基于内质网应激PERK-eIF2α信号通路的胆固醇稳态调节机制及冠心康干预效应

On the base of our previous study data and the latest progress of cholesterol homestasis. We proposed the hypothesis that PERK-eIF2α endoplasmic reticulum stress signaling pathways will be activated by intaking high cholesterol in a long-term and continuously, then, it will cause hepatic cholesterol metabolism disorders and cholesterol ectopicdeposition in the aortic intima cells. Therefore, we design the research program, firstly, to establish rats model of hypercholesterolemia and treating with Guan-xin-kang prescription. Then, the liver cholesterol metabolism molecules and the aortic cholesterol homeostasis molecules will be observed in three different time windows. Secondly, to induce high cholesterol liver cells model and macrophages model and treating with Guan-xin-kang prescription, and cholesterol homeostasis critical molecular will also be detected different time point. For example, GRP78, SREBP2, LDLR, HMG-CoAR, ABCA1 and ABCG1 gene expression in liver tissue and HepG2 cells will be detected by RT-qPCR, miR-33a and miR-122 are also be tested. GRP78, ATF4, CHOP, GADD34 and Bcl-2 gene expression in aortic tissue and macrophages will be also detected by RT-qPCR. And p-PERK, p-eIF2α, SREBP2, LDLR, CHOP et al protein level will be detected by western blot. Moreover, blood lipids, intracellular cholesterol and aortic pathological changes will also be tested. According to the overall view of TCM, we will analysis the causes of cholesterol metabolism imbalance in liver cells and cholesterol ectopicdeposition in the aortic cells during the process of hypercholesterolemia formation. And the target genes and the best treatment time point may be demonstrated by Guan-xin-kang prescription. The study will helpful for increaseing TCM theory of liver stores blood and heart leads blood, and to establish the foundation for in-depth research in the future. It is important significance in academic.

研究表明，长期的、持续性高胆固醇摄入可激活PERK-eIF2α内质网应激信号通路，进而引起肝细胞胆固醇代谢失调并异位沉积于主动脉内膜细胞。基于此，本研究拟从实验动物与细胞开展研究：建立高胆固醇血症模型大鼠，分别观察3个时间窗口内肝脏胆固醇代谢分子、主动脉胆固醇稳态分子的动态变化及其冠心康的作用；建立不同程度高胆固醇的肝细胞模型与巨噬细胞模型，观察胆固醇稳态失衡的关键分子动态变化及其冠心康的作用。采用分子生物学技术检测胆固醇稳态相关分子在mRNA水平、miRNA水平与蛋白表达水平的变化，常规检测血脂、细胞内胆固醇含量及主动脉病理变化。从中医整体观分析高胆固醇血症形成过程肝细胞胆固醇代谢失衡及其主动脉异位沉积的动态表现，阐明冠心康对胆固醇稳态失衡的可能作用靶点与最佳作用时点。该研究有助于丰富中医肝藏血、心主血脉理论，为后续深入研究奠定基础，具有有一定的学术意义。

研究表明，长期的、持续性高胆固醇摄入可激活PERK-eIF2α内质网应激信号通路，进而引起肝细胞胆固醇代谢失调并异位沉积于主动脉内膜细胞。.本研究从实验动物与细胞开展研究。(1)建立高胆固醇血症模型大鼠，分别观察3个时间窗口:造模4周、8周、12周，高胆固醇血证模型大鼠血脂、主动脉病理变化，并采用分子生物学技术观察肝脏胆固醇代谢分子、主动脉胆固醇稳态分子的动态变化及其冠心康的作用；(2)采用GepG2细胞建立高胆固醇的肝细胞模型、THP-1细胞建立巨噬细胞模型，观察细胞内胆固醇含量变化，油红染色法检测细胞内胆固醇沉积，研究胆固醇稳态失衡的关键分子动态变化及其冠心康的作用。采用分子生物学技术检测胆固醇稳态相关分子在mRNA水平、miRNA水平与蛋白表达水平的变化。.体内实验研究发现，冠心康可显著上调高脂血症大鼠肝脏Ldlr基因及蛋白表达水平，与模型组比较，P＜0.05；并于治疗4～8周和8～12周后，显著上调高脂血症大鼠肝脏Srebp2基因及蛋白表达水平，与模型组比较，P＜0.05，且随着时间延长，表达逐渐上升。冠心康治疗4周后，高脂血症大鼠肝脏Abca1基因表达显著升高，与模型组比较，P＜0.05。体外实验研究表明，成功建立胆固醇代谢细胞模型及巨噬/泡沫细胞模型基础上，冠心康可显著降低HepG2胆固醇代谢模型细胞内胆固醇沉积，并可显著降低HepG2胆固醇代谢模型细胞内胆固醇水平，与模型组比较，P＜0.01。该作用可能通过其调节PERK-eIF2α-SREBP2相关信号通路基因及蛋白，并于下调miR-33a与miR-122表达相关，与模型组比较，P＜0.01。冠心康尚可减少THP-1巨噬细胞模型细胞内胆固醇沉积，调控PERK-eIF2α-CHOP通路相关基因及蛋白，减少泡沫细胞形成，减轻内质网应激，维持细胞内胆固醇稳态。.从中医整体观分析高胆固醇血症形成过程肝细胞胆固醇代谢失衡及其主动脉异位沉积的动态表现，阐明冠心康对胆固醇稳态失衡的可能作用靶点与最佳作用时点。该研究有助于丰富中医肝藏血、心主血脉理论，为后续深入研究奠定基础，具有有一定的学术意义。