低氧预处理通过上调钙网蛋白表达减轻大鼠心肌细胞氧化应激损伤

Calreticulin upregulation induced by hypoxic preconditioning relieves oxidative stress injury in rat cardiomyocytes

本文旨在探讨钙网蛋白(calreticulin,CRT)是否参与低氧预处理(hypoxic preconditioning,HPC)对心肌细胞氧化应激损伤的保护及其信号转导过程。将原代培养的Sprague-Dawley乳鼠心肌细胞随机分为8组:氧化应激(H2O2)组、短暂低氧(HPC)组、HPC+H2O2组、SB203580(p38 MAPK特异性抑制剂)+HPC+H2O2组、干扰心肌细胞CRT表达的反义寡核苷酸(antisense oligodeoxynucleotides,AS)组、AS+H2O2组、AS+HPC+H2O2组和对照组,以细胞存活率、乳酸脱氢酶(lactate dehydrogenase,LDH)漏出及流式细胞术检测细胞损伤情况;采用RT-PCR和Westernblot分别检测CRT表达和p38MAPK磷酸化水平。结果表明:(1)HPC可减轻氧化应激损伤,与H2O2组比较,HPC+H2O2组细胞存活率增高18.0%,细胞凋亡率和LDH漏出分别降低19.4%和53.0%(均P<0.05);HPC前以SB203580预孵育可消除HPC保护作用,与HPC+H2O2组相比,SB203580+HPC+H2O2组细胞凋亡率和LDH漏出分别增高13.1%和96.0%,存活率降低7.3%(均P<0.05);(2)氧化应激明显上调CRT表达(H2O2组较对照组高7.1倍,P<0.05);HPC也诱导CRT表达上调(HPC组较对照组高2.4倍,P<0.05),但上调程度较H2O2组低59%(P<0.05);即HPC可减轻氧化应激诱导的CRT过表达;(3)AS干扰CRT表达后,HPC保护作用降低,相关性分析显示HPC诱导的CRT适度表达与细胞存活率呈正相关(r=0.8023,P<0.05);(4)HPC前SB203580预孵育可抑制CRT表达上调(分别较HPC+H2O2组和HPC组低75%和53%,均P<0.05)。上述结果提示,HPC可能通过p38MAPK信号途径诱导CRT表达上调,减轻心肌细胞氧化应激损伤。

The present study was aimed to investigate whether calreticulin （CRT） was involved in the protective effect of hypoxic preconditioning （HPC） against oxidative stress injury in rat cardiomyocytes. Neonatal cardiomyocytes were prepared from Sprague- Dawley rats aged 24 h, and cultured in DMEM medium containing 10% fetal bovine serum. The cultured cardiomyocytes were randomly divided into 8 groups as follows： （1） hydrogen peroxide stress （H2O2 group）; （2） brief hypoxic exposure for 20 min to simulate HPC （HPC group）; （3） hypoxic exposure for 20 min followed by normoxic reoxygenation for 24 h before hydrogen peroxide stress （HPC ＋ H2O2 group）; （4） SB203580 （a specific inhibitor of p38 MAPK） ＋ HPC ＋ H2O2 group; （5） CRT antisense oligonucleotide transfection （AS group）; （6） AS ＋ H2O2 group; （7） AS ＋ HPC ＋ H2O2 group; （8） control group. Morphological observation, lactate dehydrogenase （LDH） leakage and flow cytometry were employed to assess cell apoptosis and necrosis. RT-PCR and Western blot were used to detect CRT expression and activity of p38 MAPK. All experiments were repeated at least four separate times. The results obtained were as follows： （1） HPC relieved cell injury caused by H2O2. Compared with that in H2O2 group, the cell survival rate increased by 18.0% （P〈0.05）, apoptotic rate and LDH leakage in culture medium decreased by 19.4% and 53.0%, respectively （P〈0.05） in HPC ＋ H2O2 group. （2） H2O2 induced CRT over-expression （7. 1-fold increase compared with control, P〈0.05）, while HPC resulted in mild CRT up-regulation （2.4-fold increase compared with control, P〈0.05）, suggesting that HPC can relieve the over-expression of CRT induced by H2O2. （3） CRT AS transfection weakened the protection of HPC. Compared with that in HPC ＋ H2O2 group, the cell survival rate decreased by 4% （P〈0.05）, and apoptotic rate and LDH leakage in culture medium increased by 2.6% and 39.0%, respectively （P〈 0.05） in AS ＋ HPC ＋ H2O2 group. （4） The protection of HPC and HPC-induced upregulation of CRT were almost eliminated when SB203580 was administered before HPC. These results suggest that HPC up-regulates CRT expression through the p38 MAPK signaling pathway and protects cardiomyocytes from oxidative stress injury.