Background: Oxidized low-density lipoprotein (ox-LDL)-induced oxidative stress and endothelial apoptosis are essential for atherosclerosis. Our previous study has shown that ox-LDL-induced apoptosis is mediated by the protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic translation initiation factor 2α-subunit (eIF2α)/CCAAT/enhancer-binding protein homologous protein (CHOP) endoplasmic reticulum (ER) stress pathway in endothelial cells. Statins are cholesterol-lowering drugs that exert pleiotropic effects including suppression of oxidative stress. This study aimed to explore the roles of simvastatin on ox-LDL-induced ER stress and apoptosis in endothelial cells.
Methods: Human umbilical vein endothelial cells (HUVECs) were treated with simvastatin (0.1, 0.5, or 2.5 μmol/L) or DEVD-CHO (selective inhibitor of caspase-3, 100 μmol/L) for 1 h before the addition of ox-LDL (100 μg/ml) and then incubated for 24 h, and untreated cells were used as a control group. Apoptosis, expression of PERK, phosphorylation of eIF2α, CHOP mRNA level, and caspase-3 activity were measured. Comparisons among multiple groups were performed with one-way analysis of variance (ANOVA) followed by post hoc pairwise comparisons using Tukey's tests. A value of P < 0.05 was considered statistically significant.
Results: Exposure of HUVECs to ox-LDL resulted in a significant increase in apoptosis (31.9% vs. 4.9%, P < 0.05). Simvastatin (0.1, 0.5, and 2.5 μmol/L) led to a suppression of ox-LDL-induced apoptosis (28.0%, 24.7%, and 13.8%, F = 15.039, all P < 0.05, compared with control group). Ox-LDL significantly increased the expression of PERK (499.5%, P < 0.05) and phosphorylation of eIF2α (451.6%, P < 0.05), if both of which in the control groups were considered as 100%. Simvastatin treatment (0.1, 0.5, and 2.5 μmol/L) blunted ox-LDL-induced expression of PERK (407.8%, 339.1%, and 187.5%, F = 10.121, all P < 0.05, compared with control group) and phosphorylation of eIF2α (407.8%, 339.1%, 187.5%, F = 11.430, all P < 0.05, compared with control group). In contrast, DEVD-CHO treatment had no significant effect on ox-LDL-induced expression of PERK (486.4%) and phosphorylation of eIF2α (418.8%). Exposure of HUVECs to ox-LDL also markedly induced caspase-3 activity together with increased CHOP mRNA level; these effects were inhibited by simvastatin treatment.
Conclusions: This study suggested that simvastatin could inhibit ox-LDL-induced ER stress and apoptosis in vascular endothelial cells.
辛伐他汀抑制氧化型低密度脂蛋白诱导的内质网应激及内皮细胞凋亡摘要背景：氧化型低密度脂蛋白可诱导氧化应激与内皮细胞凋亡，在动脉粥样硬化的发生与进展过程中起到重要作用。既往研究表明氧化型低密度脂蛋白可通过内质网应激的PERK/eIF2α/CHOP途径诱导血管内皮细胞凋亡。他汀类药物除降低血脂水平外，还具有抗炎、抗氧化应激等多重非调脂功能。本研究将就辛伐他汀在氧化型低密度脂蛋白诱导的血管内皮细胞内质网应激和细胞凋亡过程中的作用进行探讨。 方法：对人脐静脉内皮细胞使用辛伐他汀（0.1, 0.5, 2.5 μmol/L）及100 μmol/L的DEVD-CHO（特异性caspase-3抑制剂）孵育细胞1小时后，加入含100 μg/ml氧化型低密度脂蛋白的培养液孵育内皮细胞24小时，以未处理的细胞作为对照组；采用流式细胞仪检测内皮细胞凋亡率，采用Real-time PCR法检测CHOP mRNA的表达，采用比色法检测内皮细胞内caspase-3活性，采用western-blot法检测细胞内PERK蛋白表达及eIF2α蛋白的磷酸化。多组间比较采用单因素方差分析。 结果：与对照组（4.9%）相比，氧化型低密度脂蛋白培养内皮细胞24 小时显著增加内皮细胞凋亡率（31.9%）, P<0.05，辛伐他汀（0.1 ，0.5，2.5 μmol/L）可减轻氧化型低密度脂蛋白诱导的内皮细胞凋亡（28.0%，24.7%，13.8%，F =15.039，相对于对照组，P<0.05)。以对照组为100%，氧化型低密度脂蛋白可显著增加PERK表达（499.5%,P<0.05）和eIF2α磷酸化水平（451.6%,P<0.05），而辛伐他汀（0.1，0.5，2.5 μmol/L）可抑制氧化型低密度脂蛋白诱导的PERK表达（477.4%，397.0%，194.0%，F = 10.121，相对于对照组，P <0.05）和eIF2α磷酸化水平（407.8%，339.1%，187.5%，F = 11.430，相对于对照组，P <0.05）；而DEVD-CHO对于氧化型低密度脂蛋白诱导的PERK表达和eIF2α磷酸化水平无影响。另外，氧化型低密度脂蛋白可显著增强caspase-3活性，增加CHOP mRNA水平，而辛伐他汀可抑制此氧化型低密度脂蛋白诱导的上述效果。 结论：辛伐他汀可抑制ox-LDL诱导的内皮细胞凋亡，其机制与辛伐他汀抑制内质网应激PERK/eIF2α/CHOP/caspase-3通路有关。.
Keywords: Apoptosis; Endoplasmic Reticulum Stress; Endothelial Cells; Oxidized Low-Density Lipoprotein; Simvastatin.