摘要
目的:探讨烟酸(NA)对巨噬细胞溶酶体游离胆固醇外流的作用及其机制。方法:以佛波酯诱导人单核-巨噬细胞THP-1形成的巨噬细胞为细胞模型,利用激光扫描共聚焦成像技术观察NA对经氧化型低密度脂蛋白(oxLDL)孵育的巨噬细胞溶酶体胆固醇外流的作用,以及烟酸腺嘌呤二核苷酸磷酸(NAADP)拮抗剂Ned-19、Ca2+螯合剂BAPTA、肝X受体α(LXRα)siRNA和尼曼-皮克C1蛋白(NPC1)siRNA对NA效应的影响。RT-qPCR和Western blot检测NA、Ned-19和BAPTA对LXRαmRNA和NPC1蛋白表达的影响。结果:NA呈剂量依赖性地促进oxLDL孵育的巨噬细胞溶酶体胆固醇外流;这种效应可被Ned-19和BAPTA显著抑制。NA可明显促进NPC1蛋白及LXRαmRNA表达;这种效应也可被Ned-19和BAPTA明显抑制。LXRαsiRNA可明显抑制NA对NPC1蛋白表达的促进作用。siRNA沉默LXRα和NPC1表达也可明显减弱NA促溶酶体胆固醇外流的效应。结论:NA可显著促进巨噬细胞溶酶体胆固醇外流,其机制可能与其增加NAADP的生成,进而促进溶酶体瞬时感受器电位黏脂蛋白1(TRPML1)通道Ca2+的释放,后者经LXRα促进NPC1蛋白表达有关。
AIM:To explore the effects of nicotinic acid(NA)on lysosomal free cholesterol efflux in macrophages and its underlying mechanism.METHODS:Macrophages induced from human monocytic leukemia cell line THP-1 by phorbol myristate acetate served as the cell model.Laser scanning confocal microscopy was applied to observe the effects of NA on lysosomal free cholesterol efflux in macrophages loaded with oxidized low-density lipoprotein(oxLDL).The influences of nicotinic acid adenine dinucleotide phosphate(NAADP)antagonist Ned-19,Ca2+chelator BAPTA,liver X receptorα(LXRα)siRNA and Niemann-Pick C1 protein(NPC1)siRNA on NA effects were also evaluated.RT-qPCR and Western blot were conducted to evaluate the influence of NA,Ned-19 and BAPTA on LXRαmRNA and NPC1 protein expression.RESULTS:NA dose-dependently promoted lysosomal free cholesterol efflux in macrophages.This effect was markedly inhibited by Ned-19 and BAPTA.NA increased NPC1 protein and LXRαmRNA expression.These effects were also attenuated by Ned-19 and BAPTA remarkably.LXRαsiRNA significantly inhibited the promoting effect of NA on NPC1 protein expression.Silencing of LXRαand NPC1 with siRNA remarkably abolished the effect of NA on lysosomal free cholesterol efflux.CONCLUSION:NA promotes lysosomal free cholesterol efflux in macrophages.This effect may be mediated by the increased production of NAADP,which subsequently promotes Ca2+release through lysosomal transient receptor potential mucolipin 1(TRPML1)channel and finally up-regulates NPC1 protein expression via LXRα.
作者
杨萍
张汉斌
刘钰林
方淑香
李平
许小洋
YANG Ping;ZHANG Han-bin;LIU Yu-lin;FANG Shu-xiang;LI Ping;XU Xiaoyang(The Second College of Clinical Medicine,2School of Basic Medical Sciences,Guangzhou Medical University,Guangzhou 511436,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2020年第8期1359-1367,共9页
Chinese Journal of Pathophysiology
基金
广东省自然科学基金资助项目(No.2019A1515010983)
广东省高等教育教学改革项目(No.2018-484)
广州医科大学大学生实验室开放项目(No.2018-2)。
