Zhang Zhen-Ying, Liu Xiu-Hua, Guo Xiao-Sun, Liu Feng-Ying
Department of Pathophysiology, Chinese PLA General Hospital, Beijing 100853, China.
Sheng Li Xue Bao. 2007 Oct 25;59(5):643-50.
The present study was aimed to investigate the effect of ischemic postconditioning (I-postC) on ischemia/reperfusion (I/R) injury and whether calreticulin (CRT) is involved in its intracellular signal transduction both in vivo and in cultured skeletal muscle cells. I/R injury in the right hind limb of healthy male Wistar rats was induced by clamping the right femoral artery, and the rats were randomly divided into 3 groups (n=16): I/R group (4-hour ischemia/12- or 24-hour reperfusion), ischemic preconditioning (IPC) group (3 cycles of 1-minute ischemia/1-minute reperfusion) and I-postC group (3 cycles of 5-minute reperfusion/5-minute ischemia). The left hind limb was used as control. Lactate dehydrogenase (LDH) activity in blood plasma, wet/dry weight ratio (W/D) and ultramicrostructure of skeletal muscle were detected 12 h or 24 h after reperfusion. Cultured skeletal muscle cells from neonatal Sprague-Dawley (SD) rat were divided into 6 groups: hypoxia/reoxygenation (H/R) group, hypoxic postconditioning (H-postC) group, hypoxic preconditioning (HPC) group, cyclosporine A (CsA) + H-postC group, CsA + H/R group and control group. H/R was produced by 2-hour hypoxia/24-hour reoxygenation. The survival rate and apoptotic rate of skeletal muscle cells in each group were measured. Western blot was used to detect the expressions of CRT and calcineurin (CaN). The results were as follows: (1) During in vivo experiment, compared with I/R, I-postC significantly decreased LDH activity and W/D, attenuated the ultramicrostructure injury of skeletal muscle and the apoptosis of nucleolus. 12 h and 24 h after reperfusion, compared with that in I/R group, the expression of CRT in I-postC group increased by 439% and 102%, respectively (P<0.05), and the expression of CaN increased by 196% and 63%, respectively (P<0.05). Correlation analysis indicated a positive correlation between CRT and CaN expressions (r=0.865, P<0.01). (2) In cultured skeletal muscle cells, H-postC attenuated cell injury induced by H/R. Compared with those in H/R group, CRT and CaN expressions in H-postC increased by 31.8% (P<0.05) and 6.02%, respectively. The protection of H-postC and CaN up-regulation were eliminated when CsA, the inhibitor of CaN, was added before H-postC. Both in vivo and in vitro results indicate that I-postC, similar as IPC, can protect the skeletal muscle against I/R injury, and its effects may be mediated by CRT and CaN up-regulation. The inhibition of CaN expression may also attenuate the protective effects of I-postC.
本研究旨在探讨缺血后处理（I-postC）对缺血/再灌注（I/R）损伤的影响，以及钙网蛋白（CRT）是否参与其在体内和培养的骨骼肌细胞中的细胞内信号转导。通过夹闭右侧股动脉诱导健康雄性Wistar大鼠右后肢I/R损伤，并将大鼠随机分为3组（n = 16）：I/R组（4小时缺血/12或24小时再灌注）、缺血预处理（IPC）组（3个周期，每次1分钟缺血/1分钟再灌注）和I-postC组（3个周期，每次5分钟再灌注/5分钟缺血）。以左后肢作为对照。再灌注12小时或24小时后检测血浆乳酸脱氢酶（LDH）活性、骨骼肌湿/干重比（W/D）及超微结构。将新生Sprague-Dawley（SD）大鼠的培养骨骼肌细胞分为6组：缺氧/复氧（H/R）组、缺氧后处理（H-postC）组、缺氧预处理（HPC）组、环孢素A（CsA）+H-postC组、CsA+H/R组和对照组。通过2小时缺氧/24小时复氧建立H/R模型。检测每组骨骼肌细胞的存活率和凋亡率。采用蛋白质免疫印迹法检测CRT和钙调神经磷酸酶（CaN）的表达。结果如下：（1）在体内实验中，与I/R组相比，I-postC组显著降低了LDH活性和W/D，减轻了骨骼肌超微结构损伤和核仁凋亡。再灌注12小时和24小时后，与I/R组相比，I-postC组CRT表达分别增加了439%和102%（P<0.05），CaN表达分别增加了196%和63%（P<0.05）。相关性分析表明CRT与CaN表达呈正相关（r = 0.865，P<0.01）。（2）在培养的骨骼肌细胞中，H-postC减轻了H/R诱导的细胞损伤。与H/R组相比，H-postC组CRT和CaN表达分别增加了31.8%（P<0.05）和6.02%。在H-postC前加入CaN抑制剂CsA后，H-postC的保护作用及CaN上调作用均被消除。体内和体外实验结果均表明，I-postC与IPC相似，可保护骨骼肌免受I/R损伤，其作用可能通过上调CRT和CaN介导。抑制CaN表达也可能减弱I-postC的保护作用。
