缺血后调适对骨骼肌缺血再灌注损伤的保护及其机制的初步探讨

Ischemic post-conditioning protects against ischemia-reperfusion injury in the skeletal muscle: a preliminary research on its mechanism

背景:再灌注损伤挽救激酶信号通路在缺血预调适和后调适引发的组织器官缺血再灌注损伤保护机制中均起重要作用。目前国内外有关缺血后调适对心肌缺血再灌注损伤保护通路机制的研究较多,而对骨骼肌缺血再灌注损伤保护信号通路机制研究较少。目的:探索后调适方案对大鼠骨骼肌缺血再灌注损伤的保护作用及再灌注损伤挽救激酶通路机制。方法:将18只雄性SD大鼠随机等分为缺血再灌注组、缺血后调适组和对照组。缺血再灌注组给予右侧股动脉无创动脉夹夹闭使远端肢体缺血4 h,松开再灌注24 h;缺血后调适组在缺血4 h后立即施加4个循环30 s再灌注/30 s缺血操作,再灌注24 h;对照组分离右侧股动脉不予以处理。结果与结论:(1)苏木精-伊红染色显示,与缺血再灌注组相比,缺血后调适组骨骼肌纤维结构病理改变程度较轻,少见炎性灶,肌细胞水肿程度明显改善;(2)TTC染色结果显示,缺血后调适组的梗死面积小于缺血再灌注组;(3)Western blot结果显示,缺血后调适组的磷酸化Akt、磷酸化内皮型一氧化氮合酶-S1177蛋白表达量较缺血再灌注组明显增加;而磷酸化内皮型一氧化氮合酶-Thr495的蛋白表达较缺血再灌注组显著降低;(4)Ca2+诱导线粒体通透性转换孔开放测试结果:缺血后调适组线粒体吸光度的降低程度较缺血再灌注组更为明显。(5)结果表明,缺血后调适能显著减轻大鼠骨骼肌缺血再灌注损伤,其机制可能通过激活再灌注损伤挽救激酶信号通路作用于线粒体通透性转换孔,限制其开放,从而增强大鼠骨骼肌对缺血再灌注损伤的耐受能力。

BACKGROUND： Reperfusion injury salvage kinase（RISK） pathway plays an important role in protective mechanism against ischemia reperfusion injury（IRI） induced by both ischemic pre-and post-conditioning. Many researches have been carried out on RISK pathway mechanism underlying ischemic post-conditioning conferring cardioprotection against IRI; however, there is less research about its effect on IRI in the skeletal muscle.OBJECTIVE： To investigate the protective effect of an optimized protocol of ischemic post-conditioning on IRI in rat skeletal muscle and its underlying mechanism. METHODS： Eighteen male Sprague-Dawley rats were equivalently randomized into IRI, ischemic post-conditioning and control groups. Rats were given occlusion or disocclusion of the right femoral artery of the right lower limb. Subsequently, the IRI group rats were subjected to 24 hours of reperfusion; the ischemic post-conditioning group immediately given 4 cycles of 30 seconds reperfusion/30 seconds ischemia, followed by 24 hours of reperfusion; the control group given no intervention. RESULTS AND CONCLUSION： Hematoxylin-eosin staining showed that in the ischemic post-conditioning group, the morphology of muscle fibers changed little, with fewer inflammatory lesions and milder edema compared with the IRI group. The infarct size with TTC staining in the ischemic post-conditioning group was smaller than that in the IRI group. Western blot analysis revealed that the expressions of phospho-Akt and phosphorylated endothelial nitric oxide synthase-S1177 were significantly increased, but the expression of phosphorylated type endothelial nitric oxide synthase-Thr495 was much decreased in the ischemic post-conditioning group compared with the IRI group. The measurement of mitochondrial permeability transition pore opening with Ca2＋ induction showed that the absorbance values in the ischemic post-conditioning group were significantly lower than those in the IRI group（P〈0.05）. These results indicate that ischemia-reperfusion injury can be improved by applying an optimal protocol of ischemic post-conditioning in rat skeletal muscle. The underlying mechanism may be associated with the activation of RISK signaling pathway to inhibit opening of mitochondrial permeability transition pore, thereby contributing to the enhanced tolerance to IRI in rat skeletal muscle.