Background
Atopic dermatitis (AD) is an allergic skin disease characterized by complex symptoms such as drying and thickening of the skin, and scratch marks that are frequently associated with immunoglobulin-E (IgE) hyper-responsiveness to environmental allergens. The wrist, neck, face, and the crooks of the elbows and knees are among the most frequent locations of the lesions [
1]. AD is mostly affecting children with onset before the age of five years [
2]. Environmental (house dust mites and air pollution) and genetic factors considered the causes of AD. In addition, genetic predisposition accompanied by assorted peculiar immune symptom accounts for more than 50% of reported cases [
3].
Activation of T-helper 2 (Th2) and mast cells are mentioned in the development of AD [
4]. It is associated with an increase in serum concentration of Th2 cytokines, including interleukin (IL) -4, IL-5, IL-10, and IL-13. In addition, expression of interferon- γ (IFN-γ) is also reported in cases of AD [
5,
6]. Basal keratinocytes produce thymus and activation-regulated chemokine (TARC) recruits Th2-lymphocytes and further aggravate dermatitis [
7]. Authors also suggested that the release of β-hexosaminidase from degranulated mast cells, high levels of serum immunoglobulin (Ig)E, and the expression of proinflammatory mediators such as prostaglandin E2 (PGE2) and nitrite (NO) are important determinants in the propensity of mice to AD [
8,
9].
In spite of the profound side effects caused by topical steroids and oral anti-histamines, these drugs are commonly used to treat AD [
10]. Hence, efforts have been directed towards identifying safer and effective compounds of plant origin which can modulate the pathological mechanism(s) of AD such as anti-histamine effects, inhibition of Th
2 responses and IgE production [
11]. More than 20% of the population in Korea rely on traditional medicine as the primary health care [
12]. However, only a few studies have been conducted on the efficacy and safety of medicinal plants. Therefore, a screening test was conducted using the DPPH antioxidant, β-hexoseaminidase, and NO assay on 286 plants from Gyeongbuk Forest Resource Development Institute, Republic of Korea. These assays were selected as a screening method, taking into account the multifactorial nature of AD. Accordingly,
Cornus officinalis (Family: Cornaceae),
Castanea crenata (Family: Fagaceae),
Rosa multiflora (Family: Cornaceae)
, Lespedeza bicolor (Family: Legumes), and
Platycladus orientalis (Family: Cupressaceae) were selected for further studies. Previous studies have shown that these plants produced various degrees of biological activities that are associated with AD [
13‐
17]. In addition, various parts of these plants combined in different proportions to produce ointments for inflammatory skin disorders, including AD [
18‐
23]. However, all of the studies are conducted on the activities of a single plant against inflammation or free radical activity. To the best of our knowledge, there is no scientific report available on the efficacy and safety of the most commonly used plant combinations.
Based on the results of the screening assay, a 4:1:1:1:1 ratio of
Cornus officinalis (fruit):
Rosa multiflora (stem)
, Lespedeza bicolor (aerial part),
Platycladus orientalis (leaves),
Castanea crenata (leaves) respectively, were selected for further studies. Additional investigation using the NO and β-hexosaminidase assay have demonstrated that C2RLP produced better activity than each plant extract (data not shown). Finally, a topical ointment was formulated (C2RLP) taking into account the main complaints of the disease such as pruritus, dryness, and psoriasis on the skin [
24]. Therefore, the study was aimed to evaluate the in vivo protective effects of topical application of herbal formulation, C2RLP, against 1-Chloro-2, 4-dinitrobenzene (DNCB) induced AD-like lesion in BALB/C mice. In addition, the effects of C2RLP on free radical scavenging activity and cellular mediators were evaluated using various in vitro methods.
Discussion
AD results in various histopathological and pathophysiological changes in mice, which are related to alterations in the levels of proinflammatory cytokines, IgE, and histamine [
4,
34]. These changes resulted in epidermal hyperplasia, inflammatory cell infiltration, erythema, alopecia, skin dryness and hyperkeratosis [
35] which were clearly observed in petrolatum treated control mice shown in Figs.
6 and
7. The binding of antigen activates infiltrated cells to secrete chemical mediators such as histamine, proteases, cytokines, and chemokines that are essential in the progression of dermatitis [
36]. Our study showed that C2RLP treatment suppressed the DNCB induced AD-like lesions and histopathological changes, including epidermal hyperplasia and inflammatory cell infiltration.
The Th1/Th2 cytokine imbalance is vital in the progression of atopic dermatitis, with increased production of IgE and mast cell activation in Th2-dominant AD [
37]. Studies have shown that compounds isolated from plants modulate the Th1 and/or Th2 cell response and prevents the development of AD in mice [
31,
38]. More precisely, Th2 cytokines such as IL-4, IL-5, IL-13, and IL-4 mediated increment of serum IgE and mast cells were reported in mice with symptoms of AD [
2,
39]. In this study, C2RLP significantly reduced the serum levels of IL-4 with respect to petrolatum. IL-4 is known to activate mast cells by inducing isotype switching to IgE synthesis by B cells. The binding of IgE with allergens activates the immune system and induces degranulation [
40]. In contrast, C2RLP did not affect the DNCB induced production of IFN-γ which are strong inhibitors of IgE synthesis and Th2 cell proliferation [
2]. Therefore, down-regulation of Th2 immunity could be considered as a possible mechanism for the mechanism of C2RLP against AD.
AD is commonly associated with marked infiltration of the skin by mast cells, eosinophils and macrophages [
2]. Macrophages are known to release proinflammatory mediators such as NO and PGE
2, which aggravate the inflammatory responses [
41]. Regulation NO production and iNOS expression might be essential because it is known to affect the pathogenesis of several inflammatory diseases, including AD [
42]. In the current study, C2RLP inhibited the LPS induced production of NO and PGE
2 in RAW 264.7 macrophage cells. In addition, C2RLP produced a dose-dependent attenuation of iNOs-mRNA expression.
Chemokines produced by keratinocytes can cause an imbalance in Th1/Th2 cytokines and contributes to the development of atopic lesions [
4]. The expression of TARC by keratinocytes in AD patients and in mice with atopic lesions was confirmed in previous studies. TARC is known to attract Th2 cells and aggravates the pathological changes related to AD [
41,
43,
44]. In this study, the production of TARC by TI-sensitized HaCa-T cells was reduced following treatment with 300 and 100 μg/ml of C2RLP consolidating the Th2 cell suppressing effects of C2RLP.
Degranulation of activated mast cells and release of mediators are suggested in allergic reactions associated with AD. Mast cell degranulation can be determined by measuring the amount of β-hexosaminidase released from various cell lines including RBL-2H3 [
45]. Inflammatory mediators are released from degranulated mast cells following an Fc epsilon RI (FcεRI) receptor activation, which is a high-affinity IgE receptor [
46]. In our study, C2RLP exhibited a concentration-dependent inhibition of β-hexosaminidase release from RBL-2H3 cells (IC
50 = 179.5 μg/ml) with significant inhibitory activity at 300 μg/ ml. Direct inhibition of FcεRI cascade could be one of the mechanisms of the anti-atopic activity of C2RLP.
Oxidative stress in AD is associated with an increase in lipid peroxidation and reduction in the levels of antioxidants. It promotes tissue inflammation through upregulation of genes that code for pro-inflammatory cytokines and subsequent release of free radicals [
47,
48]. Oxidative stress can also alter the integrity of epidermal keratinocytes by damaging DNA and cellular enzymes [
49]. Previous studies have confirmed higher levels of lipid peroxidation and lower levels of antioxidants in patients with inflammatory skin conditions that resembles AD such as eczema [
50] and alopecia areata [
51]. Therefore, the antioxidant activity of C2RLP could contribute to the reduction in reactive oxygen species and alleviate the oxidative stress associated with AD.
Chemical compounds are implicated directly or indirectly in the biological effects of most plant extracts. The study revealed the presence of various compounds in C2RLP, mainly Loganin, Ellagic acid, and Kaempferol 3-glucoside (Table
1). Previous studies indicated that suppression of NF-κB and MAP- kinases (mitogen-activated protein kinases) are critical to inhibit the secretion of pro-inflammatory cytokines and reduce the number of mast cells, which are involved in the inflammatory response [
52,
53]. Among the major metabolites, loganin is reported to inhibit NF-κB activation and MAP kinase [
54,
55]. The polyphenolic compound, ellagic acid, is suggested to have a diverse biological activity, including antibacterial, antioxidant, anti-inflammatory and anti-carcinogenic actions [
56]. Most importantly, Ellagic acid has been shown to inhibit activation of MAP kinases [
57] and repress NF-κB through down-regulation of the secretion of various inflammatory mediators during AD [
58]. Kaempferol-3-O-glucoside and its derivatives were also reported to produce an anti-inflammatory effect through inhibition of the activation of cyclooxygenase (COX-2) and iNOS [
59,
60]. In addition to the three major compounds, the antioxidant and anti-inflammatory effects of cornuside [
61], naringenin7-O-β-D-glucoside [
62], and quercetin [63] could also contribute to the protective effect of C2RLP in the development of AD. Therefore, suppression of AD-like lesions in C2RLP treated mice might be due to the synergistic action of these compounds.