血管老化中细胞骨架改变的机制研究及益气活血药的干预作用

Vascular aging is confirmed to be a significant risk factor for cardiovascular disease, although the mechanism of which are still under development. It is well known that the morbid state of cytoskeleton could induce series of cardiovascular pathological changes, such as the endothelial barrier permeability failing, as well as the proliferation and invasion of smooth muscle cells. Therefore, it is of great importance to study cytoskeleton in vascular aging and the corresponding Traditional Chinese Medicine Intervention on it. In this project, firstly, the models of senile vascular endothelial cell, senile vascular smooth muscle cell and natural aging rat will be settled; secondly, the pathological changes of cardiovascular cytoskeleton as microtubules, microfilaments, etc, and the abnormal expression of some cytoskeleton proteins as F-actin, VE-cadherin and SM22α will be investigated; finally, HSP27, the regulating factor of cytoskeleton protein, will be researched by gene transfection and RNA interference technology. In conclusion, this study is intended to interpret structural changes of cytoskeleton in vascular aging and the corresponding mechanism, which might be the key element inducing cardiovascular disease and the target point for Traditional Chinese Medicine Intervention. Meanwhile, the intervention with Radix Ginseng, Radix Notoginseng and Rhizoma Chanxiong, three kinds of Traditional Chinese Medicine for nourishing qi and activating blood, will be investigated, which will provide new ideas for vascular-aging prevention and cardiovascular-disease treatment.

血管老化是老龄人群心血管疾病风险增加的主要原因，但其内在机制尚不明确。细胞骨架改变介导的血管内皮通透性增加、屏障功能缺失和中膜平滑肌细胞的迁移、增殖是心血管疾病的重要病理环节；因此，研究血管老化中细胞骨架的病理变化以及益气活血药的干预作用，对于延缓血管老化、防治心血管疾病具有重要的意义。本项目通过复制血管内皮细胞、平滑肌细胞衰老模型以及自然衰老大鼠模型，从整体、心血管组织、细胞和分子水平，动态观察衰老血管细胞微管、微丝等骨架结构的改变，以及骨架蛋白F-actin、VE-cadherin和SM22α的表达，并采用基因转染技术和RNA干扰技术对骨架调控因子HSP27的作用进行深入研究。以期从细胞骨架的角度阐明血管老化的结构改变及调节机制，明确其增加心血管疾病危险的关键病理环节以及人参、三七、川芎提取物延缓血管老化的作用靶点，为益气活血中药防治心血管疾病提供新的思路。

血管老化是机体衰老过程中的重要环节，并能诱发多种心脑血管疾病。细胞骨架在维持细胞的形状、运动及物质运输等方面起重要作用，其结构改变可导致内皮细胞功能损伤或平滑肌细胞异常增殖，成为心血管疾病的重要病理环节。因此，研究血管老化中细胞骨架的病理改变，对延缓血管老化和防治心血管疾病具有重要的意义。本项目以自然衰老大鼠、复制性衰老血管内皮细胞及血管平滑肌细胞为模型，从整体、细胞和分子水平，观察衰老血管细胞骨架结构的改变，并采用RNA干扰技术对热休克蛋白27（HSP27）基因调控骨架的作用进行深入研究。结果表明自然衰老大鼠及复制性衰老的血管内皮和平滑肌细胞的骨架蛋白F-actin表达增加，而微管蛋白、平滑肌22α蛋白（SM22α）及钙粘蛋白（VE-cadherin）表达减少。给予人参三七川芎提取物干预后，可以减少血管内膜损伤，减少中膜及外膜的增生，维持弹力板结构的完整，降低胶原纤维的增生；降低细胞衰老β-半乳糖苷酶染色（SA-β-gal）的蓝染比率，提高CCK-8细胞增殖能力，促进细胞周期中S期细胞增多；降低骨架蛋白F-actin的表达，提高微管蛋白及相关骨架蛋白的表达，维持细胞骨架的结构。HSP27基因敲低后，骨架蛋白及其相关蛋白表达均降低。项目补充建立了饥饿诱导的内皮及平滑肌细胞自噬模型，药物干预后电镜观察胞质中产生大量的自噬体、自噬空泡及次级溶酶体，自噬相关基因Beclin1及微管相关蛋白1轻链3Ⅱ（LC3-Ⅱ）表达增多。本项目明确了细胞骨架参与调节血管老化进程，人参三七川芎提取物可以调节细胞骨架，并可通过此作用延缓血管老化的进程；HSP27基因参与调节细胞骨架，提示药物作用靶点有可能与HSP27/磷酸化HSP27/F-actin通路有关；自噬的研究表明药物同时有可能通过提高自噬水平从而发挥延缓血管老化的作用，为益气活血中药防治心血管疾病提供新的思路。