Effect of Polygala tenuifolia on TMA-induced atopic dermatitis aggravated by immobilization stress in mice

다국어 초록 (Multilingual Abstract)

Atopic dermatitis (AD) and stress create a vicious cycle: stress exacerbates atopic symptoms, and atopic skin disease elicits stress and anxiety. Increasing evidence suggests that stress-induced exacerbation of AD symptom is closely associated with the degranulation of skin mast cells via corticotropin-releasing factor (CRF) and substance P (SP) signaling triggered by stress. The aim of this study is to evaluate efficacy of the boiled water extract of Polygala tenuifolia Willd. (PTW) on immobilization (IMO) stress-exacerbated AD in the mouse and to identify the action mechanism at the molecular level. An in vitro model of stress-associated AD was developed by applying the stress hormone CRF (200 nM) to the human mast cell line (HMC)-1 following pre-treatment of SP (10 µM) for 48 h to induce inflammatory priming. The cells were treated with PTW (250, or 500 g/ml) 30 min before exposure to CRF. Mast cell degranulation was assessed by microscopic examination and tryptase assay 24 h after treatment. Specifically, the upstream signaling of protein kinase A (PKA) and protein kinase C (PKC) and the downstream phosphorylation of mitogen-activated protein kinase (MAPK) was examined. For the in vivo experiment, AD-like skin lesions were generated by repeated application of tetramethylammonium (TMA) to both ears of Balb/c mice. Sensitization was induced by application of 5% TMA to the dorsal skin, followed by application of 2% TMA to both ears on 5th days, and then, 1% TMA was applied once a day from 6th to 14th day (totally 9 days). The animals underwent 2 h-IMO stress after every application of 1% TMA from 9th to 14th day. Mice undergoing IMO stress received oral PTW (50, or 250 mg/kg) daily 30 min prior to the application of 1% TMA from 9th to 14th day, and the medicinal efficacy of PTW was assessed using inflammation of ear skin tissue, scratching behavior, water content of the ear tissue, lymph node weight, and serum histamine and immunoglobulin E (lgE) levels. The microscopic examination and tryptase activity assay in the in vitro experiment revealed that mast cell degranulation was significantly greater in the cells exposed to CRF to the HMC-1 cells after pre-treatment with SP for 48 h than in those exposed to either compound alone and both. Treatment with 500 g/ml PTW effectively inhibited mast cell degranulation and release of inflammatory cytokines in the HMC-1 cells. In the in vivo experiment, treatment with 250 mg/kg PTW showed a significant decrease in scratching behavior, morphological changes in ear thickness, water content, lymph node weight, and serum histamine and lgE levels. Furthermore, treatment with 250 mg/kg PTW significantly decreased the expression of tumor necrosis factor-alpha (TNF-α) and interleukin-4 (IL-4) cytokines in the mouse of IMO stress-induced AD exacerbation. Application of CRF to the HMC-1 cells after pre-treatment with SP for 48 h significantly activated upstream signaling of PKA and PKC; however, treatment with 500 g/ml PTW showed only result of decreasing of PKA activation. Moreover, the analysis of the downstream phosphorylation of MAPK revealed that p38 phosphorylation was significantly decreased by PTW. Taken together, the IMO stress-exacerbated AD was verified to be valid in an animal model and PTW inhibited mast cell degranulation in HMC-1 cell line and IMO stress-exacerbated atopic mouse. Anti-atopic effect of PTW was exerted via the modulation of p38 MAPK cell signaling rather than affecting intrinsic signaling of atopic inflammation itself. Thus, PTW may be a useful for the treatment of IMO stress-exacerbated AD. Moreover, these results provide a molecular basis for developing new therapeutics for treating various inflammatory diseases, especially those aggravated by stress.

목차 (Table of Contents)

• 1. Introduction 1
• 2. Materials and methods 7
• 2.1. Cell line 7
• 2.2. Animals 7
• 2.3. Chemicals and drugs 7
• 2.4. Experimental schedules 8
• 2.5. In vitro experiment 11
• 2.5.1. Toluidine blue staining assay 11
• 2.5.2. Tryptase activity assay 11
• 2.5.3. RNA isolation and RT-PCR analysis 12
• 2.5.4. ELISA of PKA and PKC activities 15
• 2.5.5. Western blot analysis of MAPKs phosphorylation 15
• 2.6. In vivo experiment 17
• 2.6.1. Experimental groups 17
• 2.6.2. Ear skin manifestation and hematoxylin-eosin histochemistry analysis 17
• 2.6.3. Scratching behavior and water content analysis 18
• 2.6.4. ELISA of serum histamine and IgE levels 19
• 2.6.5. RNA isolation and RT-PCR analysis 19
• 2.7. Statistical analysis 20
• 3. Results 21
• 3.1. Establishment of stress-associated AD model 21
• 3.1.1. Toluidine blue staining and tryptase activity assay of HMC-1 cells 21
• 3.1.2. Scratching behavior and serum levels of histamine and IgE in mice 23
• 3.2. In vitro selection of effective medicinal herbs for stress-associated AD 25
• 3.2.1. Expression levels of cytokine mRNAs during degranulation of HMC-1 cells 25
• 3.3. Effect of Polygala tenuifolia on IMO stress-exacerbated AD in mice 28
• 3.3.1. Effect of Polygala tenuifolia on TMA-induced mouse AD 28
• 3.3.2. Ear manifestation and hematoxylin-eosin histochemistry in IMO stress-exacerbated AD mice 31
• 3.3.3. Scratching behavior, water content, and serum histamine and IgE levels in IMO stress-exacerbated AD mice 34
• 3.3.4. TNF-α and IL-4 cytokine mRNAs in IMO stress-exacerbated AD mice 37
• 3.4. Action mechanisms of Polygala tenuifolia on stress-associated AD 39
• 3.4.1. Toluidine blue staining and tryptase activity assay of HMC-1 cells 39
• 3.4.2. Activities of PKA and PKC of HMC-1 cells 42
• 3.4.3. MAPKs cell signaling pathway of HMC-1 cells 44
• 4. Discussion 47
• 5. Conclusion 57
• References 60
• Korean abstract (국문 초록) 71