基于“浊的理论”的健脾化浊调脂颗粒调控NOX/ROS-NF-kB信号通路治疗动脉粥样硬化机制研究

Jian pi hua zhuo tiao zhi particles based on the theory of "turbidity " have used in clinical field for many years, they widely collect fine effect in the treatment of atherosclerosis,but its biological mechanism is unclear.NF-KB is a big family of transcription factors, control the variety of inflammatory factor expression.It has played an important role in the development of atherosclerosis.NOX is recognized to be able to adjust the ROS produced the special function of enzyme,and ROS can directly cause the NF-KB activation and cytokine expression. There are main cells such as vascular smooth muscle cells, endothelial cells, macrophages, etc. in the AS plaque.In the process of the development AS,there is NF-kB in vascular smooth muscle cells, endothelial cells and macrophages .. Our hypothesis is that Jian pi hua zhuo tiao zhi particles is by adjusting the NOX/ROS - NF - kB signaling pathway to delay the development of atherosclerosis,we found that preliminary experiment on endothelial cells have certain effect, this topic will have the further research.We build rabbit AS model established by high fat feedstuff,build cell model by Ox - LDL,use methods of HE staining, MTT,Flow cytometric method,RT-PCR,Western blot,etc. to have further observation of NOX, ROS,NF-kB of the influence the particles from the overall level and cell and molecular level, to explore possible mechanism of Jian pi hua zhuo tiao zhi particles by adjusting NOX/ROS-NF-kB signaling pathway,so as to provide objective evidence for the use in clinical treatment, prevention, health care .

基于“浊”的理论的健脾化浊调脂颗粒具有抗动脉粥样硬化作用，但其生物学机制尚不清楚。NF-KB是一大的转录因子家族,调控着多种炎症因子表达，在AS的发展中起到重要作用。NOX是公认的能够调节ROS产生的特殊功能酶，而ROS能直接导致NF-KB活化及细胞因子表达。AS斑块中的主要细胞有VSMC、ECs和巨噬细胞等。在参与AS发生发展的这几种细胞内均有NF-kB表达。我们假定，本方是通过调节NOX/ROS-NF-kB信号通路来延缓AS的进程，预实验发现本方对内皮细胞有一定的作用，本课题拟进一步研究，通过高脂饲料建立兔AS模型，Ox-LDL建立细胞模型，运用流式细胞术、RT-PCR、Western blot等技术，从整体水平及细胞分子水平进一步观察本方对NOX、ROS、NF-kB相关指标的影响，探索本方调控NOX/ROS-NF-kB通路可能机制，以期为本方在临床治疗、预防保健中的使用提供客观证据。

动脉粥样硬化（atherosclerosis，AS）是高血压、急性冠脉综合征、脑梗死等心脑血管疾病的主要病理基础，寻求有效的作用机制抑制AS的形成，减少心脑血管事件的发生显得尤为重要。AS的发病机制与氧化应激关系密切，其中NADPH氧化酶（NADPH oxidase，NOX）/活性氧（reaction oxygen species，ROS）/核转录因子kappa B （nuclear factor kappa B，NF-κB）信号通路是氧化应激过程中的重要通路。健脾化浊调脂颗粒是基于“浊邪”理论而创立的经验方，被广泛应用于AS的防治当中，在前期研究中，发现该方能够降低氧化应激损伤反应而起到防治AS的作用，但具体的机制尚不明确。本项目通过建立动脉粥样硬化动物模型和细胞实验，探讨该方与NOX/ROS/NF-κB信号通路之间的关联性。在细胞实验中，结果显示中药组可减少由于ox-LDL氧化应激导致的巨噬细胞损伤，细胞存活率提高，凋亡率下降，MDA和SOD水平明显下降/上升，抑制VSMCs的过度增殖，同时中药组呈浓度依赖性地降低MDA水平，提高SOD活性，降低ROS、IKK-α、IKK-β、NF-κB表达和抑制NOX4、p22phox蛋白表达（P﹤0.05）。在动物实验中，AS动物模型在中药干预后其主动脉内膜增厚及粥样斑块等改变与模型对照组比较明显改善，同时血粘度、血清中ET-1、GMP-140、ox-LDL的含量及主动脉中MDA、AOPP、ROS 等含量明显降低、NOX4、NF-κB的蛋白表达均明显下调，血清中一氧化氮、降钙素基因相关肽的含量及主动脉中总抗氧化力的含量均明显升高（P﹤0.05）。本研究结果显示健脾化浊调脂颗粒通过抑制NOX/ROS/NF-κB信号通路减少氧化损伤，提高机体的抗氧化能力从而对AS的病变起到防治的作用，为防治动脉粥样硬化提供新思路。