摘要
目的探讨蛋白磷酸酶4(PP4)和蛋白磷酸酶2A(PP2A)在棕榈酸(PA)诱导的内皮功能障碍中的作用及机制。方法采用PA处理人脐静脉内皮细胞(HUVECs)体外建立脂毒性细胞模型,用PP2A亚家族抑制剂福司曲星(FST)或冈田酸(OA)预处理和特异性小干扰RNA(siRNA)技术沉默PP2Ac和PP4c基因,用慢病毒感染过表达PP4R2基因转染HUVECs;蛋白免疫印迹分析(Western blot)检测eNOS总蛋白、eNOS Ser633和eNOS Ser1177磷酸化水平及PP4各亚基、PP2Ac亚基蛋白表达水平,DAF-FM DA荧光探针检测细胞内一氧化氮(NO)含量,划痕实验以及小管形成实验检测内皮细胞迁移能力(迁移率)和小管形成能力(管长度),免疫共沉淀(Co-IP)法观察HUVECs内eNOS、PP4c、PP4调节亚基PP4R2及PP4R3α之间的相互作用。结果与Control组相比,PA组eNOS Ser633和eNOS Ser1177磷酸化水平呈时间和浓度依赖性增加(P<0.05),但eNOS总蛋白表达水平变化不明显(P>0.05);与PA组相比,FST+PA组、OA+PA组eNOS Ser633和Ser1177的磷酸化水平增加(P<0.05),细胞内NO含量增加(P<0.05);与si-Control+PA组相比,si-PP2Ac+PA组eNOS Ser1177的磷酸化水平增加(P<0.05),si-PP4c+PA组eNOS Ser633磷酸化水平增加(P<0.05);与Control相比,PA组抑制PP4R2蛋白表达(P<0.05);与Vector+PA组相比,OE-PP4R2+PA组eNOS Ser633磷酸化水平增加(P<0.05),但eNOS Ser1177磷酸化水平无明显变化(P>0.05),同时细胞内NO产量增加,内皮细胞的迁移能力以及小管形成能力增强(P<0.05);Co-IP检测显示,eNOS蛋白与PP4c蛋白、PP4R2蛋白、PP4R3α蛋白存在共定位关系。结论PA激活PP2A和PP4分别诱导eNOS在Ser1177和Ser633去磷酸化,降低eNOS活性,导致内皮功能障碍,且PP4可能以三聚体形式(PP4R2-PP4c-PP4R3α)存在,提示在血浆游离脂肪酸增多时,调节PP4R2或PP4R3α活性可减轻细胞脂毒性,保护血管内皮细胞,改善内皮功能障碍。
Objective To investigate the roles and mechanisms of protein phosphatase 4(PP4)and protein phosphatase 2A(PP2A)in palmitic acid(PA)-induced endothelial dysfunction.Methods Human umbilical vein endothelial cells(HUVECs)were treated with PA to establish a lipotoxic cell model.PP2A subfamily inhibitor fostriecin(FST)or okadaic acid(OA)was used for pretreatment,and specific small interfering RNA(siRNA)technology was used to silence the PP2Ac and PP4c genes.HUVECs were transfected with lentivirus overexpressing PP4R2 gene.Western blot was used to detect the phosphorylation levels of eNOS total protein,eNOS Ser633,and eNOS Ser1177,as well as the protein expression levels of PP4 subunits and PP2Ac subunits.DAF-FM DA fluorescent probe was used to detect intracellular nitric oxide(NO)content.Scratch experiment and tube formation experiment were used to detect the migration ability and tube formation ability of endothelial cells.Immunoprecipitation(Co-IP)was used to observe regulatory sub-unit PP4R2 and PP4R3αinteractions in eNOS,PP4c,and PP4.Results Compared with the Control group,the phosphorylation levels of eNOS Ser633 and eNOS Ser1177 in the PA group showed a time-and concentration-dependent decrease(P<0.05),but did not affect the total protein expression level of eNOS(P>0.05).In comparison to the PA group,FST+PA group,and OA+PA group revealed an upregulated phosphorylation levels of eNOS Ser633 and Ser1177(P<0.05),leading to the increase of intracellular NO content(P<0.05).Compared with the si-Control+PA group,the si-PP2Ac+PA group showed an increase in phosphorylation level of eNOS Ser1177(P<0.05),while the si-PP4c+PA group presented an upregulated phosphorylation level of eNOS Ser633(P<0.05).The PA group significantly inhibited the protein expression level of PP4R2 compared to the Control group(P<0.05).Furthermore,compared to the Vector+PA group,OE-PP4R2+PA group showed a significantly increased phosphorylation level of eNOS Ser633(P<0.05),but no obvious changes were observed on the phosphorylation level of eNOS Ser1177(P>0.05).Simultaneously,intracellular NO production,endothelial cell migration ability,and tube formation capability were obviously promoted(P<0.05).Co-immunoprecipitation(Co-IP)assay revealed that eNOS protein had co-localization with PP4c protein,PP4R2 protein,and PP4R3αprotein.Conclusion PA activated PP2A and PP4 inducing eNOS dephosphorylation at Ser1177 and Ser633 respectively,thereby reducing eNOS activity and leading to endothelial dysfunction.Additionally,PP4 may exist in a trimeric form(PP4R2-PP4c-PP4R3α).Therefore,in the context of increased free fatty acids in the plasma,modulating PP4R2 or PP4R3αactivity can alleviate cellular lipotoxicity,protect vascular endothelial cells,and improve endothelial dysfunction.
作者
梁政伟
张俊诗
张倩
柴鑫
吕莎
邓亚竹
丁菁
陆德琴
LIANG Zhengwei;ZHANG Junshi;ZHANG Qian;CHAI Xin;LYU Sa;DEN Yazhu;DING Jing;LU Deqin(Guizhou Provincial Key Laboratory of Common Chronic Disease Etiological Mechanism and Pharmaceutical Prevention Treatment Research,Guiyang 550025,Guizhou,China;Teaching Division of Pathophysiology,School of Basic Medicine,Guizhou Medical University,Guiyang 550025,Guizhou,China)
出处
《贵州医科大学学报》
CAS
2023年第8期869-880,共12页
Journal of Guizhou Medical University
基金
国家自然科学基金(No.32160206)。
关键词
人脐静脉内皮细胞
棕榈酸
蛋白磷酸酶4
蛋白磷酸酶2A
内皮型一氧化氮合酶
内皮功能障碍
human umbilical vein endothelial cells
palmitic acid
protein phosphatase 4
protein phosphatase 2A
endothelial nitric oxide synthase
endothelial dysfunction
