钠泵功能改变及内质网应激在大鼠离体心脏缺血/再灌注损伤中的作用

目的：探讨钠泵活性改变及内质网应激（ERS）在大鼠离体心脏再灌损伤中的作用及其机制。方法：将60只雄性SD大鼠随机分为6组（n=10）：正常对照组（NC组）、缺血/再灌损伤组（I/R组）、哇巴因-缺血/再灌损伤组（OUA-I/R组）、地高辛抗血清-缺血/再灌损伤组（Anti-Dig-I/R组）、Src抑制剂PP2-哇巴因-缺血/再灌损伤组（PP2-OUA-I/R组）、PLC抑制剂U73122-哇巴因-缺血/再灌损伤组（U73122-OUA-I/R组）。建立全心缺血30 min，再灌注120min的Langendorff大鼠离体心脏缺血再灌损伤模型。检测各组相同时间点心功能恢复率、冠脉流出液中乳酸脱氢酶（LDH）和肌酸激酶（CK）活性，心肌中Na⁺-K⁺-ATP酶活性和钙离子水平。流式细胞仪检测心肌细胞凋亡率，Western blot检测心肌钠泵α1亚基、葡萄糖调节蛋白78（GRP78）、C/EBP同源蛋白（CHOP）及凋亡蛋白Bcl-2/Bax的表达。结果：与I/R组相比，给予哇巴因预处理可使心功能恢复率明显下降，心肌酶漏出增多，Na⁺-K⁺-ATP酶的活性降低，心肌细胞内钙水平升高，细胞凋亡率增多，心肌钠泵α1亚基和Bcl-2表达降低，GRP78、CHOP和Bax表达升高；而Anti-Dig-I/R组与I/R组相比各指标均明显改善；给予Src抑制剂PP2或PLC抑制剂U73122后，哇巴因对心肌的损伤作用被部分阻断，表现为心功能恢复率升高，心肌酶漏出减少，Na⁺-K⁺-ATP酶的活性明显恢复，Ca²⁺水平下降，细胞凋亡率下降，心肌钠泵α₁亚基和Bcl-2表达增多，GRP78和Bax表达减少。结论：钠泵功能改变和内质网应激共同参与大鼠离体心脏缺血再灌损伤，钠泵通路（Src和PLC）介导内质网应激是引起大鼠离体心脏缺血再灌损伤细胞凋亡机制之一。

Role of changes in sodium pump activity and endoplasmic reticulum stress in the ischemia/reperfusion induced injury of isolated hearts

Objective: To investigate the roles of change in sodium pump activity and endoplasmic reticulum stress (ERS) in ischemia/reperfusion (I/R) injury of the isolated rat hearts.Methods: Sixty male SD rats were randomly divided into 6 groups (n=10 each):normal control group (NC), I/R group (I/R), ouabain-I/R group (OUA-I/R), anti-digoxin antiserum-I/R group (Anti-Dig-I/R), PP2 (Src kinase inhibitor)-ouabain-I/R group (PP2-OUA-I/R),U73122 (PLC inhibitor)-ouabain-I/R group (PP2-OUA-I/R). The isolated rat hearts were perfused on the Langendorff apparatus. Except for NC group, all the hearts were subjected to 30 min global ischemia and followed by 120 min reperfusion. The cardiac function indexes were recorded at the same time. The coronary effluent was collected for estimating the activity of lactate dehydrogenase (LDH) and creatine kinase (CK). The activity of Na⁺-K⁺-ATPase and intracellular calcium concentration in myocardial tissue were measured. Apoptosis was evaluated by Flow cytometric analysis. The expressions of sodium pump α1 subunit, glucose regulated protein(GRP78),C/EBP homologous protein (CHOP) and Bcl-2/Bax were determined by Western blot.Results: Pretreatment with ouabain significantly reduced the recovery of cardiac function, increased the levels of CK, LDH and intracellular calcium concentration, decreased the activity of Na⁺-K⁺-ATPase. In addition, ouabain markedly increased the myocardial apoptosis index, down-regulated the expressions of sodium pump α1 subunit and Bcl-2, up-regulated the expressions of GRP78,CHOP and Bax; while these changes were significantly improved in the Anti-Dig-I/R group compared with those in the I/R group; PP2 or U73122 partially blocked the effects of ouabain on myocardial I/R injury. Compared with the OUA-IR group, the recovery of cardiac function, the activity of Na⁺-K⁺-ATPase and the expressions of sodium pump α₁ subunit and Bcl-2 were significant higher, meanwhile the leakage of CK and LDH, intracellular calcium concentration, myocardial apoptosis index and the expressions of GRP78 and Bax were significantly lower in PP2-OUA-I/R and U73122-OUA-I/R group.Conclusion: Changes in sodium pump function and endoplasmic reticulum stress all participate in the process of I/R injury. Current findings further suggest that sodium pump mediates ERS by activating signals of Src and PLC pathway, which may be one of the mechanisms of apoptosis induced by I/R injury.