허혈양상화가 허혈된 흰쥐 뼈대근육의 Akt 인산화와 SOD-1 발현에 미치는 영향
Effects of Ischemic Preconditioning on the Phosphorylation of Akt and the Expression of SOD-1 in the Ischemic-reperfused Rat Skeletal Muscles
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세포의 성장과 생존에 중요한 Akt가 인산화되면 여러 세포보호인자들이 유도되어 세포를 보호한다. 허혈양상화는 Akt 인산화를 증가시켜 허혈손상과 세포자멸사를 감소시킨다고 알려져 있다. 또한, 항산화효소 SOD-1는 허혈 및 재관류시 발생되는 활성산소기를 제거하고, Akt 인산화를 증가시켜 세포를 보호하는 것으로 알려져 있다. 본 실험에서는 허혈양상화가 허혈 및 재관류시 흰쥐의 뼈대근육에서 발현되는 Akt 인산화와 SOD-1에 어떤 영향을 주는지 알아보았다. 30주령의 SD계 수컷 흰쥐를 사용하여 대조군과 허혈양상화군, 허혈군, 허혈양상화 후 허혈군으로 나누었다. 허혈양상화는 온엉덩동맥을 혈관집게로 5분 폐색, 5분 재관류를 3회 반복하였고, 허혈은 온엉덩동맥을 4시간 동안 폐색하였다. 재관류 직후와 1시간, 3시간, 24시간 경과시 실험동물을 희생시켜 앞정강근과 가자미근을 적출하였다. Akt 인산화(Ser473)와 SOD-1의 발현은 면역조직화학염색과 Western blot 분석을 이용하여 관찰하였고, 다음과 같은 결과를 얻었다. 허혈양상화는 Akt 인산화와 SOD-1을 대조군보다 증가시켰다. 허혈시 Akt 인산화와 SOD-1은 대조군보다 감소되었고, 재관류 시간이 지남에 따라 증가되었다. 허혈양상화는 허혈 후 재관류된 뼈대근육에서 Akt 인산화와 SOD-1을 허혈군보다 증가시켰다. Akt 인산화와 SOD-1은 앞정강근보다 가자미근에서 더 많이 증가되었다. 이상의 실험결과로, 흰쥐의 뼈대근육에서 허혈양상화는 허혈 후 재관류시 나타나는 Akt의 인산화와 SOD-1의 발현을 증가시키는 것을 알 수 있었으며, 이러한 허혈양상화 효과는 백색근육인 앞정강근보다 적색근육인 가자미근에서 더 크게 나타나는 것을 알 수 있었다.
Abstract
Akt, a key protein of cell survival, can promote cell growth and survival by activations of various cellular protective factors. Ischemic preconditioning (IP) has been known to reduce ischemic injury through upregulation of phosphorylation of Akt (p-Akt). CuZn-superoxide dismutase (SOD-1), an antioxidant enzyme, scavenges reactive oxygen species and protects cell from oxidative stress by increasing the activaiton of Akt. The present study was performed to examine the effects of IP on the expression of p-Akt and SOD-1 in the ischemicreperfused rat skeletal muscles. Thirty weeks old male SD rats were divided into 4 groups, such as controls, IP, 4 hour ischemia and 4 hour ischemia with IP. For IP, commom iliac artery was occluded three times for 5 min ischemia followed by 5 min reperfusion using rodent vascular clamps. Ischemia was induced by occlusion on the same artery for 4 hours. The Tibialis anterior and Soleus were removed at 0, 1, 3, and 24 hours of reperfusion. The expressions of p-Akt (Ser 473) and SOD-1 were examined with immunohistochemistry and Western blot analysis. In the IP group, the p-Akt and SOD-1 were increased, compared to the control group. In the ischemia group, the p-Akt and SOD-1 were decreased, compared to the control group, and were more abundant when reperfusion time were increased. IP increased the p-Akt and SOD-1 after 4 hour ischemia, and the p-Akt and SOD-1 were higher in Soleus compared to Tibialis anterior. These findings suggest that IP increases p-Akt and expression of SOD-1 in the ischemic-reperfused rat skeletal muscles, and that upregulations of p-Akt and SOD-1 induced by IP were higher in the red muscle fiber, Soleus, than the white muscle fiber, Tibialis anterior.
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