Moringa concanensis L. Alleviates DNCB-Induced Atopic Dermatitis-like Symptoms by Inhibiting NLRP3 Inflammasome-Mediated IL-1β in BALB/c Mice

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by pruritus, dry skin and redness on the face and inside elbows or knees. Most patients with AD are children and youths, but it can also develop in adults. In the therapeutic aspect, treatment with corticosteroids for AD has several side effects, such as weight loss, atrophy and acne. In the current study, we examined the anti-inflammatory effect of Moringa concanensis leaves on HaCaT keratinocytes and 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis-like symptoms in BALB/c mice. We observed that M. concanensis treatment exhibited significant inhibition in the production of inflammatory mediators and proinflammatory cytokines, such as IL-1β, in LPS-induced HaCaT keratinocytes by downregulating the NLRP3 inflammasome activation. Moreover, M. concanensis inhibited the activation of JNK, AP-1 and p65, which resulted in the deformation of NLRP3 in LPS-stimulated HaCaT cells. In mice with DNCB-induced AD-like skin lesions, the administration of M. concanensis ameliorated the clinical symptoms, such as the dermatitis score, thickness of lesional ear skin and TEWL. Furthermore, M. concanensis could attenuate the activation of the immune system, such as reducing the spleen index, concentration of the IgE levels and expression of the NLRP3 inflammasome in ear tissues. Therefore, our results suggest that M. concanensis exerts anti-atopic dermatitis effects by inhibiting the NLRP3 inflammasome-mediated IL-1β.


Introduction
From various pathophysiological perspectives, atopic dermatitis (AD) is a chronic relapsing inflammatory skin disease associated with pruritus and redness, typically on the face and the inside of elbows and knees [1]. Therefore, AD is closely connected to patients' quality of life and comorbidities [2]. AD can occur at any age and its prevalence is increasing. According to one report, the number of AD patients diagnosed in one year ranged from 13.5% to 41.9%, depending on the country [3]. The main symptoms of AD are characterized by a multidimensional patient burden, including persistent itching, dryness of the skin and depression [4,5]. Although the pathogenesis of AD is still unclear, some studies have suggested that most patients with AD are affected due to sensitization to environmental allergens, genetic backgrounds and increased serum immunoglobulin E (IgE) [6,7]. Based on clinical experiments, topical corticosteroids are commonly used in patients with AD [8]. However, the current treatments for AD, especially topical steroids,  Table 1). The major phytochemicals included quinic acid (C 7 H 12 O 6 ), coumaroylquinic acid (C 16 ). It has been indicated that quinic acid derivatives, coumaric acid and quercetin suppress immune responses [21][22][23]. In particular, some studies revealed that quercetin downregulated the production of IL-1β by inhibiting the NLRP3 inflammasome [24,25].

M. concanensis Inhibits the LPS-Stimulated Inflammatory Mediators in HaCaT Cells
Recently, new insight into the pathogenesis of AD focused on abnormalities in the epidermal layer [26]. Furthermore, several studies have suggested that the downregulation of immune activation in epidermal keratinocytes plays a key role in ameliorating inflammatory skin diseases, such as AD [27,28]. Therefore, we investigated the effect of M. concanensis on LPS-stimulated inflammatory responses in keratinocytes. Firstly, an MTT assay was performed to determine the cytotoxic concentration of M. concanensis in HaCaT keratinocytes. The treatment of M. concanensis extract did not affect the viability of HaCaT cells at the concentration of 10-300 µg/mL (Figure 2A). HaCaT cells were pretreated with M. concanensis for 1 h and then treated with LPS (1 µg/mL) for 24 h. M. concanensis extract markedly reduced the production of NO and PGE 2 at 100 and 300 µg/mL concentrations ( Figure 2B,E). Moreover, M. concanensis inhibited the mRNA as well as protein expressions of iNOS and COX-2, which is related to the synthesis of NO and PGE 2 production ( Figure 2C,D,F,G). Our results suggest that M. concanensis can regulate the production of inflammatory mediators, such as NO and PGE 2 , via the inhibition of iNOS and COX-2 expressions. HaCaT cells were treated with M. concanensis (MC) for 24 h, and cell viability was determined by MTT assay (A). Cells were pretreated with MC for 1 h before LPS stimulation (1 μg/mL) for 24 h. The production of NO and PGE2 was determined by Griess reagent and ELISA kits, respectively (B,E). The level of mRNA expressions of iNOS and COX-2 was determined by RT-qPCR (C,F). The levels of iNOS and COX-2 proteins were measured by Western blotting analysis, and the quantifications were normalized to the control (D,G). The data presented are mean of three independent determinations and indicate the mean ± S.E.M. # p < 0.05, ### p < 0.001 compared to the vehicle-treated controls; * p < 0.05, ** p < 0.01 and *** p < 0.001 compared to the LPS-treated group.

M. concanensis Downregulated the Expression of Inflammatory Cytokines in LPS-Stimulated HaCaT Cells
The production of inflammatory cytokines, such as TNF-α, IL-1β and IL-6, due to the inflammatory reactions could control immune activation [29]. Therefore, we examined whether M. concanensis had an inhibitory effect on inflammatory cytokines, such as TNF- HaCaT cells were treated with M. concanensis (MC) for 24 h, and cell viability was determined by MTT assay (A). Cells were pretreated with MC for 1 h before LPS stimulation (1 µg/mL) for 24 h. The production of NO and PGE 2 was determined by Griess reagent and ELISA kits, respectively (B,E). The level of mRNA expressions of iNOS and COX-2 was determined by RT-qPCR (C,F). The levels of iNOS and COX-2 proteins were measured by Western blotting analysis, and the quantifications were normalized to the control (D,G). The data presented are mean of three independent determinations and indicate the mean ± S.E.M. # p < 0.05, ### p < 0.001 compared to the vehicle-treated controls; * p < 0.05, ** p < 0.01 and *** p < 0.001 compared to the LPS-treated group.

M. concanensis Downregulated the Expression of Inflammatory Cytokines in LPS-Stimulated HaCaT Cells
The production of inflammatory cytokines, such as TNF-α, IL-1β and IL-6, due to the inflammatory reactions could control immune activation [29]. Therefore, we examined whether M. concanensis had an inhibitory effect on inflammatory cytokines, such as TNFα, IL-1β and IL-6, in LPS-stimulated HaCaT cells. The cells were pretreated with M. concanensis for 1 h before LPS stimulation (1 µg/mL) for 24 h. The LPS-stimulated cells and mediums were collected to investigate the expression of inflammatory cytokines using RT-qPCR and ELISA kits. Figure 3A-C show that M. concanensis treatment significantly reduced the mRNA expression levels of TNF-α, IL-1β and IL-6 compared to those in the LPS-treated controls in HaCaT cells. Moreover, the secretion of TNF-α, IL-1β and IL-6 protein was markedly reduced by M. concanensis ( Figure 3D-F).

M. concanensis Reduced the Expression of IL-1β by Inhibiting the NLRP3 Inflammasome in HaCaT Cells
The NLRP3 inflammasome is a multiprotein complex that consists of NLRP3, ASC and caspase-1. The NLRP3 inflammasome initiates immune responses during exposure to a variety of stimuli, mainly pathogen and danger-related molecular patterns [30]. The activation of the inflammasome results in the secretion of cytokine IL-1β, which is correlated with chronic inflammatory diseases [31]. As shown in Figures 3B,E, we found that M. concanensis effectively reduced the expression of IL-1β in HaCaT cells stimulated with LPS. Therefore, we next examined the effects of M. concanensis on the NLRP3 inflammasome activation in LPS/ATP-stimulated HaCaT cells. The experimental data demonstrated that M. concanensis dose-dependently downregulated the expression of NLRP3 ( Figure 4B). Furthermore, M. concanensis significantly attenuated the activation of ASC and cleaved caspase-1 at 300 μg/mL ( Figure 4C,D). These data indicate that M. concanensis reduced the secretion of IL-1β by regulating the formation of the NLRP3 inflammasome.

M. concanensis Reduced the Expression of IL-1β by Inhibiting the NLRP3 Inflammasome in HaCaT Cells
The NLRP3 inflammasome is a multiprotein complex that consists of NLRP3, ASC and caspase-1. The NLRP3 inflammasome initiates immune responses during exposure to a variety of stimuli, mainly pathogen and danger-related molecular patterns [30]. The activation of the inflammasome results in the secretion of cytokine IL-1β, which is correlated with chronic inflammatory diseases [31]. As shown in Figure 3B,E, we found that M. concanensis effectively reduced the expression of IL-1β in HaCaT cells stimulated with LPS. Therefore, we next examined the effects of M. concanensis on the NLRP3 inflammasome activation in LPS/ATP-stimulated HaCaT cells. The experimental data demonstrated that M. concanensis dose-dependently downregulated the expression of NLRP3 ( Figure 4B). Furthermore, M. concanensis significantly attenuated the activation of ASC and cleaved

M. concanensis Inhibited the Phosphorylation of NF-κB, MAPK and AP-1 in HaCaT Cells Stimulated with LPS
ROS and NF-κB contribute to the mechanism underlying NLRP3 inflammasome activation [32]. Mounting evidence indicates that activated transcription factors, such as NF-κB, induce the priming of the NLRP3 inflammasome [33,34]. Hence, we studied whether M. concanensis suppressed the phosphorylation of NF-κB and its upstream MAPK in LPSstimulated HaCaT cells. As shown in Figure 5A,B, LPS treatment significantly upregulated the phosphorylation of NF-κB. However, M. concanensis treatment significantly downregulated the phosphorylation of p65. Furthermore, LPS treatment increased the phosphorylation of JNK, p38 and ERK which are subunits of MAPK, but M. concanensis only exhibited a significant reduction in the phosphorylation of JNK ( Figure 5A,C). In addition, AP-1, which is regulated by the activated MAPK family, such as JNK, can mediate the transcription of inflammatory mediators [35]. Our results show that M. concanensis also inhibited the phosphorylation of the AP-1 subunit c-fos ( Figure 5A,D). Therefore, these results demonstrate that M. concanensis had anti-inflammatory properties and inhibited the priming of the NLRP3 inflammasome via the inhibition of phosphorylated p65, JNK and c-fos signaling. . The data presented are the mean of three independent determinations and indicate the mean ± S.E.M. # p < 0.05 and ## p < 0.01 compared to the vehicletreated controls; * p < 0.05, ** p < 0.01 and *** p < 0.001 compared to the LPS-treated group.

M. concanensis Inhibited the Phosphorylation of NF-κB, MAPK and AP-1 in HaCaT Cells Stimulated with LPS
ROS and NF-κB contribute to the mechanism underlying NLRP3 inflammasome activation [32]. Mounting evidence indicates that activated transcription factors, such as NF-κB, induce the priming of the NLRP3 inflammasome [33,34]. Hence, we studied whether M. concanensis suppressed the phosphorylation of NF-κB and its upstream MAPK in LPS-stimulated HaCaT cells. As shown in Figure 5A,B, LPS treatment significantly upregulated the phosphorylation of NF-κB. However, M. concanensis treatment significantly downregulated the phosphorylation of p65. Furthermore, LPS treatment increased the phosphorylation of JNK, p38 and ERK which are subunits of MAPK, but M. concanensis only exhibited a significant reduction in the phosphorylation of JNK ( Figure 5A,C). In addition, AP-1, which is regulated by the activated MAPK family, such as JNK, can mediate the transcription of inflammatory mediators [35]. Our results show that M. concanensis also inhibited the phosphorylation of the AP-1 subunit c-fos ( Figure 5A,D). Therefore, these results demonstrate that M. concanensis had anti-inflammatory properties and inhibited the priming of the NLRP3 inflammasome via the inhibition of phosphorylated p65, JNK and c-fos signaling.

M. concanensis Improved the Clinical Symptoms in Mice with AD-like Skin Lesions Induced by DNCB
Given the anti-inflammatory properties of M. concanensis in keratinocytes, we further investigated whether M. concanensis has anti-atopic dermatitis effects in mice with ADlike skin lesions induced by DNCB. BALB/c mice were shaved using a clipper for dorsal skin. After shaving, the mice were sensitized with 1% DNCB twice every 7 days. To evaluate the effects of M. concanensis on AD, the mice were orally administered with M. concanensis (100 and 200 mg/kg) for 14 days. In addition, 0.6% DNCB was topically applied to accelerate atopic dermatitis once every 2 days. There was no difference in the body weight of the AD mice in the M. concanensis-treated group, but a significant reduction in the body weight of dexamethasone (1 mg/kg)-administered group when compared with the DNCB-treated group ( Figure 6B). A significant increase in the SCORAD index was observed in the DNCB-treated group compared with the normal group. However, we observed that the dermatitis scores were dose-dependently reduced by the administration of M. concanensis when compared with the DNCB-treated group ( Figure 6A,C). Moreover, the mice treated with M. concanensis had reduced ear thickness and TEWL compared to the DNCB-treated group ( Figure 6D,E). These results demonstrate that the M. concanensis oral administration can attenuate the clinical symptoms of AD without side effects, such as body weight loss, in a DNCB-induced AD mice model.

M. concanensis Improved the Clinical Symptoms in Mice with AD-like Skin Lesions Induced by DNCB
Given the anti-inflammatory properties of M. concanensis in keratinocytes, we further investigated whether M. concanensis has anti-atopic dermatitis effects in mice with ADlike skin lesions induced by DNCB. BALB/c mice were shaved using a clipper for dorsal skin. After shaving, the mice were sensitized with 1% DNCB twice every 7 days. To evaluate the effects of M. concanensis on AD, the mice were orally administered with M. concanensis (100 and 200 mg/kg) for 14 days. In addition, 0.6% DNCB was topically applied to accelerate atopic dermatitis once every 2 days. There was no difference in the body weight of the AD mice in the M. concanensis-treated group, but a significant reduction in

M. concanensis Ameliorated the Immunological and Histological Changes in DNCB-Challenged BALB/c Mice
To determine whether the administration of M. concanensis affects immunological activation, the weights of the cernical lymph nodes and spleen and the plasma IgE concentration were calculated after sacrifice. The indices of the lymph nodes and spleen were significantly increased in the DNCB-only group when compared with the normal group. Although the lymph node index did not significantly decrease, the spleen index was significantly reduced by the oral administration of M. concanensis when compared with the DNCB-administered group ( Figure 7A,B). In addition, it is well known that the upregulated IgE levels have been detected in patients with AD [36]. As shown in Figure 7C, M. concanensis dose-dependently inhibited the level of the plasma IgE compared to that in the DNCB-only group. These data indicate that the reduced spleen index and IgE levels could be therapeutic strategies in AD therapies.

M. concanensis Ameliorated the Immunological and Histological Changes in DNCB-Challenged BALB/c Mice
To determine whether the administration of M. concanensis affects immunological activation, the weights of the cernical lymph nodes and spleen and the plasma IgE concentration were calculated after sacrifice. The indices of the lymph nodes and spleen were significantly increased in the DNCB-only group when compared with the normal group. Although the lymph node index did not significantly decrease, the spleen index was significantly reduced by the oral administration of M. concanensis when compared with the DNCB-administered group ( Figure 7A,B). In addition, it is well known that the upregulated IgE levels have been detected in patients with AD [36]. As shown in Figure 7C, M. concanensis dose-dependently inhibited the level of the plasma IgE compared to that in the DNCB-only group. These data indicate that the reduced spleen index and IgE levels could be therapeutic strategies in AD therapies.
To investigate the histological changes, epidermal hyperplasia and mast cell infiltration in lesional dorsal skin were investigated by H&E and toluidine blue staining, respec- To investigate the histological changes, epidermal hyperplasia and mast cell infiltration in lesional dorsal skin were investigated by H&E and toluidine blue staining, respectively ( Figure 7D). A significant increase in the epidermal thickness in the DNCBadministered group was observed when compared with the normal group ( Figure 7D,E). Furthermore, mast cell infiltration was considerably increased in the DNCB-challenged group ( Figure 7D,F). However, the mice treated with M. concanensis showed a dosedependent suppression of hyperplasia and mast cell infiltration in lesional dorsal skin tissues ( Figure 7D,E,F). These data suggest that M. concanensis may regulate of the immune system activation and histological changes in DNCB-induced lesional dorsal skin.
( Figure 7D,F). However, the mice treated with M. concanensis showed a dose-dependent suppression of hyperplasia and mast cell infiltration in lesional dorsal skin tissues ( Figure  7D,E,F). These data suggest that M. concanensis may regulate of the immune system activation and histological changes in DNCB-induced lesional dorsal skin.

M. concanensis Inhibited the Activation of the NLRP3 Inflammasome in DNCB-Treated BALB/c Mice
It has been shown that the upregulation of NLRP3 inflammasome is associated with the pathogenesis of chronic dermatitis in the skin of mice [37]. To determine whether the

M. concanensis Inhibited the Activation of the NLRP3 Inflammasome in DNCB-Treated BALB/c Mice
It has been shown that the upregulation of NLRP3 inflammasome is associated with the pathogenesis of chronic dermatitis in the skin of mice [37]. To determine whether the symptoms of AD were attenuated via the inhibition of the NLRP3 inflammasome, we investigated whether the application of M. concanensis inhibited the NLRP3 inflammasome in DNCB-induced lesional ear tissues. After the sacrifice, the lesional ear tissues were collected and analyzed for the expression of NLRP3, ASC and IL-1β. As shown in Figure 8, the NLRP3, ASC and IL-1β expressions were significantly increased in the DNCB-treated group. However, similar to Figure 4, we found that the mice treated with M. concanensis had significantly reduced NLRP3, ASC and IL-1β mRNA expression compared to mice in the DNCB-treated group ( Figure 8A-C). Therefore, the results of the present study suggest that M. concanensis relieved AD-like symptoms by downregulating the expression of NLRP3, IL-1β and ASC in DNCB-induced lesional ear tissues. symptoms of AD were attenuated via the inhibition of the NLRP3 inflammasome, we investigated whether the application of M. concanensis inhibited the NLRP3 inflammasome in DNCB-induced lesional ear tissues. After the sacrifice, the lesional ear tissues were collected and analyzed for the expression of NLRP3, ASC and IL-1β. As shown in Figure 8, the NLRP3, ASC and IL-1β expressions were significantly increased in the DNCB-treated group. However, similar to Figure 4, we found that the mice treated with M. concanensis had significantly reduced NLRP3, ASC and IL-1β mRNA expression compared to mice in the DNCB-treated group ( Figure 8A-C). Therefore, the results of the present study suggest that M. concanensis relieved AD-like symptoms by downregulating the expression of NLRP3, IL-1β and ASC in DNCB-induced lesional ear tissues.

Discussion
Atopic dermatitis is known as a typical chronic inflammatory disease, and its prevalence in patients with AD has consistently increased over the last decade [38]. This inflammatory skin disease is normally demonstrated during the first year of birth, however, it can occur in adults [39]. The pathogenesis of AD involves complex factors, including environmental provocation, genetic predisposition and immunological abnormalities [40]. Based on clinical research, AD cannot be completely cured [41]. Therefore, the main management of AD involves improving the clinical symptoms and achieving long-term disease control following treatment guidelines. The drugs used in the treatment of AD, such as glucocorticosteroids, antihistamines and calcineurin inhibitors, can improve itching, edema and skin inflammation [42]. However, several studies have shown that the prolonged use of these medications could cause various adverse effects, including skin atrophy, heart failure and high blood pressure [43,44]. In this study, we first found that M. concanensis alleviated AD-like lesions in BALB/c mice induced by DNCB. Furthermore, we revealed that M. concanensis blocked NLRP3 formation by inhibiting the JNK-NF-κB and AP-1 pathways. These observations indicate that M. concanensis could be a novel candidate for preventing and treating AD.
Keratinocytes play a potential role in skin immune responses that cause immune cells to produce proinflammatory cytokines [45]. The present study exhibited the anti-inflammatory properties of M. concanensis and its underlying mechanisms in LPS-stimulated HaCaT keratinocytes. After Toll-like receptor 4 (TLR4) recognizes LPS, the TLR4 signaling cascade regulates inflammatory mediators via the phosphorylation of transcription factors, mainly NF-κB [46,47]. A recent study indicated that the NF-κB activation could positively regulate the NLRP3 inflammasome, which aggravated immune-related skin diseases, such as AD [48]. In LPS-induced HaCaT keratinocytes, it was observed that M.

Discussion
Atopic dermatitis is known as a typical chronic inflammatory disease, and its prevalence in patients with AD has consistently increased over the last decade [38]. This inflammatory skin disease is normally demonstrated during the first year of birth, however, it can occur in adults [39]. The pathogenesis of AD involves complex factors, including environmental provocation, genetic predisposition and immunological abnormalities [40]. Based on clinical research, AD cannot be completely cured [41]. Therefore, the main management of AD involves improving the clinical symptoms and achieving long-term disease control following treatment guidelines. The drugs used in the treatment of AD, such as glucocorticosteroids, antihistamines and calcineurin inhibitors, can improve itching, edema and skin inflammation [42]. However, several studies have shown that the prolonged use of these medications could cause various adverse effects, including skin atrophy, heart failure and high blood pressure [43,44]. In this study, we first found that M. concanensis alleviated AD-like lesions in BALB/c mice induced by DNCB. Furthermore, we revealed that M. concanensis blocked NLRP3 formation by inhibiting the JNK-NF-κB and AP-1 pathways. These observations indicate that M. concanensis could be a novel candidate for preventing and treating AD.
Keratinocytes play a potential role in skin immune responses that cause immune cells to produce proinflammatory cytokines [45]. The present study exhibited the antiinflammatory properties of M. concanensis and its underlying mechanisms in LPS-stimulated HaCaT keratinocytes. After Toll-like receptor 4 (TLR4) recognizes LPS, the TLR4 signaling cascade regulates inflammatory mediators via the phosphorylation of transcription factors, mainly NF-κB [46,47]. A recent study indicated that the NF-κB activation could positively regulate the NLRP3 inflammasome, which aggravated immune-related skin diseases, such as AD [48]. In LPS-induced HaCaT keratinocytes, it was observed that M. concanensis reduced iNOS and COX-2 expressions, which synthesize NO and PGE 2 , inhibiting the secretion of inflammatory mediators. Furthermore, we found that M. concanensis inhibited the level of mRNA and protein expressions of TNF-α, IL-1β and IL-6 by inhibiting the phosphorylation of JNK/AP-1/NF-κB. Moreover, we confirmed that M. concanensis reduced the expression of IL-1β by inhibiting the formation of the NLRP3 inflammasome. The genus Moringa is known as a medicinal plant that has been traditionally used for diseases such as colds and diabetes [49]. Many Moringa species have been reported to inhibit the inflammatory response [50,51]. A previous study demonstrated that the hydroethanolic extract of Moringa oleifera flowers has anti-inflammatory potentials by preventing the phosphorylation of NF-κB in RAW 264.7 macrophages stimulated with LPS [52]. Moreover, the study showed that the ethanolic extract of M. concanensis relieved pain and had anti-inflammation effects by reducing the synthesis of prostaglandin [53]. Thus, these previous studies support our results that M. concanensis had inhibitory effects on LPS-induced inflammatory responses in HaCaT cells. In addition, in DNCB-challenged BALB/c mice, we found that the mice treated with M. concanensis had significantly improved skin lesions, ear thickness and TEWL. Note that, as shown in Figure 6E, the levels of TEWL were reduced from Day 15. It is thought that the TEWL levels were decreased by hair regrowth. Moreover, our results show that the mice treated with M. concanensis had a reduced spleen index, IgE levels in plasma, epidermal thickness, mast cell infiltration and NLRP3 inflammasome expression in lesional ear tissue. Therefore, our data indicate the anti-atopic properties of M. concanensis in DNCB-challenged BALB/c mice.
Accumulating evidence suggests that inflammasomes are associated with the inflammatory response in AD [54,55]. In particular, NLRP3 inflammasome-mediated IL-1β plays a critical role in the pathological process of inflammation-mediated skin diseases [56]. Thus, inhibition of NLRP3 inflammasome-dependent IL-1β could regulate the pathogenesis of AD. The present study revealed that M. concanensis inhibited the priming signal of the NLRP3 inflammasome by restraining NF-κB phosphorylation. This evidence supports the notion that M. concanensis inhibits the priming and activating signals of the NLRP3 inflammasome. Moreover, several studies reported that extracts of M. concanensis had antioxidative properties [57,58]. In this study, various chemical compounds, including quercetin and quinic acid, were detected using a UPLC-QTOF analysis of M. concanensis (Table 1). Some studies reported that quercetin and quinic acid derivatives had anti-inflammatory properties in an animal model of colitis and microglia, respectively [59][60][61]. Notably, previous studies reported that quercetin elicited an inhibitory effect of NLRP3 inflammasome activation in macrophages and endothelial cells [62,63]. These previous reports implicate that quercetin and quinic acid in M. concanensis may contribute to the anti-inflammatory effects of M. concanensis. Therefore, we considered that quercetin in M. concanensis may alleviate the AD symptoms by reducing the activation of NLRP3 inflammasome-mediated IL-1β. Although inhibitors of the NLRP3 inflammasome have anti-inflammatory properties in mice, evidence of similar effects in human skin diseases is still lacking. Therefore, clinical studies targeting the NLRP3 inflammasome for inflammatory skin diseases are needed.

Animals
BALB/c mice (female, 6 weeks old) were procured from Orient Bio (Seongnam, Korea). All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee of the Laboratory Animal Research Center, Kangwon National University, Korea (KW-211208-5). Each cage contained four mice, which were housed under controlled conditions of 21-25 • C and a 12 h light and dark cycle. The animals were given free access to food and water throughout the experimental period.

Preparation of an Ethanolic Extract of M. concanensis
The leaves of M. concanensis were collected Coimbatore District, Tamil Nadu, India and the plant sample was authenticated (Letter No. BSI/SRC/5/23/2018/Tech-437) Botanical Survey of India, Southern Regional Centre, Coimbatore, Tamil Nadu, India. After washing with distilled water, the leaves were dried under light-shielding conditions. Then, 100 g of dried M. concanensis leaves (MC) were mixed with 1 L of 70% ethanol for extraction, twice for 2 h by using an ultrasonic bath. After the extraction, the filtrate was evaporated using a rotary vacuum evaporator. Then, the semisolid residue was lyophilized to produce the extract with 20%.

Identification of Phytochemicals in M. concanensis by UPLC-QTOF-MS/MS
The phytochemicals in M. concanensis L. were estimated by using ultra-performance liquid chromatography supplied with quadrupole time-of-flight mass spectrometry (UPLC/ QTOF-MS/MS) (WATERS XEVO GS-XS QTOF analyzer). Ten milligrams of M. concanensis were dissolved in 10 mL of 70% ethanol, and then, 2 µL of M. concanensis were injected into a Waters ACQUITY UPLC BEH C18 column (50 × 2.1 mm, 1.7 µm). The flow rate of the column was altered at 0.3 mL/min. The mobile phase contained 0.1% formic acid in water (solvent A), and 0.1% formic acid in acetonitrile (solvent B). The column conditions and the characterization of chemical components were followed according to the method described by Oh et al. [64]. The chemical components in the leaves of M. concanensis were identified from the library of traditional Chinese medicine (TCM) using UNIFI 1.8 (Waters, Milford, MA, USA) software and an in-house library.

Cell Culture
The human epidermal keratinocyte cells (HaCaT) were provided by Professor Ok-Hwan Lee from the Food Chemistry Laboratory at Kangwon National University. HaCaT cells (2 × 10 5 ) were cultured in DMEM supplemented with 10% FBS in a 5% CO 2 incubator at 37 • C. LPS was used to stimulate the HaCaT cells at the concentration of 1 µg/mL for 1 h or 24 h.

Cell Viability
The viability of HaCaT cells was determined using an MTT assay. For this purpose, the cells were pretreated with M. concanensis for 24 h and incubated with MTT solution at 5 mg/mL for 4 h to form formazan crystals. After the incubation, the supernatant in each well was replaced with 100 µL of DMSO and isopropyl alcohol (1:1). The absorbance was measured at 540 nm on SpectraMax microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Nitric Oxide Production
HaCaT cells were pretreated with different concentrations of M. concanensis (10, 30, 100 and 300 µg/mL) for 1 h and then treated with LPS (1 µg/mL) for 24 h. The production of nitric oxide (NO) was assessed by measuring the nitrite accumulation in the culture medium. The level of nitrite in the medium was measured by Griess reagent. Briefly, 100 µL of supernatant and Griess reagent were mixed and incubated for 10 min. The absorbance was measured at 540 nm on SpectraMax microplate reader (Molecular Devices, Sunnyvale, CA, USA). The level of nitrite in the culture supernatant of LPS-induced HaCaT cells was calculated using a sodium nitrite standard curve.

RNA Extraction and Real Time Quantitative Polymerase Chain Reaction (RT-qPCR)
The mRNA expression of iNOS, COX-2, TNF-α, IL-1β, IL-6, NLRP3 and ASC was measured using RT-qPCR. The total RNA was extracted using RNAiso PLUS (Takara, Otsu, Japan). Complementary DNA (cDNA) was synthesized from 1 µg of total RNA using Allin-One FirstStrand cDNA Synthesis SuperMix previously described by Ko et al. [65]. The synthesized cDNAs were used as a template for RT-qPCR using a QuantStudio 3 (Applied Biosystems, Foster City, CA, USA) system with POWER SYBR Green PCR master mix and gene-specific primers ( Table 2). A dissociation curve analysis of iNOS, COX-2, IL-1β, IL-6, NLRP3, ASC and β-actin demonstrated a single peak. The expression levels of the target genes were quantified by duplicating measurements and normalized with the 2 −∆∆CT method relative to the control β-actin. The PCR analyses were performed under the following conditions: 40 cycles of 95 • C for 15 s; 57 • C for 20 s, and 72 • C for 40 s. The expression of PGE 2 , TNF-α, IL-1β and IL-6 in the culture supernatant was measured using ELISA kits (R&D Systems, Minneapolis, MN, USA). The cells were pretreated with M. concanensis at various concentrations (10, 30, 100 and 300 µg/mL) for 1 h and stimulated with LPS (1 µg/mL) for 24 h. The expression of IgE in plasma was also measured using an ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's protocol.

2,4-Dinitrochlorobenzene (DNCB)-Induced Atopic Dermatitis Mice
BALB/c mice were topically sensitized with 200 μL of 1% DNCB diluted in a mixture of acetone and olive oil (3:1), on shaved dorsal skin and ears twice a week. The mice were divided into 5 groups (n = 8/group) as follows: an untreated group (Normal), an only DNCB-sensitized group (DNCB), a group receiving oral administration of 100 mg/kg M. concanensis (MC 100), a group receiving oral administration of 200 mg/kg M. concanensis (MC 200) and a group receiving the administration of 1 mg/kg dexamethasone (DEXA). Seven days later, the mice were stimulated with 0.6% DNCB on the dorsal skin (200 μL) and the right ear (20 μL for every 2 days). Mice with DNCB-induced AD-like skin lesions were orally treated with M. concanensis (100 and 200 mg/kg) and dexamethasone (1 mg/kg) every day (Figure 9).

Measurement of Clinical Symptoms and Histological Changes
The severity of the skin lesions in DNCB-induced AD was estimated according to the SCORAD index, which is scored from 0 (none) to 3 (severe) based on erythema, pruritus/dry skin, edema and excoriation [67]. The thickness of the ear was measured using a Digimatic micrometer (Mitutoyo, Kawasaki, Japan). GPSKIN Barrier Pro (GPpower, Hanam, Korea) was used to measure transepithelial water loss (TEWL) in the dorsal skin using the GPSKIN Research program [68]. Changes in the clinical symptoms, such as body weight, ear thickness and TEWL, in the AD mice were measured every three days. To evaluate the histological examination, the dorsal skin tissues were punched using a 5 mm biopsy punch, fixed in 10% formalin solution and embedded in paraffin [69]. Each section slice of paraffin-embedded skin tissue was stained with hematoxylin and eosin (H&E) and toluidine blue (TB). The histological changes were examined by light microscopy (Olympus, Tokyo, Japan). The epidermal thickness was observed using H&E staining at 100× magnification. The infiltration of mast cells was analyzed with TB staining and the slices were examined in four randomly selected sections.

Statistical Analysis
The statistical analyses were performed using GraphPad Prism Version 8.0 (GraphPad, La Jolla, CA, USA). All data are expressed as the mean ± S.E.M. The data were analyzed by a one-way analysis of variance (ANOVA), followed by a Student-Newman-Keuls test for multiple comparisons. p < 0.05 was considered a significant statistical value.

Conclusions
In conclusion, our results show that M. concanensis inhibited the formation of the NLRP3 inflammasome through JNK/AP-1/NF-κB signaling in HaCaT keratinocytes. Furthermore, we suggest that M. concanensis attenuated DNCB-induced AD-like symptoms in BALB/c mice by inhibiting IL-1β mediated by the NLRP3 inflammasome. Therefore, M. concanensis has therapeutic properties in chronic inflammatory skin diseases, mainly AD.