Bcl-2与PI3K/Akt/mTOR信号通路“串话”调控CSA血管内皮细胞自噬及活血定眩胶囊的干预机制研究

CSA is a common clinical disease and chronic disease, which seriously affects the physical and mental health of the citizen. One of the causes of CSA is the damage of morphology and structure of vascular endothelial cells. Autophagy plays an important role in maintaining the homeostasis of blood vessels, The regulation of autophagy is associated with multiple pathways and multiple factors. Based on the previous study, we suggest that Autophagy plays an important role in the pathogenesis of CSA, and proper autophagy at a specific baseline can maintain intravascular environmental homeostasis, Through the "crosstalk" between Bcl-2 and PI3K / Akt / mTOR signal pathway, Huoxue Dingxuan Capsule regulates the autophagy of vascular endothelial cells to reach the working hypothesis for the treatment of CSA. Therefore, we intend to carry out: ①In vitro experiments: by culturing bEnd.3 cells, Using hypoxia stimulation, siRNA interference and other methods, Using RT-PCR, Western Blot and immunoprecipitation and other techniques, To explore the regulatory mechanism of Bcl-2 and PI3K/Akt/mTOR signaling pathway between the "crosstalk" on autophagy; ②In vivo experiment: Establishing the CSA rat model, Using transmission electron microscopy, immunohistochemistry and a variety of biological detection, To explore the regulatory mechanism of Huoxue Dingxuan Capsule on the autophagy function of vascular endothelial cells. Ultimately, it provides the research ideas for the prevention and treatment of CSA by Traditional Chinese Medicine at the cell and gene level, and to guide clinical medicine.

CSA是临床常见病、慢性病，严重影响国民身心健康。CSA发病原因之一是血管内皮细胞形态结构的损伤。自噬对维持血管内环境稳态具有十分重要的作用，自噬的调节与多个通路、多种因子相关。项目组在前期研究基础上提出“自噬在CSA病理机制中具有重要作用，特定基线的适度自噬可维持血管内环境稳态，活血定眩胶囊通过Bcl-2与PI3K/Akt/mTOR信号通路间的‘串话’调控血管内皮细胞自噬水平而达到治疗CSA之目的”的工作假说。为此，拟开展：①离体培养bEnd.3细胞，采用乏氧刺激、siRNA转染等方法，运用RT-PCR、Western Blot及免疫共沉淀技术，探讨Bcl-2与PI3K/Akt/mTOR信号通路间“串话”对自噬的调控机制；②在体建立CSA大鼠模型，采用透射电镜及生物学检测探讨活血定眩胶囊对血管内皮细胞自噬功能的调节机制。最终在细胞和基因水平上为中医药防治CSA提供研究思路，指导临床用药。

椎动脉型颈椎病（CSA）是临床常见病、多发病，严重影响国民身心健康。血管内皮细胞形态结构损伤是CSA发病的重要因素之一，而自噬对维持血管内环境稳态具有重要作用。项目组认为自噬在CSA病理机制中具有重要作用，特定基线的适度自噬可维持血管内环境稳态，活血定眩胶囊通过Bcl-2与PI3K/Akt/mTOR信号通路间的‘串话’调控血管内皮细胞自噬水平来治疗CSA。本项目借助乏氧刺激、siRNA转染、RT-PCR、WB、免疫共沉淀、透射电镜等技术，研究内容如下：bEnd.3细胞自噬激活及活血定眩胶囊对其凋亡、自噬的干预作用；Bcl-2与PI3K/Akt/mTOR通路“串话”调控bEnd.3细胞自噬；乏氧环境bEnd.3自噬中Bcl-2与PI3K/Akt/mTOR通路的相互作用；Bcl-2与PI3K/Akt/mTOR通路对椎动脉内皮细胞自噬的互动效应及活血定眩胶囊的干预机制。结果：①氧糖剥夺可诱导bEnd.3细胞发生凋亡与自噬，且干预6小时为最佳诱导时间；②活血定眩胶囊含药血清可显著对抗OGD诱导的bEnd.3细胞损伤；且通过影响p62、Bcline-1、LC3相关自噬蛋白的表达水平减轻乏氧饥饿环境下bEnd.3细胞自噬，促进血管新生。③体外乏氧培养bEnd.3细胞可激活自噬，10%浓度HXDXC含药血清能增强血管内皮细胞自噬程度。PI3K/Akt/mTOR信号通路在组织缺血缺氧后调节自噬的发生发展中起着关键作用。④植骨压迫联合力学失衡建立CSA模型成功后，透射电镜观察造模组大鼠椎动脉血管内皮存在大量自噬体，说明存在自噬发生；在经HXDXC干预后，CSA大鼠椎动脉血管内皮细胞自噬的程度受到抑制；⑤CSA大鼠经LY294004和雷帕霉素干预后，透射电镜及免疫荧光检测发现，LY294004能够抑制自噬发生程度，雷帕霉素可以促进自噬的进一步发生。说明PI3K/Akt/mTOR信号通路在CSA大鼠能对自噬发生调节作用；经HXDXC干预后，自噬受到不同程度抑制或激活。⑥OGD法诱导的bEnd.3细胞自噬过程中，Bcl-2与PI3K/Akt/mTOR 信号通路存在对 bEnd.3自噬的互动效应，并且活血定眩胶囊通过干预Bcl-2与PI3K/Akt/mTOR 通路互动而调节椎动脉内皮细胞适度自噬，维持椎动脉内皮功能；本项目研究发表CSCD期刊7篇，培养硕博士研究生各2名。