基于UHPLC-Q-Exactive-MS/MS的酸枣仁总皂苷对失眠大鼠血清代谢物的影响

Effects of Ziziphi Spinosae Semen Saponins on Blood Metabolites of Insomnia Rats Based on UHPLC-Q-Exactive-MS/MS

目的采用代谢组学技术观察酸枣仁总皂苷对失眠大鼠血清代谢物的影响,探讨其改善失眠的作用机制。方法30只大鼠随机分为对照组、模型组和酸枣仁总皂苷组(ZSSS组),每组10只。连续3 d腹腔注射对氯苯丙氨酸建立失眠大鼠模型,造模后ZSSS组灌胃酸枣仁总皂苷溶液(124.8 mg/kg),对照组和模型组灌胃等量生理盐水,连续7 d。观察大鼠一般状态;戊巴比妥钠睡眠实验观察大鼠睡眠潜伏期和睡眠时间;HE染色观察下丘脑组织病理变化;超高效液相色谱-四级杆高分辨质谱(UHPLC-Q-Exactive-MS/MS)联用技术建立血清代谢组学分析方法,结合主成分分析、正交偏最小二乘判别分析筛选潜在差异代谢物,采用Metabo Analyst 5.0数据库和Cytoscape3.8.2软件分析相关代谢通路并构建代谢网络。结果与对照组比较,模型组大鼠昼夜节律紊乱,出现疲劳、精神不振和活动减少现象,睡眠潜伏期显著延长(P<0.05),睡眠时间显著缩短(P<0.05),下丘脑组织神经细胞边界模糊,排列松散,有大量碎片细胞;与模型组比较,ZSSS组大鼠兴奋性降低,攻击行为减少或消失,昼夜节律有所恢复,睡眠潜伏期显著缩短(P<0.05),睡眠时间显著延长(P<0.05),下丘脑组织神经细胞较丰富,分布较均匀,细胞形态明显恢复。模式识别分析筛选出8个与酸枣仁总皂苷治疗失眠相关的潜在差异代谢物,涉及烟酸和烟酰胺代谢、甘油磷脂代谢及酪氨酸代谢3条通路,GPCPD1、PNPLA6和PLB1等可能是其改善失眠的作用靶点。结论酸枣仁总皂苷能改善失眠大鼠睡眠情况,其作用机制与调节1-甲基烟酰胺、甘油磷酸胆碱、乙酰胆碱和3,4-二羟基苯乙二醇等代谢有关。

Objective To observe the effects of Ziziphi Spinosae Semen saponins on blood metabolites of insomnia rats;To explore its mechanism to improve insomnia.Methods Totally 30 rats were randomly divided into control group,model group,and Ziziphi Spinosae Semen saponins group(ZSSS group),with 10 rats in each group.Insomnia model was established by intraperitoneal injection of p-chlorophenylalanine solution in rats for 3 days.The ZSSS group was given the Ziziphi Spinosae Semen saponins(124.8 mg/kg)for gavage after the model was made,the control group and model group were given the same amount of normal saline for 7 consecutive days.General condition was observed;pentobarbital sodium sleep experiment was used to observe the sleep latency and sleep time of rats;HE staining was used to observe the pathological changes of hypothalamus;UHPLC-Q-Exactive-MS/MS technology was used to establish a serum metabolomics analysis method,principal component analysis and orthogonal partial least squares discriminant analysis were used to screen potential differential metabolites,and Metabo Analyst 5.0 database and Cytoscape 3.8.2 software were used to analyze related metabolic pathways and construct a metabolic network.Results Compared with the control group,the circadian rhythm of the rats in model group was disturbed,fatigue,lack of energy and reduced activity occurred,the sleep latency was significantly prolonged(P<0.05),and the sleep time was significantly shortened(P<0.05).The nerve cells in the hypothalamus had fuzzy boundaries,loosely arranged,and a large number of fragmented cells appeared.Compared with the model group,the rats in ZSSS group had reduced excitability,reduced or disappeared aggressive behavior,restored circadian rhythm,the sleep latency was significantly shortened(P<0.05),and the sleep time was significantly extended(P<0.05).The nerve cells in hypothalamus tissue were abundant and evenly distributed,and the cell morphology was obviously restored.Pattern recognition analysis screened out 8 potential different metabolites related to Ziziphi Spinosae Semen saponins in the treatment of insomnia,involving 3 pathways of nicotinic acid and nicotinamide metabolism,glycerophospholipid metabolism,and tyrosine metabolism.GPCPD1,PNPLA6 and PLB1 may be target of improving insomnia.Conclusion Ziziphi Spinosae Semen saponins can improve the sleep of insomnia rats,and its mechanism of action is related to the regulation of the metabolism of 1-methylnicotinamide,glycerophosphocholine,acetylcholine and 3,4-dihydroxyphenylglycol.