CD38经TFEB介导促进溶酶体再生而调节巨噬细胞胆固醇外流  

CD38 regulates macrophagic cholesterol efflux by promoting lysosome reformation via TFEB

作　　者：许浩 孙雪妮 吴天祺 刘进源 黄倩琳 莫蝶 王嘉馨 陈沈娴 邓伯丹 许小洋 XU Hao;SUN Xueni;WU Tianqi;LIU Jinyuan;HUANG Qianlin;MO Die;WANG Jiaxin;CHEN Shenxian;DENG Bodan;XU Xiaoyang(The Second College of Clinical Medicine,Guangzhou Medical University,Guangzhou 511436,China;School of Basic Medical Sciences,Guangzhou Medical University,Guangzhou 511436,China)

机构地区：[1]广州医科大学第二临床学院,广东广州511436 [2]广州医科大学基础医学院,广东广州511436

出　　处：《中国病理生理杂志》2024年第1期28-37,共10页Chinese Journal of Pathophysiology

基　　金：广东省基础与应用基础研究基金面上项目(No.2021A1515011335,No.2019A1515010983);国家级大学生创新创业训练计划项目(No.202310570032);广州医科大学学生创新能力提升计划项目(广医大[2022]66号);广州医科大学第二临床学院大学生科技创新项目(No.S2023A017)。

摘　　要：目的:探讨CD38对巨噬细胞溶酶体再生及胆固醇外流的影响。方法:以低密度脂蛋白(LDL)受体敲除(LDLr^(-/-))小鼠的骨髓源性巨噬细胞为细胞模型。采用活细胞成像系统观察烟酸腺嘌呤二核苷酸磷酸(NAADP)对巨噬细胞溶酶体数量的影响;利用ELISA检测巨噬细胞内NAADP的水平;细胞经NA处理后,利用RT-q PCR检测CD38 m RNA表达,利用Western blot检测CD38蛋白表达和转录因子EB(TFEB)磷酸化水平;利用激光共聚焦技术观察CD38/NAADP信号通路对溶酶体数量和胆固醇外流的影响。结果:NAADP可显著增加巨噬细胞中溶酶体的数量(P<0.05),这种效应可被NAADP拮抗剂NED-19、Ca^(2+)螯合剂BAPTA及钙调磷酸酶抑制剂Cs A明显抑制(P<0.05);CD38可明显促进巨噬细胞中NAADP的合成(P<0.05);NAADP合成底物NA可明显促进CD38 m RNA和蛋白表达(P<0.05);NA还可显著降低TFEB的磷酸化水平,且这一效应也可被NED-19、BAPTA和Cs A明显抑制(P<0.05);阻断CD38/NAADP信号通路可明显抑制NA诱导的溶酶体数量增加和溶酶体游离胆固醇及胞质胆固醇酯的外流(P<0.05);在LDLr/CD38双基因敲除巨噬细胞中,NA诱导的溶酶体数量增加和溶酶体游离胆固醇及胞质胆固醇酯的外流效应消失,CD38基因回补后,这一效应即可恢复(P<0.05)。结论:CD38可经TFEB介导,触发巨噬细胞溶酶体再生,进而促进巨噬细胞溶酶体游离胆固醇和胞质中胆固醇酯的外流。AIM:To explore the effects of CD38 on lysosome reformation and cholesterol efflux in macrophages.METHODS:Bone marrow-derived macrophages from low-density lipoprotein(LDL)receptor knockout(LDLr^(-/-))mice were cultured as cell model.Live cell imaging system was applied to evaluate the effect of nicotinic acid adenine dinucleotide phosphate(NAADP)on lysosome number.ELISA was conducted to measure NAADP level in macrophages.After the cells were treated with nicotinic acid(NA),RT-q PCR was conducted to detect CD38 mRNA expression,and Western blot was conducted to observe CD38 protein expression and phosphorylated transcription factor EB(TFEB)level.Laser scanning confocal microscopy was applied to evaluate the influence of CD38/NAADP signaling on lysosome number and cholesterol egression.RESULTS:NAADP remarkably increased lysosome number(P<0.05),and this effect was significantly inhibited by NAADP antagonist NED-19,Ca^(2+)chelator BAPTA,and calcineurin inhibitor Cs A(P<0.05).CD38 markedly enhanced NAADP synthesis in macrophages(P<0.05).NAADP synthetic substrate NA prominently elevated the expression of CD38 mRNA and protein(P<0.05).NA significantly decreased the phosphorylated TFEB level;this effect was also attenuated by NED-19,BAPTA and Cs A(P<0.05).Disrupting CD38/NAADP signaling pathway markedly inhibited NA-induced enhancement of lysosome number,lysosomal free cholesterol and cytosol cholesterol ester efflux in macrophages(P<0.05).NA-induced enhancement of lysosome number,lysosomal free cholesterol and cytosol cholesterol ester efflux abolished in LDLr/CD38 DKO macrophages(P<0.05),whereas these effects induced by NA were recovered after CD38 gene rescue.CONCLUSION:CD38 triggers lysosome reformation via TFEB and consequently promotes the efflux of lysosomal free cholesterol and cytosol cholesterol ester.

关 键 词：CD38 溶酶体再生 巨噬细胞 胆固醇 转录因子EB 

分 类 号：R329.21[医药卫生—人体解剖和组织胚胎学] R363.2[医药卫生—基础医学]