血小板生成素通过PI3K/AKT通路防止CoCl_2诱导内皮细胞凋亡

Thrombopoietin Prevents CoCl_2-inducing Apoptosis of HUVEC through the PI3K/AKT Pathway

目的:研究血小板生成素(TPO)对化学性缺氧模拟剂氯化钴(CoCl_2)诱导的人脐静脉内皮细胞(HUVEC)凋亡的作用及其机制。方法:实验分为4组,体外培养的未经处理的HUVEC为对照组,CoCl_2与HUVEC共培养的为CoCl_2组,在加入CoCl_2前用TPO预处理HUVEC为TPO+CoCl_2组,TPO单独处理HUVEC为TPO组。使用CCK-8法检测各组细胞的细胞活性,流式细胞术检测细胞凋亡率、凋亡蛋白酶Caspase-3的表达以及线粒体膜电位的变化。Western blot检测TPO对AKT通路磷酸化水平表达的影响。结果:CoCl_2可以明显抑制HUVEC的生长,细胞存活率随着CoCl_2的浓度的升高逐渐下降(r=-0.997);与对照组相比较,CoCl_2组的细胞凋亡率明显升高(P<0.001),而TPO+CoCl_2组的细胞凋亡率较前者明显下降(P<0.001),提示TPO对HUVEC有保护作用。TPO还能减少细胞凋亡蛋白酶Caspase-3的表达和抑制细胞线粒体膜电位的降低,以及刺激HUVEC PI3K/AKT通路的活化。结论:TPO具有防止化学性缺氧所致的细胞凋亡作用,其机制可能是通过活化PI3K/AKT通路,从而减少细胞凋亡蛋白酶Caspase-3的活化表达和稳定线粒体膜电位来发挥细胞保护作用。

Objective ： To investigate the effect of thrombopoietin （TPO） on chemical hypoxia-induced apoptosis of human umbilical vein endothelial cells （HUVEC）, and to explore its potential mechanism. Methods： The experiment was divided into 4 groups. The untreated HUVECs were used as normal control group. HUVECs treated with CoC12 was CoC12 group, and TPO was added into the culture medium 48 h before CoC12 treatment as TPO ＋ CoC12 group. The cells was treated with TPO alone as TPO group. The cell viability and apoptosis of each groups were tested by Cell Counter Kit 8 （CCK-8） assay and flow cytometry. The expression of Caspase-3 and mitochondrial membrane potential （MMP） were then determined by flow cytometry with Caspase-3 -PE and JC-1. The effect of TPO in PI3K/AKT pathway was detected by using Western blot. Results： CoC12 significantly inhibited the growth of HUVECs. The cell viability of HUVECs decreased gradually with enhancement of CoC12 at a gradient of chemical concentrations （ r = - 0. 997）. CoC12 dramatically increased apoptosis of HUVECs, whereas pre-treatment with TPO rescued cell apoptosis induced by COC12 （P 〈 0. 001）. Further investigation found that TPO decreased the expression of Caspase-3 and inhibited the reduction of MMP indluced by CoCl2 （ P 〈 0. 05 ）. TPO could increased the activation of PI3K/AKT pathway in HUVECs. Conclusion： TPO has a protective effect against CoC12-induced apoptosis of HUVECs through activating the PI3K/AKT pathway, thus decreasing the expression of apoptosis protease Caspase-3 and inhibiting the reduction of MMP. K