二甲双胍通过激活AMPK-Pdlim5信号通路抑制动脉粥样硬化形成的实验研究

AMP-activated protein kinase (AMPK) is generally recognized as an energy sensor. Currently, researches have revealed that AMPK also plays a key role in the regulation of cell migration. Very recently, a new substrate of AMPK called Pdlim5 is reported being phosphorylated by AMPK in augmented condition, and regulates the cytoskeleton organization and cell migration. These findings may help to explain the continuous clinical trials which reported the close relationship between diabetes and cardiovascular disease. The most difficult task is to explain why medicine, like Metformin, an AMPK activator used for diabetes therapy, has the function of anti- atherosclerosis or anti-vascular remodeling. Here, we will first focus on explain the distinct kinetics of AMPK on regulation of cell migration by vascular cells in vitro; Moreover, by using diabetes rats to establish atherosclerosis model, the effect of Metformin on preventing the development of atherosclerosis will be tested. Meanwhile, we will also prove the important role of AMPK-Pdlim5 signaling pathway in this Metformin-treatment system. This work may have profound meaning to cardiovascular diseases and may establish solid foundation for future targeted therapeutic studies.

单磷酸腺苷活化激酶（AMPK）调控细胞的迁移。我们报道过扩增激活AMPK活性使细胞骨架相关蛋白Pdlim5磷酸化，并在体外证实了该信号通路对细胞迁移的抑制作用。那么，这种现象与心血管疾病的发病机理有何联系呢？近年来，多项大规模临床试验发现，AMPK激活剂二甲双胍在治疗糖尿病的同时，对以细胞迁移为成因的动脉粥样硬化（As）的发生具有抑制倾向。预实验显示：二甲双胍确实可刺激鼠类血管平滑肌细胞Pdlim5磷酸化，并干扰肌钙蛋白骨架系统，抑制细胞迁移。为此，我们提出AMPK-Pdlim5信号通路在糖尿病动物As的形成中或起关键作用。本项目拟进一步对自发性糖尿病大鼠行高脂饮食喂养及手术干预，从而构建由糖尿病与高脂血症诱导的As模型，在动物体内评价二甲双胍在As发生中的效用，解析AMPK-Pdlim5通路及其他下游信号通过干扰细胞迁移来抑制As形成的分子生物学机制，为探讨As的靶点化治提供科学依据。

目的：对动脉粥样硬化（As）斑块中的平滑肌细胞进行分析，研究AMPK-Pdlim5信号通路在二甲双胍（Met）对抗糖尿病加速的As形成中的作用。方法与结果：制备斑马鱼脂质沉积模型，发现被降脂化合物依泽替米贝直接激活的AMPK可抑制动物血管内脂质沉积。接着，把Met给与糖尿病ApoE−/−或ApoE−/−/AMPK-α1−/− 小鼠，8周后发现糖尿病ApoE−/−小鼠Met喂养组的动脉内脂质沉积明显少于生理盐水组，主动脉瓣膜口处脂质沉积较对照组明显减少、斑块面积更局限。但Met并未逆转糖尿病ApoE−/−/AMPK-α1−/− 小鼠As的形成。为明晰Met是否对细胞表型转换起作用，将TNFa和PDGF-b作为表型转变诱导剂，观察分泌型SMC表面marker分子KLF4的变化情况。结果显示，Met不影响KLF4的表达，说明Met对平滑肌细胞的影响不涉及表型转变。接着，针对Pdlim5磷酸化位点行基因改造并制备模拟磷酸化型S177D、非磷酸化型S177A及野生型WT-Pdlim5，并在尾端连接EGFP。转染了EGFP-S177D-Pdlim5质粒的细胞表现出大量应力纤维增生、粘附斑增多、细胞的介板结构遭破坏。而S177A及WT-Pdlim5组则仍保留了原来的细胞结构骨架。EGFP-S177D-Pdlim5组细胞较WT和S177A组迁移能力下降，划痕愈合速度明显降低。最后，给与ApoE−/−糖尿病小鼠Met，WT-Pdlim5病毒组小鼠体内的AMPK-Pdlim5信号被激活，而S177A-Pdlim5病毒组动物未见磷酸化Pdlim5扩增，反之，S177D-Pdlim5病毒组动物即使没有Met喂养，同样表达磷酸化Pdlim5。病理染色显示，WT或S177A-Pdlim5病毒的小鼠，在无Met情况下血管内均有大量的脂质沉积，主动脉瓣膜口处粥样硬化形成明显。在喂养met组，WT-Pdlim5组脂质沉积和As形成明显减轻。而S177A-Pdlim5组动物即使给与Met， As形成缓解仍不明显。而S177D-Pdlim5组即使在不给予Met的情况下，As程度仍较WT-Pdlim5组减轻，说明Pdlim5的磷酸化在抑制As形成方面发挥重要作用。结论：二甲双胍通过激AMPK-Pdlim5通路干扰平滑肌细胞的迁移来抑制As形成。

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