μ受体在吗啡预处理减轻慢性心力衰竭大鼠心肌缺血再灌注损伤中的作用：离体实验

Role of ix opioid receptor in morphine preconditioning-induced reduction of myocardial ischemia.reperfusion injury in rats with chronic heart failure： an in vitro experiment

目的评价斗受体在吗啡预处理减轻慢性心力衰竭大鼠心肌缺血再灌注损伤中的作用。方法雄性成年SD大鼠，体重170～230g，采用尾静脉注射盐酸多柔比星的方法制备慢性心力衰竭模型。采用Langendorff模型制备离体心脏灌注模型。取慢性心力衰竭大鼠离体灌注模型制备成功的心脏40个，采用随机数字表法分为4组（n=10）：缺血再灌注组（I／R组）、吗啡预处理组（MP组）、“受体拮抗剂CTOP＋吗啡预处理组（CTOP＋MP组）和CTOP组。采用结扎左冠状动脉前降支30min，恢复灌注120min的方法制备离体心脏缺血再灌注损伤模型。MP组先平衡灌注K—H液15min，随后灌注含1μmol／L吗啡的K．H液5min，再次灌注K．H液5min，共3个循环，然后制备离体心脏缺血再灌注损伤模型。CTOP＋MP组于吗啡预处理前10min至缺血5min，灌注含1la,mol／LCTOP的K．H液。CTOP组于缺血前40min至缺血5min灌注含1p,mol／LCTOP的K_H液。于稳定灌注15min（基础状态）、再灌注5和10min时收集冠状动脉流出液，采用化学比色法检测LDH活性。再灌注120min时，采用1Trc染色法确定缺血危险区体积（AAR）、梗死区体积（IS）及IS／AAR百分比。再灌注10min时采用qRT。PCR法检测心肌Bcl-2和Bax的mRNA表达，并计算Bcl-2／Bax比值。结果与I／R组比较，MP组IS和IS／AAR百分比降低，冠状动脉流出液LDH活性降低，心肌BaxmRNA表达下调，Bcl．2mRNA表达上调，Bcl-2／Bax比值升高（P〈0．05），CTOP组和CTOP＋MP组IS和IS／AAR百分比差异无统计学意义（P〉0．05）：与MP组比较，CTOP＋MP组IS和IS／AAR百分比升高，冠状动脉流出液LDH活性升高，心肌BaxmRNA表达上调，Bcl-2mRNA表达下调，Bcl-2／Bax比值降低（P〈0．05）。结论吗啡预处理减轻慢性心力衰竭大鼠心肌缺血再灌注损伤的机制可能与激活心肌μ受体，从而维持Bcl-2／Bax基因表达平衡有关。

Objective To evaluate the role of μ opioid receptor in morphine preconditioningin duced reduction of myocardial ischemiareperfusion （I/R） injury in rats with chronic heart failure. Methods Adult male SpragueDawley rats, weighing 170230 g, in which chronic heart failure was induced by in jecting doxorubicin via the tail vein, were studied. The rats were sacrificed and their hearts were excised and perfused in a Langendorff apparatus with KH solution saturated with 95% 025% CO2 at 37 ℃. Forty isolated rat hearts with I/R injury were randomly divided into 4 groups （n = 10 each） ： group I/R, morphine preconditioning group （group MP ）, opioid receptor antagonist CTOP plus morphine preconditioning group （group CTOP＋MP） and CTOP group. Myocardial I/R was induced by occlusion of the left coronaryartery for 30 min followed by 120 min of reperfusion. In group MP, the hearts were perfused with KH solu tion for 15 min, with KH solution containing 1 mol/L morphine for 5 min and with KH solution for 5 min, 3 cycles in total, and then the model of myocardial I/R was established. The hearts were perfused with KH solution containing 1 Ixmol/L CTOP starting from 10 rain before morphine preconditioning until 5 min of ischemia in group CTOP ＋ MP. The hearts were perfused with KH solution containing 1 txmol/L CTOP starting from 40 min before ischemia until 5 min of ischemia in group CTOP. The coronary effluent was collected at 15 min of equilibration （baseline） and 5 and 10 rain of reperfusion to detect the activity of lactate dehydrogenase （LDH）. Myocardial infarct size （IS） and the area at risk （AAR） were measured by 2,3,5triphenyltetrazolium staining, and IS/AAR percentage was calculated. The expression of Bel2 and Bax mRNA was determined using uantitative realtime polymerase chain reaction, and the ratio of Bel2/Bax was calculated. Results Compared with group I/R, the IS and IS/AAR percentage were significantly de creased, the activity of LDH in coronary effluent was decreased, the expression of Bax mRNA was down regulated, the expression of Bcl2 mRNA was upregulated, and the Bel2/Bax ratio was increased in group MP （P〈0.05）, and no significant change was found in the IS or IS/AAR percentage in CTOP and CTOP＋ MP groups （P〉0. 05）. Compared with group MP, the IS and IS/AAR percentage were significantly in creased, the activity of LDH in coronary effluent was increased, the expression of Bax mRNA was upregu lated, the expression of Bcl2 mRNA was downregulated, and the Bcl2/Bax ratio was decreased in group CTOP＋MP （P〈0. 05）. Conclusion The mechanism by which morphine preconditioning reduces myocar dial I/R injury may be related to activating μ opioid receptors and thus maintaining the balance between Bcl 2 and Bax gene expression in the rats with chronic heart failure.