Topical Application of Cudrania tricuspidata Stem Extract Inhibits Atopic Dermatitis-Like Skin Lesions in an NC / Nga Mouse Model : An Experimental Animal Study

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by elevated immunoglobulin E (IgE), mast cell infiltration and skin lesions including pruritus, erythema and eczema. Cudrania tricuspidata extracts have been clinically administered for a long time in the East Asia including Korean and China as a home-remedy to diminish the inflammation of gastritis and hepatitis. To examine whether it works on AD or not, an AD-like animal model was experimented in this study. AD was induced by applying Dermatophagoides farinae (D. farinae) extract to the backs of 9-week old NC/Nga mice for 21 days. Following this, an ethanol extract of C. tricuspidata stems (EECT) was applied topically for 14 days to the sensitized skin, while distilled water was used as a control (EECT0 mice). Anti-AD effects of EECT were evaluated using scores for AD-like skin lesions, serum IgE levels and mast cell counts in the skin dermal layers to assess inflammation. Topically applied ethanol extract of Cudrania tricuspidata stems (EECT 7.5, 25 and 75 mg/mL) markedly reduced AD-like skin lesions after 4 days (by 30.1%, 31.4% and 38.5%, respectively) and also after Corresponding author.


Introduction
Atopic dermatitis (AD) is a chronic inflammatory skin disease accompanied by representative symptoms such as itch, erythema, excoriation, edema, dryness and scaling [1].It is very prevalent in infants and nowadays has also spread to adolescents and adults.Its causes are not fully understood, but inflammatory dysregulation of the immune system of the skin and mucous membranes in response to allergens is probably involved.This immunologic disturbance is reflected in elevated serum immunoglobulin E (IgE) resulting from increased inflammatory cell infiltration into the skin, and is dominated by type2 helper T-cells [2].Accordingly, AD is characterized by histopathologic skin changes and dermatitis lesions of the face, neck, head, arms, knees and elbows.Therefore, sensory evaluation of the disturbance caused by such skin lesions or determination of IgE levels is used to diagnose or evaluate AD [3].
We examined a natural material with anti-inflammatory effects as a new drug candidate for AD treatment.The selected natural material was extract of Cudrania tricuspidata (C.tricuspidata), a kind of mulberry tree which is native to East Asian countries such as Korea, China and Japan [4]- [7], and occurs mainly in the southern area of South Korea [5].Extracts of the fruit, leaves, root-bark and stems of C. tricuspidata have been traditionally used for hepato-protection [8]- [10], and as anti-hypertension [11], anti-diabetes [12] [13], anti-allergy [14] and antioxidation [15]- [17] treatments in Korea.Well-known physiologically active compounds of C. tricuspidata are isoflavones and flavonoids from the fruits [18], gerontoxanthone, cudraflavone B, gericudranin, cudraxanthones, isocudraxanthones, some flavanones and macluraxanthone from the roots [9] [19], kaempferol and kaempferol-D-glucopyranoside from the leaves [20], and kaempferide-D-glucopyranoside and naringenin-D-glucopyranoside from the stems [21].However, the anti-AD effects of C. tricuspidata have not been studied except for a recent report using fruit extract [22].Therefore, we examined the therapeutic effect of ethanol extract of C. tricuspidata stems (EECT) on AD symptoms when administered topically in an NC/Nga mice AD model induced by Dermatophagoides farinae (D. farinae) extract.

Preparation of the EECT
Dried stems of C. tricuspidata were finely powdered, and biologically active compounds were extracted with 95% ethanol (1:10, v/v) for 24 h, in a 60˚C water bath, then filtered through filter paper (ADVANTEC No.2, 150 mm) under vacuum.The filtrate was concentrated by evaporation at 45˚C, then deep-frozen at −70˚C after reconstitution in 70% ethanol in a concentration range of 100 -200 mg/mL.The frozen filtrate was diluted appropriately after thawing to room temperature.Three concentrations of the ethanol extract (EECT) namely 7.5, 25 and 75 mg/mL, were prepared by diluting with distilled water, and tested topically.

Induction of AD-Like Skin Lesions in an NC/Nga Mouse Model
Twenty-eight NC/Nga mice (9 week-old, male) were purchased from Central Exp.Animals Co. Ltd. (South Ko-rea), and all animal procedures were approved by the Institutional Animal Care and Use Committee of Hanyang University (No: HY-IACUC-10-062).After one-week of adaptation, the mice were anesthetized with ether and their heads, necks and shoulders shaved with razors and waxing cream.Then twenty-four of the mice were topically administered 100mg of D. farinae (house mite) extract-ointment (Biostar AD ointment, Japan) on their shaved skin regions six times over 21 days to induce AD-like skin lesions [23].4% SDS solution was used to brush their shaved skin instead of waxing cream after the first administration of house mite extract [23].On the 21st day, the severity of the AD-like skin lesions was evaluated.Eight mice died during the AD induction period during waxing and sensitization, and the remaining twenty mice were divided into five groups.

Treatment on the AD-Like Skin Lesions with EECT
On the 21st day EECT was administered to three experimental groups of 4 mice receiving 7.5 mg/mL, 25 mg/ml and 75 mg/mL EECT, respectively, for 14 days.The EECT was topically applied with a small flat brush once a day on all the treated skin areas.Distilled water was administered to the control group (EECT0, n = 4) in the same manner, while the "normal" group (normal, n = 4) received no treatment.

Changes of AD-Like Skin Lesions
The changes of the AD-like skin lesions were assessed visually by two trained investigators on the 21st day of sensitization, and after the 4 th , 10 th and 14 th day of EECT treatment.The severity was scored as the sum of the AD scores for each category, namely itch, edema, erythema/hemorrhage, excoriation/erosion and dryness/ scaling, graded as 0 (no symptoms), 1 (mild symptoms), 2 (moderate symptoms) and 3 (severe symptoms) [24]- [26].Then the AD score for each group was converted to "AD reduction (%)" calculated from the equation {(AD score at baseline − AD score at the 4th, 10th or 14th day of EECT treatment)/AD score at baseline} × 100.

Changes of Inflammation Measured from Serum IgE Levels
Orbit blood was collected under ether anesthesia on the final day of AD induction, then on the 4 th , 10 th and 14 th days of treatment.Sera were deep-frozen at −70˚C after centrifugation of blood at 3000 rpm, 4˚C for 20 min.Serum IgE levels were determined using a Mouse IgE ELISA Assay kit (XpressBio Life Science Products, Catalog No: 595-700, Express Biotech International, Thurmont, USA) at 450 nm expressed as {(serum IgE level at baseline − serum IgE level on the 4th, 10th or 14th day of EECT treatment)/serum IgE level at baseline} × 100.

Histopathologic Evaluation
After the 14 days of EECT treatment, all the mice were killed under ether anesthesia after evaluation of the AD-like skin lesions and orbit blood sampling, and the skin was removed from the affected regions of the animals' heads, necks and shoulders.The skin was fixed with 10% formaldehyde, embedded in paraffin, and thin sections were made.The sections were stained with toluidine blue O, and microscopic examination was carried out at ×200; the total numbers of mast cells including granulated and degranulated forms were counted at five sites (400 μm 2 ) chosen at random in each skin sample [26]-[28].

Statistical Evaluation
The measurements on the five groups were compared by one-way ANOVA and by post-hoc Duncan or Scheffe test at p < 0.05 using SPSS ver.21.0.Eight mice died due to excess anesthesia during the AD induction period.

The Therapeutic Effects of EECT on AD-Like Skin Lesions
The changes of AD-like skin lesions from the baseline at the end of AD induction to the 14 days after the EECT treatment are presented in Figure 1(a).AD-like skin lesions were reduced by the 14-day EECT treatment (Figure 1(b)), and significant reductions compared to EECT 0 were noted after 4 days of treatment only with 25 and 75 mg/mL EECT (p = 0.0001), even though EECT 7.5, 25 and 75 mg/mL were equally effective (30.1% ± 2.4% vs. 31.4%± 6.7% vs. 38.5% ± 2.7%).The extent of the AD reduction increased by 14 days of EECT treatment (63.6% ± 9.8% vs. 66.1% ± 6.3% vs. 49.6% ± 12.9%, respectively), and to a lesser extent (from 17.8% ± 5.4% (4 days) to 38.7% ± 9.9% (14 days)) in the EECT0 group.In spite of the higher AD reduction in the EECT-treated groups, no significant differences were found between the EECT0 and EECT-treated groups after 14 days of EECT treatment.Importantly, the AD-like skin lesions were improved most when the mice were treated with EECT 25 mg/mL for 14 days, and the therapeutic effect of EECT 25 mg/mL was significantly greater than that seen in the EECT0 group in the early stage of treatment.

Effects on Mast Cell Counts Stained with Toluidine Blue O
Numbers of mast cells (both granulated and degranulated forms) in the skin dermis layers stained with toluidine BlueO were examined by optical microscopy (200×) (Figure 3) as described in Materials and Methods.Total mast cell counts were significantly increased after sensitization by D. farinae extract when compared to normal mice (p = 0.003; (Figure 4): normal (153.0 ± 7.5) vs. EECT0 (659.0 ± 129.3), EECT7.5 (469.5 ± 39.2), EECT25 (541.0 ± 80.2) and EECT75 (544.0 ± 48.8)).However, total mast cell counts tended to increase less in   the EECT-treated groups, especially the EECT7.5 group, than in the distilled water-treated (EECT0) group.This tendency was particularly evident in the skin dermis of heads and necks.The granulated and degranulated forms of mast cells were also counted separately, and only the latter increased significantly after AD induction (p = 0.0001; normal (45.0 ± 8.1) vs. EECT0 (484.8 ± 60.8)) while degranulated mast cells increased significantly less when treated with EECT (EECT7.5 (315.0 ± 36.6),EECT25 (289.0 ± 19.6) and EECT75 (362.0 ± 18.2)) than with distilled water (Figure 4).The topical use of EECT 7.5, 25 and 75 mg/mL on the AD-like skin lesions for 14 days was effective in reducing numbers of degranulated mast cells even though they did not reach the numbers in the normal group (p = 0.0001).

Discussion
In the current study, topically applied EECT had a significant effect on the severity of AD-like skin lesions, serum IgE levels and on mast cell counts in the skin dermis.The anti-AD effects of EECT were detectible when EECT 7.5, 25 and 75 mg/mL were applied topically for 14 days to the mice (Figure 1), and these therapeutic effects are presumed to be due to diminution of systemic inflammatory reactions (Figure 2) and histopathological changes (Figure 3 and Figure 4).Natural recovery from the AD-like skin lesions was also evaluated from the results in the EECT0 group treated with distilled water.The therapeutic efficacies of EECT 7.5, 25 and 75 mg/mL on the AD-like skin lesions were 1.7 (30.1%), 1.8 (31.4%) and 2.2 (38.5%) times higher than that of EECT 0 (17.8%) after 4 days (p = 0.0001), and 1.6 (63.6%), 1.7 (66.1%) and 1.3 (49.6%) times higher than that of EECT 0 (38.7%) after 14 days of EECT treatment (Figure 1).The anti-inflammatory efficacies of EECT 7.5, 25 and 75 mg/mL on serum IgE levels were also higher than those of EECT 0 (Figure 2).The anti-AD efficacy of EECT seemed to be more significant in the early stage (after 4 days) of EECT treatment than at the later stage of treatment.
AD appears to be associated with inflammatory immune dysregulation of the skin, impaired skin barrier function, and IgE-mediated sensitization to food and environmental allergens such as microbial pathogens [29] [30].Such immunologic disturbance is reflected in elevated serum IgE levels due to increased inflammatory cell infiltration into the skin, which is type2 helper T-cell-dominated, and, according to the immune dysregulation hypothesis, the defective skin barrier is interpreted as a consequence of inflammation [2].Regardless of how to explain disease progression, the acute eczematous lesions exhibit spongiosis, hyperkeratosis and parakeratosis, and the chronic lesions are characterized by acanthosis and perivascular infiltration of lymphocytes and mast cells, and by lichenification of the skin, infiltration of Th1 cells, and tissue remodeling with increased collagen deposition and dermal thickening [30].From this point of view, the elevation of serum IgE levels in this NC/ Nga mouse AD model was related to the onset of AD-like skin lesion development [31] [32], and also similar to that reported in AD patients with IgE hyperproduction [33].Therefore, topically applied EECT might be very effective in reducing systemic inflammatory reactions and diverse AD symptoms such as itch, edema, erythema/ hemorrhage, excoriation/erosion, and dryness/scaling in the early stage of AD treatment.This suggests that patients with AD could obtain relief from the severe distress of AD symptoms more easily in the very highly induced AD state.
Many skin disorders like AD seem to be related to infiltration and activation of mast cells, resulting in skin inflammation [30] [34].In the current study, total mast cell counts increased significantly after 21 days of sensitization (Figure 4).EECT treatment at 7.5, 25 and 75 mg/mL showed a tendency to suppress the mast cell production in the skin dermis layers of the head, neck and shoulder of the mice.This tendency to reduce dermal mast cells, specially degranulated mast cells, was significant in the EECT-treated groups (p = 0.0001), when compared to the EECT 0 group (Figure 4).The degranulated to granulated mast cell ratio (de-G/G) was greatly increased after AD induction, and the de-G/G ratio was lower after EECT treatment than after treatment with distilled water (data not shown) though the difference was not statistically significant (p = 0.162; normal (0.4070 ± 0.042) vs. EECT0 (6.914 ± 4.062) vs. EECT7.5(2.049 ± 0.257) vs. EECT25 (1.308 ± 0.235) vs. EECT75 (2.1880 ± 0.357)).It has also been reported that stress-induced skin disorders due to infiltration and activation of mast cells in the skin dermis resulted in significant increases of granulated and degranulated mast cells in rats [34]- [36].In allergic or stressed conditions, mast cells increase their IgE production [37], and degranulated mast cells that bind IgE increase [38] with a subsequent rise in histamine production, which generates severe itch and scratching of skin lesions [39].Degranulated mast cells can initiate an acute inflammatory response and contribute to the progression of chronic diseases.Therefore, a decrease of mast cells, degranulated forms in particular, or of the deG/G ratios, in the skin is correlated with reductions of IgE and inflammatory responses, and could be a key to reducing the severity of AD-like skin lesions.Mast cells in the skin dermis of head and neck were considerably decreased by EECT 7.5, 25 and 75 mg/mL (Figure 4).This is of interest as the necks of AD patients were found to have higher number of mast cell profiles than any other region such as elbow, scalp and nates [3].
The current study has a few limitations.Thus, the considerable variability of one group resulted in limited statistical power of the comparison with the other groups.It may have been caused by the small sample size of the groups.If the sample size had been larger, the anti-AD effect of EECT 7.5, 25 and 75 mg/mL might have been more significant at 14 days of EECT treatment than that of distilled water.
In spite of the above limitation, the current study's strengths include the fact that the EECT was applied topically to the AD-like skin lesions as an ointment.Most traditional oriental natural substances, such as mulberry tree extract and ginseng are orally administered.Secondly, NC/Nga mice develop AD-like skin lesions spontaneously under normal conditions, and they are a suitable model for some aspects of human AD, with IgE hyperproduction which is influenced by environmental factors, in part, due to interleukin-4 and -5 released by mast cells of the affected skin [31].Therefore, the NC/Nga mouse model is useful for developing therapies for AD.In addition, it is very important to control and weaken the inflammation and dermatitis symptoms of AD, given that these unbearable AD symptoms are severer at night and prevalent in younger subjects.Because, AD is an intractable chronic disease there should be as much focus on its control as on its complete cure.From this point of view, EECT may be a possible solution to reduce AD symptoms while seeking a cure for the disease.

Conclusion
Ethanol extract of C. tricuspidata stems (EECT) administered topically to an NC/Nga mouse AD model induced by D. farinae extract decreased serum IgE levels and degranulated mast cell counts in the dermis of the skin, and consequently improved the AD-like skin lesions.The anti-AD effect was especially significant when EECT 7.5 and 25 mg/mL were applied for 14 days, and the therapeutic effect was more obvious in the early stage of EECT treatment when AD was strongly induced than later in the treatment.

Figure 1 .
Figure 1.The changes of AD-like skin lesions from baseline at the end of AD induction.(a) The AD reductions (%) in each group were compared with the EECT0 group by one-way ANOVA with Duncan's posthoc test.† (p < 0.001); (b) Representative photographs of AD-like skin lesions before and after EECT treatment.EECT = ethanol extract of C. tricuspidata stems; Normal = no AD induction and no EECT treatment; EECT0 = AD induction and distilled water treatement (control); EECT7.5 = AD induction and 7.5 mg/mL EECT treatment; EECT25 = AD induction and 25 mg/mL EECT treatment; EECT75 = AD induction and 75 mg/mL EECT treatment (mean ± SEM, n = 4).

Figure 2 .
Figure 2. The changes of serum IgE from baseline at the end of AD induction.The IgE reductions (%) in each group were compared with the EECT0 group by one-way ANOVA with Duncan's posthoc test ‡ (p < 0.0001).EECT = ethanol extract of C. tricuspidata stems; Normal = no AD induction and no EECT treatment; EECT0 = AD induction and distilled water treatement (control); EECT7.5 = AD induction and 7.5 mg/mL EECT treatment; EECT25 = AD induction and 25 mg/mL EECT treatment; EECT75 = AD induction and 75 mg/mL EECT treatment (mean ± SEM, n = 4).

Figure 3 .
Figure 3.The representative optical microscopic images (200×) of mast cells.Granulated (◁) and degranulated (◀) mast cells in the skin dermis stained with toluidine Blue O after 14 days of EECT treatment.Other details are the same as in Figure 1 and Figure 2.

Figure 4 .
Figure 4. Mast cell counts at five sites (400 μm 2 ) chosen at random in the skin samples.Mean mast cell counts in each group were compared with the EECT0 group by one-way ANOVA with Duncan's posthoc test, and statistical differences among the groups were presented as different alphabetical scripts on the bar charts of each group at p < 0.05 (mean ± SEM, n = 4).Other details are the same as in Figure 1 and Figure 2.