促红细胞生成素后处理对大鼠再灌注损伤肺细胞凋亡的干预及机制

The interventive effects and mechanisms of erythropoietin postconditioning on celluar apoptosis of lung ischemia/reperfusion injury in rats

目的:探讨促红细胞生成素后处理对再灌注损伤肺细胞凋亡的干预作用及其机制。方法:健康雄性成年SD大鼠40只,随机分为假手术对照组(C组)、缺血/再灌注组(I/R组)、促红细胞生成素+缺血/再灌注组(EPO组)、促红细胞生成素+溶剂对照组1(0.4%DMSO的PBS溶液)(D组)和促红细胞生成素+U0126(U组)。对比观察各组肺组织湿/干重比值(W/D)的变化;原位缺口末端标记法(TUNEL)检测肺组织细胞凋亡情况,计算凋亡指数(AI);免疫组织化学方法测定肺组织Bcl-2、Bax蛋白的相对含量,RT-PCR法检测肺组织Bcl-2、Bax mRNA表达;光镜下观察肺组织的病理变化及测定肺泡损伤数(IQA)。结果:与C组比较,I/R组肺组织W/D显著升高,I/R组IQA显著升高,AI显著升高,Bcl-2蛋白和Bcl-2 mRNA表达明显下降,Bax蛋白和Bax mRNA表达明显上调,Bcl-2/Bax的比值降低(P均<0.01),肺组织形态学发生异常改变;与I/R组比较,EPO组、D组、U组的W/D显著降低,IQA显著降低,AI显著降低,Bcl-2蛋白和Bcl-2mRNA表达增强,Bax蛋白和Bax mRNA表达减弱,Bcl-2/Bax的比值增高(P<0.05或P<0.01),肺组织形态学结构异常改变有所减轻;D组的W/D、IQA、AI、Bcl-2蛋白和Bcl-2 mRNA、Bax蛋白和Bax mRNA及Bcl-2/Bax的比值与EPO组变化相近(P>0.05);与EPO组比较,U组的W/D升高,IQA升高,AI显著升高,Bcl-2蛋白和Bcl-2mRNA表达下降,Bax蛋白和Bax mRNA表达上调,Bcl-2/Bax的比值降低(P<0.05或P<0.01),U组的肺组织形态学结构损伤较EPO组加重。结论:促红细胞生成素后处理能减轻肺缺血/再灌注损伤,其机制可能通过激活ERK1/2信号转导通路,上调凋亡抑制因子Bcl-2的表达,下调促凋亡基因Bax的表达,提高Bcl-2/Bax比值,使肺组织细胞凋亡减少。

Objective： To investigate the intervention effects and mechanisms of erythropoietin postconditioning on celluar apoptosis of lung ischemia/reperfusion injury in rats. Methods： Adult male Sprague-Dawley rats were randomly divided into 5 groups based upon the intervention （n=8）： control group （C group）, Lung ischemia reperfusion group （I/R group）, I/R＋EPO（EPO group）, EPO＋solvent control 1（0.4%DMSO of the PBS solution）（D group）, EPO＋U0126（U group）. At the end of the experiment, the wet to dry wight ratio（W/D） of the lung tissue was tested; The pneumocyte apoptosis index（AI） was achieved by terminal deoxynucleotidyl transferase mediated dUTP nick end abeling（TUNEL） ; The content of Bcl-2 and Bax in cytoplasm was determined with immunohistochemistry, Bcl-2 mRNA and Bax mRNA in lung tissues were determined by RT-PCR. The pathological changes of lung tissue were observed under light microscope and the index of quantitative assessment（IQA） of histologic lung injury was counted by HE staining. Results： Compared with C group, W/D, IQA and AI of lung tissue were increased in I/R group, the expression of Bcl-2 protein and Bcl-2 mRNA were decreased, the expression of Bax protein and Bax mRNA were increased, the ratio of Bcl-2/Bax was decreased.There were abnormal morphological changes under the light microscope; Compared with I/R group, W/D, IQA and AI of lung were decreased in EPO, D and U groups, the expression of Bcl-2 protein and Bcl-2 mRNA were increased but the Bax protein and Bax mRNA were decreased, the ratio of Bcl-2/Bax was increased. The morphological changes under the light microscope markedly reversed in EPO, D and U groups;There was no statistical difference between D group and EPO group ; Compared with EPO group , W/D, IQA and AI of lung were increased in U group, the expression of Bcl-2 protein and Bcl-2 mRNA were decreased, the expression of Bax protein and Bax mRNA were increased, the ratio of Bcl-2/Bax was decreased. There were abnormal morphological changes under the light microscope in U group than EPO group. Conclusion： Erythropoietin postconditioning may attenuate pneumocyte apoptosis in LIRI by activating ERK1/2 signal transduction pathway, and up-regulating expression of Bcl-2/ Bax ratio.