Thymus vulgaris L. ameliorated atopic dermatitis symptoms through controlling histamine release

다국어 초록 (Multilingual Abstract)

Thymus vulgaris L., commonly known as thyme, is an aromatic herb widely used worldwide. T. vulgaris has antioxidant, anti-inflammatory, and immunomodulatory activities as well as skin anti-photoaging and protective effects. However, there have been no studies regarding the inhibitory effects of T. vulgaris on AD. Therefore, the present study aimed to demonstrate the effects of T. vulgaris on improving AD symptoms.
The active components of T. vulgaris were detected by HPLC analysis. In the in vitro study, the anti-atopic effects of T. vulgaris were examined in LPS-stimulated Raw264.7 cells, DNP-IgE/BSA-stimulated RBL-2H3 cells, and TNF-α/IFN-γ-stimulated HaCaT cells using ELISA kits, RT-PCR, and western blotting. In the in vivo study, the mitigating effects of T. vulgaris against AD were investigated by measuring morphological and physiological changes, scratching behavior, AD symptom-related protein expression, and histological changes in DFE-induced NC/Nga mice.
In the in vitro results, T. vulgaris significantly inhibited NO production in LPS-treated Raw264.7 cells. When T. vulgaris was treated with at a concentration of 250 μg/mL, the release of β-hexosaminidase and histamine was reduced by 52.7% and 38.0%, respectively, than those of only DNP-IgE/BSA-induced RBL-2H3 cells. This was confirmed to be due to the regulation of calcium-related signaling pathway. Furthermore, the production of TARC and MDC was remarkably suppressed by 35.5% and 64.5% at 100 μg/mL of T. vulgaris, respectively, compared with those of only TNF-α/IFN-γ-stimulated HaCaT cells. T. vulgaris also influenced the decrease in mRNA expression of pro-inflammatory cytokines and chemokines, such as IL-6, TARC, MDC, RANTES, and IL-8, via the modulation of MAPKs, STAT1, and NF-κB pathways.
The results of the in vivo study confirmed that topical application of T. vulgaris improved AD symptoms. Treatment with T. vulgaris led to a decline in the severity of dermatitis, TEWL, erythema index. In addition, the number of scratches was significantly decreased by T. vulgaris. This was affected by suppression of serum IgE level and histamine release. Histological analysis results showed that T. vulgaris was involved in the reduction of epidermal thickness as well as the reduction of mast cell infiltration. On the other hand, the skin density and hydration level aggravated by DFE were recovered by topical application of T. vulgaris.
Taken together, this study revealed the ameliorating effects of T. vulgaris on AD-induced in vitro and in vivo models. The results suggest that T. vulgaris can be applied as a natural product-derived therapeutic agent in both cosmetic and pharmaceutical industries as it attenuates itching by controlling histamine release, thereby improving symptoms of AD.

국문 초록 (Abstract)

백리향으로 알려진 타임은 아로마 허브 중 하나로 전 세계적으로 사용되고 있다. 타임은 항산화, 항염 및 면역조절 활성이 있는 것으로 보고되었으며, 특히 피부와 관련하여 광노화 억제 및 피부 보호 효과에 대해 보고되었다. 그러나 타임의 아토피 피부염 억제 효과에 대해서는 과학적으로 검증된 바 없다. 따라서 본 연구는 타임의 아토피 피부염 증상 개선 효과를 검증하기 위하여 수행되었다.
타임의 활성 성분은 HPLC 분석법에 의해 확인되었다. In vitro 연구에서는 ELISA kits, RT-PCR 및 western blot법을 통하여 LPS로 자극된 Raw264.7 세포주, DNP-IgE/BSA로 자극된 RBL-2H3 세포주 및 TNF-α/IFN-γ로 자극된 HaCaT 세포주에서 타임의 아토피 피부염 억제 효과가 확인되었다. In vivo 연구에서는 집먼지 진드기로 아토피 피부염이 유도된 NC/Nga 동물에서 형태학적 및 생리학적 변화, 긁는 행동, AD 증상과 관련된 단백질 발현 및 조직학적 변화를 측정하여 타임의 아토피 피부염 개선 효과를 확인하였다.
In vitro 실험 결과, 타임이 LPS로 자극된 Raw264.7 세포주에서 NO 생성량을 유의적으로 억제시켰다. 250 μg/mL의 농도로 타임이 처리되었을 때 베타헥소사미니다제 및 히스타민의 분비가 각각 52.7% 및 38.0% 감소되었다. 이는 칼슘 관련 경로의 조절에 의한 것으로 확인되었다. 게다가, TNF-α/IFN-γ로 자극된 HaCaT 세포주와 비교하여 타임은 100 μg/mL의 농도에서 TARC와 MDC의 생성을 각각 35.5% 및 64.5% 억제시켰다. 또한, 타임이 MAPKs, STAT1 및 NF-κB 경로의 조절을 통해 IL-6, TARC, MDC, RANTES 및 IL-8과 같은 염증성 사이토카인 및 케모카인의 유전자 발현의 감소에 영향을 미치는 것을 확인하였다.
In vivo 실험 결과 타임의 국소 도포가 아토피 증상을 개선하는 것을 확인하였다. 타임은 피부염의 중증도, 경피수분손실량, 홍반 지수의 감소를 초래하였다. 게다가, 타임에 의해 긁는 횟수가 유의적으로 감소되었다. 이는 혈청 IgE 및 히스타민 분비의 억제에 의해 영향을 받았다. 조직학적 분석 결과는 타임이 비만 세포의 침윤 뿐만 아니라 표피 두께의 감소에도 관여한다는 것을 나타내었다. 반면에 DFE에 의해 악화된 피부치밀도와 수분량이 타임의 국소 도포에 의해 회복되었다.
종합적으로 본 연구는 아토피 피부염이 유도된 in vitro 및 in vivo 모델에서 타임의 아토피 피부염 개선 효과를 검증하였다. 이러한 결과는 타임이 히스타민 분비를 조절하여 가려움증을 완화시킴으로써 아토피 피부염 증상 개선에 효과가 있음을 나타내므로 화장품 및 의약품 산업에서 천연물 유래의 소재로서 활용될 수 있음을 시사한다.

목차 (Table of Contents)

• Ⅰ. Introduction 1
• 1. Thymus vulgaris L. 1
• 2. Atopic dermatitis 8
• 2.1. Epidemiology of atopic dermatitis 9
• 2.2. Causes of atopic dermatitis 9
• 2.3. Diagnosis of atopic dermatitis based on clinical features 10
• 2.4. Pathogenesis of atopic dermatitis 11
• 2.4.1. Imbalance of Th1/Th2 immune response 11
• 2.4.2. Skin barrier dysfunction 12
• 2.5. Itching in atopic dermatitis 15
• 2.5.1. Role of histamine in itching 15
• 2.5.2. Psychological stress and itching 21
• 3. Mechanism of atopic dermatitis 24
• 3.1. Mast cells activation 24
• 3.2. Inflammatory response in keratinocytes 28
• 4. Therapeutic agents of atopic dermatitis and allergic response 31
• 4.1. Ketotifen fumarate 33
• 4.2. Tacrolimus 34
• 4.3. Natural products-derived therapeurics 36
• 5. Purpose of this study 40
• Ⅱ. Materials and Methods 42
• 1. Materials 42
• 2. Sample preparation 43
• 3. HPLC analysis 45
• 4. In vitro study 46
• 4.1. Cell culture 46
• 4.2. Sample treatment 46
• 4.3. Measurement of cell viability 47
• 4.4. Measurement of NO production 47
• 4.5. β-hexosaminidase release assay 48
• 4.6. Histamine release assay 49
• 4.7. Measurement of intracellular calcium 49
• 4.8. Enzyme-linked immunosorbent assay (ELISA) 49
• 4.9. Reverse transcription-polymerase chain reaction (RT-PCR) 50
• 4.10. Western blot analysis 52
• 5. In vivo study 53
• 5.1. Experimental animals 53
• 5.2. Induction of AD-like skin lesion and topical application 53
• 5.3. Evaluation of AD-like skin symptoms 54
• 5.4. Measurement of physiological skin alteration 56
• 5.4.1. Video scope 56
• 5.4.2. High resolution ultrasound 56
• 5.4.3. Hydration 56
• 5.4.4. TEWL 57
• 5.4.5. Erythema 57
• 5.5. Evaluation of scratching behavior 57
• 5.6. Measurement of serum IgE level 58
• 5.7. Histopathological analysis 58
• 6. Statistical analysis 59
• Ⅲ. Results 60
• 1. The identification of active components from T. vulgaris 60
• 2. Effects of T. vulgaris on atopic dermatitis and histamine response: in vitro 63
• 2.1. Effects of T. vulgaris on atopic dermatitis inflammation in Raw 264.7 cells 63
• 2.1.1. Cell viability 63
• 2.1.2. NO production 64
• 2.2. Effects of T. vulgaris on histamine related allergic response in RBL-2H3 cells 66
• 2.2.1. Cell viability 66
• 2.2.2. β-hexosaminidase release 67
• 2.2.3. Histamine release 68
• 2.2.4. Intracellular calcium levels 70
• 2.2.5. Signaling pathways in histamine release 72
• 2.2.5.1. CRHR1 and CRHR2 expression 72
• 2.2.5.2. phosphorylation of PLCγ 74
• 2.2.5.3. phosphorylation of IP3R 76
• 2.2.5.4. calcium channel protein expressions 78
• 2.3. Effect of T. vulgaris on atopic dermatitis inflammation in HaCaT cells 80
• 2.3.1. Cell viability 80
• 2.3.2. Chemokines production 81
• 2.3.3. mRNA expression of pro-inflammatory cytokines and chemokines 83
• 2.3.4. Signaling pathways in inflammatory response 85
• 2.3.4.1. MAPKs activation 85
• 2.3.4.2. NFκB and STAT1 activation 87
• 3. Effects of T. vulgaris on atopic dermatitis and histamine response: in vivo 90
• 3.1. Morphological characteristics 90
• 3.1.1. Body weight 90
• 3.1.2. Spleen weight 92
• 3.1.3. Skin morphology 94
• 3.2. Physiological characteristics 97
• 3.2.1. Skin density 97
• 3.2.2. Skin hydration 99
• 3.2.3. TEWL 100
• 3.2.4. Erythema index 101
• 3.3. Itching related characteristics 103
• 3.3.1. Scratching behavior 103
• 3.3.2. Serum IgE level 105
• 3.3.3. β-hexosaminidase and histamine release 107
• 3.4. Signaling pathways related atopic dermatitis symptoms 109
• 3.4.1. CRHR1 and CRHR2 expression 109
• 3.4.2. MAPKs activation 111
• 3.4.3. IκBα and NFκB activation 113
• 3.4.4. Filaggrin expression 115
• 3.5. Histological characteristics 117
• 3.5.1. Epidermal thickness 117
• 3.5.2. Accumulation of mast cells 119
• Ⅳ. Discussion 121
• Ⅴ. Conclusion 134
• Ⅵ. References 135
• 국 문 초 록 146