棕榈酸降低内皮型一氧化氮合酶Ser633位点磷酸化水平对内皮细胞功能影响的分子机制  

Molecular Mechanisms of palmitic acid(PA) inhibiting endothelial nitric oxide synthase Ser633 phosphorylation from affecting endothelial cell

作　　者：李小宇 梁政伟 秦思 张倩 张俊诗 丁菁 陆德琴 LI Xiaoyu;LIANG Zhengwei;QIN Si;ZHANG Qian;ZHANG Junshi;DING Jing;LU Deqin(Guizhou Provincial Key Laboratory of Pathogenesis and Drug Research on Common Chronic Diseases,Guizhou Medical University,Guiyang 550025,Guizhou,China;Department of Pathophysiology,School of Basic Medicine Sciences,Guizhou Medical University,Guiyang 550025,Guizhou,China)

机构地区：[1]贵州省常见慢性疾病发病机制及药物防治研究重点实验室,贵州贵阳550025 [2]贵州医科大学基础医学院病理生理学教研室,贵州贵阳550025

出　　处：《贵州医科大学学报》2023年第2期125-136,共12页Journal of Guizhou Medical University

基　　金：国家自然科学基金(32160206);贵州医科大学国基培育项目(20NSP015)。

摘　　要：目的探讨棕榈酸(PA)降低内皮型一氧化氮合酶(eNOS)Ser633位点磷酸化水平对内皮细胞功能影响的分子机制。方法将人脐静脉内皮细胞(HUVECs)随机分为Control组、PA处理组、FST预处理组、FST预处理+PA组、si-Control组、si-Control+PA组、si-PP2Ac组、si-PP4c组、si-PP2Ac+PA组、si-PP4c+PA组、Vector组、OE-PP4R2组、Vector+PA组、OE-PP4R2+PA组、NAC预处理组、APO预处理组、NAC预处理+PA组、APO预处理+PA组、si-gp91phox组及si-gp91phox+PA组,用Western blot检测eNOS总蛋白、eNOS Ser633磷酸化水平,蛋白磷酸酶2催化亚基(PP2Ac)、蛋白磷酸酶4催化亚基(PP4c)及其负性调节亚基(PP4R2)和NADPH氧化酶(Nox)催化亚基gp91phox蛋白表达水平;将HUVECs随机分为Control组、PA处理组、FST预处理组、FST预处理+PA组、Vector组、OE-PP4R2组、Vector+PA组及OE-PP4R2+PA组,用DAF-FM DA荧光探针检测细胞内NO含量;将HUVECs随机分为Control组、PA处理组、Vector组、OE-PP4R2组、Vector+PA组、OE-PP4R2+PA组,细胞划痕实验检测内皮细胞迁移能力;将HUVECs随机分为Control组、PA处理组、NAC预处理组及APO预处理组,DCFH-DA荧光探针检测细胞内ROS含量。结果PA诱导HUVECs内eNOS Ser633磷酸化水平降低呈时间依赖和浓度依赖方式(P<0.05),但对eNOS总蛋白表达水平无影响(P>0.05);PA下调PP4R2蛋白表达水平、上调gp91phox蛋白表达水平(P<0.05),但不影响PP4c蛋白表达水平(P>0.05);PP4抑制剂FST预处理可逆转PA诱导的eNOS Ser633磷酸化水平降低(P<0.05),恢复细胞内NO产量(P<0.05);敲低PP4c亚基可逆转PA诱导的eNOS Ser633磷酸化水平降低(P<0.05),而敲低PP2Ac亚基对eNOS Ser633磷酸化水平无影响(P>0.05);过表达PP4R2可使eNOS Ser633磷酸化水平升高(P<0.05),细胞内NO生成增加(P<0.05),内皮细胞的迁移能力增强(P<0.05);抗氧化剂NAC和APO预处理,细胞内ROS产量减少(P<0.05),PP4R2亚基蛋白表达增加(P<0.05),同时eNOS Ser633磷酸化水平增加(P<0.05);特异性siRNA�Objective To explore the molecular mechanism of the effect of palmitic acid(PA)on endothelial cell function by decreasing the phosphorylation level of endothelial nitric oxide synthase(eNOS)at Ser633.Methods Human umbilical vein endothelial cells(HUVECs)were randomly divided into following groups:Control,PA,FST,FST+PA,si-Control,si-Control+PA,si-PP2Ac,si-PP4c,si-PP2Ac+PA,si-PP4c+PA,Vector,OE-PP4R2,Vector+PA,OE-PP4R2+PA,NAC,APO,NAC+PA,APO+PA,si-gp91phox group,and si-gp91phox+PA group.Western blot was applied to detect the protein levels of eNOS,phosphorylation of eNOS at Ser633(phospho-Ser633-eNos),protein phosphatase 2A catalytic subunit(PP2Ac),Protein phosphatase 4 catalytic subunit(PP4c),protein phosphatase-4 regulatory subunit 2(PP4R2),and the catalytic subunit gp91phox of NADPH-oxidase(Nox).Intracellular NO content was measured using DAF-FM DA fluorescent staining in control,PA,FST,FST+PA,Vector,OE-PP4R2,Vector+PA,and OE-PP4R2+PA groups.Wound healing assay was used to examine HUVECs migration capacity in Control,PA,Vector,OE-PP4R2,Vector+PA,and OE-PP4R2+PA groups.Intracellular ROS content was measured using DCFH-DA fluorescent staining in control,PA,NAC,and APO groups.Results PA reduced phospho-Ser633-eNOS level in a time-and concentration-dependent manners in HUVECs(P<0.05),but not total eNOS protein level(P>0.05).PA downregulated PP4R2 protein expression and upregulated gp91phox protein expression(P<0.05),but did not affect PP4c protein expression(P>0.05).Pretreatment with FST,a PP4 inhibitor,reversed the decrease of PA-induced phospho-Ser633-eNOS level(P<0.05)and restored intracellular NO production(P<0.05).Silencing PP4c reversed the decrease of PA-induced phospho-Ser633-eNOS level(P<0.05),while PP2Ac knockdown had no effect on phospho-Ser633-eNOS level(P>0.05).PP4R2 overexpression increased phospho-Ser633-eNOS level(P<0.05)and intracellular NO production(P<0.05),enhanced HUVECs migration(P<0.05).Pretreatment with antioxidant NAC and APO reduced the production of ROS in cells(P<0.05),increased PP4R2 prote

关 键 词：棕榈酸 内皮型一氧化氮合酶 蛋白磷酸酶2A 蛋白磷酸酶4 NADPH氧化酶 内皮功能障碍 

分 类 号：R363.21[医药卫生—病理学]