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在低氧预处理延迟心肌保护中钙网蛋白表达升高

徐菲菲, 付艳, 刘凤英, 祝筱梅, 刘秀华

解放军总医院病理生理研究室.北京 100853

摘要

该文分别在整体实验和细胞培养条件下研究钙网蛋白（calreticulin，CRT）在低氧预处理（hypoxic preconditioning，HPC）延迟心肌保护中的表达及其信号转导机制。（1）整体实验时Wistar大鼠随机分为3组：假手术（sham）组、仅结扎冠状动脉的心肌缺血（myocardial infarction，MI）组和HPC后再结扎冠状动脉的HPC+MI组，分别于术后24h、14d和28d观察HPC对缺血后心功能和梗死区、危险区面积的影响；采用Western blot检测CRT表达以及p38丝裂素活化蛋白激酶（p38 mitogen--activated protein kinase，p38MAPK）、应激活化蛋白激酶（stress--activated protein kinase，SAPK）活性。（2）原代培养Sprague--Dawley乳鼠心肌细胞，随机分为6组：低氧，复氧（hypoxia／reoxygenation，H／R）组、HPC组、HPC+H，R组、p38 MAPK抑制剂SB203580+HPC+H／R（SB+HPC+H／R）组、SAPK抑制剂SP600125+HPC+H/R（SP+HPC+H／R）组和正常对照组；采用台盼蓝排斥实验、乳酸脱氢酶（1actate dehydrogenase，LDH）活性检测及流式细胞仪检测各组细胞损伤情况；采用Western blot检测CRT表达及p38 MAPK、SAPK的磷酸化水平。主要结果如下：（1）整体动物实验结果表明，HPC改善缺血对心肌左室压力最大上升，下降速度（±dp／dt_(max)）的抑制，限制心肌梗死面积；HPC后CRT表达呈动态变化：术后24h时HPC+MI组CRT表达增高106％（{sl P}<0.05 vs MI组），以危险区最为显著；14d至28d表达逐步降低。相关分析显示，术后24h时CRT表达量与心功能呈正相关（{sl r}=0.9867，{sl P}<0.05），与梗死面积呈负相关（{sl r}=--0.9709，{sl P}<0.05）。（2）细胞培养实验结果表明，HPC可减轻H／R诱导的心肌细胞LDH漏出，增加心肌细胞存活率，降低细胞凋亡；单纯HPC可诱导CRT表达轻度增加（222％，{sl P}<0.05 vs 对照组），而损伤性H／R诱导CRT过表达（503％，{sl P}<0.05 vs 对照组），HPC可降低H／R诱导CRT表达升高的幅度；p38 MAPK活性与HPC诱导的CRT表达呈正相关（{sl r}=0.9021，{sl P}<0.05），而SAPK活性与其呈负相关（{sl r}=--0.8211，{sl P}<0.05）。由此得出结论：（1）整体实验中HPC可明显改善缺血后心脏的收缩与舒张功能，限制心肌梗死范围，促进危险区心肌恢复；心肌梗死早期，HPC诱导CRT表达上调，参与心肌保护；（2）细胞培养实验中HPC可诱导CRT适度表达，增强原代培养乳鼠心肌细胞对H／R损伤的抵抗力；p38 MAPK可能介导HPC诱导的CRT表达，而SAPK激活可能不利于心肌保护。

关键词： 钙网蛋白; 低氧; 缺血预处理; 丝裂素活化蛋白激酶

Calreticulin expression increases during delayed cardioprotection induced by hypoxic preconditioning

Xu Feifei, Fu Yan, Liu Fengying, Zhu Xiaomei, Liu Xiuhua

Department of Pathophysiology, Chinese People's Liberation Army General Hospital.Beijing 100853

Abstract

Both in rive and cultured cardiomyocyte experiments were performed to investigate the alteration of expression of calreticulin （CRT） during the delayed cardioprotection induced by hypoxic preconditioning （HPC） and the intracellular signal transduction mechanisms of the alteration. （1） Wistar rats wore randomly divided into three groups： sham operation group （Sham）, myocardial infarction （MI） group induced by left coronary artery ligation and HPC+MI group （4-hour HPC 24 h before MI）. Twenty-four hours, 14 d and 28 d after left coronary artery ligation, myocardial function, infarction size and the area at risk were measured. Western blot was used to detect the expression of CRT, the activity of p38 mitogen-activated protein kinase （MAPK） and stress-aedvated protein kinase （SAPK）. （2） Cultured cardiomyocytes from neonatal Sprague-Dawley （SD） rat were divided into six groups： hypoxia/ reoxygenation （H/R）, HPC, HPC+H/R, p38 MAPK inhibitor SB203580+HPC+H/R （SB+HPC+H/R）, SAPK inhibitor SP600125+HPC+H/R （SP+HPC+H/R） and control. Survival rate and apoptosis rate of cardiomyocytes 6 h after H/R and activities of lactate dehydrogenase （LDH） in culture medium in each group were measured. Western blot was used to detect the expression of CRT and activities of p38 MAPK and SAPK. The results are as follows： （1） During in vivo experiment, compared with MI group, HPC significantly improved ±dp/dt_(max) and -dp/dt_(max), reduced infarction size and the area at risk. HPC dramatically changed the expression of CRT. CRT expression in HPC+MI group was 206% of that in MI group （P〈0.05） 24 h after infarction, especially in the area at risk. However, 28 d after operation, the expression of CRT decreased by 57%. Correlation analysis indicated a positive correlation between CRT expression and myocardial function （r = 0.9867, P〈0.05）, and negative correlation between CRT expression and infarction size （r = -0.9709, P〈0.05）. （2） In cultured cardiomyocytes, HPC attenuated cell injury induced by H/R. CRT expression increased moderately to 222% of control （P〈0.05） during HPC, but increased dramatically to 503% of control （P〈0.05） after H/R. HPC reduced H/R-induced CRT up-regulation to 56% of that in H/R group （P〈0.05）. Correlation analysis indicated that CRT expression induced by HPC had a positive correlation with p38 MAPK activity （r = 0.9021, P〈0.05）, but a negative correlation with SAPK activity （r = -0.8211, P〈 0.05）. Both in vivo and in vitro results indicate that HPC protects myocardium from ischemia or H/R injury, p38 MAPK is possibly involved in the up-regulation of CRT induced by HPC, while SAPK has a negative influence.

Key words: calreticulin;Hypoxia;ischemic preconditioning;Mitogen-activated protein kinase

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引用本文：

徐菲菲, 付艳, 刘凤英, 祝筱梅, 刘秀华. 在低氧预处理延迟心肌保护中钙网蛋白表达升高[J]. 生理学报 2006; 58 (6): .

Xu Feifei, Fu Yan, Liu Fengying, Zhu Xiaomei, Liu Xiuhua. Calreticulin expression increases during delayed cardioprotection induced by hypoxic preconditioning. Acta Physiol Sin 2006; 58 (6): (in Chinese with English abstract).