艾塞那肽通过抑制黄嘌呤氧化酶-活性氧类降低内皮细胞缺氧复氧损伤

摘要

目的 探究原代心肌微血管内皮细胞(CMEC)在缺氧复氧损伤(H/R)模型条件下的损伤信号通路,以及艾塞那肽(Exe)的保护机制.方法 双酶消化法体外分离培养SD大鼠CMEC,CD31和Ⅷ因子鉴定纯度.建立H/R模型并筛选Exe的最佳干预浓度.Fluo-3AM荧光探针检测H/R条件下胞内Ca2+变化.Western blotting检测黄嘌呤氧化酶(XO)表达量.DCFH-DA标记的活性氧探针检测胞内活性氧类(ROS)水平.共聚焦显微镜观察JC-1染色和细胞色素-C(Cyt-C)释放.予以Exe干预和使用小干扰RNA(siRNA)干扰技术抑制XO的表达,同步观察各指标变化情况.计量资料以均数±标准差(±s)表示,两组间比较采用t检验,多组间比较采用单因素方差分析.结果 10 nmol/L为Exe的最佳抗凋亡浓度.H/R导致胞浆内Ca2+荧光强度和Ca2+依赖的XO表达量升高,予以Exe处理可显著降低Ca2+荧光(P<0.05).予以Exe处理或使用siRNA干扰技术可显著抑制XO的表达、降低XO-ROS生成、降低H/R损伤造成的线粒体膜电位、减少Cyt-C的释放,并伴随caspase-3和caspase-9活性的降低(P<0.05).结论 H/R通过激活Ca2+-XO-ROS损伤信号通路,导致CMEC的线粒体结构功能障碍,最终诱导细胞凋亡;而予以Exe处理可通过抑制上述信号通路保护CMEC,降低H/R模型引起的细胞凋亡.%Objective To explore the specific injured signal pathways under the condition of hypoxia/reoxygenation (H/R) injury in primary cardiac microvascular endothelial cells (CMEC), and investigate the protective mechanisms of exenatide.Methods Primary CMEC cells were islolated from SD rats with double-enzyme digestion and cultured in vitro.Immunocytochemical staining of CD31 antibody and Ⅷ factors were used to identify the purity of the obtained cells.The injury model of H/R was established.MTT assay was used to detect cell viability for the optimal concentrations of exenatide.The changes of intracellular calcium under H/R condition was detected with the aid of Fluo-3AM fluorescence probe.Western blotting was employed to measure the expression of xanthine oxidase (XO).The level of intracellular reactive oxygen species (ROS) was measured by DCFH-DA labeled ROS probe.Confocal microscopy was performed to observe the JC-1 staining and cytochrome C(Cyt-C) release.After XO siRNA and exenatide pre-treatment were applied to intervent the cells, the above indices were studied again.The measurement data were expressed as mean ±standard deviation (±s).Student's t test was used for mutual groups comparison, and one-way ANOVA for multiple groups comparison.Results The optimal anti-apoptotic concentration of exenatide was 10 nmol/L.H/R injury induced the increases in the intracellular calcium level and calcium-dependent XO expression level, while exenatide pre-treatment could decrease the calcium level (P<0.05).Both siRNA and exenatide pre-treatment significantly suppressed the expression of XO, reduced the level of XO-induced ROS, decreased the H/R injury induced nitochrondrial membrane potential, reduced Cyt-C release, and decreased caspase-3 and caspase-9 activities (P<0.05).Conclusion H/R activates Ca2+-XO-ROS signaling pathway, leads to mitochondrial structural dysfunction, and finally induces cell apoptosis.exenatide pre-treatment can protect the CMEC cells by suppressing the signaling pathway and reduce apoptosis induced by H/R injury.