慢性应激在GR介导下影响动脉粥样硬化易损斑块的机制及调肝方药干预作用的研究

Previous studies have found that chronic stress could affect the unstability of the atherosclerotic vulnerable plaques. In early research of our team we had found that Xiaoyaosan Decoction had obvious therapeutic effect on the chronic stress. Recently studies demonstrated.that heat shock protein(HSP) can affect the stability of the atherosclerotic vulnerable plaques through Glucocorticoid receptor(GR) pathway. Therefore we propose that Xiaoyaosan Decoction may regulate the stability of the atherosclerotic vulnerable plaques during the chronic stress through the HSP/GR signaling pathway. The combine model of atherosclerosis (AS) and chronic stress animal models as well as cell model will be established, then the behavioral and morphological data in vitro and in vivo will be determined. We will test the protein and mRNA expression of HSPs, GR in these models using Western Blot, RT-PCR and other methods. Co-immunoprecipitation will be used to detecte the protein binding. Dual-Luciferase Reporter Assay will tell us that how HSPs regulate GR’s nuclear transcription. By all these methods the effect of Xiaoyaosan Decoction will be demonstrated from multiple perspective and levels.We will confirm our hypothesis about Xiaoyaosan Decoction on the regulation of the stability of the atherosclerotic vulnerable plaques worsen by the chronic stress, and detect the inner molecular mechanism. So as to provide experimental evidences for novel treatment targets of chronic stress combined with AS.

研究发现慢性应激可引起动脉粥样硬化（AS）易损斑块失稳。课题组前期发现疏肝解郁方药逍遥散治疗慢性应激疗效显著。最新研究指出热休克蛋白（HSP）在糖皮质激素受体（GR）的介导下，可对AS易损斑块的稳定性产生影响。因此，我们提出基于疏肝解郁治法，逍遥散可通过HSP /GR信号通路调节慢性应激下AS小鼠易损斑块稳定性的假说。本研究拟建立慢性应激与AS动物及细胞复合模型，观察体内外逍遥散改善易损斑块稳定性的作用；检测其行为学及形态学指标，釆用Western Blot、RT-PCR等方法，从蛋白、mRNA水平检测逍遥散对HSPs、GR表达的影响；以免疫共沉淀法检测相关蛋白的结合情况，双荧光素酶报告基因检测的方法探讨HSPs对GR转录、定位的调节作用。从多个角度、多个层面论证我们的假说。本课题将进一步揭示逍遥散调节慢性应激下AS小鼠易损斑块稳定性的分子机制，为该疾病寻求防治的新靶点提供理论和实验依据。

本课题以ApoE-/-小鼠、大鼠腹腔巨噬细胞为研究对象，建立慢性不可预知性应激与动脉粥样硬化（AS）易损斑块复合ApoE-/-小鼠的体内动物模型、皮质酮致巨噬细胞损伤的体外细胞模型，采用逍遥散及其含药血清进行干预研究。通过检测动物行为学、形态学、血脂及皮质酮等指标，RT-PCR检测主动脉组织HSPs、GR的mRNA表达，Western blot及免疫共沉淀方法检测HSPs、GR的蛋白表达及相互作用，从体内层面研究逍遥散延缓慢性应激致AS易损斑块的机制；通过检测细胞形态学、胆固醇酯等指标，RT-PCR、Western blot及免疫共沉淀方法检测细胞HSPs、GR的表达及相互作用，激光共聚焦显微镜观察细胞GR核转位以及双荧光素酶报告基因检测GR对靶基因反应元件的影响，从体外层面研究逍遥散含药血清抑制巨噬细胞泡沫化的机制。实验结果：（1）逍遥散能够降低慢性应激引起的高总胆固醇、甘油三酯、低密度脂蛋白水平、提升高密度脂蛋白水平，抑制慢性应激引起的高皮质酮和糖皮质激素水平，延缓慢性应激致AS易损斑块的病理进程；机制研究上发现逍遥散可通过HSPs/GR信号通路，调控小鼠动脉组织中HSPs、GR基因表达、促进HSPs与GR结合从而抑制GR向核转位；（2）逍遥散含药血清能够改善皮质酮对细胞的损伤、降低细胞内胆固醇酯水平，抑制皮质酮引起的巨噬细胞泡沫化形成；机制研究上发现逍遥散含药血清通过HSPs/GR信号通路，调控细胞中HSPs和GR基因表达、促进HSPs与GR结合、抑制GR向核内转位从而减弱GR对ABCA1反应元抑制作用，进而促进了ABCA1的表达以减少巨噬细胞对胆固醇酯的摄入，抑制泡沫细胞的形成，进一步解释逍遥散稳定AS易损斑块的作用机制。综上研究从体内和体外层面证实了逍遥散可通过调控HSPs/GR信号通路延缓慢性应激致AS易损斑块的病理进程，为AS易损斑块临床治疗靶点提供了新思路，体现了中医药独特的辩证治疗优势。