磷酸化PKM2调控糖尿病内皮依赖性血管舒张功能  

Phosphorylated PKM2 regulates endotheliumdependent vasodilation in diabetes

作　　者：鲁斌 唐磊 李乐 周晓煜 冷一平 全承炫[1,4] LU Bin;TANG Lei;LI Le;ZHOU Xiaoyu;LENG Yiping;QUAN Chengxuan(Vascular Disease and Transformation Medical Center,Third Xiangya Hospital,Central South University,Changsha 410013;Department of Cardiology,Affiliated Hospital of Southwest Medical University,Luzhou Sichuan 646000;Research Center for PhaseⅠClinical Trials,Affiliated Changsha Central Hospital,University of South China,Changsha 410004;Department of Anesthesiology,Third Xiangya Hospital,Central South University,Changsha 410013,China)

机构地区：[1]中南大学湘雅三医院血管疾病与转化医学中心,长沙410013 [2]西南医科大学附属医院心内科,四川泸州646000 [3]南华大学附属长沙中心医院I期临床研究中心,长沙410004 [4]中南大学湘雅三医院麻醉科,长沙410013

出　　处：《中南大学学报（医学版）》2023年第5期663-670,共8页Journal of Central South University ：Medical Science

基　　金：长沙市中心医院院内科研项目(YNKY202105)。

摘　　要：目的:内皮依赖性血管舒张功能障碍是糖尿病性大血管病变的病理基础,内皮细胞对于高糖的利用及适应性改变决定了内皮细胞的功能状态。糖酵解途径是内皮细胞的主要能量来源,糖酵解异常在高糖诱导的内皮依赖性舒张功能障碍中发挥重要作用。M2型丙酮酸激酶(pyruvate kinase isozyme type M2,PKM2)是糖酵解途径关键酶之一,磷酸化可使其活性下降从而影响葡萄糖的糖酵解过程。TEPP-46可使PKM2稳定在四聚体形态,从而抑制其二聚体的形成和磷酸化。本研究采用TEPP-46作为抑制PKM2磷酸化的工具药探讨高糖条件下磷酸化PKM2(phosphorylated PKM2,p-PKM2)对内皮依赖性血管舒张功能的影响和潜在机制,以期为寻找糖尿病性大血管病变的新干预靶点提供理论依据。方法:将小鼠分为3组,其中野生型组(对照组,C57BL/6小鼠)和糖尿病组(db/db组,db/db小鼠)用羧甲基纤维素钠溶液每日灌胃1次;TEPP-46组(治疗组,db/db小鼠)用TEPP-46(30 mg/kg)和羧甲基纤维素钠溶液每日灌胃1次。各组小鼠分别处理12周后,检测胸主动脉p-PKM2和PKM2的蛋白质表达和血浆一氧化氮(nitric oxide,NO)水平以及胸主动脉内皮依赖性血管舒张功能。高糖(30 mmol/L)伴或不伴TEPP-46(10μmol/L)、甘露醇孵育人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)72 h后,检测上清液中NO水平、细胞内NO含量及p-PKM2和PKM2的蛋白质表达水平。最后,在细胞水平和动物水平检测TEPP-46对内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)磷酸化的影响。结果:与野生型组相比,糖尿病组小鼠胸主动脉p-PKM2表达增加(P<0.05)。糖尿病组小鼠胸主动脉对乙酰胆碱(acetylcholine,ACh)的反应性较对照组降低了47%(P<0.05),TEPP-46组小鼠胸主动脉对ACh的反应性较糖尿病组提高了28%(P<0.05);3组小鼠胸主动脉对硝普钠(sodium nitroprusside,SNP)的反应性差异无统计学意义(P>0.05)。与对照�Objective:Endothelium-dependent vasodilation dysfunction is the pathological basis of diabetic macroangiopathy.The utilization and adaptation of endothelial cells to high glucose determine the functional status of endothelial cells.Glycolysis pathway is the major energy source for endothelial cells.Abnormal glycolysis plays an important role in endothelium-dependent vasodilation dysfunction induced by high glucose.Pyruvate kinase isozyme type M2(PKM2)is one of key enzymes in glycolysis pathway,phosphorylation of PKM2 can reduce the activity of pyruvate kinase and affect the glycolysis process of glucose.TEPP-46 can stabilize PKM2 in its tetramer form,reducing its dimer formation and phosphorylation.Using TEPP-46 as a tool drug to inhibit PKM2 phosphorylation,this study aims to explore the impact and potential mechanism of phosphorylated PKM2(p-PKM2)on endothelial dependent vasodilation function in high glucose,and to provide a theoretical basis for finding new intervention targets for diabetic macroangiopathy.Methods:The mice were divided into 3 groups:a wild-type(WT)group(a control group,C57BL/6 mice)and a db/db group(a diabetic group,db/db mice),which were treated with the sodium carboxymethyl cellulose solution(solvent)by gavage once a day,and a TEPP-46 group(a treatment group,db/db mice+TEPP-46),which was gavaged with TEPP-46(30 mg/kg)and sodium carboxymethyl cellulose solution once a day.After 12 weeks of treatment,the levels of p-PKM2 and PKM2 protein in thoracic aortas,plasma nitric oxide(NO)level and endothelium-dependent vasodilation function of thoracic aortas were detected.High glucose(30 mmol/L)with or without TEPP-46(10μmol/L),mannitol incubating human umbilical vein endothelial cells(HUVECs)for 72 hours,respectively.The level of NO in supernatant,the content of NO in cells,and the levels of p-PKM2 and PKM2 protein were detected.Finally,the effect of TEPP-46 on endothelial nitric oxide synthase(eNOS)phosphorylation was detected at the cellular and animal levels.Results:Compared with the control grou

关 键 词：糖尿病 M2型丙酮酸激酶 内皮依赖性血管舒张功能 内皮型一氧化氮合酶 一氧化氮 

分 类 号：R587.1[医药卫生—内分泌]