원거리 허혈양상화가 흰쥐의 심장근육에서NOS와 Akt 발현에 미치는 영향
Effects of Remote Ischemic Preconditioning on the Expressionsof NOS and Akt in the Rat Myocardium
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원거리 허혈양상화는 생명유지와 밀접한 관련이 없는 장기에서 짧은 허혈과 재관류를 반복하는 과정으 로, 심장의 기능 장애에 의한 허혈손상을 감소시킨다고 알려져 있고, 원거리 허혈양상화는 iNOS를 감소시키고 eNOS를 증가시키며, Akt를 활성화시켜 심장근육을 보호한다고 알려져 있다. 본 실험은 원거리 허혈양상화가 흰쥐 의 심장근육에서 관찰되는 iNOS와 eNOS, Akt의 발현 및 Akt의 활성화 (phospho-Akt, p-Akt)에 미치는 영향을 알 아보고자 실시하였다. 35주령의 SD 수컷 흰쥐를 사용하여 대조군과 원거리 허혈양상화군으로 나누었고, 원거리 허혈양상화의 횟수에 따라 3회와 6회, 10회군으로 나누었다. 원거리 허혈양상화는 온엉덩동맥을 5분 허혈과 5분 재관류를 반복하여 실 시하였고, 재관류 0시간과 3시간, 6시간, 24시간, 72시간에 심장을 적출하여 iNOS와 eNOS, Akt, p-Akt의 면역조직 화학염색과 Western blot 분석을 시행하여 다음과 같은 결과를 얻었다. iNOS는 원거리 허혈양상화에 의하여 증가되었고, 10회군에서는 3회와 6회군보다 증가되었다. eNOS는 원거리 허혈양상화에 의하여 증가 또는 감소되었으나, 횟수에 따른 eNOS 발현의 차이는 없었다. Akt는 원거리 허혈양상 화 후 재관류 24시간과 72시간에 감소되었고, 횟수에 따른 Akt 발현의 차이는 없었다. p-Akt는 원거리 허혈양상화 에 의하여 증가되었으나, 횟수에 따른 p-Akt 발현의 차이는 없었다. 이상의 실험결과로, 흰쥐 뒷다리에서 실시한 원거리 허혈양상화로 증가된 세포보호단백질 eNOS와 p-Akt가 심 장근육세포의 보호현상에 관련이 있을 것으로 생각되며, 10회 이상의 원거리 허혈양상화는 iNOS를 증가시켜 심 장근육의 허혈손상을 일으키는 것으로 생각된다.
Abstract
Remote ischemic preconditioning (IP), brief tolerating cycles of ischemia and reperfusion in remote non-vital organs, can reduce ischemic injury of the heart. IP induces cardiac protection by down-regulating iNOS or up-regulating eNOS. In addition, Akt has been known to protect myocardium against ischemia-reperfusion injury. This study was undertaken to observe the expression of iNOS, eNOS, Akt and phospho-Akt (p-Akt) in the rat myocardium after IP. Thirty-five weeks-old male Sprague-Dawley rats were divided into control and IP groups. The IP group was further subdivided into 3 groups based on the number of cycles of IP. For IP, left commom iliac artery was occluded 3, 6 and 10 cycles for 5 min of ischemia alternating with 5 min of reperfusion. The rat were sacrificed at 0, 3, 6, 24 and 72 hours of IP and the heart was removed. The expression of iNOS, eNOS, Akt and p-Akt in the rat myocardium was examined by immunohistochemical staining and Western blot analysis. The expression of iNOS was increased by IP and was higher in 10IP groups than 3IP and 6IP group. The expression of eNOS was increased or decreased by IP and was showed no difference with increasing episode of IP. The expression of Akt was decreased by IP at 24 and 72 hours after reperfusion, and showed no differences with increasing episode of IP. The expression of p-Akt was increased by IP and showed no difference with increasing episode of IP. These results suggest that hind limb ischemic preconditioning provides cardiac protection through up-regulation of eNOS and phosphorylation of Akt, however excessive episodes of remote preconditioning may induce the myocardial ischemic injury through overexpression of iNOS.
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