目的: 以代谢组学技术研究辛伐他汀对小鼠肝脏中脂类物质代谢的影响。方法: 采用超高效液相色谱-高分辨飞行时间质谱(UPLC-Q TOF MS)联用技术对给予药物辛伐他汀后小鼠的肝脏样品进行代谢物指纹信息采集,使用Acquity BEH C18色谱柱,流动相为水-乙腈,梯度洗脱,流速0.5 mL·min-1,柱温35 ℃,进样室温度4 ℃,进样量10 μL;电喷雾离子源,正离子检测模式,扫描范围m/z 100~1 000,毛细管电压为3.1 kV,锥孔电压为30 V,采用100 pg·mL-1亮氨酸-脑啡肽(leucine-enkephalin,LE)溶液为质量校正溶液。通过数据采集进行代谢轮廓分析。读取总离子流色谱图中峰信息,利用主成分分析筛选出候选的差异代谢物后,通过一级、二级质谱碎片信息,比对数据库鉴别差异代谢物的结构。利用免疫印迹、同位素放射代谢流分析,验证辛伐他汀对细胞脂质合成的影响。结果: 给予辛伐他汀后,在肝组织发现8个代谢差异物,各剂量组溶血磷脂的含量增多,其中LysoPE(18∶1)在低、中、高剂量组中分别是对照组的1.14、1.24、1.31倍,LysoPC(16∶0)在、中、高剂量组分别是对照组的1.58、2.05、2.32倍。花生四烯酸、岩芹酸、O-花生四缩水甘油、17(S)-HETE、PC(16∶0/O-1∶0)、DG(16∶1/17∶1/0∶0)等脂肪酸和磷脂的含量减少,并呈现出剂量相关性。其中PC(16∶0/O-1∶0)的下降幅度最大,低、中、高剂量组中分别是对照组的0.68、0.43、0.38倍。通过同位素示踪验证辛伐他汀对脂质合成的影响并呈现出剂量相关性,对脂质合成限速酶具有调节作用。证实辛伐他汀可以抑制肝脏脂质的合成,最终导致脂类总量的降低。结论: 研究结果提示通过调节肝脏脂类代谢是辛伐他汀实现降脂作用的途径之一。
Objective: To study the effects of simvastatin on lipid metabolism of liver samples in mice by metabolomics. Methods: The metabolite fingerprint information of liver samples of mice treated with simvastatin was collected by ultra-performance liquid chromatography-high resolution time-of-flight mass spectrometry (UPLC-Q TOF MS) using Acquity BEH C18 column, water-acetonitrile gradient eluted mobile phase, flow rate was 0.5 mL·min-1, column temperature was 35 ℃.The injection chamber temperature was 4 ℃, and the injection volume was 10 μL. The conditions of mass spectrometer were ectrospray ion source, positive ion detection mode, scanning range m/z 100-1 000, capillary voltage 3.1 kV and cone-hole voltage of 30 V,100 pg·mL-1 leucine enkephalin (LE) solution as quality correction solution. Metabolic profile analysis was performed by data acquisition. The peak information in the total ion flow chromatogram was read. The acquired data was processed by using principal component analysis. After screening for candidate differential metabolites, the structure of the differential metabolites was identified by comparing the primary and secondary mass spectrometry fragment information with the database. The effect of simvastatin on lipid synthesis was verified by Western blot and isotope radio metabolic flow analysis. Results: After administration of simvastatin, eight metabolic differentials were found in liver tissues. The content of lysophospholipids increased in each dose group, with LysoPE (18∶1) being 1.14, 1.24, and 1.31 times compared to the control group in the low, medium and high dose groups, respectively. LysoPC (16∶0) was 1.58, 2.05, and 2.32 times compared to the control group in the low, medium and high dose groups, respectively. The contents of fatty acids and phospholipids, such as arachidonic acid, petroselinic acid, O-arachidonoylglycidol, 17(S)-HETE, PC(16∶0/O-1∶0), DG(16∶1/17∶1/0∶0) were reduced and showed a dose correlation. The largest decrease was observed in PC (16∶0/O-1∶0), which was 0.68, 0.43 and 0.38 times of the control group in the low, medium and high dose groups, respectively. Through isotope tracing, the effect of simvastatin on lipid synthesis was verified and showed a dose correlation, and regulated the rate-limi ting enzyme of lipid synthesis. It was confirmed that the administration of simvastatin inhibited the synthesis of hepatic lipids, which ultimately led to a decrease in total lipid. Conclusion: The results suggest that modulation of hepatic lipid metabolism is one of the ways in which simvastatin achieve its lipid-lowering effects.
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