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Molecular Mechanism of Nuciferine's Atopic Dermatitis Improvement Effect

Nuciferine의 아토피 피부염 개선효과에 대한 분자기전

  • 신현희 (Hyeon Hui Shin, 강릉원주대학교 일반대학원)

초록/요약 도움말

Atopic dermatitis (AD) is a chronic and recurrent inflammatory skin disease caused by an irregularity in the body's immune system. The prevalence of AD is concentrated in infancy or childhood, at more than 28%, and recently, adult AD has also become a social problem. It is usually characterized by severe itching, dry skin, and eczema. It is believed to be caused by a combination of genetic and immunologic factors, as well as abnormalities in skin barrier function. Nelumbo nucifera is a plant with several physiological properties, including anti-inflammatory and antioxidant properties. Compounds from this plant are also known to have positive effects on the treatment of atopic dermatitis, including anti-inflammatory and antioxidant properties, but no studies have been reported. Therefore, we conducted an anti-atopic screening of three compounds from this plant and selected Nuciferine, which is highly effective, for the study. The mechanisms of skin barrier function improvement and immune function improvement have not been clearly reported in atopic dermatitis studies. Therefore, we confirmed the allergic inflammatory response through IgE treatment of RBL-2H3 cells, which are like mast cells, in vitro and confirmed the effect of Nuciferine on molecular mechanisms of skin barrier function and immune regulation through HaCaT cells, which are keratinocytes. We also confirmed the in vivo effect of Nuciferine by conducting mouse experiments on DNCB-induced atopic-like dermatitis. In the in vitro results of this study, Nuciferine inhibited IL-4 production in RBL-2H3 cells induced by PMA and Inomycin. Nuciferine reduced the release of β-hexosaminidase, a marker for assessing degranulation of DNP-IgE and DNP-BSA-induced RBL-2H3 cells. Nuciferine also inhibited the phosphorylation of the signaling molecules PLCγ1, Lyn, and Syk in RBL-2H3 cells, and suppressed the production of IL-4, IL-13, and TNF-α by inhibiting the phosphorylation of the Mitogen-Activated Protein Kinase (MAPK) proteins extracellular signal-regulated kinase (ERK), c-Jun N-terminal protein kinase (JNK), and p38. Nuciferine upregulated the skin barrier factors Filaggrin (FLG), Involucrin (IVL) and Loricrin (LOR) by inhibiting the signaling of The Janus Kinases/ signal transducers and activators of transcription (JAK/STAT) pathway in HaCaT cells and suppressed the mRNA expression of IL-1β, CCL2, CCL5 and CCL26. In vivo results showed that in DNCB (2,4-Dinitrochlorobenzene)-induced atopic dermatitis mouse model (SKH-1), it improved morphology such as erythema and hemorrhage, decreased TEWL (Transepidermal Water Loss), and increased skin hydration. In addition, Hematoxylin & Eosin (H&E) tissue staining showed decreased dorsal skin thickness in mice, and Toluidine blue tissue staining showed decreased mast cell number. In the blood, IL-4 and IgE, which are major atopic triggers, were reduced. Therefore, these results suggest that Nuciferine may help improve atopic dermatitis by improving skin barrier function, suppressing IgE-mediated responses and immune responses caused by inflammatory cytokines.

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목차 도움말

Abstract 10
Ⅰ. Introduction 15

Ⅱ. Materials and Methods 18
2.1. Materials 18
2.2. Cell lines and cell culture 18
2.3. Cell proliferation assay 19
2.4. β-Hexosaminidase release activity 19
2.5. Real-time PCR (RT-PCR) 20
2.6. Protein analysis by Western blot 21
2.7. HET-CAM assay 21
2.8. Animal studies 22
2.9. DNCB induction and drug treatment 22
2.10. Clinical Skin Score 23
2.11. TEWL and Hydration Measurements 23
2.12. H&E (Hematoxylin & Eosin) staining 23
2.13. Toluidine blue staining 24
2.14. Measurement of total serum IgE and IL-4 levels by ELISA 24
2.15. Statistical analysis 24

Ⅲ. Results 26
3.1. Influence of Nelumbo nucifera Compounds on the Cell Viability of HaCaT, RBL-2H3 Cells 26
3.2. Screening of Nelumbo nucifera Compounds in RBL-2H3 Cells 29
3.3. Inhibitory effects of Nuciferine on IL-4 expression and degranulation in RBL-2H3 cells 32
3.4. Effect of Nuciferine on suppression of activation signal molecule and MAPK protein expression in IgE-induced RBL-2H3 Cells 33
3.5. The impact of Nuciferine on cytokine mRNA expression in IgE-induced RBL-2H3 cells 40
3.6. The impact of Nuciferine on JAK/STAT signaling in IL-4-induced HaCaT cells 43
3.7. Effect of Nuciferine on skin barrier in IL-4-induced HaCaT cells 44
3.8. Effect of Nuciferine on the cytokine in IL-4-induced HaCaT cells 51
3.9. Effect of Nuciferine on the chemokine in IL-4-induced HaCaT cells 52
3.10. Photographs from the HET-CAM test of Nuciferine 57
3.11. Observation of SKH-1 mouse skin induced by DNCB 59
3.12. The effect of Nuciferine on skin moisture content in DNCB-induced SKH-1 Mouse skin 62
3.13. The effect of Nuciferine on immune-related organs in a DNCB-induced Atopic Dermatitis-like SKH-1 mouse model 65
3.14. Histopathological effects of Nuciferine in Atopic Dermatitis-like mouse model induced by DNCB 70
3.15. The Effect of Nuciferine on IgE and IL-4 levels in the serum of DNCB-induced AD model mouse 74
Ⅳ. Conclusion 78

Ⅴ. References 83
 
List of Tables
Table 1. Primer sequences used in real-time PCR 21

List of Figures
Figure 1. Cell viability of Nelumbo nucifera compound in HaCaT and RBL-2H3 cells 27
Figure 2. Screening of Nelumbo nucifera compounds for RBL-2H3 cell selection 30
Figure 3. Effects of Nuciferine on IL-4 mRNA expression and β-hexosaminidase release in RBL-2H3 Cclls 33
Figure 4. Inhibitory effect of Nuciferine on IgE activation signaling molecule and MAPK protein expression in RBL-2H3 cells 36
Figure 5. Effect of Nuciferine on inhibition of cytokine production in IgE-induced RBL-2H3 cells 41
Figure 6. The influence of Nuciferine on JAK/STAT signaling in IL-4 induced HaCaT Cells 44
Figure 7. Nuciferine's Effect on skin barrier in IL-4 Induced HaCaT Cells 48
Figure 8. Inhibitory effects of Nuciferine on chemokine expression in HaCaT Cells 52
Figure 9. Inhibitory effects of Nuciferine on cytokine expression in HaCaT Cells 55
Figure 10. Toxicity Assessment of Nuciferine: HET-CAM Test 58
Figure 11. Improvement effects of Nuciferine on DNCB-induced skin conditions in mice 60
Figure 12. Effect of Nuciferine on improving moisture content of AD mouse skin 63
Figure 13. Effects of Nuciferine on immune-related organs in an atopic dermatitis SKH-1 mouse model 66
Figure 14. Histopathological effects of Nuciferine in an AD-like mouse model 71
Figure 15. Effects of Nuciferine on serum IgE and IL-4 levels in an atopic dermatitis mouse model 75
Figure 16. Atopic Dermatitis pathway of Nuciferine 77

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