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

Xu Feifei, Liu Xiuhua, Zhu Xiaomei

Department of Pathophysiology, Chinese PLA General, Hospital.Beijing 100853

Abstract

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 （H_(2)O_(2) 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 + H_(2)O_(2) group）; （4） SB203580 （a specific inhibitor of p38 MAPK） + HPC + H_(2)O_(2) group; （5） CRT antisense oligonucleotide transfection （AS group）; （6） AS + H_(2)O_(2) group; （7） AS + HPC + H_(2)O_(2) 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 H_(2)O_(2). Compared with that in H_(2)O_(2) 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 + H_(2)O_(2) group. （2） H_(2)O_(2) 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 H_(2)O_(2). （3） CRT AS transfection weakened the protection of HPC. Compared with that in HPC + H_(2)O_(2) 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 + H_(2)O_(2) 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.

Key words: calreticulin;oxidative stress;Hypoxic preconditioning;mitogen-activated protein kinases

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Citing This Article：

Xu Feifei, Liu Xiuhua, Zhu Xiaomei. Calreticulin upregulation induced by hypoxic preconditioning relieves oxidative stress injury in rat cardiomyocytes. Acta Physiol Sin 2008; 60 (1): 29-37 (in Chinese with English abstract).