基于mTORC1信号通路研究益气凉血生肌方对人脐静脉内皮细胞损伤修复的干预机制

Modern Research consider that the drugs of Drug Eluting Stents(DSE) such as rapamycin(RAPA) can delay endothelial repairation of the patients who accepted percutaneous coronary intervention(PCI), is the start link of the postoperative restenosis and major adverse cardiac event (MACE), and also is lack of effective countermeasures. Yiqi Liangxue Shengji Prescriptions is built on the theory of injure repairation of TCM, the Previous studies show that this prescriptions can reduce oxidative stress, improve the energy shortage and promote proliferation and migration of the vascular endothelial cells. In consider of RAPA is mTORC1 inhibitor, the oxidative stress and energy shortage is the upstream signal of mTORC1 Signaling，we propose the hypothesis that Yiqi Liangxue Shengji Prescriptions will reduce oxidative stress, improve the energy shortage which can repair the injure of HUVECs by mTORC1 Signaling Pathway. We are planning to take the method of vivo experiment, Copy anoxia/reoxygenation ischemia-reperfusion HUVECs model, take the Atorvastatin as contrast, intervene with the medicated sera of TCM，to research the repair mechanism of the HUVECs proliferation and migration before and after RAPA intervention from the aspect of cells, molecules and signaling pathways, with the methods such like live cell imaging system, transmission electron microscope, Ultra high performance liquid chromatography-mass spectrometry（UP-LC-MS），molecular biology etc.The project will further clarify the mechanism of traditional Chinese medicine regulating mTORC1 Signaling, and provide a basis for resolving the problem of the delay of endothelial repair of DES.

现代研究认为涂层支架药物如雷帕霉素延迟血管内皮修复是PCI术后晚期再狭窄及不良心血管事件的启动环节，且缺乏有效防治手段。益气凉血生肌方基于中医创伤修复理论而成，前期研究证实该方具有改善氧化应激及能量障碍、促进内皮细胞增殖与迁移的作用。基于雷帕霉素是mTORC1抑制剂，且氧化应激及能量不足是mTORC1通路的上游信号，提出益气凉血生肌方是通过减少氧化应激，改善能量代谢来调节mTORC1信号通路并促进人脐静脉内皮细胞(HUVECs)增殖迁移的假说。拟采用体外实验的方法，复制缺氧/复氧缺血再灌注HUVECs模型，以他汀药物作为对照，使用中药含药血清进行干预，运用活细胞工作站、透射电镜、超高效液相色谱-质谱、分子生物学等技术从细胞、分子及信号通路层面研究该方对雷帕霉素诱导前后HUVECs增殖及迁移的干预机制。项目将进一步阐明中药如何调节mTORC1通路，并为解决涂层支架内皮修复延迟的难题提供依据

现代研究认为DES延迟血管内皮的修复是PCI术后晚期再狭窄及不良心血管事件的启动环节。益气凉血生肌方基于中医创伤修复理论而成，前期研究证实该方具有改善氧化应激及能量障碍、促进血管内皮增殖与迁移的作用。据此提出益气凉血生肌方促进损伤血管内皮细胞修复的机制可能是通过减少氧化应激、改善能量代谢，从而调节mTORC1信号传导通路促进内皮细胞增殖的假说。研究内容：实验1：对人脐静脉内皮细胞（HUVECs）进行缺氧/复氧（H/R）造模，检测细胞增殖率、迁移距离、超微结构变化、活性氧自由基（ROS）含量、丙二醛（MDA）含量、超氧化物歧化酶（SOD）活力、ATP、AMP含量，western检测蛋白及磷酸化表达水平。实验2：对HUVECs进行H/R造模，并在复氧时给予RAPA，按照干预情况分为对照组、模型组、RAPA组、中药+RAPA组、西药+RAPA组、中西药+RAPA组；检测细胞增殖率、迁移距离，western检测蛋白及磷酸化表达水平。研究结果:实验1：①药物干预组内皮细胞细胞增殖率均升高，且中西药组细胞增殖率高于中药组和西药组；②划痕实验结果表明，中西药组细胞迁移距离加大；③透射电镜观察结果表明，药物干预组损伤内皮细胞细胞核边界更清楚，线粒体结构更完整；④药物干预组细胞ROS、MDA含量下降，SOD含量显著升高；⑤中药组、中西药组细胞ATP/AMP明显升高，且中西药组效果优于西药组；⑥western结果表明，中药组、中西药组p-AMPK/AMPK明显降低，中西药组较西药组更低，药物干预组p-mTOR/mTOR，p-p70S6K/p70S6K，p-4E-BP1/4E-BP1升高。实验2：①中西药+RAPA组内皮细胞增殖率显著高于RAPA组；②中药+RAPA组、中西药+RAPA组与RAPA组相比p-AMPK/AMPK明显降低，但中药+RAPA组、西药+RAPA组、中西药+RAPA组与RAPA组相比p-mTOR/mTOR，p-p70S6K/p70S6K，p-4E-BP1/4E-BP1没有统计学差异。结论：益气凉血生肌方可改善H/R损伤内皮细胞能量代谢、减少氧化应激，抑制AMPK激活，调节mTORC1信号通路，发挥保护内皮细胞并促进细胞增殖的作用。