基于代谢组学研究痰热内扰型慢性失眠病人血清代谢物及中药干预作用

Serum Metabolites of Chronic Insomnia with Phlegm-heat Internal Disturbance and the Intervention of Traditional Chinese Medicine

目的基于代谢组学技术寻找痰热内扰型慢性失眠病人的差异性血清代谢物,分析慢性失眠痰热内扰证病人经黄连温胆汤治疗前后血清代谢物的变化,初步揭示黄连温胆汤对痰热内扰型慢性失眠的干预作用。方法选取2019年8月—2020年12月自愿参加本研究的痰热内扰型慢性失眠病人36例作为慢性失眠组,选取同期健康受试者36名作为健康对照组,其中,慢性失眠组脱落3例,最终纳入33例。慢性失眠组给予黄连温胆汤免煎颗粒(组方:黄连、法半夏、竹茹、橘红、麸炒枳实、茯神、甘草、生姜、大枣),每日1剂,上下午分服,治疗4周。健康对照组不予干预。慢性失眠病人治疗前后及健康受试者均进行空腹静脉采血,采用液相色谱-质谱联用(LC/MS)技术对收集的血清样本进行检测,使用多元统计分析法寻找血清代谢物,利用MetaboAnalyst 4.0软件筛选出差异性代谢物及其涉及的代谢通路。结果与健康对照组比较,慢性失眠组病人血清中上升的代谢物质有L-乙酰肉碱、L-亮氨酸、L-缬氨酸、溶血磷脂酰胆碱(LysoPC)[20∶3(5Z,8Z,11Z)]、LysoPC(17∶0)、组胺、磷脂酰胆碱(PC)[18∶1(9Z)/20∶4(5Z,8Z,11Z,14Z)];下降的物质有肌酸、L-苯丙氨酸、L-色氨酸、鞘磷脂(SM)[d18∶0/18∶1(11Z)]、油酸、PC[18∶3(6Z,9Z,12Z)/16∶0]、二十二碳六烯酸。与治疗前比较,经黄连温胆汤治疗后,慢性失眠组病人血清中上升的物质有LysoPC[18∶2(9Z,12Z)]、LysoPC[20∶4(8Z,11Z,14Z,17Z)]、LysoPC[18∶1(11Z)]、L-苯丙氨酸、L-亮氨酸、肌酸、L-色氨酸;下降的物质有PC[16∶1(9Z)/20∶3(8Z,11Z,14Z)]、PC[16∶1(9Z)/22∶2(13Z,16Z)]、L-肉碱、SM[d18∶0/18∶1(11Z)]、PC[18∶1(9Z)/20∶4(5Z,8Z,11Z,14Z)]、SM[d18∶0/16∶1(9Z)]、PC[o-16∶1(9Z)/20∶4(8Z,11Z,14Z,17Z)]、2-羟基丁酸、L-乙酰肉碱、SM(d18∶1/16∶0)。结论痰热内扰型慢性失眠病人体内存在磷脂代谢、氨基酸代谢、能量代谢、脂肪酸代谢等异常,黄连温胆汤对痰热内扰型慢性失眠干预作用与调节氨基酸代谢、脂质代谢、能量代谢有关。

Objective Based on metabonomic technology,the serum metabolites in patients with phlegm-heat internal disturbance type chronic insomnia were analyzed before and after treatment with Huanglian Wendan Decoction.Methods A total of 33 patients with phlegm-heat internal disturbance chronic insomnia were selected as the chronic insomnia group,and 36 healthy subjects were selected as the healthy control group.The chronic insomnia group was given Huanglian Wendan Decoction,one dose a day,for 4 weeks.Healthy control group did not intervene.Fasting venous blood samples were collected before and after treatment.Liquid chromatography-mass spectrometry(LC/MS)was used to detect the collected serum samples.Multivariate statistical analysis was used to find serum metabolites.MetaboAnalyst 4.0 software was used to screen out different metabolites and the related metabolic pathways.Results Compared with healthy control group,in chronic insomnia group L-acetylcarnitine,L-leucine,L-valine,LysoPC[20∶3(5Z,8Z,11Z)],LysoPC(17∶0),histamine,and PC[18∶1(9Z)/20∶4(5Z,8Z,11Z,14Z)]were increased;the creatine,L-phenylalanine,L-tryptophan,SM[d18∶0/18∶1(11Z)],oleic acid,PC[18∶3(6Z,9Z,12Z)/16∶0],and docosahexaenoic acid were decreased.Compared with data before treatment,after treatment with Huanglian Wendan Decoction,serum substances in chronic insomnia group increased were LysoPC[18∶2(9Z,12Z)],LysoPC[20∶4(8Z,11Z,14Z,17Z)],LysoPC[18∶1(11Z)],L-phenylalanine,L-leucine,creatine,L-tryptophan;the descending substances were PC[16∶1(9Z)/20∶3(8Z,11Z,14Z)],PC[16∶1(9Z)/22∶2(13Z,16Z)],L-carnitine,and SM[d18∶0/18∶1(11Z)],PC[18∶1(9Z)/20∶4(5Z,8Z,11Z,14Z)],SM[d18∶0/16∶1(9Z)],PC[0-16∶1(9Z)/20∶4(8Z,11Z,14Z,17Z)],2-hydroxybutyric acid,L-acetylcarnitine,and SM(d18∶1/16∶0).Conclusion Chronic insomnia patients with phlegm-heat internal disturbance shows abnormalities in phospholipid metabolism,amino acid metabolism,energy metabolism,fatty acid metabolism,etc.The intervention effect of Huanglian Wendan Decoction is related to the regulation of amino acid metabolism,lipid metabolism and energy metabolism.