促红细胞生成素对脓毒症大鼠心肌损伤的保护作用

The mechanisms of protective effects of erythropoietin （EPO） on sepsis induced myocardial injury in rat

目的 探讨促红细胞生成素（EPO）对脓毒症大鼠心肌损伤的保护作用及其机制.方法 通过盲肠结扎穿孔引流术（CLP）建立脓毒症大鼠模型.健康SD大鼠96只,随机（随机数字法）分为3组：假手术组（Sham组,n=32）、脓毒症组（CLP组,n=32）、EPO组（n=32,脓毒症模型基础上EPO 1000 IU/kg腹腔内注射）.分别于造模术后3、6、12、24 h记录心脏血流动力学指标变化,测定血清炎症反应因子水平及心肌酶变化,应用流式细胞仪检测心肌细胞线粒体膜电位、心肌细胞凋亡及心肌组织核因子-κB （NF-κB p65）表达水平；光镜下观察大鼠心肌细胞的病理学变化.所有数据以均数±标准差（-x±s）表示,采用SPSS 13.0统计软件分析,组间比较采用t检验,以P ＜0.05为差异具有统计学意义.结果 （1）与Sham组相比,CLP组大鼠左室收缩压（LVSP）、左室舒张末压（LVEDP）、左室压力上升最大速度（＋dp/dtmax）、左室压力下降最大速度（-dp/dtmax）均有不同程度恶化（P ＜0.05,P＜0.01）,EPO组各指标降低幅度小于CLP组（P＜0.05,P＜0.01）；（2）与Sham组相比,CLP组各时间点血清肿瘤坏死因子-α（TNF-α）、白介素-6 （IL-6）、C-反应蛋白水平（CRP）、肌钙蛋白I（cTNI）、肌酸激酶（CK）、乳酸脱氢酶（LDH）和谷草转氨酶（AST）水平及心肌组织NF-κB p65表达水平等指标均有不同程度升高（P＜0.05）,而EPO组上述各指标低于CLP组（P＜0.05）,与之相反,EPO组血清抗炎因子白介素-10 （IL-10）各时间点均高于CLP组；（3）与Sham组相比,CLP组心肌细胞凋亡率升高（P＜0.01）；（4）心肌细胞线粒体膜电位较Sham组降低（P＜0.01）,与CLP组相比,EPO组心肌细胞凋亡率明显降低,线粒体膜电位升高（P＜0.叭）；（5）应用EPO干预后,心脏组织损害的病理改变较脓毒症组减轻.结论 脓毒症大鼠中,外源性EPO可阻抑心肌细胞线粒体膜电位降低,抑制心肌细胞凋亡；同时抑制NF-κB活化,减少促炎因子生成,减轻心脏组织的炎症损伤.以上两种机制发挥保护受损心肌、改善心功能的作用.

Objective To investigate the protective effects of erythropoietin （EPO） on myocardium injury after sepsis in rats in order to clarify the mechanisms.Methods The rat models of sepsis were produced by cecal ligation and perforation method （CLP）.Ninty-six healthy SD rats were randomly （random number） divided into 3 groups：the sham operation group （Sham group,n =32）,the sepsis group （CLP group,n =32）,and the EPO treatment group （EPO group,n =32） treated with EPO 1000 IU/kg intraperitoneal injection after the CLP.The observation intervals were set at 3 h,6 h,12 h and 24 h after the surgery.The cardiac hemodynamics of the CLP group were measured.Plasma levels of inflammatory factors and myocardial enzyme indicators were determined by enzyme-linked immunosorbent assay （ELISA） ; Membrane potential levels of chondriosome of myocardial cells,cell apoptosis rates and expressions of nuclear factor-κB p65 of myocardium tissue were detected by flow cytometer; Then the pathological change of myocardium with HE staining was observed under light microscopy.Results （1） Compared with the CLP group,left ventricle systolic pressure （LVSP）,left ventricle diastolic end pressure （LVEDP）,the maximum rate of left ventricle rise and fall （＋ dp/dtmax and-dp/dtmax） in the EPO group improved （P ＜0.05,P＜0.01）; （2） Compared with the Sham operation group,plasma levels of tumor necrosis factoralpha （TNF-α）,interleukin-6 （IL-6）,C-reactive protein （CRP）,cardiac troponin-I （cTNI）,creatine kinase （CK）,lactate dehydrogenase （LDH） and glutamine-oxaloacetic transaminase （GOT） in the CLP group increased at each interval （P ＜ 0.05）,and those biomarkers in the EPO group were lower than those in the CLP group （P ＜ 0.05） ; On the contrary,plasma level of anti-inflammatory factor IL-10 in EPO group was higher than that in the CLP group （P ＜ 0.01） ; （3） Compared with the Sham operation group,the cell apoptosis rates in the CLP group increased significantly （P ＜ 0.01）,and it decreased obviously in the EPO group （P ＜ 0.01）; （4） Compared with the Sham group,membrane potential levels of chondriosome of myocardial cell in the CLP group decreased （P ＜0.01）,while it increased in the EPO group in comparison with the CLP group （P ＜ 0.01） ; （5） Pathological changes of myocardium after the CLP could be lessened by the EPO treatment.Conclusions EPO may increase membrane potential levels of chondriosome and decrease the apoptosis rates of myocardial cell in rats after sepsis,and it may reduced the production of inflammatory factors to protect the myocardial cell by down-regulating NF-κB p65.Both increased membrane potential level of chonriosome and decreased inflammatory factor may implicate in myocardium protection thereby improving cardiac function after sepsis.