从AMPK/mTOR通路探讨高糖诱导血管衰老的机制及益气活血药的干预作用

Vascular aging belongs to the basic pathological change of vascular complications of diabetis. Adenosine Monophosphate Activated Protein Kinase (AMPK) / mammalian target of rapamycin (mTOR) pathway is closely associated with aging and glycometabolism, but the effect of AMPK/mTOR pathway is not clear in the vascular aging induced by diabetes. High glucose-induced vascular senescence and the change of AMPK pathwayare closely related to oxidative stress and AMPK could regulate the cell life by PPAR gamma and p53. Our previous studies found that Radix Ginseng, Radix Notoginseng and Rhizoma Chuanxiong extracts, one of Yiqi huoxue Chinese Herbal Medicine, could delay the vascular aging of the mice by inhibiting the oxidative stress and regulating the PPAR gamma and p53 pathway. But the functional mechanism of this Chinese Herbal Medicine remained unclear whether it could regulate the change of AMPK/mTOR pathway in the oxidative stress in order to delay the vascular aging induced by diabetes. In this study we set up the models of the vascular aging mice and cell senescence in high-glucose environments, and realize gene silencing of AMPK and overexpression of mTOR on senescent cells by RNA interference gene and transfection technology in order to discuss the action of AMPK/mTOR pathway for the vascular senescence induced by high glucose, ascertain the mechanism of this medicine to delay vascular aging by AMPK/mTOR pathway and illuminate the scientific connotation of yiqi huoxue Chinese Herbal Medicine on removing stasis and promoting coronary circulation.

血管衰老是糖尿病血管并发症的基本病理改变。腺苷酸活化蛋白激酶(AMPK)和哺乳动物雷帕霉素靶蛋白(mTOR)均与衰老及糖代谢密切相关，但AMPK/mTOR通路在糖尿病诱导的血管衰老中作用机制不明。高糖诱导的血管衰老及AMPK/mTOR通路变化均与氧化应激关系密切，且AMPK可通过PPARγ、p53调控细胞寿命。前期研究发现，益气活血药人参三七川芎提取物可抑制氧化应激并调节PPARγ与p53通路从而延缓小鼠血管衰老，其是否通过调节氧化应激下AMPK/mTOR通路的改变延缓糖尿病引发的血管老化尚不明确。本研究建立高糖环境下的血管衰老小鼠与细胞衰老模型，采用RNA干扰和基因转染技术实现衰老细胞AMPK基因沉默和mTOR过表达，探讨AMPK/mTOR通路对高糖诱导的血管衰老的影响，明确益气活血药通过AMPK/mTOR通路延缓血管老化的作用机制，阐明益气活血药祛瘀生新、通脉活络的科学内涵。

血管衰老是糖尿病血管并发症的基本病理改变。腺苷酸活化蛋白激酶(AMPK)/哺乳动物雷帕霉素靶蛋白(mTOR)是生物能量代谢的关键通路，并与衰老密切相关。因此，探讨高糖环境下AMPK/mTOR通路与血管衰老的关系，对延缓血管老化及防治糖尿病血管并发症具有重要意义。本项目以STZ诱导的糖尿病小鼠主动脉衰老、高糖诱导的人主动脉血管内皮及主动脉平滑肌细胞衰老为研究模型，并采用药物或RNA干扰技术特异性抑制AMPK基因，观察AMPK/mTOR通路的变化，从整体、细胞和分子水平探讨AMPK/mTOR 通路与高糖诱导的血管衰老之间的关系。结果表明糖尿病小鼠主动脉组织及高糖诱导衰老的血管内皮和平滑肌细胞的AMPK、p-AMPK、LKB1、p-LKB1表达减少，而mTOR、p-mTOR、P70S6K、p-P70S6K表达减少。给予人参三七川芎提取物干预后，可以减少血管内皮损伤程度，抑制血管中内膜增生，维持弹力板结构完整性，降低胶原纤维增生；降低细胞衰老β-半乳糖苷酶（SA-β-gal）蓝染比例；减少血管内皮细胞线粒体活性氧，提高线粒体膜电位，激活线粒体自噬；改善血管平滑肌细胞增殖迁移能力、细胞周期、表型转化；增加p-AMPK/AMPK、p-LKB1/LKB1蛋白表达，并减少p16、p21、p53、p-p53、p-mTOR/mTOR、p-p70s6k/p70s6k蛋白表达；药物和RNA干扰AMPK基因表达后，人参三七川芎提取物的上述作用均被抑制。本项目明确了AMPK/mTOR通路参与了高糖诱导的病理性血管衰老的进程，人参三七川芎提取物可通过调节该通路达到延缓血管组织和血管细胞衰老的作用，为益气活血药延缓血管衰老以防治衰老相关疾病引起的血管病变提供科学依据。