载脂蛋白E基因敲除对小鼠脂蛋白上鞘氨醇-1-磷酸分布的影响

Effects of apolipoprotein E deficiency on sphingosine-1-phosphate distribution in plasma and lipoproteins of mice

目的 研究载脂蛋白E基因敲除（Apo E-/-）对小鼠体内鞘氨醇-1-磷酸（S1P）在脂蛋白上分布的影响及作用机制.方法 分别给予普通生长繁殖饲料喂养Apo E-/-及野生型雄鼠、雌鼠各5只,饲养小鼠至周龄32周,处死小鼠并收集血浆,采用超速离心技术分离出血浆脂蛋白组分[极低密度脂蛋白脂蛋白（VLDL）、低密度脂蛋白（LDL）、高密度脂蛋白（HDL）],采用BCA试剂盒检测各组分的蛋白浓度,液相色谱-串联质谱法（LC-MS/MS）检测小鼠血浆S1P和脂蛋白组分上S1P的浓度,用Western bolt检测S1P特异性配体载脂蛋白M（Apo M）的蛋白表达.比较Apo E-/-组与野生型组中S1P在血浆及脂蛋白组分上的水平;比较两组中脂蛋白组分标准化后的S1P的水平及两组中Apo M在血浆、肝脏、肾脏的表达.结果 Apo E-/-小鼠血浆S1P水平高于野生型组[雄鼠组：（535.7±78.5） nmol/L比（263.3±22.0）nmol/L;雌鼠组：（601.1±64.0） nmol/L比（279.0±33.9）nmol/L;P均＜0.01].Apo E-/-组小鼠S1P在非-HDL（合并LDL及VLDL组分）的水平高于野生型组[雄鼠组（504.9±52.8）比（28.7±9.0） nmol/L;雌鼠组：（427.7±27.4）比（27.8±4.7） nmol/L;P均＜0.01;蛋白标准化后,雄鼠组：（385.0±41.2） pmol/mg蛋白比（71.4±6.6） pmol/mg蛋白;雌鼠组：（330.2±22.0）pmol/mg蛋白比（67.2±12.1）pmol/mg蛋白;P均＜0.01].Apo M在Apo E-/-组小鼠血浆、肝脏及肾脏的表达均明显高于野生型组（P＜0.05）.结论 Apo E-/-可导致小鼠血浆中非-HDL上S1P的水平上升,其作用机制可能是Apo M携带S1P从HDL转移至非-HDL.Apo M从HDL到非-HDL的转移可能导致Apo E-/-小鼠血浆中非-HDL上S1P水平增加.

Objective To investigate the effects of apolipoprotein E deficiency （Apo E-/-） on plasma and lipoprotein distribution of sphingosine-1-phosphate （S1P） in mice.Methods Five male or female Apo E-/-or wild type （WT） mice were fed with chow diet and sacrificed at 32-week-age and plasma was collected.The constituents of lipoprotein（very low density lipoprotein （VLDL）,low density lipoprotein （LDL）,high density lipoprotein （HDL）） were separated by ultracentrifuge.The protein concentration of constituents was detected by BCA protein quantitative kit,and the S1P concentration in plasma and various lipoprotein constituents was detected by liquid chromatography-tandem mass spectrometry （LC-MS/MS）.Western blot was used to determine the plasma,liver,and kidney protein expression of apolipoprotein M （Apo M）,which is considered as specific ligand of S1P.The S1P concentration in plasma and various constituents of lipoprotein in the Apo E-/-mice was compared to respective WT mice.Results （1） Plasma S1P content was significantly higher in the Apo E-/-groups than that of WT groups （male：（535.7 ± 78.5） nmol/L vs.（263.3 ±22.0）nmol/L;female：（601.1 ±64.0）nmol/L vs.（279.0 ± 33.9） nmol/L;all P ＜ 0.01）.（2） Compared with WT mice,S1P content in non-HDL（LDL ± VLDL） was significantly higher in Apo E-/-mice （male：（504.9 ±52.8） nmol/L vs.（28.7 ±9.0） nmoL/L;female：（427.7 ±27.4） vs.（27.8 ± 4.7） nmol/L;after standardization of protein concentration,male：（385.0 ± 41.2） pmol/mg protein vs.（71.4 ±6.6） pmol/mg protein;female：（330.2 ± 22.0） pmol/mg protein vs.（67.2 ± 12.1） pmol/mg protein;all P ＜ 0.01）.（3） The expression of Apo M in plasma,liver and kidney was significantly higher in Apo E-/-groups than that of WT groups （all P ＜ 0.05）.Conclusion The deficiency of Apo E could lead to upregulated S1P expression in the non-HDL,the underlying mechanism might be the increased transfer of HDL into the non-HDL by Apo M-S1P.