Zhou Yi-jun, Wang Jia-he, Zhang Jin
Department of Endocrinology and Metabolism, the First Affiliated Hospital, China Medical University, Shenyang 110001.
Chin Med Sci J. 2006 Mar;21(1):6-10.
To investigate the effects of hepatocyte growth factor (HGF) on vascular endothelial cells apoptosis induced by advanced glycation end products (AGEs) and its possible mechanism.
Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and intervened by different concentrations of AGEs and HGF. The cell inhibitory rates of each group with different culture time (12, 24, 48, and 72 hours) were measured by methyl thiazolyl tetrazolium (MTT) assay. The early stage apoptosis was detected by flow cytometry with Annexin V-FITC/PI double staining, morphology of cell apoptosis was observed by hoechst 33258 fluorescence staining, and the expression of apoptosis-associated genes Bax and Bcl-2 were determined by Western blotting. The activity of caspase-3 was detected by enzyme-linked immunosorbent assay (ELISA).
Morphological observation indicated that high concentration of AGEs induced characteristic apoptotic changes in HUVECs. Within a certain concentration range, HUVECs apoptosis inducing rates by AGEs were in both dose- and time-dependent manners. HGF significantly inhibited the apoptosis of HUVECs induced by AGEs (P < 0.05). AGEs significantly promoted expression of Bax protein, but not Bcl-2. Whereas HGF significantly promoted the expression of Bcl-2 (P < 0.01) and decreased the activity of caspase-3 (P < 0.05) without affecting Bax level.
AGEs can induce the apoptosis of endothelial cells in vitro. HGF may effectively attenuate AGEs-induced endothelial cells apoptosis through upregulating Bcl-2 gene expression and inhibiting caspase-3 activation.
探讨肝细胞生长因子（HGF）对晚期糖基化终末产物（AGEs）诱导的血管内皮细胞凋亡的影响及其可能机制。
体外培养人脐静脉内皮细胞（HUVECs），用不同浓度的AGEs和HGF进行干预。采用甲基噻唑基四氮唑（MTT）法检测不同培养时间（12、24、48和72小时）各组的细胞抑制率。通过Annexin V-FITC/PI双染流式细胞术检测早期凋亡，用hoechst 33258荧光染色观察细胞凋亡形态，通过蛋白质免疫印迹法检测凋亡相关基因Bax和Bcl-2的表达。采用酶联免疫吸附测定（ELISA）法检测caspase-3的活性。
形态学观察表明，高浓度的AGEs可诱导HUVECs出现特征性凋亡变化。在一定浓度范围内，AGEs诱导HUVECs凋亡的比率呈剂量和时间依赖性。HGF显著抑制AGEs诱导的HUVECs凋亡（P<0.05）。AGEs显著促进Bax蛋白表达，但不影响Bcl-2表达。而HGF显著促进Bcl-2表达（P<0.01）并降低caspase-3的活性（P<0.05），且不影响Bax水平。
AGEs可在体外诱导内皮细胞凋亡。HGF可能通过上调Bcl-2基因表达和抑制caspase-3激活有效减轻AGEs诱导的内皮细胞凋亡。
