二氢杨梅素通过Prohibitin2介导的线粒体自噬抑制血管内皮细胞NLRP3炎性体活化的作用机制研究

NLRP3 inflammasomes are closely related to the pathogenesis of atherosclerosis (AS). Previous studies found that mitochondrial dysfunction could induce NLRP3 inflammasome activation. However, mitophagy can selectively remove excess or damaged mitochondria to maintain mitochondrial homeostasis, and then block the activation of NLRP3 inflammasome. Moreover, the latest research reveals that mitochondrial membrane protein Prohibitin 2 (PHB2) could interact with LC3 and then trigger mitophagy when mitochondria are damaged. In our previous study, our group found that dihydromyricetin (DHM) obviously inhibited NLRP3-mediated pyroptosis in vascular endothelial cells. Meanwhile, our previous study indicated that DHM significantly increased the expression of LC3 and the formation of autophagosome in vascular endothelial cells. Therefore, we presumed that “ DHM could inhibit NLRP3 inflammasome activation through inspiring PHB2-mediated mitophagy in the vascular endothelial cells, and thereby inhibit the progression of AS” We plan to test the hypothesis in vitro and in vivo. Firstly, we will clarify the function of mitophagy in the process that DHM inhibit NLRP3 inflammasome activation, and the signaling mechanism that DHM regulates mitophagy in the primary endothelial cells. Secondly, we will use C57BL/6 mice with PHB2 gene knockdown by plasmids, and assess the effects of DHM on the inhibition of the NLRP3 inflammasome activation and the prevention of AS by regulating the mitophagy through PHB2 signaling pathway. This project will provide a new scientific evidence for the prevention and treatment of atherosclerosis with plant chemicals or drugs interventions.

NLRP3炎性体与动脉粥样硬化（AS）的发生密切相关。研究表明线粒体功能障碍是NLRP3炎性体活化的关键，而线粒体自噬可通过清除多余或受损的线粒体，维持线粒体稳态，从而阻断NLRP3炎性体活化。最新研究揭示线粒体内膜蛋白Prohibitin2（PHB2）在线粒体受损时与LC3相互作用，激活线粒体自噬。本课题组前期研究发现二氢杨梅素（DHM）能抑制NLRP3调控的内皮细胞焦亡，另外发现DHM可明显增加内皮细胞LC3表达以及自噬体形成。据此，本项目提出假说“DHM可通过上调PHB2介导的线粒体自噬，抑制血管内皮细胞NLRP3炎性体活化，从而抑制AS的发生发展”。该项目拟通过基于原代细胞和小鼠体内基因敲减的研究，阐明线粒体自噬在DHM抑制内皮细胞NLRP3炎性体活化中的作用，并进一步明确PHB2在DHM调控线粒体自噬中的关键作用及机制。本研究为防治AS的膳食植物化学物或药物干预提供新的科学依据。

NLRP3炎性体与动脉粥样硬化（AS）的发生密切相关。研究表明线粒体功能障碍是NLRP3炎性体活化的关键，而线粒体自噬可通过清除多余或受损的线粒体，维持线粒体稳态，从而阻断NLRP3炎性体活化。最新研究揭示线粒体内膜蛋白Prohibitin2（PHB2）在线粒体受损时与LC3相互作用，激活线粒体自噬。本课题组前期研究发现二氢杨梅素（DHM）能抑制NLRP3调控的内皮细胞焦亡，另外发现DHM可明显增加内皮细胞LC3表达以及自噬体形成。据此，本项目提出假说“DHM可通过上调PHB2介导的线粒体自噬，抑制血管内皮细胞NLRP3炎性体活化，从而抑制AS的发生发展”。为此，我们开展了以下二个方面的研究工作：（1）利用原代培养的人脐静脉内皮细胞，明确DHM通过调控线粒体自噬抑制血管内皮细胞NLRP3炎性体活化，并进一步阐明PHB2在DHM调控线粒体自噬中的关键作用及机制。（2）运用ApoE-/-基因敲除小鼠，并以高脂膳食诱导建立小鼠AS模型，研究 DHM在体内防治AS的作用及机制。最终，我们证实（1）游离脂肪酸（棕榈酸，PA）可明显诱导血管内皮细胞NLRP3炎性体活化及其调控的细胞焦亡，其作用机制主要与PA上调血管内皮细胞线粒体活性氧（mtROS）水平有关。（2）PA可明显降低线粒体膜电位，损伤线粒体，但对线粒体自噬无显著影响；另外，上调线粒体自噬水平可明显改善PA诱导的NLRP3炎性体活化。（3）DHM可通过Prohibitin 2促进线粒体自噬，或下调线粒体活性氧水平，抑制内皮细胞NLRP3炎性体活化及其调控的细胞焦亡，从而阻止或延缓AS进程。综上，本研究揭示了动脉粥样硬化发生发展过程中血管内皮细胞的作用，明确DHM通过调控线粒体自噬抑制血管内皮细胞NLRP3炎性体活化，并阐明Prohibitin 2在DHM调控线粒体自噬中的关键作用机制。该研究为AS的研究提供了新干预靶点，同时为防治AS的膳食植物化学物或药物干预提供新的科学依据。