线粒体钙单向转运体在模拟失重所致巨噬细胞极化改变中的调控作用研究

Regulatory roles of mitochondrial calcium uniporter in simulated microgravity-induced macrophage polarization

目的探究线粒体钙单向转运体(mitochondrial calcium uniporter,MCU)在模拟失重状态下对巨噬细胞极化的调控作用及其机制。方法体外培养小鼠骨髓来源巨噬细胞(bone marrow derived macrophage,BMDM),采用配对比较法将野生型(wild ty pe,WT)小鼠的BM DM分为2D回转模拟失重组和同步对照组,2D回转模拟失重组细胞在2D细胞回转仪中回转24 h模拟失重效应,同步对照组细胞在2D细胞回转仪中静置24 h;按照小鼠基因型将2D回转模拟失重的细胞分为LysM Cre、MCU f l/f l与同窝对照MCU f l/f l组,2组均在2D细胞回转仪中回转24 h模拟失重效应。采用实时荧光定量核酸扩增检测系统(realtime quantitative PCR detecting system,Q-PCR)和流式细胞术检测BMDM向M1、M2型巨噬细胞极化的能力;蛋白免疫印迹检测B M D M在模拟失重状态下巨噬细胞线粒体融合分裂蛋白的表达水平;实时荧光定量核酸扩增检测系统检测B M D M线粒体D N A拷贝数;转染线粒体特异性绿色荧光钙质粒Ad-4 m t-G C a M P 6检测B M D M线粒体钙信号;D C F H-DA活性氧探针检测BMDM活性氧(reactive oxygen species,ROS)水平。结果在24 h回转模拟失重处理后,WT小鼠的BMDM向M1型巨噬细胞极化的能力减弱(t=6.409、4.800、7.808、6.506,P均<0.01),向M2型巨噬细胞极化的能力增强(t=3.265、4.458,P均<0.05);线粒体融合蛋白Mfn2和OPA1表达下降,分裂蛋白Drp1表达增加;线粒体DNA拷贝数增加(t=10.06,P<0.01),ATP数目下降(t=15.6,P<0.01);线粒体Ca2+与ROS水平上升;BMDM MCU mRNA与蛋白表达水平都显著升高(t=7.824,P<0.01);敲除MCU后的BM DM向M2型巨噬细胞极化被抑制(t=8.574、5.155,P均<0.05),ATP水平显著下降(t=3.317,P<0.05),线粒体Ca2+与ROS水平的升高也受到抑制。结论在模拟失重状态下,MCU作为线粒体Ca2+内流的关键调控蛋白介导线粒体钙超载导致ROS水平上升,从而促进了巨噬细胞向M2型巨噬细胞极化。

Objective To investigate the regulatory effects and mechanism of the mitochondrial calcium uniporter(MCU)on macrophage polarization during simulated microgravity.Methods 2-D clinostat was used to simulate microgravity in murine bone marrow derived macrophage(BMDM)in vitro.The BMDMs from wild type(WT)mice were randomly divided into 2 groups:the 2-D clinorotation group and control group.According to the genotype of mice,the cells with 2-D clinorotation were divided into LysM Cre,fl/fl fl/fl MCU and littermate control MCU group without MCU knockout.Macrophage M1/M2 polarization of BMDMs was detected via Q-PCR and flow cytometry.Western blot was used to detect the expression levels of mitochondrial fusion/fission proteins.Q-PCR-based determination of copy numbers of mitochondrial DNA was performed.Ad-4mt-GCaMP6 and DCFH-DA were used to detect mitochondrial calcium signals and reactive oxygen species(ROS)respectively.Results The macrophages from WT mice were polarized toward M2 macrophages with inhibited M1(t=6.409,4.800,7.808,6.506,all P<0.01)and activated M2 macrophages(t=3.265,4.458,both P<0.05).The expressions of mitochondrial fusion proteins Mfn2 and OPA1 decreased,while that of mitochondrial fission protein Drp1 increased.The copy number of mitochondrial DNA increased(t=10.06,P<0.01),while the number of 2+ATP decreased(t=15.6,P<0.01).The levels of mitochondrial Ca and ROS increased.The mRNA and protein expression levels in BMDM MCU were significantly increased(t=7.824,P<0.01).Knock out MCU from BMDM,M2 macrophage polarization was inhibited during simulated microgravity(t=8.574,5.155,both P<0.05),ATP levels significantly decreased 2+(t=3.317,P<0.05),while the levels of mitochondrialCa and ROS decreased.Conclusion MCU as a key regulatory 2+2+protein for mitochondrial Ca influx mediates mitochondrial Ca overload and increases production of ROS,thereby promoting the polarization of macrophages toward M2 macrophages.