Neonatal Medicine

The Korean Society of Neonatology (대한신생아학회)
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Neonatal Rat Necrotizing Enterocolitis Model Adopting Oral Endotoxin and Hypoxia Exhibits Increased Apoptosis through Caspase-3 Activation

경구 내독소와 저산소로 유발된 신생쥐의 괴사성 장염모델에서 caspase-3 활성화를 통한 세포자멸사의 증가

  • Lee, Yun-Kyoung (Department of Pediatrics, Seoul National University College of Medicine) ;
  • Kim, Ee-Kyung (Department of Pediatrics, Seoul National University Children's Hospital) ;
  • Kim, Ji-Eun (Department of Pathology, Seoul National University College of Medicine) ;
  • Kim, Yoon-Joo (Department of Pediatrics, Seoul National University Children's Hospital) ;
  • Son, Se-Hyung (Department of Pediatrics, Seoul National University Children's Hospital) ;
  • Kim, Han-Suk (Department of Pediatrics, Seoul National University Children's Hospital) ;
  • Kim, Beyong-Il (Department of Pediatrics, Seoul National University College of Medicine) ;
  • Choi, Jung-Hwan (Department of Pediatrics, Seoul National University College of Medicine)
  • 이윤경 (서울대학교 의과대학 소아과학교실) ;
  • 김이경 (서울대학교 어린이병원 소아청소년과) ;
  • 김지은 (서울대학교 의과대학 병리학교실) ;
  • 김윤주 (서울대학교 어린이병원 소아청소년과) ;
  • 손세경 (서울대학교 어린이병원 소아청소년과) ;
  • 김한석 (서울대학교 어린이병원 소아청소년과) ;
  • 김병일 (서울대학교 의과대학 소아과학교실) ;
  • 최중환 (서울대학교 의과대학 소아과학교실)
  • Published : 2010.05.31
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Abstract

Purpose : The aim of this study was to develop a model for necrotizing enterocolitis (NEC) in the neonatal rat using endotoxin and hypoxia, a plausible insult in a neonatal intensive care and to investigate the role of apoptosis as the underlying mechanism. Methods : Newborn rats were given oral endotoxin and intermittent 8% hypoxia$\pm$caspase inhibitor. The intestinal histology was evaluated using hematoxylin-eosin staining. Apoptosis was analyzed with TUNEL staining and by measuring the caspase 3 activity in the intestinal lysates. IEC-6 cells were assessed for apoptosis and the expression of Bax, Bcl-2, Fas and FasL was measured after treatment with endotoxin and hypoxia. Results : Oral endotoxin (5 mg/kg) and exposure to 8% hypoxia of 60-min duration twice induced human NEC-like lesions in the rat intestine. Intestinal tissue revealed increased apoptosis and caspase-3 activity. After caspase inhibitor treatment, the grades of both apoptosis and NEC were significantly reduced. IEC-6 cells exhibited increased apoptosis and caspase 3 activity after endotoxin and hypoxia treatment and significantly increased Bax/Bcl- 2 ratio compared to control cells. Conclusion : This neonatal rat model of NEC which was induced by oral endotoxin and intermittent hypoxia showed increased apoptosis of intestinal epithelial cells that was mediated by caspase 3 activation. Our model has a advantage in the study of NEC because the use of much more clinically plausible insults may provide a suitable model for the investigation of its pathophysiology and therapeutic trials.

목 적 : 경구 내독소와 저산소 자극을 이용하여 신생쥐에서 실험적 괴사성 장염(necrotizing enterocolitis, NEC)을 유발하고 그 병태생리에서 세포자멸사의 역할을 알아본다. 방 법:신생쥐에 경구로 내독소(5 mg/kg)를 투여하고 8% 저산소 자극을 주어 NEC를 유발하고 성공적으로 NEC가 유발된 군에 caspase 억제제를 전처치하고 비교하였다. 장 조직의 병리학적 분석은 hematoxylin-eosin 염색한 슬라이드로 하였고 세포자멸사는 TUNEL 염색과 장 조직 caspase-3 활성으로 분석하였다. IEC-6세포주에 내독소와 저산소를 처리한 후 세포자멸사와 Bax, Bcl-2, Fas, FasL의 발현을 분석하였다. 결 과:경구 내독소(5 mg/kg)와 반복적으로 60분간 2회 투여된 8% 저산소 자극에 의해서 NEC와 유사한 장병변이 신생쥐에서 유발되었다. 장 조직은 세포자멸사와 caspase-3 활성의 증가를 보여주었다. Caspase 억제제 전처치에 의해서 세포자멸사와 NEC의 중증도가 모두 감소하였다. IEC-6 세포주도 내독소와 저산소 자극에 의해서 세포자멸사와 caspase-3 활성의 증가를 보여주었으며 Bax/Bcl-2 ratio가 유의하게 증가하였다. 결 론:경구 내독소와 저산소를 이용한 본 NEC 신생쥐 모델은 caspase-3 활성에 의해 매개되는 장 상피세포의 세포자멸사가 병태생리에 관여함을 보였다. 본 동물모델은 임상에서 흔히 접할 수 있는 자극을 적용하였기 때문에 그 병태생리와 치료적 시도의 연구에 더욱 유용하리라고 본다.

Keywords

References

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