Clinical and Experimental Pediatrics

The Korean Pediatric Society (대한소아청소년과학회)
권호 표지

The neuroprotective effect of mycophenolic acid via anti-apoptosis in perinatal hypoxic-ischemic brain injury

주산기 저산소성 허혈성 뇌손상에서 항세포자멸사를 통한 mycophenolic acid의 신경보호 효과

  • Kim, Ji Young (Department of Pediatrics, School of Medicine, Catholic University of Daegu) ;
  • Yang, Seung Ho (Department of Pediatrics, School of Medicine, Catholic University of Daegu) ;
  • Cha, Sun Hwa (Department of Pediatrics, School of Medicine, Catholic University of Daegu) ;
  • Kim, Ji Yeun (Department of Neurology, School of Medicine, Catholic University of Daegu) ;
  • Jang, Young Chae (Department of Pathology, School of Medicine, Catholic University of Daegu) ;
  • Park, Kwan Kyu (Department of Pathology, School of Medicine, Catholic University of Daegu) ;
  • Kim, Jin Kyung (Department of Pediatrics, School of Medicine, Catholic University of Daegu) ;
  • Chung, Hai Lee (Department of Pediatrics, School of Medicine, Catholic University of Daegu) ;
  • Seo, Eok Su (Department of Opthalmology, College of Medicine, Dongguk University) ;
  • Kim, Woo Taek (Department of Pediatrics, School of Medicine, Catholic University of Daegu)
  • 김지영 (대구가톨릭대학교 의과대학 소아과학교실) ;
  • 양승호 (대구가톨릭대학교 의과대학 소아과학교실) ;
  • 차선화 (대구가톨릭대학교 의과대학 소아과학교실) ;
  • 김지언 (대구가톨릭대학교 의과대학 신경과학교실) ;
  • 장영채 (대구가톨릭대학교 의과대학 병리학과교실) ;
  • 박관규 (대구가톨릭대학교 의과대학 병리학과교실) ;
  • 김진경 (대구가톨릭대학교 의과대학 소아과학교실) ;
  • 정혜리 (대구가톨릭대학교 의과대학 소아과학교실) ;
  • 서억수 (동국대학교 의과대학 안과학교실) ;
  • 김우택 (대구가톨릭대학교 의과대학 소아과학교실)
  • Received : 2007.04.13
  • Accepted : 2007.05.02
  • Published : 2007.07.15
Download PDF
⟨ Previous Next ⟩

Abstract

Purpose : Mycophenolic acid (MPA), the active metabolite of mycophenolate mofetil (MMF), is a potent inhibitor of inosine-monophosphate dehydrogenase (IMPDH), a new immunosuppressive drug used. It was reported that MPA protected neurons after excitotoxic injury, induced apoptosis in microglial cells. However, the effects of MPA on hypoxic-ischemic (HI) brain injury has not been yet evaluated. Therefore, we examined whether MPA could be neuroprotective in perinatal HI brain injury using Rice-Vannucci model (in vivo) and in rat brain cortical cell culture induced by hypoxia (in vitro). Methods : Cortical cells were cultured using a 18-day-pregnant Sprague-Dawley (SD) rats and incubated in 1% $O_2$ incubator for hypoxia. MPA ($10{\mu}g/mL$) before or after a HI insult was treated. Seven-day-old SD rat pups were subjected to left carotid occlusion followed by 2 hours of hypoxic exposure (8% $O_2$). MPA (10 mg/kg) before or after a HI insult were administrated intraperitoneally. Apoptosis was measured using western blot and real-time PCR for Bcl-2, Bax, caspase-3. Results : H&E stain revealed increased brain volume in the MPA-treated group in vivo animal model of neonatal HI brain injury. Western blot and real-time PCR showed the expression of caspase-3 and Bax/Bcl-2 were decreased in the MPA-treated group In in vitro and in vivo model of perinatal HI brain injury, Conclusion : These results may suggest that the administration of MPA before HI insult could significantly protect against perinatal HI brain injury via anti-apoptotic mechanisms, which offers the possibility of MPA application for the treatment of neonatal HI encephalopathy.

목 적 : Mycophenolate mofetil (MMF)의 활성 대사산물인 (MPA는 IMPDH의 잠재적인 반응 억제제로써 새로운 면역치료제로 사용되고 있다. 이러한 MPA는 신경계에서 흥분독성 손상 후 뇌세포를 보호하고, 미세아교세포에서는 세포사멸사(apoptosis)를 유도하지만, 저산소성 허혈성 뇌질환에서 MPA의 효과는 아직 알려지지 않아, 본 연구에서 Rice-Vannucci 모델을 이용한 신생 백서의 저산소성 허혈성 뇌 손상과 저산소 상태의 태아 백서 뇌세포 배양에서 MPA의 뇌보호 효과를 알아보고자 실험하였다. 방 법 : 생후 7일된 백서의 좌측 총 경동맥을 결찰한 후 저산소 (8% $O_2$) 상태에서 2시간 노출하여, 저산소성 허혈성 뇌 손상을 유발하고 뇌 손상 전후에 MPA(10 mg/kg)를 투여하여 대조군과 비교하였다. 또한, 재태기간 18일된 태아 백서의 대뇌피질 세포를 배양하여 1% $O_2$ 배양기에서 저산소 상태로 세포손상을 유도하여 저산소군, 손상 전후 MPA 투여군($10{\mu}g/mL$)으로 나누어 정상산소군과 비교하였다. 세포사멸사와의 관련을 알아보기 위해서 Bcl-2, Bax, caspase-3 항체로 western blotting하였고 Bcl-2, Bax, caspase-3 primer를 이용하여 real-time PCR을 하였다. 결 과 : 형태학적으로 H&E 염색상 MPA를 투여한 군에서 뇌 보호 효과를 보였다. Western blotting과 real-time PCR을 이용한 저산소성 허혈성 뇌손상 동물 모델뿐만 아니라 저산소 상태로 태아 백서 뇌세포 배양 실험에서도 MPA 투여한 경우 caspase-3의 발현과 Bax/Bcl-2의 비율이 감소함을 보였다. 결 론 : 본 연구에서 MPA가 anti-apoptosis 작용을 통하여 주산기 저산소성 허혈성 뇌 손상에 뇌보호 역할을 하는 것을 알 수 있었고 향후 신생아 저산소성 허혈성 뇌병증의 치료에 임상적 적용이 가능하리라 생각된다.

Keywords

Acknowledgement

Supported by : 대구가톨릭대학교 의과학연구소

References

  1. Volpe JJ. Neurology of the newborn. 4th ed. Philadelia : WB saunders Co, 1995;314-69
  2. Vannucci RC, Connor JR, Mauger DT, Palmer C, Smith MB, Towfighi J, et al. Rat model of perinatal hypoxicischemic brain damage. J Neurosci Res 1999;55:158-63 https://doi.org/10.1002/(SICI)1097-4547(19990115)55:2<158::AID-JNR3>3.0.CO;2-1
  3. Vannucci RC, Perlman JM. Interventions for perinatal hypoxic-ischemic encephalopathy. Pediatr 1997;100:1004-14 https://doi.org/10.1542/peds.100.6.1004
  4. Gunn AJ, Gunn TR. The 'pharmacology' of neuronal rescue with cerebral hypothermia. Early Hum Dev 1998;53:19-35 https://doi.org/10.1016/S0378-3782(98)00033-4
  5. Matute C, Sanchez-Gomez MV, Martinez-Millan L, Miledi R. Glutamate receptor-mediated toxicity in optic nerve oligodendrocytes. Proc Natl Acad Sci USA 1997;94:8830-5 https://doi.org/10.1073/pnas.94.16.8830
  6. Cheng Y, Deshmukh M, D'Costa A, Demaro JA, Gidday JM, Shah A, et al. Caspase inhibitor affords neuroprotection with delayed administration in a rat model of neonatal hypoxic-ischemic brain injury. J Clin Invest 1998;101: 1992-9 https://doi.org/10.1172/JCI2169
  7. Kawasaki H, Morooka T, Shimohama S, Kimura J, Hirano T, Gotoh Y, et al. Activation and involvement of p38 mitogen-activated protein kinase in glutamate-induced apoptosis in rat cerebellar granule cells. J Biol Chem 1997;272: 18518-21 https://doi.org/10.1074/jbc.272.30.18518
  8. Feng Y, Fratkin JD, LeBlanc MH. Treatment with tamoxifen reduces hypoxic-ischemic brain injury in neonatal rats. Eur J Pharmacol 2004;484:65-74 https://doi.org/10.1016/j.ejphar.2003.10.048
  9. Dehghani F, Hischebeth GT, Wirjatijasa F, Kohl A, Korf HW, Hailer NP. The immunosuppressant mycophenolate mofetil attenuates neuronal damage after excitotoxic injury in hippocampal slice cultures. Europ J Neuro 2003;18:1061- 72 https://doi.org/10.1046/j.1460-9568.2003.02821.x
  10. Daina E, Schieppati A, Remuzzi G. Mycophenolate mofetil for the treatment of takayasu arteritis: report of three cases. Ann Intern Med 1999;130:422-6 https://doi.org/10.7326/0003-4819-130-5-199903020-00013
  11. Waiser J, Budde K, Braasch E, Neumayer HH. Treatment of acute c-ANCA-positive vasculitis with mycophenolate mofetil. Am J Kidney Dis 1999;34:e9
  12. Langford CA, Talar-Williams C, Sneller MC. Mycophenolate mofetil for remission maintenance in the treatment of Wegener's granulomatosis. Arthritis. Rheum. 2004;51:278-83 https://doi.org/10.1002/art.20240
  13. Hu WX, Liu ZH, Chen HP, Tang Z, Li LS. Mycophenolate mofetil treatment for refractory severe lupus nephritis. J Nephrol Dialy Transplant 1998;7:511-4
  14. Thomas-Golbanov C, Sridharan S. Novel therapies in vasculitis. Expert Opin Investig Drugs 2001;10:1279-89 https://doi.org/10.1517/13543784.10.7.1279
  15. Allison AC, Eugui EM. Mycophenolate mofetil and its mechanism of action. Immunopharmacology. 2000;47:85-118 https://doi.org/10.1016/S0162-3109(00)00188-0
  16. Choi DW. Excitotoxic cell death. J Neurobiol 1992;23:1261-76 https://doi.org/10.1002/neu.480230915
  17. Rice JE, Vannucci RC, Brierley JB. The influence of immaturity on hypoxic-ischemic brain damage in the rat. Ann Neurol 1981;9:131-41 https://doi.org/10.1002/ana.410090206
  18. Brewer GJ. Isolation and culture of adult rat hippocampal neurons. J Neurosci Meth 1997;71:143-55 https://doi.org/10.1016/S0165-0270(96)00136-7
  19. Williams RH, Lively DH, Delong DC, Cline JC, Sweeny MJ. Mycophenolic acid: antiviral and antitumor properties. J Antibiot(Tokyo) 1968;21:463-4 https://doi.org/10.7164/antibiotics.21.463
  20. Mitsui A, Suzuki S. Immunosuppressive effect of mycophenolic acid. J Antibiot 1969;22:358-63
  21. Carr SF, Papp E, Wu JC, Natsumeda Y. Characterization of human type I and type II IMP dehydrogenases. J Biol Chem 1993;268:27286-92
  22. Mathieu P, Carrier M, White M, Pellerin M, Perrault L, Pelletier G, et al. Effect of mycophenolate mofetil in heart transplantation. Can J Surg 2000;43:202-6
  23. Allison AC, Kowalski WJ, Muller CJ, Waters RV, Eugui EM. Mycophenolic acid and brequinar, inhibitors of purine and pyrimidine synthesis, block the glycosylation of adhesion molecules. Transplant Proc 1993;25:67-70
  24. Cohn RG, Mirkovich A, Dunlap B, Burton P, Chiu SH, Eugui E, et al. Mycophenolic acid increases apoptosis, lysosomes and lipid droplets in human lymphoid and monocytic cell lines. Transplantation 1999;68:411-8 https://doi.org/10.1097/00007890-199908150-00014
  25. Pardo-Mindan FJ, Errasti P, Panizo A, Lozano MD, de Alava E. Decrease of apoptosis rate in patients treated with mycophenolate mofetil. Nephron 1999;82:232-7 https://doi.org/10.1159/000045407
  26. Edwards AD, Yue X, Cox P, Hope PL, Azzopardi DV, Squier MV, et al. Apoptosis in the brains of infants suffering intrauterine cerebral injury. Pediatr Res 1997;42:684-9 https://doi.org/10.1203/00006450-199711000-00022
  27. Ota K, Yakovlev AG, Itaya A, Kameoka M, Tanaka Y, Yoshihara K. Alteration of apoptotic protease-activating factor-1 (APAF-1)-dependent apoptotic pathway during development of rat brain and liver. J Biochem 2002;131:131-5 https://doi.org/10.1093/oxfordjournals.jbchem.a003067
  28. Merry DE, Veis DJ, Hickey WF, Korsmeyer SJ. Bcl-2 protein expression is widespread in the developing nervous system and retained in the adult. PNS Development 1994;120:301-11
  29. Vekrellis K, McCarthy MJ, Watson A, Whitfield J, Rubin LL, Ham J. Bax promotes neuronal cell death and is down-regulated during the development of the nervous system. Development 1997;124:1239-49