허혈 후 재관류 시점에 따른 뼈대근육과 혈청 내 cytokine의 변화

Abstract

뼈대근육의 허혈(ischemia)은 각종 사고, 격렬한 운동에 의한 외상, 임상적 치료 목적으로 시행되는 일시적 혈류차단이나 혈관 질환들에 의해서 발생되며 허혈시간과 기간에 따라 급성염증 및 조직의 손상 등이 유발된다. 허혈 손상의 주된 원인은 재관류 시 생성되는 활성산소기(reactive oxygen species)로 알려져 있다. 증가된 활성산소기의 영향으로 활성화된 조직 내 다양한 면역세포들은 염증반응의 주요 매개인자가 되는 cytokine을 분비 자극하여 조직을 손상시킨다. 이에 저자는 허혈 후 재관류 시점에 따라 뼈대근육과 혈청에서 나타나는 cytokine의 변화 양상을 myokine, 친염증성 cytokine, 통증연관 cytokine, 항염증성 cytokine으로 분류하여 알아보기 위하여 본 실험을 실시하였다. 12주령의 ICR계 수컷 생쥐를 사용하여 Sham군, 허혈 후 재관류 시간에 따라 재관류 0, 0.5, 1, 2, 4, 8, 16, 24시간군으로 분류하였다. 허혈 처치는 생쥐용 혈관집게(vascular clamp)를 사용하여 왼온엉덩동맥(left common iliac artery)을 2시간 동안 차단하였다. 재관류 시간 경과에 맞추어 실험동물을 마취시킨 후 심장의 오른 심방에서 혈액을 채취하고 경추탈골로 희생시킨 뒤 장딴지근(gastrocnemius muscle)을 적출하였다. 혈액에서 얻은 혈청과 뼈대근육에서 cytokine(Eotaxin, GM-CSF, IFNγ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, MCP-1, MDC, MIP-1α, RANTES, TARC, TCA-3, TNFα)의 변화를 확인하기 위하여 ELISA 분석을 시행하였으며 다음과 같은 결과를 얻었다.

1. 허혈 후 재관류 시점에 따른 뼈대근육 내 전체 cytokine의 변화양상은 유사하게 나타났고(Cronbach’s alpha : 0.9915), 혈청에서는 Sham군, 재관류 0, 1, 2, 4, 8, 16시간군에서 cytokine들 사이의 발현양의 차이가 유의한 것으로 관찰되었다(p＜.05).

2. 허혈 후 재관류 시점에 따른 뼈대근육 내 myokine(GM-CSF, IL-6, MCP-1)의 발현은 재관류 16시간군에서 가장 높았다. 혈청에서는 재관류 2, 4, 16시간군에서 cytokine들 사이의 발현양의 차이가 유의한 것으로 관찰되었고(p＜.05), 특히 IL-6는 4시간군에서, MCP-1은 16시간군에서 다른 cytokine보다 유의하게 높은 농도로 관찰되었다.

3. 허혈 후 재관류 시점에 따른 뼈대근육 내 친염증성 cytokine(Eotaxin, IFNγ, IL-1α, IL-1β, IL-2, IL-3, IL-5, IL-6, MCP-1, MDC, MIP-1α, RANTES, TARC, TCA-3, TNFα)의 발현은 재관류 16시간군에서 가장 높았다. 혈청에서는 Sham군과 재관류 0, 1, 2, 4, 8, 16시간군에서 cytokine들 사이의 발현양의 차이가 유의한 것으로 관찰되었고(p＜.05), 특히 IL-6는 재관류 4시간군에서, Eotaxin, MCP-1, TARC는 재관류 16시간군에서 다른 cytokine보다 유의하게 높은 농도로 관찰되었다.

4. 허혈 후 재관류 시점에 따른 뼈대근육 내 통증연관 cytokine(IL-1β, IL-2, IL-4, IL-6, TNFα)의 발현은 재관류 16시간군에서 가장 높았다. 혈청에서는 Sham군, 재관류 0, 1, 2, 4, 8, 16시간군에서 cytokine들 사이의 발현양의 차이가 유의한 것으로 관찰되었다(p＜.05).

5. 허혈 후 재관류 시점에 따른 뼈대근육 내 항염증성 cytokine(IL-4, IL-10)의 발현은 재관류 16시간군에서 가장 높았다. 혈청에서는 재관류 4시간군에서 cytokine들 사이의 발현양의 차이가 유의한 것으로 관찰되었다(p＜.05).

이상의 실험결과로 허혈 후 cytokine들의 변화양상은 재관류 시점에 따라 유사하게 나타나는 것으로 관찰되었으며, 뼈대근육에서는 재관류 16시간에서, 혈청에서는 재관류 4시간과 16시간에서 증가하는 친염증성 cytokine인 IL-6, Eotaxin, MCP-1, TARC의 변동 폭이 크게 나타나 염증성 반응이 촉진될 것으로 생각된다. 이중 혈청에서 재관류 4시간에서 관찰되는 IL-6와 재관류 16시간에서 관찰되는 MCP-1은 친염증성 cytokine의 표지인자라고 결론지을 수 있다.| Ischemic injury of skeletal muscle results from various accidents and traumas caused by strenuous exercises, temporary obstruction of blood vessel for therapeutic purposes, and blood vessel diseases. Acute inflammation and damage to the tissue occurs depending on the time of onset and duration of ischemia. The reactive oxygen species(ROS) generated during reperfusion has been known to be the main cause of an ischemic injury. The increased ROS activates immunce cells to secrete cytokines, a major mediators of inflammation, and these cytokines damage the tissues. The author conducted the study to identify the changes of cytokines in the skeletal muscle and the serum according to the time of reperfusion after ischemic injury by comparing of myokine, pro-inflammatory cytokine, pain-related cytokine, and anti-inflammatory cytokine. By using 12-week-old male ICR strain mice, a total of 9 groups were assigned: Sham group (SC), 0-hour after reperfusion group (0H), 0.5-hour after reperfusion group (0.5H), 1-hour after reperfusion group (1H), 2-hour after reperfusion group (2H), 4-hour after reperfusion group (4H), 8-hour after reperfusion group (8H), 16-hour after reperfusion group (16H), and 24-hour after reperfusion group (24H). For the purposes of ischemic injury, left common iliac arteries of mice were clamped for 2 hours. After anesthetizing the experimental animals, blood samplings were obtained from the mice hearts on the times of 0-hour, 0.5-hour, 1-hour, 2-hours, 4-hours, 8-hours, 16-hours and 24-hours after reperfusion. After sacrificing the mice via dislocation of cervical vertebra, their calf muscles (gastrocnemius muscle) were excised. To analyze the cytokines (Eotaxin, GM-CSF, IFNγ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, MCP-1, MDC, MIP-1α, RANTES, TARC, TCA-3, TNFα) in the skeletal muscle and the serum, an ELISA test was performed, and the following results were obtained. 1. There were similarities in the changes of total cytokines in the skeletal muscles according to the time of reperfusion after ischemic injury(Cronbach’s alpha : 0.9915). In the serum, there were statistically significant differences of changes of cytokines in the Sham group, 0-hour, 2-hour, 4-hour, 8-hour and 16-hour reperfusion groups(P<0.05).

2. The myokines(GM-CSF, IL-6, MCP-1) in the skeletal muscle according to the time of reperfusion after ischemic injury had a highest levels in the 16-hour reperfusion group. In the serum, there were significant differences of changes of cytokines on 2-hour, 4-hour and 16-hour after reperfusion(P<0.05). IL-6 had a highest level in the 4-hour group and MCP-1 had a highest level in the 16-hour group, respectively.

3. The pro-inflammatory cytokines(Eotaxin, IFNγ, IL-1α, IL-1β, IL-2, IL-3, IL-5, IL-6, MCP-1, MDC, MIP-1α, RANTES, TARC, TCA-3, TNFα) in the skeletal muscles according to the time of reperfusion after ischemic injury had a highest levels in 16-hour reperfusion group. In the serum, there were significant differences of changes of cytokines in the Sham, 0-hour, 1-hour, 2-hour, 4-hour, 8-hour, 16-hour groups(P<0.05). Eotaxin, MCP-1, MDC and TARC had a highest levels in the 16-hour reperfusion group.

4. The pain-related cytokines(IL-1β, IL-2, IL-4, IL-6, TNFα) in the skeletal muscles according to the time of reperfusion after ischemic injury had a highest levels in the 16-hour reperfusion group. In the serum, there were significant differences of changes of cytokines in the Sham, 0-hour, 1-hour, 2-hour, 4-hour, 8-hour and 16-hour reperfusion groups(P<0.05).

5. The anti-inflammatory cytokines(IL-4, IL-10) in the skeletal muscles according to the time of reperfusion after ischemic injury had a highest levels in the 16-hour reperfusion group. In the serum, there were significant differences of changes of cytokines in the 4-hour groups(P<0.05).

It was concluded that the post-ischemic cytokines in the skeletal muscle and the serum have been changed similarly according to the time of reperfusion after ischemic injury. The pro-inflammatory cytokines(IL-6, Eotaxin, MCP-1, TARC) had highest levels on 16-hour in skeletal muscles and on 4-hour and 16-hour in the serum and those cytokines were considered to facilitate the inflammatory responses. IL-6 observed on 4-hour after reperfusion in the serum, MCP-1 on 16-hour in the serum could be considered as were concluded the main indicator of inflammatory cytokines.; Ischemic injury of skeletal muscle results from various accidents and traumas caused by strenuous exercises, temporary obstruction of blood vessel for therapeutic purposes, and blood vessel diseases. Acute inflammation and damage to the tissue occurs depending on the time of onset and duration of ischemia. The reactive oxygen species(ROS) generated during reperfusion has been known to be the main cause of an ischemic injury. The increased ROS activates immunce cells to secrete cytokines, a major mediators of inflammation, and these cytokines damage the tissues. The author conducted the study to identify the changes of cytokines in the skeletal muscle and the serum according to the time of reperfusion after ischemic injury by comparing of myokine, pro-inflammatory cytokine, pain-related cytokine, and anti-inflammatory cytokine. By using 12-week-old male ICR strain mice, a total of 9 groups were assigned: Sham group (SC), 0-hour after reperfusion group (0H), 0.5-hour after reperfusion group (0.5H), 1-hour after reperfusion group (1H), 2-hour after reperfusion group (2H), 4-hour after reperfusion group (4H), 8-hour after reperfusion group (8H), 16-hour after reperfusion group (16H), and 24-hour after reperfusion group (24H). For the purposes of ischemic injury, left common iliac arteries of mice were clamped for 2 hours. After anesthetizing the experimental animals, blood samplings were obtained from the mice hearts on the times of 0-hour, 0.5-hour, 1-hour, 2-hours, 4-hours, 8-hours, 16-hours and 24-hours after reperfusion. After sacrificing the mice via dislocation of cervical vertebra, their calf muscles (gastrocnemius muscle) were excised. To analyze the cytokines (Eotaxin, GM-CSF, IFNγ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, MCP-1, MDC, MIP-1α, RANTES, TARC, TCA-3, TNFα) in the skeletal muscle and the serum, an ELISA test was performed, and the following results were obtained. 1. There were similarities in the changes of total cytokines in the skeletal muscles according to the time of reperfusion after ischemic injury(Cronbach’s alpha : 0.9915). In the serum, there were statistically significant differences of changes of cytokines in the Sham group, 0-hour, 2-hour, 4-hour, 8-hour and 16-hour reperfusion groups(P<0.05).

2. The myokines(GM-CSF, IL-6, MCP-1) in the skeletal muscle according to the time of reperfusion after ischemic injury had a highest levels in the 16-hour reperfusion group. In the serum, there were significant differences of changes of cytokines on 2-hour, 4-hour and 16-hour after reperfusion(P<0.05). IL-6 had a highest level in the 4-hour group and MCP-1 had a highest level in the 16-hour group, respectively.

3. The pro-inflammatory cytokines(Eotaxin, IFNγ, IL-1α, IL-1β, IL-2, IL-3, IL-5, IL-6, MCP-1, MDC, MIP-1α, RANTES, TARC, TCA-3, TNFα) in the skeletal muscles according to the time of reperfusion after ischemic injury had a highest levels in 16-hour reperfusion group. In the serum, there were significant differences of changes of cytokines in the Sham, 0-hour, 1-hour, 2-hour, 4-hour, 8-hour, 16-hour groups(P<0.05). Eotaxin, MCP-1, MDC and TARC had a highest levels in the 16-hour reperfusion group.

4. The pain-related cytokines(IL-1β, IL-2, IL-4, IL-6, TNFα) in the skeletal muscles according to the time of reperfusion after ischemic injury had a highest levels in the 16-hour reperfusion group. In the serum, there were significant differences of changes of cytokines in the Sham, 0-hour, 1-hour, 2-hour, 4-hour, 8-hour and 16-hour reperfusion groups(P<0.05).

5. The anti-inflammatory cytokines(IL-4, IL-10) in the skeletal muscles according to the time of reperfusion after ischemic injury had a highest levels in the 16-hour reperfusion group. In the serum, there were significant differences of changes of cytokines in the 4-hour groups(P<0.05).

It was concluded that the post-ischemic cytokines in the skeletal muscle and the serum have been changed similarly according to the time of reperfusion after ischemic injury. The pro-inflammatory cytokines(IL-6, Eotaxin, MCP-1, TARC) had highest levels on 16-hour in skeletal muscles and on 4-hour and 16-hour in the serum and those cytokines were considered to facilitate the inflammatory responses. IL-6 observed on 4-hour after reperfusion in the serum, MCP-1 on 16-hour in the serum could be considered as were concluded the main indicator of inflammatory cytokines.