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

• 1:贵州医科大学
• 2: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
• 3:贵州医科大学基础医学院病理生理学教研室

摘要(Abstract)：

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敲低gp91phox亚基，可增加PP4R2亚基蛋白表达(P<0.05),提高eNOS Ser633磷酸化水平(P<0.05)。结论 PA可通过Nox/ROS通路抑制PP4调节亚基PP4R2蛋白表达，激活PP4,进而降低eNOS Ser633磷酸化水平，细胞内NO产生减少，导致内皮功能障碍。
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 protein expression(P<0.05) and phospho-Ser633-eNOS level(P<0.05). Knockdown of gp91phox subunit by specific siRNA increased PP4R2 protein expression of(P<0.05) and phospho-Ser633-eNOS level(P<0.05). Conclusion PA may inhibit PP4R2 expression through Nox/ROS pathway to activate PP4, and then reduce phospho-Ser633-eNOS level and intracellular NO content, resulting in endothelial dysfunction.

关键词(KeyWords)： 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
palmitic acid(PA);endothelial nitric oxide synthase(eNOS);protein phosphatase 2A(PP2A);protein phosphatase 4(PP4);NADPH oxidase;endothelial dysfunction

Abstract:

Keywords:

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

作者(Author): 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

DOI: 10.19367/j.cnki.2096-8388.2023.02.001

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