AMPK 通过VSMC细胞骨架F/G-actin调控下肢浅静脉曲张发病的机制研究

Varicose vein of lower extremity is a very common chronic venous disease, which induces serious impact on life quality of patients and society. In addition to common causes such as vascular smooth muscle cells (VSMC) and wall extracellular matrix dysfunction, the imbalance of VSMC cytoskeletal F/G-actin homeostasis plays an important role in the pathogenesis of varicose vein , but the molecular mechanisms how F/G-actin homeostasis is regulated is still unclear. AMPK is the "energy regulator" and ubiquitous kinase in whole body. The applicant previous published studies have found that AMPK can control resistant artery or small artery constriction and relaxation by regulating VSMC cytoskeleton F/G-actin dynamics. The pre-experimental results of this project show that AMPK activation is obviously increased and F/G -actin dynamics is changed in varicose vein of lower extremity. So, we suppose that AMPK may play an important role in the pathogenesis of varicose vein of lower extremity. In this project, we plan to use superficial varicose animal models (including AMPK gene knockout mouse) and primary cultured human and mouse superficial vein VSMCs, and combined the treatment of AMPK stimulators or inhibitors to study the molecular mechanisms of AMPK on the regulation of the occurrence and development of lower extremity varicose veins. At the same time, we also plan to confirm the results from clinical varicose vein specimens. We finally clarify whether AMPK can regulate the occurrence and development of varicose veins of lower extremities, and the related specific molecular mechanisms through cytoskeletal F/G-actin. The results of this study will offer a new potential therapeutic option for AMPK on the treatment of lower extremity varicose veins.

下肢浅静脉曲张是血管外科常见疾病，除了血管平滑肌细胞（VSMC）和管壁细胞外基质功能障碍外，VSMC细胞骨架F/G-actin稳态失衡在其发病中起重要作用，但调控F/G-actin稳态失衡的分子机制仍不清楚。申请人既往研究证实AMPK可通过VSMC的F/G-actin调节小动脉舒缩。本课题前期结果提示下肢曲张浅静脉中AMPK显著活化，F/G-actin稳态发生了改变。因此，我们推测AMPK可能通过VSMC的F/G-actin进而调节此疾病的发生。本课题拟采用小鼠浅静脉曲张动物模型（包括AMPK基因组敲除小鼠）、原代浅静脉VSMC等，并联合应用AMPK激动剂或抑制刺激处理，研究AMPK通过VSMC的F/G-actin在下肢浅静脉曲张发生中的作用及病理生理机制；并在临床标本中验证。最后阐明AMPK是否可通过F/G-actin调控下肢浅静脉曲张的分子机制，为其防治下肢浅静脉曲张提供新的药物靶点。

下肢浅静脉曲张是一种常见疾病，给患者生活质量及社会带来严重影响。其具体发病机制仍需研究，也缺乏良好的动物模型。静脉管壁的改变是浅静脉曲张的主要异常，其VSMC 细胞骨架 F/G-actin 失衡能促进下肢浅静脉曲张的发生与发展。申请人前期研究证实AMPK可通过调控 VSMC 胞浆游离钙和 F/G-actin 稳态来调节小动脉舒缩功能。因此，本项目研究思路为“AMPK 是否通过调控下肢浅静脉VSMC 细胞骨架 F/G-actin 稳态在下肢浅静脉曲张的发生与发展中发挥一定的作用”为课题主线。. 其主要研究内容包括：第一：通过原代培养静脉VSMC细胞，证实AMPK调控细胞骨架稳态及其可能机制；第二：建立稳定的小鼠背部浅静脉曲张模型，并进一步研究AMPK调控浅静脉曲张发生发展的分子机制；第三: 采用临床患者曲张静脉标本研究AMPK 通过 VSMC 细胞骨架 F/G-actin 在下肢浅静脉曲张发生中的作用及病理生理机制。. 细胞水平：我们顺利培养了正常浅静脉的原代VSMC，并实验证实AMPK可以通过调节cofilin活性调控细胞骨架F/G-actin稳态。. 动物实验方面：普通小鼠曲张静脉模型建立成熟，一般在建模后7天获得典型的曲张静脉模型，相关病理及分子生物学检测也获得一定成果，正在整理成文，等待发表。受疫情影响，目前基因敲除小鼠相关实验仍在进行中。. 临床标本结果：AMPK可能通过增强HSP20的磷酸化水平，增加与p-cofilin和螯合蛋白14-3-3的结合，从而使p-cofilin去磷酸化增加，进而增加cofilin促进F-actin去聚化，最终导致血管壁张力降低，而促进静脉曲张的发生和发展。相关结果在2022年国际生理学年会获得大会收录，目前文章也在SCI杂志投稿。. 本项目建立了稳定有效的小鼠背部曲张静脉模型，为曲张静脉发病机制研究提供坚实的基础，同时为防治下肢浅静脉曲张提供新的提供理论依据和药物靶点。. 依托本项目，发表了3篇SCI文章、一篇中文核心期刊文章；另外相关结果在2022年国际生理学年会获得大会收录,并经完成2名硕士研究生培养。