Effects of apple mint extract on signal transduction of platelet aggregation = 애플 민트 추출물의 항혈소판 기전에 관한 연구

국문 초록 (Abstract)

애플민트 추출물 (APE)이 콜라겐으로 유도한 혈소판 응집반응 및 그 신호전달 기전에 미치는 영향력을 검토하였다. APE는 콜라겐으로 유도한 사람 혈소판 응집을 억제시켰으며, 고성능액체크로마토그래피에서 APE는 rosmarinic acid (RA)를 가장 많이 포함하고 있는 것으로 나타났다. APE와 RA는 혈소판 응집을 강하게 촉진시키는 물질인 thromboxane A2 (TXA2) 생성을 억제시켰으며, 이러한 결과는 APE와 RA가 TXA2 생성을 유도하는 TXA2 synthase (TXAS) activity를 억제시킨 결과임을 확인하였다. 또한, APE와 RA는 Fura-2 이 loading된 사람 혈소판에서 콜라겐으로 유도된 세포질내 칼슘 동원을 농도 의존적으로 감소시켰으며, 이는 phospholipase C (PLC)γ2의 인산화 억제에 의한 결과로 나타났다. 반면, APE는 혈소판 응집을 억제시키는 물질인 cyclic adenosine monophosphate (cAMP) 농도를 증가시켰으며, 이는 cAMP 생성을 촉진하는 adenylyl cyclase의 활성을 유도시킨 결과로 확인되었다. 또한, APE는 혈소판 응집을 억제시키는 물질인 vasodilator-stimulated phosphoprotein (VASP)의 Ser157 부위 인산화를 촉진시켰으며, 이는 cAMP-의존성 단백 인산화 효소 억제제인 Rp-8-Br-cAMPs를 처리하였을 때 억제되었다. 이러한 결과는 APE에 의해 상승된 cAMP 농도가 VASP의 Ser157 부위의 인산화 촉진에 기여했음을 의미한다. RA는 cyclase의 활성화 없이 cAMP와 cyclic guanosine monophosphate (cGMP)의 농도를 증가시켰다. RA는 VASP Ser157 부위와 Ser239 부위의 인산화를 촉진시켰으며, 이는 Rp-8-Br-cAMPs 및 cGMP-의존성 단백 인산화 효소 억제제인 Rp-8-Br-PET-cGMP를 처리하였을 때 억제되었다. 이러한 결과는 RA에 의해 상승된 cAMP 및 cGMP의 농도가 VASP Ser157 및 Ser239 부위의 인산화 촉진에 기여했음을 의미한다. 또한, 동물 실험에서 APE을 2주간 위내 투여하였을 때, 고지방식이로 유도된 과도한 혈소판 응집반응을 억제시켰다. 콜라겐과 ADP를 처리하여 급성 폐색전증을 유도한 마우스에서 APE와 RA의 섭취가 폐사율을 감소시켰으며, 이는 폐 조직 내 미세 혈전 생성 억제 및 순환 혈소판 수 증가로 증명되었다. 결론적으로, APE와 RA는 사람 혈소판 및 동물실험에서 항혈전 효과를 나타냄을 확인하였다. 본 연구 결과를 통하여 APE와 RA는 혈전성 질환의 새로운 치료제로 사용될 수 있음을 제시하는 바이다.

다국어 초록 (Multilingual Abstract)

Apple mint (Mentha suaveolens) has been reported to have many biological activities, such as anti-inflammation and anti-oxidant properties. However, the effects of apple mint extract (APE) on human platelet aggregation stimulated by collagen and the signal transduction of platelet aggregation remain obscure. In the present study, I investigated the effect of APE on platelet aggregation. APE inhibited platelet aggregation induced by collagen. High performance liquid chromatography (HPLC) analysis of APE revealed that APE contained rosmarinic acid (RA). APE and RA down-regulated thromboxane A2 (TXA2) production by blocking TXA2 synthase activity, but not cyclooxygenase-1 (COX-1). APE and RA also reduced intracellular Ca2+ mobilization ([Ca2+]i) and the phosphorylation of phospholipase C (PLC) γ2. On the other hand, APE increased cyclic adenosine monophosphate (cAMP) level via activation of adenylyl cyclase but not cyclic guanosine monophosphate (cGMP). APE also phosphorylated vasodilator-stimulated phosphoprotein (VASP) Ser157 which was inhibited by Rp-8-Br-cAMPs, cAMP-dependent protein kinase inhibitor, indicating that APE-elevated cAMP contributed to the phosphorylation of VASP Ser157. In addition, RA elevated both cAMP and cGMP levels without activation of cyclases. RA phosphorylated VASP Ser157 and Ser239 which are suppressed by Rp-8-Br-cAMPs and Rp-8-Br-PET-cGMPs (cGMP-dependent protein kinase inhibitor), indicating that RA-increased both cAMP and cGMP contributed to the phosphorylation of VASP Ser157 and Ser239, respectivley. Moreover, After two weeks of APE treatment (500 mg/kg and 1000 mg/kg), the platelet aggregation rate in the treated groups was significantly less than that in the high fat diet (HFD)-fed control group. Also, APE and RA were effective in reducing the mortality of collagen plus ADP-induced acute pulmonary thromboembolism in mice. This study demonstrates that APE and RA have anti-platelet effects in vitro and in vivo. Therefore, I suggest APE and RA may have a high therapeutic potential for prevention of platelet-derived vascular diseases.

목차 (Table of Contents)

• Ⅰ. INTRODUCTION 1
• Ⅱ. PURPOSE 5
• Ⅲ. MATERIALS AND METHODS 5
• A. MATERIALS 6
• B. METHODS 8
• Ⅳ. RESULTS 18
• A. The mechanism of APE and RA on human platelet aggregation induced by collagen 18
• 1. Effects of APE on platelet aggregation and TXA2 production 18
• 2. Effects of APE on COX-1 and TXAS activities 22
• 3. Effects of APE on collagen-stimulated intracellular Ca2+ mobilization 25
• 4. Effects of APE on phosphorylation of PLCγ2 and Syk 27
• 5. Effects of APE on cyclic nucleotides 30
• 6. Effects of APE on adenylyl and guanylyl cyclases 33
• 7. Effects of APE on VASP phosphorylation 37
• 8. Analysis of RA in APE with HPLC 40
• 9. Effects of RA on platelet aggregation and TXA2 production 43
• 10. Effects of RA on COX-1 and TXAS activities 46
• 11. Effects of RA on collagen-stimulated intracellular Ca2+ mobilization 49
• 12. Effects of APE and RA on phosphorylation of PLCγ2 and Syk 51
• 13. Effects of RA on cyclic nucleotides 54
• 14. Effects of RA on adenylyl and guanylyl cyclases 57
• 15. Effects of RA on VASP phosphorylation 61
• 16. Effects of APE and RA on cell viability 64
• B. The effects of anti-vascular disorders and anti-thrombus of APE on collagen induced platelets in vivo 66
• 17. Effects of APE on high fat diet induced obese rats 66
• 18. Effects of APE on serum lipoprotein levels and hepatotoxicity 68
• 19. Effects of APE on platelet aggregation and TXA2 production in HFD-fed obese rats 71
• 20. Effects of APE on [Ca2+]i in HFD-fed obese rats 74
• C. Effects of APE and RA on mouse pulmonary thromboembolism in vivo 76
• 21. Effects of APE and RA on mouse pulmonary thromboembolism 76
• 22. Effects of APE on pulmonary microvessels 78
• 23. Effects of RA on pulmonary microvessels 80
• 24. Effects of APE and RA on platelet counts after pulmonary thromboembolism 82
• Ⅴ. DISCUSSION 84
• Ⅵ. CONCLUSION 92
• Ⅶ. REFERENCES 96
• SUMMARY 111
• 국문요약 113
• ACKNOWLEDGEMENT 115