Nymphoides peltata Root Extracts Improve Atopic Dermatitis by Regulating Skin Inflammatory and Anti-Oxidative Enzymes in 2,4-Dinitrochlorobenzene (DNCB)-Induced SKH-1 Hairless Mice

Nymphoides peltata is widely used pharmacologically in Traditional Chinese Medicine and Ayurvedic medicine as a diuretic, antipyretic, or choleretic and to treat ulcers, snakebites, and edema. Previous studies have shown that phytochemicals from N. peltata have physiological activities such as anti-inflammatory, anti-tumor, and anti-wrinkle properties. Nevertheless, research on the anti-atopic dermatitis (AD) effect of N. peltata extract is limited. This study was undertaken to assess the in vitro and in vivo anti-atopic and antioxidant activities of a 95% EtOH extract of N. peltata roots (NPR). PI-induced RBL-2H3 cells and two typical hapten mice (oxazolone-induced BALB/c mice and 2,4-dinitrochlorobenzene (DNCB)-induced SKH-1 hairless mice) were used to investigate the effect of NPR extract on AD. The expressions of AD-related inflammatory cytokines, skin-related genes, and antioxidant enzymes were analyzed by ELISA, immunoblotting, and immunofluorescence, and skin hydration was measured using Aquaflux AF103 and SKIN-O-MAT instruments. The chemical composition of NPR extract was analyzed using an HPLC-PDA system. In this study, NPR extracts were shown to most efficiently inhibit IL-4 in PI-induced RBL-2H3 cells and AD-like skin symptoms in oxazolone-BALB/c mice compared to its whole and aerial extracts. NPR extract markedly reduced DNCB-induced increases in mast cells, epidermal thickness, IL-4 and IgE expressions, and atopic-like symptoms in SKH-1 hairless mice. In addition, NPR extract suppressed DNCB-induced changes in the expressions of skin-related genes and skin hydration and activated the Nrf2/HO-1 pathway. Three phenolic acids (chlorogenic acid, 3,5-dicaffeoylquinic acid, and 3,4-dicaffeoylquinic acid) were identified by HPLC-PDA in NPR extract. The study shows that NPR extract exhibits anti-atopic activities by inhibiting inflammatory and oxidative stress and improving skin barrier functions, and indicates that NPR extract has potential therapeutic use for the prevention and treatment of AD.


Introduction
Atopic dermatitis (AD) is a chronic relapsing inflammatory skin disease with an increasing incidence in industrialized countries. AD can start in infancy but commonly begins in adulthood [1,2]. The main symptoms of AD include pruritus, eczema, dryness, keratosis, lichenification, and erythema, which can markedly reduce the quality of life [2]. AD is

Plant Material and Extraction
Whole N. peltata and its aerial portion and roots were obtained from the Hantaek Botanical Garden foundation in Yongin-si, Gyeonggi-do, Republic of Korea, and authenticated by Dr. Jung Hwa Kang (Researcher Director). Voucher specimens (PNU-0040) were deposited in the Medicinal Herb Garden, Pusan National University. Whole plants (59 g), aerial parts (51.5 g), or roots (38.5 g) were air-dried at room temperature, ground to a powder, and then suspended in 2 L volumes of 95% ethanol (EtOH), and extracted ultrasonically twice for 90 min at room temperature. Extracts were then filtered and evaporated under reduced pressure in vacuo at 40 • C and freeze-dried to obtain N. peltata whole extract (NP, 3.9236 g, 6.65% extract yield), N. peltata aerial extract (NPA, 3.2513 g, 6.31% extract yield), and N. peltata root extract (NPR, 3.0271 g, 7.86% extract yield).

Animal Studies
BALB/c and SKH-1 hairless mice (6 weeks old) were purchased from Orient Bio (Seoul, Republic of Korea) and maintained under standard conditions (22 ± 3 • C and relative humidity 55 ± 5% under a 12 h light/dark cycle with food and water available ad libitum). All animal experiments procedures were reviewed beforehand and approved by the Institutional Animal Care and Use Committee of the Korea Institute of Science and Technology (KIST) (IRB code No. 2016-011, 2020-001; KIST).

Evaluation of Ear Swelling in the Oxazolone-Induced BALB/c Mice
The shaved inner and outer surfaces of both ears of BALB/c mice were sensitized by a single application of 20 µL of 1% oxazolone in a mixture of acetone/olive oil = 3:1 on experiment day 7. Ears were then challenged with 20 µL of 0.1% oxazolone from experiment day 8 on alternate days for 3 weeks. 1% NP, NPA, and NPR indicate that the amount of N. peltata extract is 1% (10 mg/mL) compared to the solution (propylene glycol/EtOH = 7:3). The mice were treated with 1% N. peltata extracts (NP, NPA, and NPR) twice a day for 3 weeks. Ear swelling and skin inflammation were measured on experiment day 28. On the last day of the experiment, mice were sacrificed, and ear skin was divided into two pieces for additional analysis. One piece was fixed in 3.7% formaldehyde (Sigma-Aldrich, St. Louis, MO, USA), and the other was collected by freezing at −80 • C.

Evaluation of AD Lesion Severity in the 2,4-Dinitrochlorobenzene (DNCB)-Induced SKH-1 Hairless Mice
The dorsal skin of SKH-1 hairless mice was sensitized with a single application of 200 µL of 1% DNCB in a mixture of acetone/olive oil = 3:1 on experiment day 7. Then, 200 µL of 0.1% DNCB with 1% NPR extract was administered from experiment day 8, 3 times/week for 2 weeks (experiment day 21). NPR extracts were treated at 4 h intervals after DNCB induction to exclude interaction between DNCB and samples. A 1% NPR extract indicates that the amount of N. peltata root extract is 1% (10 mg/mL) compared to the solution (propylene glycol/EtOH = 7:3). Transepidermal water loss (TEWL) and skin hydration were measured at the beginning and the end of the experiment under standard conditions using Aquaflux AF103 (Biox systems, London, UK) and SKIN-O-MAT (Cosmomed, Ruhr, Germany) instruments. On the last day of the experiment, mice were sacrificed, and skin and blood samples were collected for further analysis. The dorsal skin was divided into two pieces. One piece was fixed in 3.7% formaldehyde (Sigma-Aldrich), and the other was collected by freezing at −80 • C.

Histological Examination
Ear and dorsal skins of BALB/c and SKH-1 hairless mice were fixed in 10% formaldehyde (Sigma-Aldrich) for 24 h and embedded in paraffin wax. To measure changes in epidermal thicknesses and mast cell counts, tissues were serially sectioned at 4 µm and stained with hematoxylin and eosin (H&E) or toluidine blue. Epidermal thicknesses were determined using HK Basic software (KOPTIC, Seoul, Republic of Korea), and the number of mast cells infiltrating skin layers were counted in three randomly selected sections per mouse.

Measurement of Serum IL-4 and IgE by ELISA
Blood samples were collected from the abdominal aortas, and serum was obtained by centrifuging (8000 rpm, 15 min, 4 • C), and then stored. Total serum IL-4 and IgE levels were determined using enzyme-linked immunosorbent assay (ELISA) kits (eBioscience, San Diego, CA, USA).
The high-performance liquid chromatography-photodiode array (HPLC-PDA) analysis was performed using an e2695 separation module and a 2998 PDA detector (Waters Corporation, Milford, MA, USA). Data were collected using Empower ® 3 Chromatography Software. NPR extract was dissolved in MeOH/H 2 O = 1:1 (v/v) at a concentration of 10 mg/mL as a sample solution. Three major compounds (chlorogenic acid, 3,5dicaffeoylquinic acid, and 3,4-dicaffeoylquinic acid) were isolated and purified from NPR extract and identified by NMR, and MeOH/H 2 O = 1:1 (v/v) at a concentration of 1 mg/mL was used as a reference solution. Extract analysis was performed using a Sun Fire ® C18 column (4.6 × 150 mm, 5 µm, Waters) at 40 ± 2 • C using solvent A (0.1% formic acid in ACN) and solvent B (0.1% formic acid in H 2 O) using the following protocol: 0 to 5 min, 5 to 10% A; 5 to 10 min, 10 to 20% A; 10 to 15 min, 20 to 30% A; 15 to 20 min, 30 to 40% A; 20 to 25 min, 40 to 5% A). All injections were 10 µL in volume, and the column flow rate was set at 1.0 mL/min. The PDA acquisition wavelength range was 210 to 400 nm.

Statistical Analysis
The significances of differences between the untreated control and treatment groups were determined by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test. Results are presented as the means ± standard deviations (SDs) or standard errors (SEs) of 2 to 3 independent experiments. Statistical significance was accepted for p values < 0.05.

Inhibitory Effects of N. peltata Extracts (NP, NPA, and NPR) on IL-4 in RBL-2H3 Cells
IL-4 overexpression induced by PI treatment in RBL-2H3 cells was used to compare the anti-AD effects of the three different N. peltata extracts (NP, NPA, and NPR). In the result of qPCR analysis, the expression of the IL-4 mRNA gene was 0.084 ± 0.09 in the untreated control group, whereas it increased by approximately 12.5-fold in the PI treatment group (1 ± 0.36) compared to the control group. The treatment groups with NP, NPA, or NPR extracts showed a decrease in the expression of the IL-4 mRNA gene compared to the PI treatment group, with values of 0.477 ± 0.09, 0.169 ± 0.18, and 0.097 ± 0.13, respectively ( Figure 1). untreated control group, whereas it increased by approximately 12.5-fold in the PI treatment group (1 ± 0.36) compared to the control group. The treatment groups with NP, NPA, or NPR extracts showed a decrease in the expression of the IL-4 mRNA gene compared to the PI treatment group, with values of 0.477 ± 0.09, 0.169 ± 0.18, and 0.097 ± 0.13, respectively ( Figure 1).

Inhibitory Effects of N. peltata Extracts (NP, NPA, and NPR) on Oxazolone-Induced Histological Changes BALB/c Mice
Oxazolone (OX) administration produced typical AD-like symptoms, which included erythema, edema, and dryness, but NPA or NPR extracts treatment improved these symptoms, and NPR extract was more effective than NPA ( Figure 2A). In H&E staining, OX showed an increased mean ear (0.42 mm) and epidermal thicknesses (39.48 µm). However, the application of NPA or NPR extracts reduced ear thickness to 0.39 mm and 0.32 mm, respectively, and epidermal thicknesses to 31.32 µm and 29.86 µm, respectively ( Figure 2B-D). These results showed that NPR extract was the most active extract, and thus it was used in the DNCB-SKH-1 hairless mice study. Oxazolone (OX) administration produced typical AD-like symptoms, which included erythema, edema, and dryness, but NPA or NPR extracts treatment improved these symptoms, and NPR extract was more effective than NPA (Figure 2A). In H&E staining, OX showed an increased mean ear (0.42 mm) and epidermal thicknesses (39.48 µm). However, the application of NPA or NPR extracts reduced ear thickness to 0.39 mm and 0.32 mm, respectively, and epidermal thicknesses to 31.32 µm and 29.86 µm, respectively ( Figure 2B-D). These results showed that NPR extract was the most active extract, and thus it was used in the DNCB-SKH-1 hairless mice study.

Effects of NPR Extract on Histological Changes in DNCB-SKH-1 Hairless Mice
To evaluate the effect of NPR extract on AD-like skin lesions, DNCB was repeatedly applied to the dorsal skin of SKH-1 hairless mice. As shown in Figure 3A, AD-like symptoms such as skin thickening, severe erythema, edema, bleeding, excoriation, dryness, scarring, and erosion were observed in the DNCB group, but these symptoms were significantly alleviated by NPR extract application. In addition, DNCB application increased epidermal thickness and mast cell infiltration by 3.8-and 3.2-fold, respectively, versus the untreated control group. However, NPR extract treatment significantly inhibited DNCB-induced increases in epidermal thickness and mast cell infiltration by 2.2-and 1.5-fold, respectively ( Figure 3B-E).

Effects of NPR Extract on Histological Changes in DNCB-SKH-1 Hairless Mice
To evaluate the effect of NPR extract on AD-like skin lesions, DNCB was repeatedly applied to the dorsal skin of SKH-1 hairless mice. As shown in Figure 3A, AD-like symptoms such as skin thickening, severe erythema, edema, bleeding, excoriation, dryness, scarring, and erosion were observed in the DNCB group, but these symptoms were significantly alleviated by NPR extract application. In addition, DNCB application increased epidermal thickness and mast cell infiltration by 3.8-and 3.2-fold, respectively, versus the untreated control group. However, NPR extract treatment significantly inhibited DNCBinduced increases in epidermal thickness and mast cell infiltration by 2.2-and 1.5-fold, respectively ( Figure 3B-E).

Effects of NPR Extract on Serum Concentrations of IL-4 and IgE and on Skin Barrier Function in DNCB-SKH-1 Hairless Mice
We examined whether NPR extracts affected serum IL-4 and IgE concentration and TEWL and skin hydration to evaluate the anti-AD effect of NPR extract. The expression levels of serum IL-4 and IgE were increased by DNCB treatment but significantly decreased by NPR extract treatment by 34% and 58%, respectively, versus the DNCB group ( Figure 4A,B). In addition, DNCB treatment increased TEWL to 74.7 g/m 2 /h versus the untreated control group (31.7 g/m 2 /h). TEWL in the NPR extract group was 55.8 g/m 2 /h, which was 25% lower than that in the DNCB group ( Figure 4C). Skin moisture was 52% lower for AD mice than the untreated control group. NPR extract application improved skin moisture by 27% compared to the DNCB group ( Figure 4D).

Effect of NPR Extract on Skin Barrier Function Associated Proteins in DNCB-SKH-1 undefinedHairless Mice
Immunohistochemical staining for KLK5 and FLG in the epidermis and stratum corneum showed that NPR extract treatment decreased or increased staining intensities and areas, respectively, versus DNCB-treated mice ( Figure 5A,B). In DNCB-treated mice, IL-4 and KLK5 protein levels were both increased by 67% versus the untreated control group, but NPR extracts treatment significantly reduced IL-4 and KLK5 protein expression levels by 57% and 73%, respectively, versus DNCB-treated mice ( Figure 5C,D).

Effects of NPR Extract on Serum Concentrations of IL-4 and IgE and on Skin Barrier Function in DNCB-SKH-1 Hairless Mice
We examined whether NPR extracts affected serum IL-4 and IgE concentration and TEWL and skin hydration to evaluate the anti-AD effect of NPR extract. The expression levels of serum IL-4 and IgE were increased by DNCB treatment but significantly decreased by NPR extract treatment by 34% and 58%, respectively, versus the DNCB group ( Figure 4A,B). In addition, DNCB treatment increased TEWL to 74.7 g/m 2 /h versus the untreated control group (31.7 g/m 2 /h). TEWL in the NPR extract group was 55.8 g/m 2 /h, which was 25% lower than that in the DNCB group ( Figure 4C). Skin moisture was 52% lower for AD mice than the untreated control group. NPR extract application improved skin moisture by 27% compared to the DNCB group ( Figure 4D).

Effect of NPR Extract on Skin Barrier Function Associated Proteins in DNCB-SKH-1 Hairless Mice
Immunohistochemical staining for KLK5 and FLG in the epidermis and stratum corneum showed that NPR extract treatment decreased or increased staining intensities and areas, respectively, versus DNCB-treated mice ( Figure 5A,B). In DNCB-treated mice, IL-4 and KLK5 protein levels were both increased by 67% versus the untreated control group, but NPR extracts treatment significantly reduced IL-4 and KLK5 protein expression levels by 57% and 73%, respectively, versus DNCB-treated mice ( Figure 5C,D).

Effect of NPR Extract on Antioxidant Enzyme Expressions DNCB-SKH-1 Hairless Mice
The activations of the transcription factors pNrf2, Nrf2, and HO-1 were evaluated by immunoblotting to investigate whether NPR extract activates antioxidant enzymes. DNCB treatment decreased the expressions of pNrf2 and Nrf2, but NPR extracts pretreatment significantly increased their expressions by 2.1-and 1.5-fold, respectively, versus the DNCB group ( Figure 6B,C). In addition, DNCB treatment reduced the expression of HO-1 by 15%, but NPR extract pretreatment increased HO-1 expression by 38% vs. DNCBtreated mice ( Figure 6D).

Effect of NPR Extract on Antioxidant Enzyme Expressions DNCB-SKH-1 Hairless Mice
The activations of the transcription factors pNrf2, Nrf2, and HO-1 were evaluated by immunoblotting to investigate whether NPR extract activates antioxidant enzymes. DNCB treatment decreased the expressions of pNrf2 and Nrf2, but NPR extracts pretreatment significantly increased their expressions by 2.1-and 1.5-fold, respectively, versus the DNCB group ( Figure 6B,C). In addition, DNCB treatment reduced the expression of HO-1 by 15%, but NPR extract pretreatment increased HO-1 expression by 38% vs. DNCB-treated mice ( Figure 6D).

Isolation of Compounds from NPR Extract
The NPR extract was applied to silica gel column chromatography and RP HPLC to obtain three known phenolic acids. The isolated compound was identified by NMR analysis and comparison to the literature (Table 1 and Figure 7).

Isolation of Compounds from NPR Extract
The NPR extract was applied to silica gel column chromatography and RP HPLC to obtain three known phenolic acids. The isolated compound was identified by NMR analysis and comparison to the literature (Table 1 and Figure 7).  -1), and a carbonyl carbon resonance at 176.01 (C-7) are characteristic of quinic acid. The above NMR data are consistent with the literature on chlorogenic acid [27]. Consequently, compound 1 was concluded as chlorogenic acid.

HPLC-PDA Analysis of NPR Extract
HPLC was performed using solvent A (0.1% formic acid in ACN) and solvent B (0.1% formic acid in H2O) using the profile 0 to 20 min, 5 to 40% A and then 20 to 25 min, 40 to 5% A at a wavelength of 325 nm. HPLC-PDA analysis, and the tR and absorption maxima (λmax) of the three phenolic acids are as follows: (1) chlorogenic acid (tR 9.76 min, λmax 241.   -7) showed the presence of a quinic acid moiety. The above NMR data are consistent with the literature on 3,5-dicaffeoylquinic acid [28,29]. Consequently, compound 2 was concluded as 3,5-dicaffeoylquinic acid.

Discussion
Extracts of plant parts contain different metabolites and have significantly different activities [30,31]; thus, researchers in the medicinal and pharmaceutical fields tend to investigate the activities of specific parts. In recent years, plants and their extracts have emerged as alternative treatments for acute and chronic diseases, including AD [32,33]. Furthermore, numerous human clinical studies have demonstrated plant extracts and herbal products to have anti-AD effects and no noticeable side effects [33]. N. peltata is an aquatic perennial and a valuable medicinal herb in Ayurvedic medicine [20]. Although N. peltata extracts have been reported to possess significant anti-inflammatory, anti-tumor, and anti-wrinkle effects [19,25,26], no previous study has examined its anti-AD effect. Our present study showed that the extracts of whole N. peltata extract and its aerial and root

Discussion
Extracts of plant parts contain different metabolites and have significantly different activities [30,31]; thus, researchers in the medicinal and pharmaceutical fields tend to investigate the activities of specific parts. In recent years, plants and their extracts have emerged as alternative treatments for acute and chronic diseases, including AD [32,33]. Furthermore, numerous human clinical studies have demonstrated plant extracts and herbal products to have anti-AD effects and no noticeable side effects [33]. N. peltata is an aquatic perennial and a valuable medicinal herb in Ayurvedic medicine [20]. Although N. peltata extracts have been reported to possess significant anti-inflammatory, anti-tumor, and anti-wrinkle effects [19,25,26], no previous study has examined its anti-AD effect. Our present study showed that the extracts of whole N. peltata extract and its aerial and root extracts efficiently inhibited IL-4 induced in RBL-2H3 cells in vitro and attenuated AD-like skin symptoms in our oxazolone-BALB/c mice model in vivo, and that the NPR extract was the most active extract. Here, we report the inhibitory effect of NPR extract on AD-related inflammation and oxidative stress, and its beneficial effect on skin barrier function in AD-induced SKH-1 hairless mice.
AD is caused by an immune imbalance associated with increases in Th2 cells or immune cells and a Th2 response predominance [3], which results in the secretion of various allergen mediators from basophils or mast cells via IL-4 production and IgE isotype conversion [3,6]. In AD, these allergen mediators penetrate skin tissue and cause skin barrier dysfunction and epidermal changes, such as skin thickening or epidermal and dermal proliferation [6,34,35]. Furthermore, these Th2 responses and epidermal changes have been suggested as measures of pruritus severity and novel therapeutic targets in AD [36,37]. In the current study, we repeatedly applied DNCB to the dorsum skin of SKH-1 hairless mice to induce AD-like skin symptoms, such as severe erythema, lichenification, mast cell infiltration, and the overexpressions of serum IL-4 and IgE [38,39]. Notably, despite repeated DNCB application, topical NPR extract significantly inhibited the development of AD-like symptoms and downregulated the DNCB-induced overexpressions of serum IL-4 and IgE. Our results demonstrate that NPR extract can ameliorate the symptoms of AD and suppress the effects of inflammatory cytokines.
Severe disruption of the epidermal barrier function in AD reduces skin hydration, which makes it susceptible to inflammation [40]. This reduction in skin moisture can be assessed using TEWL levels, which are commonly used to evaluate skin barrier function and AD severity clinically [41]. TEWL increases are explained by KLK5 activation or an FLG deficiency or null mutation, as FLG is closely related to IL-4 and contributes to skin hydration and normalizing the pH gradient by producing a natural moisturizing factor (NMF) [42,43]; whereas, KLK5 plays an essential role in stratum corneum desquamation [44]. Thus, we confirmed the direct impact of NPR extract on epidermal barrier function in DNCB-induced mice. DNCB-induced AD reduced skin hydration and FLG expression and upregulated the expressions of TEWL, KLK5, and IL-4, but topical NPR extract significantly restored skin barrier function and epidermal protein homeostasis. Therefore, our results suggest that NPR extract ameliorates AD by regulating inflammatory cytokines and epidermal barrier homeostasis.
The Nrf2/HO-1 signaling pathway is an essential defense against oxidative stress [45]. Nrf2 prevents or protects against oxidative stress-mediated cellular damage and promotes the expressions of antioxidant-related factors, such as HO-1 [46]. Moreover, the Nrf2/HO-1 signaling pathway provides a defense mechanism that regulates chronic inflammatory responses induced by oxidative stress [47][48][49]. Previous studies suggest that the activation of the Nrf2/HO-1 pathway enhances the inflammatory response and epidermal homeostasis from oxidative stress in hapten-induced AD mice [47,50]. In our study, the topical application of NPR extract increased the expressions of the antioxidant enzymes pNrf2, Nrf2, and HO-1, which regulate DNCB-induced excessive oxidative stress. Overall, our results suggest that NPR extract ameliorates AD-related inflammation and oxidative stress by activating the Nrf2/HO-1 defense pathway.
Phenolic acids are secondary metabolites present in various foods [51]. These compounds have been reported to have various physiological activities [52,53], and chlorogenic acid and di-caffeoylquinic acid have been demonstrated to have potent free radical scavenging, anti-inflammatory, and antioxidant activities [53][54][55]. In the present study, HPLC-PDA analysis showed that chlorogenic acid, 3,5-dicaffeoylquinic acid, and 3,4-dicaffeoylquinic acid are three major components of NPR extract. Therefore, we suggest that the anti-AD effects of these three compounds be further investigated.

Conclusions
The present study demonstrates for the first time that the 95% EtOH extract of N. peltata (NPR) root has anti-AD effects. Of the three different parts of N. peltata examined (whole plant and aerial and root parts), NPR extracts most effectively suppressed in vitro PI-induced IL-4 and in vivo OX-induced AD-like symptoms. Furthermore, NPR extract application attenuated AD-like symptoms and decreased serum IL-4 and IgE levels in DNCB-induced SKH-1 hairless mice, and also improved skin barrier function and activated the Nrf2/HO-1 signaling pathway. These activities of the NPR extract were probably derived from the presence of chlorogenic acid, 3,5-dicaffeoylquinic acid, and 3,4-dicaffeoylquinic acid found by HPLC in the NPR extract. Our results suggest that NPR extract inhibits inflammatory responses and activates antioxidant enzymes, and that this extract provides new insights into the prevention and treatment of AD.