허혈양상화 후 본태성고혈압 흰쥐의 뼈대근육에서 관찰되는 iNOS와 SOD의 발현
Expressions of iNOS and Superoxide Dismutase in the Skeletal Muscle of the Spontaneously Hypertensive Rat after Ischemic-preconditioning
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초록
활성산소기의 생성과 제거 사이의 균형은 심혈관계 항상성 유지에 중요하며, 이들의 균형은 고혈압 발병에 영향을 준다고 알려져 있다. 산화자극이 세포에 가해질 때 증가되는 산화질소합성효소(inducible NOS, iNOS)는 허혈-재관류 손상을 유도할 뿐 아니라 고혈압의 발생과도 연관이 있다. 뼈대근육의 허혈-재관류 손상 역시 활성산소기에 의해서 일어나며, iNOS의 과도한 증가는 근육세포의 허혈손상을 증가시킬 수 있다. 항산화효소(superoxide dismutase, SOD)는 활성산소기를 제거하는 강력한 효소로 알려져 있다. 본 연구에서는 반복적인 허혈양상화가 정상혈압 흰쥐와 본태성고혈압 흰쥐의 백색근육과 적색근육에서 iNOS와 CuZnSOD 및 MnSOD의 발현에 영향을 주는지 알아보았다. 9주령의 정상혈압 흰쥐(WKY)와 본태성고혈압 흰쥐(SHR)를 대조군과 허혈양상화군으로 나누었고, 짧은 허혈과 재관류 반복 횟수에 따라 허혈양상화 3회군과 10회군으로 나누었다. 허혈양상화는 흰쥐용 혈관집게를 이용하여 왼온엉덩동맥에 각각 5분 허혈-5분 재관류를 3회 및 10회 반복하여 실시하였고, 마지막 허혈 후 재관류 0시간과 0.5시간, 1시간, 3시간에 실험동물을 희생하여 앞정강근과 가자미근을 적출하였다. iNOS와 CuZnSOD 및 MnSOD를 면역조직화학염색과 Western blot assay 실시하여 다음과 같은 결과를 얻었다. 허혈양상화 처치 후, iNOS는 앞정강근에서 발현되었으나, 가자미근에서는 발현되지 않았다. 앞정강근에서 iNOS는 WKY와 SHR 모두에서 증가되었고, SHR에서 WKY보다 더 높았다. 허혈양상화 처치 후, CuZnSOD와 MnSOD는 앞정강근과 가자미근에서 모두 관찰되었으며, 가자미근에서 더 높았다. CuZnSOD와 MnSOD는 WKY와 SHR에서 모두 증가하였고, WKY에서 SHR보다 더 높았다. iNOS의 높은 발현과 SOD의 낮은 발현을 고려할 때, 고혈압과 백색근육섬유는 뼈대근육에 가해지는 허혈손상에 더 민감할 것이라 생각되었다.
Abstract
A balance between production and degradation of reactive oxygen species has an important role in the cardiovascular homeostasis, and is known to contribute to hypertension. Under oxidative stress, an upregulation of inducible NOS (iNOS) induces ischemic-reperfusion injury, and is involved in the pathophysiology of the hypertension. Ischemic-reperfusion injury of the skeletal muscle results from reactive oxygen species, and overexpression of iNOS in the skeletal muscle increases the ischemic injury. Superoxide dismutase (SOD), antioxidant, is a major enzyme for degradation of reactive oxygen species (ROS). The purpose of this study was to observe the effect ischemic preconditioning (IP) of the lower limb on the expression of iNOS, CuZnSOD and MnSOD in the white and red muscle of the spontaneously hypertensive rat (SHR). Nine weeks old male normotensive rat (Wistar-Kyoto rat, WKY) and SHR were divided into control and IP groups. The IP group was further divided into 3 (3IP) and 10 (10IP) times of IP. Left common iliac artery was occluded 3 and 10 times for 5 min of ischemia-5 min of reperfusion using rodent vascular clamp. The animals were sacrificed at 0, 0.5, 1, and 3 hours after reperfusion and the Tibialis anterior and Soleus were removed. The expressions of iNOS, CuZnSOD and MnSOD in the skeletal muscle were examined with immunohistochemical methods and Western blot analysis. iNOS was expressed in Tibialis anterior, but in Soleus after IP. The expression of iNOS was increased in both WKY and SHR, it was higher in SHR than WKY. CuZnSOD and MnSOD were expressed in Tibialis anterior and Soleus, higher in Soleus, after IP. The expression of CuZnSOD and MnSOD were increased in both WKY and SHR, higher in WKY than SHR. It is consequently suggested that hypertensive individual and white muscle are more sensitive to ischemic injury of the skeletal muscle as considering their high expression of iNOS and low expression of SODs.
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