预适应对肢体缺血再灌注后骨骼肌的保护效应

The protective effect of preconditioning on skeletal muscle withlimb ischemia- reperfusion

目的观察缺血预适应对肢体缺血再灌注后骨骼肌损伤的保护效应,探讨一氧化氮(NO)在该过程中的作用。方法雄性Wistar大鼠32只,随机分为4组(n=8):假手术组仅进行假手术,双后肢松弛环绕橡皮圈,不阻断血流;再灌注组结扎大鼠双后肢根部4 h,恢复血流灌注4 h;预适应组预先阻断双后肢血流5 min,恢复血流灌注5 min,反复4次,其后进行缺血再灌注;Nω-硝基-L-精氨酸甲酯(L-NAME)处理组松带前15 min给予L-NAME 10 mg/kg,其他操作同预适应组。观察各组血浆NO、内皮素-1(ET-1)及NO/ET-1比值的变化;观察骨骼肌组织中NO、ET-1、NO/ET-1比值、丙二醛(MDA)、髓过氧化物酶(MPO)、DNA双链百分率的改变,采用免疫组织化学方法检测腓肠肌组织中凋亡相关基因Bcl-2、Bax和凋亡蛋白酶Caspase-3的蛋白表达情况,利用原位脱氧核糖核甘酸末端转移酶介导的缺口末端标记法检测各组腓肠肌细胞的凋亡情况。结果再灌注组与假手术组比较,血浆及腓肠肌组织NO、ET-1含量明显升高,NO/ET-1比值减小,腓肠肌组织MDA、MPO含量升高,DNA双链百分率值降低,Bcl-2/Bax比值减小,Caspase-3蛋白表达明显上调。凋亡指数(AI)明显升高(P<0.01)。预适应组与再灌注组比较,血浆及腓肠肌组织NO含量升高,ET-1下降,NO/ET-1比值升高;腓肠肌组织MDA、MPO含量下降,DNA双链百分率值升高,Bcl-2/Bax比值升高,Caspase-3蛋白表达水平明显减弱,AI下降(P<0.01)。L-NAME处理组与预适应组比较,血浆及骨骼肌NO含量明显下降,NO/ET-1比值减小,腓肠肌组织MDA、MPO含量升高,DNA双链百分率值降低,Bcl-2/Bax比值减小,Caspase-3蛋白表达明显上调。AI明显升高(P<0.01)。结论缺血预适应可明显改善肢体缺血再灌注后骨骼肌组织损伤及凋亡,NO参与缺血预适应的保护效应。

Objective To investigate the protective effect of ischemie preconditioning （IPC） on skeletal muscle injury after limbs ischemia -reperfusion, and to investigate the role of nitrogen monoxide（NO） in this process. Methods Thirty - two male Wistar rats were randomly divided into four groups （ n = 8 ） ： sham group （ sham operation alone, Group S）, limbs ischemia -reperfusion group （subjected to 4 h of hindlimb ischemia and 4 h reperfusion, Group M）, preconditioning group（ four cycles of 5 minutes occlusion and reperfusion of both hindlimbs before ischemia- reperfusion, Group P） and Nω - nitro - L - arginine methylester treated group （ pretreated with L - NAME 10 mg/kg 15 minutes before ischemia - reperfusion, Group T）. The plasma NO, endothelin - 1 （ ET - 1 ） and NO/ET - 1 in plasma, and the NO, ET -1, NO/ET- 1, malondialehyde （MDA）, myeloperoxide （MPO） and the ratio of DNA double chain in gastrocnemius tissue were measured. The immunohistochemistry （IHC） method was used to assessthe expressions of Bcl -2, Bax and Caspase -3, while TUNEL was applied for apoptosis analysis. Results Significant elevation in NO and ET - 1 wasob- served in both plasma and gastrocnemius tissue in Group M whencompared with those in Group S, with significant reduc- tion in NO/ET- 1. Meanwhile, significant increase of MDA, MPO, Caspase- 3 and apoptosis index （AI） and reduction in the ratio of DNA double chain and Bcl - 2/baxin gastrocnemius tissues in Group M. Significant elevation of NO and the NO/ET - 1 and reduction in ET - 2 were observed in both plasma and gastrocnemius tissues in Group P when compared with those in Group M. Meanwhile, significant reduction in MDA, MPO, Caspase -2 and AI, and increase in the ratio of DNA double chain and Bcl-2/Bax in gastrocnemius tissues were also observed in Group P than Group M （P 〈0.01 ）. Significant reduction in NO and NO/ET - 1 in both plasma and gastrocnemius tissues was observed in Group T than those in Group P. Furthermore, significant increase of MDA, MPO, Caspase - 3 and AI, and reduction in the ratio of DNA double chain and Bcl - 2/Bax in gastrocnemius tissues were revealed in Group T than those in Group P （ P 〈 0. 01 ）. Conclusion PC can preventthe gastrocnemius injury from hindlimbs ischemia- reperfusion, which maybe correlated within- crease of NO.