缺氧后处理对缺氧/复氧心肌细胞的保护作用及其机理研究

The Protective Effect of Hypoxic Postconditioning on Ischemia/reperfusion-Induced Myocardial Cell Injury and Its Mechanism

目的缺血后处理(ischemic postconditioning,I-postC)是近年发现的机体重要内源性保护机制。本实验在心肌细胞缺氧/复氧(hypoxia/reoxygenation,H/R)模型上观察H/R及缺氧后处理(hy-poxic postconditioning,H-postC)对GRP78、CRT表达以及caspase-12活化的影响,探讨内质网应激(endo-plasmic reticulum stress,ERS)在H-postC保护机制中的意义及其细胞信号转导机制。方法原代培养的Sprague-Dawley乳鼠心肌细胞随机分为6组:H/R组、H/R+H-postC组、HPC+H/R组、SB203580组、SP600125组和对照组。以细胞存活率、乳酸脱氢酶(lactate dehydrogenase,LDH)漏出及流式细胞术分析细胞凋亡率反映细胞损伤情况;RT-PCR检测GRP78mRNA表达水平;Western blot方法检测CRT表达和caspase-12活化以及p38MAPK、JNK磷酸化水平。结果(1)H-postC具有细胞保护作用,与H/R组比较,H/R+H-postC组细胞凋亡率和LDH漏出分别低3.3%和62.2%,存活率高10.3%;H-postC前以特异性p38MAPK抑制剂SB203580预孵育减弱H-postC的保护作用,与H/R+H-postC组相比,细胞凋亡率和LDH漏出分别高1.9%和2.8倍,存活率降低11.0%,JNK特异性抑制剂SP600125预孵育对H-postC的保护作用无明显影响。(2)H/R可明显上调GRP78mRNA水平(较对照组高1.6倍)、CRT蛋白水平(较对照组高3.0倍)和caspase-12活化(较对照组高7.9倍);H-postC减低CRT过表达程度(较H/R低34.6%)及caspase-12活化水平(较H/R低50.1%)。(3)缺氧后H-postC前应用p38MAPK抑制剂,可轻度增高caspase-12的活化水平,并抑制CRT表达上调;应用SP600125抑制JNK活化可明显减低caspase-12活化水平(较H/R+H-postC组低68.4%),并减低H-postC对H/R诱导CRT过表达的抑制作用(其CRT蛋白相对水平较H/R+H-postC组高47.2%)。结论H-postC可调控ERS反应程度,抑制H/R诱导的过度ERS,减轻内质网凋亡信号介导的细胞凋亡的发生。p38MAPK及JNK信号途径在H/R诱导ERS反应及H-postC减轻H/R后ERS反应过激程度中具有重要意义。

Objective Ischemic postconditioning（I-postC） is an important endogenous protective mecharfism newly found to protect the tissue from ischemia/reperfusion injury（I/R）. This study tested the roles of endoplasmic reticulum stress（ERS） in eardioprotection of hypoxic postconditioning（ H-postC）. Methods Neonatal cardiomyocytes were prepared from Sprague Dawley rats aged 24 hours, and cultured in DMEM medium containing 10% fetal bovine serum, and then randomly divided into six groups as follows： H/R, H/R ＋ H-postC, HPC ＋ H/R, SB203580（the specific inhibitors of p38 MAPK）, SP600125（ the specific inhibitors of JNK）, and control. H/R was produced by H-2 h/R-14 h, and H-postC by 3 circles of R-5 min/H-5 min at the beginning of reoxygenation after 2 hours＇ hypoxia, and HPC by H-20 min/R-24 h before H/R. The inhibitors were added to the medium at the beginning of H-postC. Morphological studies, estimation of lactate dehydrogenase（LDH） leakage, and Flow Cytometry were employed to assess cell apoptosis and necrosis. The level of GRP78 mRNA was detected by reverse transcription polymerase chain reaction（RT-PCR）. CRT expression and caspase-12 activation, levels of phospho-p38 MAPK and phospho-JNK were detected by Western blot analysis. All experiments were repeated at least four separate times. Variables were analyzed by one-way ANOVA for multiple comparisons, and a P value 〈 0.05 was considered significant. Results （1） H-postC relieved cell injury caused by H/R. Compared with that of H/R group, cells＇ survive rate of H/R ＋ H-postC group increased 10.3%, and apoptosis rate and LDH in the cell culture medium decreased 3.3% and 62.2%, respectively. （2）H/R induced up-expression of GRP 78（ 1.6-fold increase）, CRT（3.0-fold increase） and caspase-12 activation（ 7.9-fold increase）. H-postC was found to relieve the over-expression of CRT induced by H/R（34.6% decrease）, and to inhibit the activation of caspase-12（50.1% decrease）. （3）When the inhibitor of p38 MAPK was present before H-postC, the protection of H-postC and the inhibition of CRT overexpression and caspase-12 activation were restrained. Using inhibitor of JNK before H-postC resulted in lower level of activated caspase-12, but had no markedly effect on the cell apoptosis rate. Conclusion These results suggest that H-postC protects the neonatal cardiomyocytes from H/R injury, partly through regulating ERS response and relieving severe ERS-induced apoptosis during H/R. P38 MAPK activation and JNK inhibition induced by H-postC serve as the important pathways of signal transduction.