Antipruritic effects of peimine through modulation of pruritus-related receptor

다국어 초록 (Multilingual Abstract)

Peimine, derived from the Fritillaria genus, has long been recognized for its anti- inflammatory properties. In the present study, we investigated the potential of peimine as an antipruritic and anti-inflammatory agent using a combination of in vitro, ex vivo, and in vivo experimental approaches. In vitro calcium imaging assays demonstrated that peimine inhibited key pruritus related receptors, including TRPM3, MRGPRX2, and MRGPRB2, while exerting minimal or no effects on TRPA1, TRPV1, and TRPV4. These findings indicate a degree of selectivity toward itch-related signaling pathways. Ex vivo experiments using dorsal root ganglion neurons further confirmed that peimine significantly suppressed TRPM3 mediated calcium responses. In addition, β-hexosaminidase release assays showed that peimine inhibited MRGPRB2 mediated degranulation in peritoneal mast cells and MRGPRX2 mediated degranulation in human mast cells HMC 1.2. Consistent with these cellular effects, in vivo models of acute itch revealed that peimine markedly reduced scratching behavior induced by pregnenolone sulfate, a TRPM3 agonist, as well as compound 48/80, an MRGPRB2 agonist. Furthermore, peimine attenuated inflammatory swelling in a compound 48/80 induced paw edema model, supporting its anti-inflammatory activity. Collectively, these results identify peimine as a promising therapeutic candidate for the management of pruritus. Key words: Peimine, pruritus, calcium imaging, TRP, MRGPRX2, MrgprB2, compound 48/80, mast cells, itch

목차 (Table of Contents)

• List of Figures viii
• List of Abbreviations x
• Abstract 1
• 1. Introduction 2
• 1.1 Pruritus and its clinical significance 3
• 1.2 Neural and molecular mechanisms of itch 4
• 1.3 Histaminergic and non-histaminergic itch pathways 4
• 1.4 Transient receptor potential (TRP) channels in itch signaling 5
• 1.5 TRPM3 and itch signaling. 6
• 1.7 TRPV1 and TRPV4 in itch signaling 7
• 1.8 G-protein–coupled receptors (GPCRs) in itch signaling 7
• 1.9 MRGPRX2 and Mrgprb2 in mast cell–mediated itch 10
• 1.10 Mast cell degranulation and MRGPRX2/Mrgprb2 signaling 10
• 1.11 Edema as a downstream consequence of MRGPRX2/ Mrgprb2 activation
• 13
• 1.12 Peimine: chemical and pharmacological background 13
• 1.13 Anti-inflammatory properties of peimine and Its Potential Role in Itch-
• Related Signaling 15
• 1.14 Rationale and hypothesis 16
• 2. Materials and Methods 17
• 2.1 Reagents 17
• 2.2 Animals 17
• 2.3 Cell seeding and Transfection 18
• 2.4 Genes 18
• 2.5 Peritoneal Mast cell (PMC) primary culture 18
• 2.6 Dorsal Root Ganglia (DRG) primary culture 19
• 2.7 Calcium Imaging. 20
• 2.8 β-hexosaminidase release assay 21
• 2.9 In vivo scratching behavior test 22
• 2.10 Mouse paw edema model 23
• 2.11 MTT Cell Viability Assay 24
• 2.12 Statistical Analysis 25
• 3. Results 26
• 3.1 Effect of Peimine on TRPM3-mediated calcium responses. 26
• 3.2 Quantification of TRPM3-mediated calcium influx following Peimine
• treatment. 28
• 3.3 IC50 determination of Peimine on TRPM3 mediated calcium response 30
• 3.5 Quantification of MRGPRB2-mediated calcium influx following peimine
• treatment. 33
• 3.6 IC50 determination of peimine on Mrgprb2 mediated calcium response .. 35
• 3.7 Effect of Peimine on MRGPRX2-mediated calcium responses 36
• 3.8 Quantification of MRGPRX2-mediated calcium responses following
• peimine treatment 38
• 3.9 IC₅₀ determination of peimine on MRGPRX2-mediated calcium response.
• 40
• 3.10 Peimine showed no measurable effect on TRPV1, TRPA1, or TRPV4
• activity 41
• 3.11 Effect of peimine on TRPM3-mediated calcium responses in DRG
• neurons. 45
• 3.12 Peimine inhibits mast cell degranulation in peritoneal and human mast
• cells 46
• 48
• 3.13 Effect of peimine on PS-induced acute itch behavior (TRPM3-mediated)
• 49
• 3.14 Effect of peimine on c48/80-induced acute itch behavior (MrgprB2-
• mediated). 51
• 3.15 Effect of Peimine on C48/80-induced paw edema via MrgprB2 53
• 3.16 Peimine significantly decreases paw thickness induced by C48/80 56
• 3.17 Peimine significantly decreases evans blue dye extravasation 57
• 3.18 Peimine does not affect cell viability 59
• 4. Discussion 60
• 5. Conclusion 64
• References 65
• Acknowledgement 70
• viii