腺苷酸活化蛋白激酶活化cofilin诱导小动脉舒张的机制研究  

Mechanism of adenosine monophosphate-activated protein kinase inducing arteriolar relaxation via cofilin activation

作　　者：张永梅 曾贤德 曾雄 闵曦曦 陈炜 邱结华[2] ZHANG Yongmei;ZENG Xiande;ZENG Xiong;MIN Xixi;CHEN Wei;QIU Jiehua(Department of Laboratory Medicine,the Second Affiliated Hospital of Nanchang University,Nanchang 330006,China;Department of Vascular Surgery,the Second Affiliated Hospital of Nanchang University,Nanchang 330006,China)

机构地区：[1]南昌大学第二附属医院检验科,江西南昌330006 [2]南昌大学第二附属医院血管外科,江西南昌330006

出　　处：《中国普通外科杂志》2022年第6期799-805,共7页China Journal of General Surgery

基　　金：国家自然科学基金资助项目(81860095;82060097);江西省自然科学基金资助项目(20202ACB216001);江西省卫生健康委科技计划基金资助项目(202210616)。

摘　　要：背景与目的:腺苷酸活化蛋白激酶(AMPK)对循环系统的调控作用已获得大量研究,并证实AMPK可以通过调控血管平滑肌细胞(VSMC)内游离Ca2+浓度来调节小动脉舒缩。前期研究发现,AMPK可通过增加cofilin活性导致细胞骨架链状肌动蛋白(F-actin)降解为单体肌动蛋白(G-actin)而扩张血管,本研究目的为进一步探讨AMPK活化cofilin的分子机制。方法:取C57BL6/N小鼠肠系膜上动脉的Ⅱ、Ⅲ级分支,在压力myograph模型上,检测离体动脉血管对肾上腺素和乙酰胆碱的反应,取收缩率>30%且舒张率>90%的血管用于实验;然后将血管分为两组,在预处理及高钾MOPS溶液预收缩后分别加入AMPK激活剂PT1 (PT1组)和溶剂DMSO (对照组),比较两组血管的扩张情况。然后采用Western blot法与免疫荧光Western blot法检测两组动脉组织中磷酸化AMPK (p-AMPK)及AMPK下游相关蛋白的表达。结果:所有实验动脉血管活性均符合要求,且两组血管之间的收缩与舒张能力差异无统计学意义(均P>0.05)。两组动脉血管直径在预处理及预收缩时差异均无统计学意义(均P>0.05)。在相应的处理后,PT1组血管逐渐扩张,而对照组无明显扩张,60 min后,PT1组血管扩张至(196.6±11.5)μm,而对照组直径为(136.1±8.1)μm,差异有统计学意义(P<0.001)。与对照组比较,PT1组血管组织中p-AMPK、G-actin水平升高,分别为对照组的(3.25±0.52)倍、(2.26±0.64)倍,而磷酸化cofilin (p-cofilin)水平降低,为对照组的(0.48±0.19)倍,差异均有统计学意义(均P<0.05);总热休克蛋白20 (t-HSP20)无明显变化(P>0.05),但磷酸化HSP20 (p-HSP20)水平升高,为对照组的(2.45±0.52)倍,差异有统计学意义(P<0.001)。结论:本研究结果提示,HSP20参与协助了AMPK对cofilin的活化,机制可能为活化的AMPK通过增加p-HSP20水平,竞争结合p-cofilin位点,促进其去磷酸化,增加cofilin活性,从而降低细胞骨架actin稳态,致血管舒张。Background and Aims:A large number of studies have been carried out on the regulatory action of adenosine monophosphate-activated protein kinase(AMPK)on circulation system,and it is confirmed that AMPK can modulate the relaxation and contraction of arterioles by regulating the concentration of free calcium ions in the vascular smooth muscle cells(VSMCs).Previous studies found that AMPK can help the arterial relaxation through increasing cofilin activity that can cause the depolymerization of cytoskeletal protein filamentous actin(F-actin)into the monomer,globular actin(G-actin).Therefore,this study was conducted to further investigate the molecular mechanism for AMPK-induced cofilin activation.Methods:The second-and third-order branches of the superior mesenteric artery of C57BL6/N mice were harvested.Using the pressure myograph model,the responses of the isolated arteries to epinephrine and acetylcholine were determined,and the vessels with a contraction rate>30%and a relaxation rate>90%were used for the experiment.Then,the vessels were divided into two groups and added with AMPK activator PT1(PT1 group)or vehicle DMSO(control group)after pretreatment and precontraction with high potassium MOPS solution.The vascular relaxation responses of the two groups of vessels were compared.After that,the expressions of phosphorylated AMPK(p-AMPK)and other relevant downstream proteins of AMPK in the vascular tissues of the two groups were detected by Western blot or immunofluorescentWestern blot.Results:The vascular activity of all experimental arteries met the requirements.There were no significant differences in contraction and relaxation abilities between the two groups of vessels(both P>0.05),and there were no significant differences in blood vessel diameters during pretreatment and precontraction between the two groups of vessels(both P>0.05).After corresponding treatment,the blood vessels in PT1 group were gradually relaxed,while those in control group showed no obvious change,and 60 min later,the average vessel diame

关 键 词：小动脉 AMP活化蛋白激酶类 肌动蛋白解聚因子类 HSP20热休克蛋白质类 

分 类 号：R654.3[医药卫生—外科学]