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Calreticulin (CRT), an important Ca2+-binding molecular chaperone in the endoplasmic reticulum (ER), and caspase-12, a pivotalmolecule mediating ER-initiated apoptosis, are involved in the ER stress (ERS). Using primary cultured neonatal cardiomyocytes, CRTand caspase-12 expression and activation during hypoxic preconditioning (HPC) and hypoxia/reoxygenation (H/R) were studied toexplore the role of ERS in cardioprotection by HPC. And by using SB203580 and SP600125 [the specific inhibitors of p38 mitogenactivatedprotein kinase (MAPK) and c-Jun N-terminal kinase (JNK) ] separately, the role of p38 MAPK in HPC-induced ERS wasalso detected. Neonatal cardiomyocytes were prepared from Sprague-Dawley rats aged 24 h, and cultured in DMEM medium containing10% fetal bovine serum, and then randomly divided into six groups as follows: H/R, HPC+H/R, SB203580+HPC+H/R, SP600125+HPC+H/R, HPC and control groups. H/R was produced by 2-hour hypoxia/14-hour reoxygenation, and HPC by 20-minutehypoxia/24-hour reoxygenation. Morphological studies, estimation of lactate dehydrogenase (LDH) leakage and flow cytometry wereemployed to assess cell apoptosis and necrosis. CRT and caspase-12 expression and activation, levels of phospho-p38 MAPK andphospho-JNK were detected by Western blot. All experiments were repeated at least four separate times. The results obtained are asfollows: (1) HPC relieved the cell injury caused by H/R. Compared with that in H/R group, cells’ survival rate in HPC+H/R goup increasedby 6.4%, and the apotosis rate and LDH leakage in the cell culture medium decreased by 6.6% and 70.0%, respectively. (2) H/R inducedcaspase-12 activation (33.2-fold increase in comparison with control) and CRT expression (8.1-fold increase in comparison with control).HPC itself resulted in mild CRT up-regulation (2.6-fold increase in comparison with control), but the extent of up-regulation was lowerthan that induced by H/R. HPC before H/R was found to relieve the over-expression of CRT induced by H/R (72.4% decrease), and toinhibit the activation of caspase-12 (59.6% decrease). (3) The protection of HPC and HPC-induced up-expression of CRT andinhibition of caspase-12 activation were almost eliminated when the inhibitor of p38 MAPK, not of JNK, was present before HPC.These results suggest that HPC protects the neonatal cardiomyocytes from severe ERS-induced apoptosis during sustained H/Rthrough pre-invoking proper ERS response. Mild up-expression of CRT and inhibition of caspase-12 activation induced by HPC,which are important protection factors, are mediated by p38 MAPK, not by JNK.