Anti-pruritic potential of luteolin through regulation of TRPM3 and MRGPRX2

다국어 초록 (Multilingual Abstract)

Luteolin (LUT), a natural flavonoid in many plants, exhibits anti- inflammatory, antioxidant, and neuroprotective effects. It reduces itch by inhibiting mast cell activation and MRGPRX2 signaling, thereby limiting the release of histamine and other itch mediators. Using calcium imaging, LUT was found to significantly inhibit TRPM3 channel activity in HEK293T cells. Similar inhibitory effects were also confirmed in functional assays targeting MRGPRX2 and MrgprB2 receptors. Furthermore, behavioral studies in mice further evaluated LUT's anti-pruritic effects. Pregnenolone sulfate (PS)-induced mice exhibited pronounced scratching behavior, and LUT treatment significantly reduced the frequency of scratching. Furthermore, in calcium imaging of murine dorsal root ganglia (DRG) neurons revealed that LUT significantly inhibits TRPM3 channel function, attenuating agonist-induced calcium responses. In addition, LUT treatment downregulated β-hexosaminidase release in peritoneal mast cells (PMCs), suggesting that it may inhibit mast cell degranulation. These results suggest that LUT may exert its anti-pruritic and anti- inflammatory effects by regulating targets strongly associated with itch signaling, such as TRPM3 and MRGPRX2/MrgprB2, providing experimental evidence for its potential application in allergic or inflammatory skin diseases. Keywords: Luteolin, TRPM3, MRGPRX2, MrgprB2, calcium imaging, itch, PMC, DRG

목차 (Table of Contents)

• List of figures iv
• List of abbreviations vii
• Abstract 1
• 1. Introduction 3
• 1.1. Itch 3
• 1.2. Ion channels in pruritus 4
• 1.3. Transient receptor potential (TRP) channels 5
• 1.3.1. TRPM3 6
• 1.3.2. TRPM8 7
• 1.3.3. TRPV4 8
• 1.3.4. TRPV1 9
• 1.4. GPCRs in pruritus 11
• 1.5. Mas-related G protein-coupled receptor member (MRGPR) ..
• 12
• 1.5.1. MRGPRX1 14
• 1.5.2. MRGPRX2 15
• 1.5.3. MrgprB2 16
• 1.6. Mast cell 18
• 1.7. Dorsal Root Ganglion (DRG) 19
• 1.8. Luteolin (LUT) 21
• 1.9. Aim of the study 23
• 2. Material and Methods 24
• 2.1. Reagents 24
• 2.2. Genes 24
• 2.3. Animals and scratching behavior tests 25
• 2.4. Acute pruritus mouse models 25
• 2.5. Cell culture and Transfection 26
• 2.6. Mouse dorsal root ganglion (DRG) primary culture 26
• 2.7. Mouse peritoneal mast cells (PMCs) primary culture 27
• 2.8. β-hexosaminidase release assay 28
• 2.9. Induction of AD-like condition by MC903 30
• 2.10. H&E staining 30
• 2.11. Statistical analysis 31
• 3. Results 32
• 3.1. Luteolin suppresses TRPM3 activation 32
• 3.2. Luteolin inhibits MRGPRX2-mediated activity 37
• 3.3. Luteolin blocks MrgprB2-mediated response 42
• 3.4. Luteolin suppresses PS-induced TRPM3 activation in DRG
• neurons 48
• 3.5. Luteolin suppresses C48/80-induced degranulation in mouse
• PMCs 51
• 3.6. Luteolin reduces PS-induced acute itch in mice 53
• 3.7. Luteolin blocks C48/80-induced acute itch in mice 55
• 3.8. Luteolin improves MC903-induced AD-like skin inflammation
• 57
• 4. Discussion 64
• 5. Conclusion 68
• References 69
• ACKNOWLEDGEMENTS 75
• 감사의 글 79
• 致谢 84