低氧性微损伤时大鼠腓肠肌抗肌营养不良蛋白和结蛋白的变化

Changes of dystrophin and desmin in rat gastrocnemius under micro-damage induced by hypoxia

为了观察低氧性微损伤时大鼠腓肠肌抗肌营养不良蛋白(dystrophin)和结蛋白(desmin)的变化特征及其调节机制,本实验将雄性Sprague-Dawley大鼠40只随机分为常氧对照组和10%氧浓度的低氧刺激1、2、4、7d组(n=8),每组各取2只大鼠进行伊文氏蓝(EBD)腹腔注射来检测肌纤维膜通透性,同时用异染ATP酶法鉴别EBD阳性肌纤维的类型;其余6只采用Western blot、RT-PCR和荧光法进行蛋白含量和基因表达等测定。结果显示,低氧第1天即出现EBD阳性纤维,主要为ⅡA和ⅡB型。各低氧组的EBD阳性细胞率和平均光密度均大于常氧对照组(P<0.05)。低氧第1天dystrophin和desmin蛋白显著增加或有增加趋势,第2天有所下降,第4天又急剧增加,第7天恢复至接近对照组水平,其变化呈波动性起伏;低氧组基因表达在第2天开始大幅度增加,与蛋白含量变化趋势相对应。除了低氧第2天外,其余各组钙激活中性蛋白酶(calpain)总活性均高于常氧对照组。热休克蛋白HSP70和HSP90分别在低氧第4天和第7天增加(P<0.05)。上述结果表明,机械应力并非细胞膜完整性破坏的唯一原因,但是仅靠低氧引起腓肠肌微损伤时的dystrophin和desmin变化与离心运动不一样,二者蛋白含量并没有出现明显的丢失。低氧性肌损伤的表现形式可能与肌纤维动员类型以及calpain活性有密切关系。

To explore the changes and regulation mechanism of dystropin and desmin under muscle injury without mechanic stress,40 male Sprague-Dawley rats were randomly divided into 5 groups,which included normoxia control and hypoxia groups for 1,2,4 and 7 d with 10% O2.Two rats from each group were examined for sarcolemma integrity using Evans blue dye（EBD） and EBD-positive fiber typing by metachromatic dye-ATPase method.The rest six rats from each group were analyzed for the changes of protein content and gene expression using Western blot,RT-PCR and fluorescence assays.The results showed that the EBD-positive muscle fibers,mainly type IIA and type IIB,appeared at 1 d after hypoxia exposure.Both the ratio of EBD-positive cell and the mean fluorescence density were significantly higher in hypoxia groups than those in control group（P0.05）.The contents of dystrophin and desmin fluctuated after hypoxia exposure,increased at 1 d,decreased at 2 d,increased dramatically again at 4 d,and returned to a normal level at 7 d.Consistently,the gene expression began to increase significantly after 2 d.The total activity of calpain was significantly higher in hypoxia groups at 1,4 and 7 d.Significantly higher levels of HSP70 and HSP90 were also observed at 4 and 7 d,respectively（P0.05）.These results suggest that the mechanical stress is not the only cause of damage of sarcolemma membrane integrity.In contrast to eccentric contraction,hypoxia-induced muscle damage is not accompanied by the loss of dystrophin and desmin.The types of muscle fibers recruited by motor units and the activities of calpain may be important in hypoxia-induced damage of sarcolemma membrane integrity.