Oleanolic acid (OA) is a pentacyclic triterpenoid, abundantly found in plants of the Oleaceae family, and is well known for its beneficial pharmacological activities.
Allergic inflammation is characterized by pathophysiological or hypersensitivity disorders, including allergic asthma, allergic rhinitis, anaphylaxis, and atopic dermatitis (AD), after exposure to allergens 
. AD is a chronic inflammatory skin disease that arises from the complicated interaction of innate and adaptive immune responses based on genetics, environmental factors, immune abnormalities, and skin barrier functions 
. The characteristic features of AD include itchy, swollen, red, and cracked skin with inflammatory cell accumulation in AD skin lesions. Although the pathogenesis of AD is not clear, it is known that several cells and factors are associated with its development. The pathological processes of AD are thought to be mediated by Th1/ Th2 balance, which is skewed toward Th2 in AD. Th2 cells are mainly activated in the acute phase of AD, while Th1 cells mediate the alteration of expression in chronic AD 
. The standard treatment for AD involves the application of topical corticosteroids or the administration of immunosuppressive agents; however, protracted use of these agents can cause various side effects, such as skin atrophy, bleeding, vasodilation, and organ toxicity. For this reason, medicines originating from herbal sources may be preferred to steroids, and may be used in combination with other methods, such as enhancing immunity, reducing house mite dust, and dietary restrictions 
Numerous intracellular signal transduction triggered by ligand–cell surface receptor binding is mediated by transcription factors. Nuclear factor (NF)-κB and the signal transducer and activator of transcription (STAT)−1 are pivotal transcription factors associated with the allergic inflammatory response 
. Upon stimulation, the inhibitor κB (IκB)-α protein is phosphorylated, leading to the ubiquitination and proteasomal degradation of IκB. Sequentially activated NF-κB and interferon (IFN)-γ-activated STAT 1 in the cytoplasm translocate into the nucleus, where they engage in the expression of numerous pro-inflammatory mediators. Thus, these transcription factors are important pharmacological targets for the discovery of novel therapeutics to treat allergic disorders 
Oleanolic acid (OA) is a pentacyclic triterpenoid that is abundant in plants of the Oleaceae
family, such as Olea europaea
. OA is ubiquitously found in food and plants, where it exists as a free acid or as an aglycone of triterpenoid saponins, such as ursolic acid, moronic acid, and betulinic acid 
. To date, various reports have described the pharmacological activities of OA, including its antioxidant 
, anti-inflammatory 
, anti-asthmatic 
, anti-diabetic 
, anti-tumor 
, hepatoprotective 
, immunomodulatory 
, anti-parasitic 
, and anti-hypertensive 
properties. Despite the fact that OA is a well-known active component contained in various plants, studies on its effect on AD are insufficient. As it is important to study natural materials that are effective against allergic diseases, we focused on OA that exhibits a wide range of biological activities, such as anti-inflammatory and anti-asthmatic effects, as a feasible active compound for allergic diseases. Previously, we reported the anti-allergic effect of OA, demonstrating that OA exerted an inhibitory effect on mast cell-mediated allergic inflammation in vivo and in vitro 
. Allergic response and inflammation can trigger AD and worsen the condition, thus, controlling allergic and inflammatory reaction could be important strategy in the manage of AD. These results prompted us to investigate its potential effect on other allergic diseases, such as AD. As AD is mainly the beginning of a series of allergic disorders, we hypothesized that OA would attenuate AD-like symptoms.
2. OA Attenuated AD Lesions in DNCB-Induced AD Mice
The repeated topical application of DNCB on the dorsal skin of mice induces AD skin symptoms. DNCB is a “contact sensitizer” that induces contact hypersensitivity of the skin in mice, which is considered to be a cell-mediated response 
. To investigate the remedial effects of OA on AD mice, we administered OA following the induction of AD mouse skin. The experimental procedure is summarized in Figure 1
A. On the day of sacrifice, severe AD-like lesions, such as erythema, edema, hemorrhage, scarring, dryness, excoriation, and erosion were observed on the dorsal skin of DNCB-induced AD mice. However, topical application of dexamethasone (10 μM), a well-known therapeutic agent for AD, and OA (10 and 50 μM) for 3 weeks significantly alleviated these AD skin symptoms compared to the DNCB group (p
< 0.001) (Figure 1
Figure 1. Effects of OA on DNCB-induced AD skin lesions in ICR mice. (A) Experimental schedule for the induction of AD. (B) Effect of OA on clinical features of DNCB-induced AD skin lesions. White arrows indicated DNCB treatment. (C) Effects of OA on dermatitis score. Densitometric analysis was performed using Bio-Rad Quantity One® Software. The data shown represent mean ± S.D. (n = 6) of three independent experiments. ### p < 0.001 vs. the control group; *** p < 0.001 vs. DNCB-treated group.
3. OA Improved the Histological Observations and Histamine Release in DNCB-Induced AD Mice
Histological alterations, such as epidermal hyperplasia and infiltration of lymphocytes and mast cells in the skin, are the main hallmarks of AD 
. Improvements in clinical skin conditions following OA treatment were confirmed by histological examination. Histological analysis was performed on atopic skin tissues. The excised skin from each group was stained with hematoxylin and eosin (H&E) or toluidine blue, and histological alterations were observed microscopically. H&E-stained tissue sections revealed that the thickness of epidermal and dermal tissues was greater in the DNCB-treated group (91.84 ± 7.60, 474.66 ± 43.65 μm, respectively, p
< 0.001) than the control group due to edema, hyperkeratosis, and hyperplasia (Figure 2
A). However, treatment with 10 and 50 μM OA markedly attenuated the epidermal (47.16 ± 5.98 and 52.65 ± 9.56 μm, p
< 0.001) and dermal thickening (258.65 ± 17.56 and 292.65 ± 25.61 μm, p
< 0.001) (Figure 2
B,C). In the toluidine blue-stained tissue sections, mast cell infiltration, an indicator of inflammation, was noticeably increased in the DNCB-treated group compared to the control group (73.67 ± 12.06 cells, p
< 0.001). Treatment with 10 and 50 μM OA attenuated the infiltration of inflammatory cells, particularly mast cells, as evidenced by toluidine blue staining (28.5 ± 6.98, 25.5 ± 3.73 cells, respectively, p
< 0.001) (Figure 3
A,B). As mast cells are sources of histamine, which is the most potent mediator involved in AD symptoms 
, histamine levels in the serum were also examined. The results showed that treatment with 50 μM OA remarkably inhibited histamine release (271.91 ± 35.75 ng/mL, p
< 0.001) compared to the DNCB-treated group (411.81 ± 60.12 ng/mL, p
< 0.001) (Figure 3
Figure 2. Effect of OA on epidermal and dermal thickness in DNCB-induced AD skin lesions. (A) H&E stained AD mouse skin lesions (scale bar = 500 μm). (B) Determination of epidermal thickness and (C) dermal thickness. Epidermal and dermal thickness in H&E stained sections were measured under a microscope. The data shown represent mean ± S.D. (n = 6) of three independent experiments. ### p < 0.001 vs. the control group; *** p < 0.001 vs. DNCB-treated group.
Figure 3. Effect of OA on mast cell infiltration and serum histamine level in DNCB-induced AD skin lesions. (A) Toluidine blue stained AD mouse skin lesions (scale bar = 100 μm). Black arrows indicated stained mast cells. (B) Number of mast cells per mm section. Mast cell infiltration in toluidine blue stained sections is expressed as the average total count in five fields. (C) Histamine release in mouse serum was measured using an ELISA kit. The data shown represent mean ± S.D. (n = 6) of three independent experiments. ### p < 0.001 vs. the control group; ** p < 0.01 and *** p < 0.001 vs. DNCB-treated group.
4. OA Suppressed the mRNA Expression of AD-Related Cytokines and Activation of IκB and STAT1 in DNCB-Induced AD Mice
Next, we investigated whether OA inhibited the signature cytokines of AD in the dorsal tissues of DNCB-induced AD mice. Above all, thymus and activation-regulated chemokine (TARC)/CCL17, are members of the CC chemokine subfamily and are involved in the recruitment of Th2 lymphocytes and the continuation of Th2 immune responses 
. In addition, thymic stromal lymphopoietin (TSLP) is known to provoke dendritic cell-mediated Th2 responses and is highly expressed in activated mast cells and skin of AD, which triggers allergic inflammation. Therefore, these cytokines are considered mediators of inflammatory skin diseases, such as AD . As shown in Figure 4
A, the mRNA expression levels of TSLP and TARC were markedly (p
< 0.001) increased by repetitive treatment with DNCB, while OA reduced the expression levels of TSLP and TARC by approximately basal levels (p
< 0.001). In line with these results, Th2-type cytokines, including IL-4, IL-5, and IL-13, were downregulated by OA treatment compared to DNCB-induced AD mice (p
< 0.001) (Figure 4
Figure 4. Effect of OA on AD cytokines and IκB, STAT1 activation in DNCB-induced AD skin lesions. Total RNA prepared from the dorsal tissue, and the level of (A) TARC, TSLP, (B) IL-4, IL-5, and IL-13 were determined by quantitative qRT-PCR. Expression of IκB (C) and STAT1 (D) was determined by Western blot analysis using specific antibodies. Densitometric analysis was performed using Bio-Rad Quantity One® Software. The data shown represent mean ± S.D. (n = 6) of three independent experiments. ### p < 0.001 vs. the control group; ** p < 0.01 and *** p < 0.001 vs. DNCB-treated group.
To investigate the signaling pathways involved in the inhibitory effect of OA on cytokine production, we examined the phosphorylation and degradation of IκB and activation of STAT1 in DNCB-induced AD mice. The results demonstrated that the phosphorylation and degradation of IκB induced by DNCB were significantly (p < 0.001) inhibited by treatment with OA (Figure 4C). In addition, OA inhibited the DNCB-induced phosphorylation of STAT1 at residues Ser727 and Tyr701 with significance (Figure 4D). Considering our results, it can be presumed that the NF-κB and STAT1 signaling pathways are involved in the inhibitory effect of OA on the cytokine profiles of DNCB-induced AD-like skin.