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Year : 2012  |  Volume : 44  |  Issue : 4  |  Page : 469--474

Platycodon grandiflorus alleviates DNCB-induced atopy-like dermatitis in NC/Nga mice

Sang-Joon Park1, Hyang-Ae Lee2, Jong Woo Kim3, Byoung-Seok Lee4, Eun-Joo Kim4,  
1 College of Veterinary Medicine, Kyungpook National University, Buk-gu, Daegu, Korea
2 Korea Institute of Toxicology, Korea Research Institute of Chemical Technology, Yuseong-gu, Daejeon; Department of Physiology, Seoul National University, Jongro-gu, Seoul, Korea
3 B and C Biopharm Co. Ltd., Yeongtong-gu, Suwon, Korea
4 Korea Institute of Toxicology, Korea Research Institute of Chemical Technology, Yuseong-gu, Daejeon, Korea

Correspondence Address:
Eun-Joo Kim
Korea Institute of Toxicology, Korea Research Institute of Chemical Technology, Yuseong-gu, Daejeon
Korea

Abstract

Objective: To investigate the therapeutic effect of crude extract from Platycodon grandiflorum (PG) roots on atopic dermatitis (AD)-like skin lesions in NC/Nga mice. Materials and Methods: To develop atopic dermatitis-like lesions, 200 μl of 0.3% 1-chloro-2, 4-dinitro benzene (DNCB) in acetone/olive oil (3:1) was applied 3 times a week for 2 weeks on the shaved skin of their backs. PG extract was dissolved in saline and orally administrated at concentrations of 300 and 500 mg/kg every day for 2 weeks. The therapeutic effect of PG on AD-like skin lesions was assessed by measuring skin severity scores and epithermal thickness, serum total immunoglobulin (Ig) E, histopathological findings for inflammatory cells including mast cells, macrophage and T cells, and mRNA expression of various cytokines related to the inflammatory and allergic response. The significance of inter-group differences was analyzed using the ANOVA test. Data were considered to be significant when P < 0.05 or P < 0.01. Results: Oral treatment of PG suppressed AD-like skin lesions according to the assessment of skin severity and epithermal thickness in the DNCB-treated NC/Nga mice. This alleviation was further correlated with a reduction of elevated serum total IgE or cytokine mRNA in the PG-treated group compared with vehicle-treated positive group. In addition, infiltrated inflammatory cells decreased on the skin lesions compared with vehicle-treated group. Conclusion: These results suggest that PG may have a potential therapeutic effect for AD via the inhibition of both inflammatory and allergic reaction.


How to cite this article:
Park SJ, Lee HA, Kim JW, Lee BS, Kim EJ. Platycodon grandiflorus alleviates DNCB-induced atopy-like dermatitis in NC/Nga mice.Indian J Pharmacol 2012;44:469-474


How to cite this URL:
Park SJ, Lee HA, Kim JW, Lee BS, Kim EJ. Platycodon grandiflorus alleviates DNCB-induced atopy-like dermatitis in NC/Nga mice. Indian J Pharmacol [serial online] 2012 [cited 2023 Jun 7 ];44:469-474
Available from: https://www.ijp-online.com/text.asp?2012/44/4/469/99306


Full Text

 Introduction



Atopic dermatitis (AD) is a chronic inflammatory skin disease presenting cutaneous hyper-reactivity which progresses by the activation of inflammatory cells related to various allergic immune responses. In AD patients, accumulated protein allergens in the epidermis provoke itch-induced scratching and finally cause secondary skin infection by purulent bacteria or viruses. Mechanical destruction of the skin barrier system is repaired by the self-renewal of basal epithelial cells in the stratum corneum with the infiltration of inflammatory cells related to wound healing such as neutrophils, macrophages, and fibroblasts, but skin lesions repeatedly exposed to allergens activate allergic responses by attracting specialized cell clones such as mast cells, eosinophils, dendritic cells, and T cells. Especially, T-help 2 (Th2) cells, producing IL-4, IL-5, and IL-13, have been known as effect cells responsible for the onset and development of AD.

Currently, therapy of atopic dermatitis has been applied by a topical or systemic treatment with therapeutic substances such as emollients, glucocortisteroids, [1] tacrolimus [2],[3],[4] and pimecrolimus, [5],[6],[7] antihistamines, and immunosuppressants. [1],[2],[3],[4],[5],[6],[7],[8],[9] Despite the widespread use of these drugs, there is demand for new treatment which specifically targets atopic dermatitis. It has been accepted that the best option is to create new efficacious drugs from plant extracts. As a representative example, specific oat extracts alleviate atopic dermatitis in infants, reducing the use of topical corticosteroid. [10] Also, anti-inflammatory agents containing vitis vinifera extracts have been demonstrated to be effective in the treatment of atopic dermatitis.

Platycodi Radix, the root of Platycodon grandiflorum (PG), is used extensively as an anti-inflammatory agent in the treatment of respiratory ailments such as coughs and colds in Oriental areas. Triterpenoid saponins, which are saponin glycosides with an attachment of various sugar molecules to the triterpene unit, have been known to be a major component of Platycodi Radix and can be easily cleaved off in the gut by bacteria. This ability allows them permeate into cell membranes and potentially activate many signaling molecules attached to the membrane. Some saponins are separated from this root: platycodins (A, D, D2, and D3), polygalacin D2, platyconic acid A, and platycosides (A, B, C, D, E and F). Among them, platycodin D has been known to be effective for the inhibition of COX-2 induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) with suppression of prostaglandin E2 (PGE2) in rat peritoneal macrophages. [11] Another group reported that various platycodin saponins inhibit iNOS and COX-2 gene expression by blocking NF-kappaB activation in LPS-induced RAW 264.7 cells. [12] In addition, Han et al. suggested that saponins derived from roots of PG inhibit anaphylactic reaction and IgE-mediated allergic response in mast cells. [13] Recently, Kim et al. reported that PG inhibits the development of AD-like skin lesions by reducing the Th2 cell response and increasing the Th1 cell on data from serum and isolated splenocytes of atopy-induced NC/Nga mice. [14]

In the present study, we investigated the therapeutic effect of PG extract on AD-like skin lesions induced by 1-chloro-2,4-dinitro benzene (DNCB) in NC/Nga mice. In addition, immune cell types regulated by PG were characterized using specific inflammatory cell markers.

 Materials and Methods



Animals and treatment

Male 8-week-old NC/Nga mice were purchased from Japan SLC Inc. (Hamamatsu, Japan). The animals were kept in a storage room under a constant temperature of 23 ± 3°C, relative humidity of 50 ± 10%, and illumination with 12 h light/dark cycles until the initiation of the experiment. All animals were fed with standard animal chow daily and had access to drinking water ad libitum. After 1-week acclimation, 200 μl of 0.3% DNCB in acetone/olive oil (3:1) was repeatedly applied on the shaved back skin 3 times a week for 2 weeks to develop atopic dermatitis-like lesions. PG extract was dissolved in saline and orally administrated at either 300 or 500 mg/kg every day for 2 weeks. The animal were then divided into five groups: Group I was treated with vehicle (normal control, NC), group II was treated with the vehicle and DNCB (positive control, PC), group III was treated with PG extract (300 mg/kg) and DNCB (PG1), group IV was treated with PG extract (500 mg/kg) and DNCB (PG2), and group V was treated with dexamethasone (1.5 mg/kg) and DNCB (DEX). All animal experiments were approved by the Institutional Animal Care and Use Committee of the Korean Institute of Toxicology (KIT).

Clinical observation of atopic dermatitis

The severity of AD was evaluated by clinical signs and symptoms once a week for 4 weeks as described previously by Matsubara et al.[15] The total clinical severity score was defined as the sum of each score grades: 0 (none), 1 (mild), 2 (moderate), and 3 (severe) for each of the five manifestations: itching, erythema, hemorrhage, scaling, and dryness.

Histological examination

The dorsal skin of each mouse was removed and fixed in 10% formalin. Fixed skin tissues were embedded in paraffin and each section was stained with hematoxylin-eosin (H and E) and toluidine blue. Degranulated mast cells were counted in toluidine blue staining slides. To measure epidermal thickness, 3 μm sections were stained with H and E and the thickness of the epidermis was determined via light microscopic (Nikon, Japan). For each individual mouse, three nonsequential sections were evaluated and for each section the epidermal thickness was determined by 10 sequential measurements with intervals of 200 μm.

Total serum IgE levels

Blood samples were collected from the mice after sacrifice, and serum samples were obtained by centrifugation (3500 rpm, 20 min). The levels of total serum IgE were measured by a mouse IgE ELISA kit (Shibayagi, Japan), following the manufacturer's instructions.

Reverse transcription polymerase chain reaction (RT-PCR)

Total RNA was extracted from the skin tissues with RNA-Bee (TEL-TEST, Inc., Friendswood, TX) according to the manufacturer's instructions. 20 μg of total RNA was used as a template for the reverse transcription (RT) reaction to test several cytokines at the same condition. For the semi-quantitative polymerase chain reaction (PCR) of pro-inflammatory cytokines, the primers were synthesized as follows: TNF-a forward, 5'-GGCAGGTCTACTTTGGAGTCATTGC- 3'and reverse, 5'-ACATTCGAGGCTCCAGTGAATTCGG-3'; IL- 1b forward, 5'-TCATGGGATGATGATGATAACCTGCT-3' and reverse, 5'-CCCATACTTTAGGAAGACACGGATT-3'; IL-4 forward, 5'-TCATCGGCATTTTGAACGAG-3' and reverse, 5'-GAATCCAGGCATCGA AAA GC-3'; IL-6 forward, 5'-CTGGTGACAACCACGGCCTTCCCTA-3' and reverse, 5'-ATGCTTAGGCATAACGCACTAGGTT-3'; IL- 13R1a forward, 5'-GCTGAAGTAACAGAACAGGC-3' and reverse, 5'-GAATTTGAGCGTCTCTGTCGAA-3'; IL-13R2a forward, 5'-GGTTATGCCAAATGCACTTGAG-3' and reverse, 5'-ATGGCTTTTGTGCATATCAGAT-3'. The PCR cycles comprise denaturation at 94°C for 30 s, annealing at 58°C for 30 s and extension at 72°C for 45 s for 27 cycles. b-actin was also amplified as an internal PCR control using the following primers, forward, 5'-GGGCATTGTGATGGACTCCG-3' and reverse, 5'-TGAGGCCAAGATGGAGCCAC-3'. The PCR for b-actin was carried out under the same conditions. PCR products were separated by electrophoresis through a 2% agarose gel, stained with ethidium bromide, and then detected using UV light. For semi-quantitative analysis of PCR bands, the density of each band was measured with a computer imaging device and accompanying software (Bio-Rad, Hercules, CA).

Immunohistochemistry

Skin sections were deparaffinized with xylene and washed in serial ethanol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 5 min. To enhance the exposure of the skin tissues to the antigen, a heat-activated method on the skin section using a decloaking chamber (Biomedical care) was applied. Slides were then blocked with 1% bovine serum albumin in PBS for 1 hr. Primary antibodies for cytokeratin-10 (C10), MHC class II cell surface receptor encoded by the human leukocyte antigen complex (HLA-DR), CD3 and ED1 were then applied and incubated at 4°C overnight. After washing in PBS, slides were treated with biotinylated secondary antibodies for 20 min, streptoavidin-horse radish peroxidase substrate (Zymed Laboratories, South San Francisco, CA). Slides were counterstained with hematoxyline (Sigma-Aldrich, St. Louis, MO) and finally covered using a mounting medium (Sigma-Aldrich, St. Louis, MO).

Statistical analysis

Numerical data obtained during the study was subjected to calculation of group means and standard error of the mean. The significance of inter-group differences was analyzed using the ANOVA test. Data was considered to be significant when P < 0.05 or P < 0.01.

 Results



Atopic dermatitis-like skin lesions

To induce AD-like skin lesions in NC/Nga mice, 200 μL of 0.3% DNCB was applied to the backs of mice 3 times a week for 2 weeks. Clinically, topical exposure of DNCB results in skin symptoms of itching, erythema, and hemorrhage after a week, then it rapidly progresses into edema, superficial erosion, deep excoriation, scaling, and dryness of the skin within two weeks following the repeated treatment with DNCB. [Figure 1]A and B present the dermatitis scores and gross findings. The clinical signs of dermatitis were significantly reduced after 2 weeks in the PG-treated groups and DEX-treated group compared to positive control group [Figure 1]A. At scarifying, DNCB-induced AD-like skin lesion was recovered to almost healthy skin on gross findings. In addition, a similar inhibitory effect of PG was assessed on H and E staining [Figure 2]B and immunohistochemical findings for cytokeratin-10 [Figure 2]C in the measurement of epidermal thickness [Figure 2]A.{Figure 1}{Figure 2}

IgE level in serum

IgE in AD functions in the activation of key effector cell types involved in allergic inflammation. Clinically, the level of IgE represents the prevalence of allergic sensitization and severity of AD. Thus, it was investigated whether oral treatment with PG affects the serum level of IgE. The total serum IgE level of the positive control group was significantly increased in comparison to normal control [Figure 3]A. But, the enhancement of IgE was significantly reduced in PG-treated groups compared to the positive control group. These results mean that PG has an inhibitory effect in the serum IgE production.{Figure 3}

Mast cell infiltration

Mast cells have the ability to be activated by certain inflammatory biomolecules such as neuropeptides and cytokines, and they also produce pro-inflammatory mediators without degranulation in the prolonged progression of AD; thus, mast cells display immunomodulatory activity via cross-talk with various inflammatory cells. Therefore, the effect of PG in the infiltration of mast cells, one of the most important effector cells involved in AD, was investigated. As shown [Figure 3]B, the DNCB-treated positive control group showed excessive mast cell infiltration in the dermis of NC/Nga mice, but enhanced mast cells were more reduced in the PG-treated group than in the positive control group. This effect was compatible with the loss of mast cell numbers in the DEX-treated group. The inhibitory effect of mast cell infiltration by PG may have multiple functions in alleviating allergic inflammation.

Pro-inflammatory cytokine expression

Skin lesions of AD contains various cellular sources responsible for the production of inflammatory and allergic mediators. Among them, Th2- and pro-inflammatory cytokines are critical for allergic inflammation. In the present study, it was examined whether total extract of PG suppressed the expression of pro-inflammatory cytokines (IL-1β, TNF-α and IL-6) and th2 cytokines (IL-4, IL-13r1a and IL-13r2a). Treatment with PG strongly inhibited mRNA expression of IL-1b, IL-6, and IL-13r1a [Figure 4]a, but slightly inhibited mRNA expression of TNF-a, IL-4 and IL-13r2a. Thus, it is considered that PG has broad effects in the suppression of both mRNA expression of pro-inflammatory cytokines, as well as Th2 cytokines in DNCB-induced AD-like skin lesions.{Figure 4}

Immunohistochemistry for major effector cells of atopic dermatitis

In the progression of atopic dermatitis, various inflammatory cells are recruited into allergen exposure sites with cutaneous hyper-reactivity. In particular, AD is associated with T-cell mediated chronic inflammatory skin disorder. In this study, the types of infiltrated inflammatory cells affected by the treatment of PG were examined. Pan T cells and ED1 + macrophages were prevalent in the DNCB-induced atopic dermatitis-like lesions with increased localization of HLA-DR in the epidermis and dermis. Pan T cells and ED1 + macrophages represented a high percentage of the dermis, and some T cells were also observed in the epidermis [Figure 4]b. However, this increase was significantly less pronounced in PG- or DEX-treated group [Figure 4]b. Infiltrated T-cells using were further characterized CD4 and CD8 specific antibodies. In the experimental AD model, CD4 + and CD8 + T-cells appeared with similar frequency in the AD-like lesions (Data not shown). The similar increases of both CD4 + and CD8 + T-cells suggests the possibility of common involvement of Th1- and Th2-type T cells.

 Discussion



AD is a chronic skin disease characterized by allergic inflammation. The immunological response to AD includes increased and dysregulated production of inflammatory cytokines, elevated serum IgE levels, and inflammatory cell infiltration. [16] The role of IgE in AD is expected to relate to activation of key effector cell types involved in allergic inflammation. In the present study, we investigated whether oral treatment with PG significantly alleviates skin thickness and serum IgE levels in DNCB-induced AD-like skin lesions of NC/Nga mice models. Histologically, PG treatment markedly reduced excessive differentiation of keratinocytes in the epidermis and infiltration of inflammatory cells into the DNCB-induced AD-like skin lesions. Mast cells are suggested to be among key effector cells involved in allergic inflammation by the increase in the mast cell number and mast cell degranulation in AD lesions. [17] In our study, repeated oral treatment of PG inhibited extensive mast cell infiltration in the dermis of NC/Nga mice, supporting the criteria of an AD animal model. [18] The mast cell infiltration in the PG-treated groups was more suppressed than in the DNCB-alone group. Han et al. (2011) demonstrated that saponins isolated from the root of Platycodi Radix inhibit the degranulation of mast cells. It is probable that systemic treatment with PG has significant suppressive effects on local mast cells.

Multiple cytokines are critical for intracellular signal transmission of allergic inflammation. [19] Among them, IL-4 is essential for IgE production and promotes the switch from naive T cells to the allergic type Th2 cells. [20],[21],[22] TNF-a is a potent inflammatory mediator in the cytokine family. TNF-a is produced at the initiation stage of AD induce the expression of a variety of chemokines and adhesion molecules which direct the recruitment, proliferation, and survival of leukocytes within the skin. [23] In our study, PG significantly suppressed DNCB-induced elevation of not only pro-inflammatory cytokines such as IL-1β and IL-6 mRNA expression, but also allergic inflammatory cytokines including IL-4, IL-13r1a and IL-13r2a in the skin of NC/Nga mice. Recently, Kim et al. reported that PG inhibits the development of AD-like skin lesions of NC/ Nga mice by decreasing the serum level of IL-4 and IgG1 and increasing the serum level of IL-12p40 and IgG2a. [14] In addition, they demonstrated that treatment with PG reduces the Th2 cell response such as IL-4 and IL-5 and increase the Th1 cell response such as IL-12p40 and IFN-γ on isolated splenocytes of a 2,4-dinitrofluorobenzene-induced mice model. It is considered that PG, in allergic immune response, has multiple inhibitory effects via broad regulation of various inflammatory cells.

The accumulation of allergens within keratinocytes and mechanical disruption of the skin barrier by scratching elicits activation of cell sources responsible for innate immune response, as well as allergic inflammation in the lesional epidermis and dermis. In particular, it has been known to have close interactions between keratinocytes and infiltrated inflammatory cells in the AD-like pathological condition. Among infiltratory inflammatory cells, AD-like skin lesions are associated with T-cell-mediated chronic inflammatory skin disorder. The imbalance between Th1 and Th2 cytokines leads to various immune-related diseases, including allergic disorders. [24] In the present study, PG also down-regulates the overexpression of both Th1 and Th2 cytokines in AD-like skin lesions caused by treatment with DNCB. It is likely that PG alleviate AD-like skin lesion via inhibition of both Th1 and Th2-type cytokines. In the present study, we characterized infiltrated inflammatory cells in the AD-like skin lesion to evaluate the anti-atopic effect of PG. The results of the immunohistochemical study of inflammatory cells showed that PG suppressed all inflammatory cell types including CD3 (pan T cells), HLA-DR (MHC class II cell surface receptor) s and ED1 (macrophages) that were elevated in AD-like skin lesions of NC/Nga mice. This inhibition can be explained by PG having a broad effect in the innate cell response as well as allergic inflammation. Consequently, it is considered that oral treatment of PG has a systemic inhibitory property via suppression of pro-inflammatory responses, as well as Th1 and Th2 responses. In addition, our data present evidence that PG can be applied as a useful drug in the therapeutic aspect of AD.

 Acknowledgment



This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ 008017), Rural Development Administration, Republic of Korea.

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