促红细胞生成素预防内皮细胞凋亡时对天冬氨酸特异性半胱氨酸蛋白酶3亚家族的影响

Effect of erythropoietin on the caspase-3 subfamily in preventing apoptosis of endothelial cell

目的探讨重组人促红细胞生成素（rhEPO）在预防氧化低密度脂蛋白（ox—LDL）诱导人脐静脉内皮细胞（HUVEcs）凋亡过程中对天冬氨酸特异性半胱氨酸蛋白酶3（caspases一3）亚家族的影响。方法体外培养HUVECs，将细胞随机分组，分别加入caspase一3抑制剂DEVD—CHO、caspase一8抑制剂z—IETD—fmk、caspase-9抑制剂z-LEHD—Imk各25μmol／L预孵育24h后，再加入100mg／LOX—LDL处理48h。平行实验中，加入6．25、25．00、100．00kU／L的rhEPO预处理HUVECs24h，再加入100mg／LOX—LDL处理48h。用比色法检测caspase一3、caspase一8、caspase一9活性；用四甲基偶氮唑盐（MTT）比色法检测HUVECs活性；用流式细胞术检测caspase一3阳性表达率和HuVECs凋亡率。结果caspase一3抑制剂、caspase～8抑制剂分别降低各自对应的caspase活性，caspase一9抑制剂可同时降低caspase一3、caspase一9活性（P均〈O．05）。加入rhEPO预处理后，OX—LDL诱导内皮细胞caspase一3、caspase一9活性及caspase一3阳性表达率降低，且呈剂量依赖性（P均〈0．05），而caspase一8活性无明显变化，HUVECs的细胞凋亡率也呈剂量依赖性降低（P均〈0．05）。结论rhEPO可呈剂量依赖性抑制OX—LDL诱导HUVECs凋亡时caspase一3、caspase一9活性，提示OX—LDL诱导HUVECs凋亡信号通路涉及caspase一3、caspase一9，而与caspase一8无关。

Objeetive To explore effect of erythropoietin on the easpase-3 subfamily in preventing apoptosis of human umbilical vein endothelial cells （HUVECs） induced by oxidized-low density lipoprotein （ox-LDL）. Methods Third- sixth passages of HUVECs were used. Two experiments were conducted. In the first experiment, there was a blank control group, ox-LDL control group （100 mg/L, incubated for 48 hours）, and low, medium, and high recombinant human erythropoietin （rhEPO） groups （6.25, 25.00, 100.00 kU/L rhEPO incubated for 24 hours ＋ 100 mg/L ox-LDL ineubated for 48 hours）. Another experimental protocol consisted of groups of the cells pretreated with either easpase-3 inhibitor DEVD-CHO, or easpase-8 inhibitor z-IETD-fmk, or easpase-9 inhibitor z-LEHD-fmk of 25 μmol/L for 24 hours, then HUVECs were exposed ox-LDL （100 rag/L） incubated for 48 hours. The activity of easpase-3, easpase-8, or easpase 9 was determined by easpase eolorimetrie assay. The cell survival rate was assessed with methyl thiazolyl tetrazolium （MTT） method. The positive expression rate of easpase-3 and apoptotic rate were measured by flow eytometer. Results The activity of easpase-3 was significantly decreased and cell survival rate was increased in the easpase-3 inhibitor group （both P〈0.05）. The activity of easpase-8 was decreased in the easpase-8 inhibitor group （P〈0. 05）, but the cell survival rate was not signifieantly different from that of ox-LDL group （P〉0. 05）. The activity of easpase-3 or easpase-9 was lower and cell survival rate was higher in the easpase-9 inhibitior group than that of ox-LDL group （all P〈0.05）. The pretreatment with rhEPO led to decreased activity of caspase-3, easpase-9, positive expression rate of caspase-3 and apoptotie rate in a dose-dependent manner compared with ox-LDL group （all P〈0. 05）, but the aetivity of easpase-8 showed no significant differenee from rhEPO pretreatment groups （all P〉0. 05）. Conclusion These results demonstrate that rhEPO can significantly inhibit the activity of easpase-3 or caspase-9 in endothelial cell apoptosis in a dose-dependent manner. Activation of easpase-3 or easpase-9 is involved in ox-LDL-induced HUVECs apoptotic signaling pathway, but caspase-8 is not involved.