Inhibitory Effects of Luteolin 7-Methyl Ether Isolated from Wikstroemia ganpi on Tnf-A/Ifn-Γ Mixture-Induced Inflammation in Human Keratinocyte

Plants of the genus Wikstroemia are traditionally used in China to treat various inflammatory diseases. The purpose of this study was to isolate the components of Wikstroemia ganpi (Siebold & Zucc.) Maxim., to evaluate their anti-atopic activities and to identify candidates with anti-atopic therapeutics. A total of 24 compounds were isolated by bioassay-guided separation, including one novel compound, which was tilianin 5-methyl ether. The anti-atopic activities of the isolated compounds were determined using TNF-α-treated RBL-2H3 cells and HaCaT cells. The mRNA expressions of IL-4, IL-6, GM-CSF, G-CSF and TRPV1 were reduced by luteolin 7-methyl ether. The study shows that the luteolin 7-methyl ether isolated from W. ganpi is a potential therapeutic agent for the treatment of atopic dermatitis.


Introduction
Atopic dermatitis (AD) is a multifactorial chronic inflammatory skin disease. The most common symptoms of AD are erythema, psoriasis, vesicles, skin tightness and itching accompanied by painful skin lesions [1]. The present study shows that the predominant manifestations of childhood-onset AD include lichenified and/or exudative flexural dermatitis, whereas adult-onset AD s more often characterized by prurigo with highly pruriginous papules and nodules [2]. Itching, combined with scratching, damages the skin barrier, and affected skin reacts sensitively to external allergens, which causes immune defects in inflammatory cells [3]. Immune defects in most AD patients are characterized by upregulating Th2 cytokine and IgE release from mast cells [4]. Representative Th2 cytokines IL-4 and IL-13 suppress filaggrin expression, which plays an important role in the construction of the skin barrier, thereby weakening the function of the skin barrier [5]. IL-31 (Th2-associated cytokine) has also been reported to be an endogenous cause of itching [6], and AD is closely related to pro-inflammatory cytokine (TNF-α, IL-6, GM-CSF, G-CSF and others) released by keratinocytes, dendritic cells, macrophages and other immune cells [3,7]. Furthermore, interactions between pro-inflammatory cytokines and T cell-associated cytokines lead to repeated vicious cycles of itching and chronic inflammatory response [7,8]. To inhibit these inflammatory mediators and reduce itching, topical corticosteroid and topical calcineurin inhibitors are commonly used to treat AD [9]. However, although these AD agents are effective at ameliorating inflammatory immune response and skin conditions, their long-term use may cause side effects [10]. Most recently, crisaborole, a topical phosphodiesterase-4 inhibitor, and dupilumab, an inhibitor of interleukin (IL)-4/13, were FDA-approved, with studies showing an excellent safety profile for chronic treatment in AD [11].
Flavonoids are the major secondary metabolites of plants and have a range of pharmacological effects, which include anti-inflammatory, anti-allergic, and antioxidant effects [12]. These pharmacological properties are attributed to the structural features of flavonoids such as the presence of a C2-C3 double bond, hydroxylation, O-methylation, glycosylation and other substitutions and conjugations [13]. Numerous studies have shown that flavonoids inhibit the expressions of a variety of inflammatory mediators [14,15], and are effective treatments for chronic inflammatory diseases [16]. The inhibition of inflammatory reactions provides a strategy for treating AD [17], and many that investigated the anti-atopic effects of flavonoids and have suggested their potential uses as a natural treatment for AD [18][19][20][21].
Wikstroemia ganpi (Siebold & Zucc.) Maxim. is a deciduous shrub and member of the Wikstroemia (Thymelaeaceae) genus. It is distributed in Japan, Australia and Korea, and in Korea, is called 'Geomundodaknamu' [22]. The plants of the Wikstroemia genus are widely used in traditional Chinese medicine to treat a variety of diseases such as syphilis, arthritis and cancer [23]. According to phytochemical reports, members of the Wikstroemia genus contain flavonoids, coumarins and lignans, with anti-inflammatory, anti-allergic, antioxidant, anti-viral and other properties [24,25]. However, no study has examined the bioactivity of W. ganpi. Recently, we found that W. ganpi EtOH extract inhibited inflammatory mediators in DNCB-induced AD mice and attenuated AD-like symptoms [26]. In the present study, we isolated one novel compound, twelve known flavonoids, seven known coumarins, three known lignans, one phenylpropanoid and one phenolic compound from W. ganpi, and performed anti-atopic activity screening testing on the isolated bioactive compounds in the hope of identifying a potential anti-atopic agent.

Plant Material and Extraction
The aerial parts of W. ganpi (Siebold and Zucc.) Maxim. were collected from Geumsa-ri, Yeongnam-myeon, Goheung-gun, Jeollanam-do, Republic of Korea. Plants were authenticated by Dr. Jin-Hyub Paik (International Biological Material Research Center, Korea Research Institute of Bioscience and Biotechnology), and a voucher specimen (#PNU-0027) was deposited in the College of Pharmacy, Pusan National University. W. ganpi samples (~4.27 kg dried plants) were extracted by sonication in 30 L of 95% MeOH for 90 min and left overnight. This process was repeated twice, and the extract obtained was then filtered through Advantec No. 2 filter paper (Advantec, Toyo Roshi Kaish, Ltd., Tokyo). The filtered extracts were concentrated under a vacuum at 35-40 • C using a rotary evaporator, and were then freeze-dried to produce W. ganpi MeOH extract (483 g, yield 11.3%).

Quantitative Real-Time PCR Analysis of IL-4 mRNA Expression in RBL-2H Cells
RBL-2H3 cells (a rat basophilic leukemia cell-line) were purchased from the Korean Cell Line Bank (Seoul, Korea) and maintained in DMEM (Dulbecco s Modified Eagle Medium). They were supplemented with 10% FBS (fetal bovine serum; HyClone Laboratories Inc., Logan, UT, USA) and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin; Invitrogen, Carlsbad, CA, USA) at 37 • C in a 5% humidified CO 2 atmosphere. Cells were sensitized with DMSO or compounds 1~24 at a concentration of 10 µM 1 h and then stimulated with PI (phorbol 12-myristate 13-acetate (PMA) and ionomycin (IOM)) for 9 h. Total RNA was isolated using a RNeasy mini kit (Qiagen, Hilden, Germany). Isolated RNA was reverse transcribed using a RevertAid First Strand cDNA Synthesis Kit (Invitrogen) and IL-4 mRNA levels were assessed by RT-PCR. The sequences of the primers used were as follows: IL-4, forward 5 -ACC TTG CTG TCA CCC TGT TC -3 and reverse 5 -TTG TGA GCG TGG ACT CAT TC -3 ; β-actin, forward 5 -TCA TCA CCA TCG GCA ACG-3 and reverse 5 -TTC CT GAT GTC CAC GTC GC-3 . Transcript levels were normalized versus β-actin. HaCaT cells (an immortalized human keratinocyte cell-line) were provided by professor Seong-Gyu Ko of Kyunghee University. Cells were seeded in 100 mm culture dishes at a density of 1 × 10 6 cells/mL and incubated for 24 h at 37°C, and then treated with 12.5 per second. After real-time quantitative analysis, the results were analyzed using an analysis program. Relative mRNA levels of genes were normalized versus GAPDH mRNA. All experiments were repeated 3 times.

Statistical Analyses
The significances of differences were determined by one-way analysis of variance (ANOVA) and Tukey's multiple comparisons test. Results are presented as means ± standard errors, and statistical significance was accepted for p values < 0.05.

Effects of Luteolin 7-Methyl Ether (4) on the Pruritus-Related Inflammatory Mediators in HaCaT cells
The effects of luteolin 7-methyl ether (4) on the mRNA expressions of IL-31 and TRP channel were evaluated. The expressions of TRPA1, TRPV1 and IL-31 mRNA in HaCaT cells were increased by TNF-α treatment and partially decreased by luteolin 7-methyl ether (4) treatment. Luteolin 7-methyl ether (4) treatment had no significant inhibitory

Effects of Luteolin 7-Methyl Ether (4) on the Pruritus-Related Inflammatory Mediators in HaCaT Cells
The effects of luteolin 7-methyl ether (4) on the mRNA expressions of IL-31 and TRP channel were evaluated. The expressions of TRPA1, TRPV1 and IL-31 mRNA in HaCaT cells were increased by TNF-α treatment and partially decreased by luteolin 7-methyl ether (4) treatment. Luteolin 7-methyl ether (4) treatment had no significant inhibitory effect on TNF-α-induced TRPA1 overexpression, but at a concentration of 12.5 uM significantly inhibited the overexpression of TRPV1 by about 33% as compared with the HT_NC group (ANOVA, p < 0.05) ( Figure 5A,B). Luteolin 7-methyl ether (4) treatment reduced TNF-αinduced IL-31 mRNA expression, but this was not significant ( Figure 5C).

Discussion
The main clinical symptoms of AD are pruritus, dry skin and lichenification. AD is a common chronic inflammatory skin disease and its prevalence is gradually increasing [7]. AD patients exhibit a characteristic immunological imbalance, in which Th2 cells dominate Th1 cells. IL-4 is a representative cytokine secreted by Th2 cells and plays an important role in the pathogenesis of AD. Elevated IL-4 levels promote antibody isotype switching to IgE, which is involved in inflammatory response [17]. Recently, several studies have indicated that IL-31 is importantly related to atopic dermatitis, and that production of IL-31 is increased by IL-4. Furthermore, in patients with atopic dermatitis, elevated IL-4 and IL-31 levels stimulate sensory nerves and cause itching [6,49]. Topical corticosteroids are being used as first-line treatments to address the immunological imbalance that has profound effects on the induction of AD. However, due to the side effects of topical corticoids, which include skin atrophy and burning and stinging sensations, the longterm use of topical corticoids was prohibited, and thus there is an urgent need for new

Discussion
The main clinical symptoms of AD are pruritus, dry skin and lichenification. AD is a common chronic inflammatory skin disease and its prevalence is gradually increasing [7]. AD patients exhibit a characteristic immunological imbalance, in which Th2 cells dominate Th1 cells. IL-4 is a representative cytokine secreted by Th2 cells and plays an important role in the pathogenesis of AD. Elevated IL-4 levels promote antibody isotype switching to IgE, which is involved in inflammatory response [17]. Recently, several studies have indicated that IL-31 is importantly related to atopic dermatitis, and that production of IL-31 is increased by IL-4. Furthermore, in patients with atopic dermatitis, elevated IL-4 and IL-31 levels stimulate sensory nerves and cause itching [6,49]. Topical corticosteroids are being Nutrients 2021, 13, 4387 9 of 11 used as first-line treatments to address the immunological imbalance that has profound effects on the induction of AD. However, due to the side effects of topical corticoids, which include skin atrophy and burning and stinging sensations, the long-term use of topical corticoids was prohibited, and thus there is an urgent need for new therapeutic agents.
In a previous study, we investigated the anti-atopic effects of W. ganpi in RBL-2H3 mice with DNCB-induced AD, finding that W. ganpi EtOH extract (WGE) effectively alleviated AD symptoms and that flavonoids and coumarins are major components of W. ganpi using HPLC-PDA [26]. Since the anti-inflammatory and anti-allergic activities of flavonoids have been well demonstrated, it was suggested that the anti-atopic activity of WGE is probably derived from flavonoids [12,15,18]. Based on these studies, we isolated compounds in W. ganpi with the aim of identifying a potential therapeutic agent for AD. In total, 11 flavonoids, 8 coumarins, 3 lignans, 1 phenylpropanoid and 1 phenolic compound were isolated using an activity-oriented chromatography, and tilianin 5-methyl ether (11) was isolated for the first time. RBL-2H3 cells were each treated with the 24 isolated compounds and then treated with PI -which, when administered alone, induced IL-4 mRNA expression -to determine to what extent degranulation was inhibited. These experiments showed that four compounds, namely, luteolin 7-methyl ether (4), pilloin (5), pilloin 5-O-glucopyranoside (9) and pinoresinol (20), significantly inhibit PI-induced IL-4 mRNA upregulation.
In addition, we also evaluated the anti-atopic activity of luteolin 7-methyl ether in TNF-α treated HaCaT cells (4). TNF-α treatment increased IL-6 expression in HaCaT cells, and this increase was reduced by luteolin 7-methyl ether pretreatment (4). IL-6 is involved in cell-mediated inflammation and immune response and enhances B-cell differentiation and T-cell proliferation [50]. Pretreatment with luteolin 7-methyl ether (4) also significantly inhibited the TNF-α-induced expressions of G-CSF and GM-CSF in HaCaT cells, and G-CSF and GM-CSF are highly expressed in the keratinocytes of AD patients [51]. To investigate the potential effect of luteolin 7-methyl ether on itching, we examined the expression levels of TRPA1, TRPV1 and IL-31 in HaCaT cells treated with TNF-α. Pretreatment with luteolin 7-methyl ether (4) was found to significantly decrease TNF-α-induced TRPV1 expression, and to slightly decrease the TNF-α-induced expressions of TRPA1 and IL-31.
To summarize, 24 compounds were isolated by activity-guided bioassay from the methanol extract of the aerial parts of W. ganpi, and these compounds included the flavonoid tilianin 5-methyl ether (4), which is reported here for the first time. When RBL-2H3 cells were pretreated with the isolated compounds and then treated with TNF-α, luteolin 7-methyl ether (4) was found to significantly inhibit the expression of IL-4, which is known to be closely related to AD. In addition, luteolin 7-methyl ether (4) also inhibited the expressions of cytokines and ion channels involved in inflammation and pruritus in TNF-α-induced HaCaT cells. Our experimental results suggest that luteolin 7-methyl ether (4), the main active ingredient of WGE, has potential as a treatment for AD.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.