(A) study on the analgesic effect of carvacrol, a TRPM7 inhibitor, after spinal cord injury

국문 초록 (Abstract)

신경병증성 통증은 척수손상 후 발생하는 주요 사건 중 하나로, 척수손상 환자의 약 80% 이상 발생한다고 보고된다. 신경병증성 통증에 관한 많은 연구가 진행되었음에도 불구하고, 척수손상 후 신경병증성 통증을 완화하기 위한 효과적인 치료 약물은 미비한 실정이다. 또한 성상세포 같은 척수 신경교세포의 활성 상태는 척수 L4-L5 dorsal horn Lamina I-II 신경세포의 과활성과 관련되어 척수손상 후 신경병성 통증을 유발한다. 그러나 활성화된 신경교세포와 신경병성 통증의 관계를 뒷받침하는 세포 및 분자 메커니즘은 명확히 밝혀진 바가 없다. 그래서 본 연구에서는 비 선택적 양이온 채널로 알려진 TRPM7(transient receptor melastatin 7)이 척수 손상 후 신경병증성 통증을 매개하고, TRPM7을 타겟으로 하는 통증 조절 메커니즘 및 통증 완화 효과를 조사하였다. 척수손상 후 4주까지 신경병증성 통증이 유발되었고, 특히, TRPM7이 척수 내 L4-L5 dorsal horn Lamina I-II의 성상세포에서 발현되었다. TRPM7 억제제인 Carvacrol 처리 시, 기계적 이질통과 열적 통각 과민 반응이 완화되었다. Carvacrol의 처리는 척수손상 후 성상세포에서 mTOR (mammalian target of rapamycin) 신호전달의 활성화를 억제하였고, mTOR 억제제인 Rapamycin를 처리했을 때, 신경병증성 통증을 상당히 억제된 것을 확인하였다. Carvacrol과 Rapamycin의 처리는 성상세포의 활성화 및 성상세포에서 활성화되는 것으로 알려진 JNK와 c-Jun을 억제하였다. 이를 통해 TRPM7-mTOR 신호전달을 억제함으로써 성상세포의 활성화가 감소되었음을 확인하였다. 마지막으로, TNF-α, IL-6, IL-β, CCL-2, CCL-3, CCL-4, CCL-20, CXCL-1 및 MMP-9과 같은 통증 관련 사이토카인, 케모카인, 기질금속 단백질 분해효소인자의 발현 또한 유의하게 억제됨을 확인하였다. 이러한 결과는 척수손상 후 성상세포 활성화에 TRPM7-mTOR 신호전달의 중요한 역할을 하며, TRPM7이 척수손상 후 만성 신경병증성 통증의 새로운 치료 표적이 될 수 있음을 시사한다.

다국어 초록 (Multilingual Abstract)

In this study, we investigated whether transient receptor melastatin 7 (TRPM7), known as a non-selective cation channel, inhibits neuropathic pain after spinal cord injury (SCI) and how TRPM7 regulates neuropathic pain. Neuropathic pain was developed 4 weeks after moderate contusive SCI and TRPM7 was markedly upregulated in astrocytes in the lamina I and II of L4-L5 dorsal horn. In addition, both mechanical allodynia and thermal hyperalgesia were significantly alleviated by a TRPM7 inhibitor, carvacrol. In particular, carvacrol treatment inhibited mammalian target of rapamycin (mTOR) signaling, which was activated in astrocytes. When rats were treated with rapamycin, an inhibitor of mTOR signaling, neuropathic pain was significantly inhibited. Furthermore, blocking TRPM7 and mTOR signaling by carvacrol and rapamycin inhibited astrocyte activation in lamina I and II of dorsal spinal cord and reduced the level of p-JNK and p-c-Jun, which are known to be activated in astrocyte. Finally, inhibiting TRPM7/mTOR signaling also significantly downregulated the production of pain-related factors such as TNF-α, IL-6, IL-1β, CCL-2, CCL-3, CCL-4, CCL-20, CXCL-1, and MMP-9, which are known to be involved in the induction and/or maintenance of neuropathic pain after SCI. These results suggest an important role of TRPM7-mediated mTOR signaling in astrocytes activation and thereby induction and/or maintenance of chronic neuropathic pain after SCI.

목차 (Table of Contents)

• ABSTRACTS ⅶ
• INTRODUCTION 1
• 1. Neuropathic pain 1
• 1) Type of pain 1
• 2) Characteristics of neuropathic pain 4
• 3) Neuropathic pain after spinal cord injury (SCI) 6
• (1) SCI 6
• (2) Neuropathic pain in SCI patients 6
• (3) Neuropathic pain pathology after SCI 8
• 2. Association between glial cells and neuropathic pain 10
• 1) Glial cells and neuropathic pain 10
• 2) Astrocyte in neuropathic pain 10
• 3. Transient receptor potential melastatin 7 (TRPM7) 15
• 1) TRPM7 15
• (1) Role of TRPM7 15
• (2) Distribution of TRPM7 16
• (3) Pharmacological inhibitor of TRPM7 18
• 2) Association between TRPM7 and neuropathic pain 21
• 3) Association between TRPM7 and astrocytes 21
• 4. Mammalian target of rapamycin (mTOR) 22
• 1) mTOR 22
• (1) Role of mTOR 22
• (2) Distribution of mTOR 22
• (3) Pharmacological inhibitor of mTOR signaling 25
• 2) Association between mTOR and neuropathic pain 27
• 3) Association between mTOR and astrocytes 27
• 5. Association of TRPM7 with mTOR 28
• 6. Summary and Hypothesis 30
• MATERIAL AND METHOD 32
• 1. Spinal cord injury 32
• 2. Behavioral tests 34
• 3. Drug administration 39
• 4. Tissue preparation 39
• 5. Immunohistochemistry 39
• 6. RNA isolation and RT-PCR 40
• 7. Western blot 41
• 8. Statistical analysis 42
• RESULT 43
• 1. TRPM7 expression is increased in astrocytes after SCI 43
• 2. TRPM7 inhibition by carvacrol alleviates mechanical allodynia and thermal hyperalgesia after SCI 45
• 3. mTOR signaling pathway is activated in astrocytes after SCI 47
• 4. Rapamycin, an inhibitor of the mTOR signaling pathway, alleviates neuropathic pain
• induced by SCI 51
• 5. TRPM7 inhibitor carvacrol blocks mTOR signaling pathway and astrocyte activation in
• spinal cord dorsal horn after SCI 53
• 6. Pro-inflammatory cytokines, chemokines, and MMP-9 secreted by reactive astrocytes are
• reduced when TRPM7 and mTOR are inhibited 55
• DISCUSSION 58
• REFERENCE 61
• 국문 초록 72