베타-이오논과 알로인이 마우스 삼차신경 척수감각핵 미측소핵 아교질 신경세포에서 구강악안면 통증 전달 조절에 미치는 영향 = Effect of beta-ionone and aloin on the modulation of orofacial nociceptive transmission in substantia gelatinosa neurons of the trigeminal subnucleus caudalis in mice

다국어 초록 (Multilingual Abstract)

The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) plays a central role in integrating and modulating nociceptive signals arising from the orofacial region. This area contains dense networks of excitatory and inhibitory interneurons that shape pain transmission through a delicate balance of synaptic signaling. Disruption of this balance contributes to abnormal pain perception and the development of chronic orofacial pain. Natural bioactive compounds with neuromodulatory potential have recently gained attention as promising candidates for novel analgesic development. Among them, beta-ionone (a monoterpenoid compound derived from plant carotenoids) and aloin (an anthraquinone glycoside isolated from Aloe vera) have been recognized for diverse pharmacological activities including anti-inflammatory and antioxidant effects. However, their electrophysiological actions on central nociceptive circuits, particularly on the SG neurons of the Vc, have not been previously elucidated. This thesis constitutes the first study to provide electrophysiological evidence regarding the effects of beta-ionone and aloin on the SG neurons, aiming to clarify their cellular mechanisms of pain modulation and to explore their potential as natural analgesic candidates.
In the first part of the study, whole-cell patch-clamp recordings were performed on the SG neurons from juvenile mouse brainstem slices to investigate the membrane actions of beta-ionone. Under a high-chloride pipette solution, beta-ionone induced consistent inward currents that persisted in the presence of tetrodotoxin, 6-cyano-7-nitro-quinoxaline-2,3-dione, and D-2-Amino-5-phosphonovaleric acid, indicating that its effects were independent of action potential generation and glutamatergic synaptic transmission. The inward currents were, however, significantly attenuated by strychnine and picrotoxin, suggesting that beta-ionone exerts gamma-aminobutyric acid (GABA)- and glycine-mimetic actions. Moreover, beta-ionone enhanced glycine- and GABA-induced currents while suppressing spontaneous neuronal firing, demonstrating its inhibitory modulation on the SG neuronal excitability. In behavioral experiments using the formalin-induced orofacial pain model, beta-ionone produced significant, dose-dependent antinociceptive effects in both the early (neurogenic) and late (inflammatory) phases, further supporting its functional inhibitory action in nociceptive processing.
In the second part, the effects of aloin on glutamate receptor-mediated responses were examined using whole-cell patch-clamp electrophysiology combined with calcium imaging. Aloin selectively inhibited N-Methyl-D-aspartic acid (NMDA) receptor-mediated inward currents without affecting alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid - or kainate-induced responses. This NMDA-specific inhibition was accompanied by a marked reduction in NMDA-evoked intracellular Ca²⁺ influx, indicating suppression of calcium-dependent excitotoxic mechanisms. The effects occurred independently of voltage-gated sodium channel activity, confirming that aloin acts directly on receptor-mediated signaling rather than action potential propagation. In addition, aloin significantly decreased the spontaneous firing rate of SG neurons, suggesting its broader role in dampening neuronal hyperexcitability.
Collectively, these findings reveal novel mechanisms by which beta-ionone and aloin modulate nociceptive signaling within the trigeminal system. Beta-ionone primarily enhances inhibitory synaptic activity, whereas aloin selectively attenuates excitatory NMDA receptor-mediated signaling and calcium influx. Together, they provide important insights into the neurophysiological basis of the antinociceptive properties of these natural compounds under pathological conditions. This work highlights their potential to uncover new cellular mechanisms of pain modulation and to serve as natural candidates for developing innovative, mechanism-based analgesic strategies for orofacial pain management.

목차 (Table of Contents)

• I. INTRODUCTION 1
• 1. Orofacial pain 1
• 1.1. Definition and classification of orofacial pain 1
• 1.2. Clinical relevance and impact 1
• 2. Anatomical and functional role of the trigeminal subnucleus caudalis in orofacial pain transmission 2
• 2.1. Overview of the trigeminal nociceptive pathway 2
• 2.2. The trigeminal subnucleus caudalis: Structure and function 3
• 2.3. Ascending and descending pathways 3
• 3. The role of the substantia gelatinosa (SG) neurons in pain modulation 4
• 3.1. Anatomy and function of SG neurons 4
• 3.2. SG neurons in orofacial pain transmission 5
• 3.3. Therapeutic potential targeting the SG neurons 5
• 4. Neurotransmitters involved in orofacial pain processing 6
• 4.1. Glutamate: The principal excitatory neurotransmitter 6
• 4.2. GABA and glycine: major inhibitory neurotransmitters 7
• 5. Natural compounds as potential modulators of orofacial pain 8
• 5.1. Beta-ionone 8
• 5.2. Aloin 10
• 6. Study objectives 12
• 7. Figures 13
• II. MATERIALS AND METHODS 20
• 1. Animal and brain slice preparation 20
• 2. Electrophysiology 21
• 3. Formalin-induced orofacial pain test 22
• 4. Fluorescence Ca2+ imaging 24
• 5. Chemicals 25
• 6. Data and statistical analysis 25
• 7. Figures 26
• III. RESULTS 33
• 1. Effect of beta-ionone on the SG neurons of the Vc 33
• 1.1. The response of SG neurons to beta-ionone exposure exhibited a dependence on concentration 33
• 1.2. Beta-ionone elicited non-desensitizing and repeatable responses in the SG neurons 33
• 1.3. Beta-ionone exerted a direct effect on the SG neurons 34
• 1.4. The response elicited by beta-ionone was not facilitated via the activation of ionotropic glutamate receptors 35
• 1.5. Beta-ionone-induced responses were inhibited by antagonists of GABAA and/or glycine receptors 35
• 1.6. Beta-ionone enhanced the responses induced by GABA and glycine 36
• 1.7. Beta-ionone suppressed the neuronal activity of SG neurons 37
• 1.8. Beta-ionone reduced formalin-induced orofacial nociceptive responses 38
• 2. Effect of aloin on the SG neurons 39
• 2.1. Aloin suppressed the spontaneous firing activity on the SG neurons of the Vc 39
• 2.2. Aloin attenuated the glutamate-induced response in the SG neurons of the Vc 40
• 2.3. Aloin exerted a direct effect on the SG neurons 41
• 2.4. Aloin's modulatory effects on glutamate receptor agonist-induced responses in the SG neurons 42
• 2.5. The impact of aloin on sodium ion influx mediated by NMDA receptors 44
• 2.6. The impact of aloin on calcium ion influx mediated by NMDA receptors 45
• 2.7. The impact of aloin on calcium ion signaling 47
• IV. DISCUSSION 49
• 1. Effect of beta-ionone on the SG neurons 49
• 1.1. Electrophysiological evidence demonstrated the direct effect of beta-ionone on the SG neurons 49
• 1.2. The formalin-induced orofacial pain model demonstrated the antinociceptive properties of beta-ionone 52
• 2. Effect of aloin on the SG neurons 54
• 2.1. Electrophysiological evidence demonstrated the direct effect of aloin on the SG neurons 54
• 2.2. Calcium imaging demonstrated aloin's modulatory effects on NMDAR-mediated calcium signaling 56
• V. FIGURE AND LEGENDS 59
• VI. CONCLUSION 87
• VII. REFERENCES 88
• VIII. 국문초록 101
• IX. ACKNOWLEDGEMENTS 104
• LIST OF PUBLICATION 106