PCSK9通过线粒体凋亡途径介导硝基化LDL促HUVEC凋亡

PCSK9 mediates N-LDL-induced apoptosis of HUVEC via the mitochondrial apoptotic pathway

摘要:

目的]探究前蛋白转化酶枯草溶菌素9(PCSK9)在硝基化低密度脂蛋白(N-LDL)诱导的人脐静脉内皮细胞凋亡中的作用及其分子机制。 [方法]利用公开可获得的转录组数据集开展差异表达分析,以评估相关疾病中N-LDL水平与PCSK9表达的相关性。通过N-LDL处理人脐静脉内皮细胞诱导细胞凋亡,并采用Hoechst33342/碘化丙啶(PI)染色法进行检测。运用Western blot检测凋亡相关分子的表达情况。利用二氢乙啶(DHE)和JC-1探针分别检测活性氧(ROS)的生成水平及线粒体膜电位变化。采用油红O染色法评估人脐静脉内皮细胞内的脂质蓄积状况。通过siRNA技术实现PCSK9的敲低。 [结果]N-LDL处理人脐静脉内皮细胞后,与对照组和LDL组相比,细胞活力显著降低(P＜0.01)；Western blot结果显示,促凋亡蛋白Caspase-9、Caspase-3和Bax的表达显著升高(P＜0.05),而抗凋亡蛋白Bcl-2的表达则显著降低(P＜0.01)；Hoechst33342/PI荧光双染结果显示,PI阳性细胞数量增多,提示细胞发生凋亡。N-LDL以剂量依赖性方式上调胆固醇代谢关键转录因子固醇调节元件结合蛋白2(SREBP2)及其下游靶蛋白PCSK9的表达(P＜0.05),且二者变化趋势一致。油红O染色结果表明,经N-LDL处理后,细胞内脂质蓄积显著增加。流式细胞术和细胞荧光检测结果表明,经N-LDL处理后,细胞内ROS生成增多,线粒体膜电位显著降低(P＜0.05),提示线粒体功能障碍的发生。沉默PCSK9后,N-LDL诱导的人脐静脉内皮细胞线粒体功能障碍及凋亡均得到显著逆转:ROS生成减少,线粒体膜电位恢复；Caspase-9、Caspase-3和Bax的表达下调,Bcl-2的表达上调；PI阳性细胞数减少(P＜0.05)。 [结论]N-LDL通过上调SREBP2的表达,促进PCSK9的表达,进而激活线粒体凋亡通路,最终诱导人脐静脉内皮细胞凋亡。

Abstract:

Aim To investigate the role of proprotein convertase subtilisin/kexin type 9 (PCSK9) in the apoptosis of human umbilical vein endothelial cells induced by nitrated low density lipoprotein (N-LDL) and its molecular mechanism. Methods Differential expression analysis was performed using publicly available transcriptome datasets to assess the correlation between N-LDL and PCSK9 expression in relevant diseases. Human umbilical vein endothelial cells were treated with N-LDL to induce apoptosis, which was detected by Hoechst33342/propidium iodide (PI) staining. Expression of apoptosis-related molecules was assessed by Western blot. Reactive oxygen species (ROS) production and mitochondrial membrane potential were measured using dihydroethidium (DHE) and JC-1 probes, respectively. Lipid accumulation in human umbilical vein endothelial cells was evaluated by oil red O staining. PCSK9 knockdown was achieved using siRNA. Results Following N-LDL treatment of human umbilical vein endothelial cells, CCK-8 assay results demonstrated a significant decrease in cell viability compared with both the control and LDL groups (P＜0.01). Western blot analysis showed markedly increased expression of the pro-apoptotic proteins Caspase-9, Caspase-3 and Bax, accompanied by a significant reduction in the anti-apoptotic protein Bcl-2 (P＜0.01). Hoechst33342/PI double staining revealed an increased number of positive cells, indicating the occurrence of apoptosis. N-LDL upregulated the protein expression of the key cholesterol metabolism transcription factor sterol regulatory element-binding protein 2 (SREBP2) and its downstream target protein PCSK9 in a concentration-dependent manner (P＜0.05), with a consistent trend of both. Oil red O staining demonstrated that N-LDL treatment promoted intracellular lipid accumulation. Moreover, flow cytometry and fluorescence analyses showed that N-LDL increased intracellular reactive oxygen species (ROS) production and significantly reduced mitochondrial membrane potential (P＜0.05), indicating mitochondrial dysfunction. Notably, silencing PCSK9 significantly reversed these N-LDL-induced mitochondrial dysfunction and apoptotic alterations in human umbilical vein endothelial cells, as evidenced by reduced ROS production, restoration of mitochondrial membrane potential, decreased expression of Caspase-9 , Caspase-3 and Bax, upregulated Bcl-2 expression, and a decreased number of PI-positive cells (P＜0.05). Conclusion N-LDL upregulates SREBP2 expression, promotes the expression of PCSK9, thereby activating the mitochondrial apoptosis pathway, and ultmately inducing apoptosis in human umbilical vein endothelial cells.