrated that puerarin and paeoniflorin might exert their effects by targeting AD-related targets, pathways, and biological processes. Puerarin and paeoniflorin also exerted anti-inflammatory effects by reducing the production of inflammatory chemokines and cytokines in HaCaT and HMC-1 cells. ese results suggest that puerarin and paeoniflorin may play a role in the regulation of skin inflammation and depression-like behavior in AD under SD conditions.       ere are several limitations to our study. First, the extraction method utilized in clinical application and the extraction method employed in this animal experiment are different, and accurate dose conversion between animals and human is challenging. In clinical applications, SMGGT is utilized as a water extract; however, in this animal trial, it was employed as a 30% ethanol extract. Second, the higher dose of SMGGT more effectively alleviated the immobility time and serum CRH and CORT levels than the low dose but not in skin symptoms and inflammatory cytokines level. Hence, further research on the safe and effective concentration of SMGGT in terms of bioavailability and tissue distribution is needed. ird, it is required to investigate whether the increase in serum melatonin caused by SMGGT treatment is due to improved sleep quality and circadian rhythms.

Conclusion
SMGGT ameliorates SD-exacerbated cutaneous inflammation and depression-like behavior in an AD mice model. is study suggests that SMGGT with puerarin and paeoniflorin as main bioactive components is a promising candidate for treatment in patients with AD under SD conditions.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
Ly i Huong Nguyen performed the experiment and formal analysis and prepared the manuscript draft with the support of Min-Jin Choi. Heung-Mook Shin and In-Jun Yang conceptualized the study and reviewed and edited the manuscript. Evidence-Based Complementary and Alternative Medicine 13