中医病证相符的大鼠心肾不交失眠模型的建立及其血清代谢组学研究

Establishment of rat heart-kidney insomnia model consistent with traditional Chinese medicine syndrome and its serum metabolomics

运用酶联免疫吸附法(ELISA)和代谢组学对2种心肾不交失眠动物模型进行分析比较,以期探究出一种较为理想的动物模型,并初步探讨心肾不交失眠本质。基于临床心肾不交失眠的证候特征和疾病特征,采用腹腔注射对氯苯丙氨酸(PCPA)和多因素共同作用的方法复制心肾不交失眠动物模型,采用行为学观测、酶联免疫吸附法、代谢组学从多个层次对病证结合的动物模型进行评价。Wistar大鼠随机分成正常组、PCPA组、复合模型(FH)组,记录大鼠行为学、体质量、肾上腺指数、脾脏指数;利用ELISA检测血清中促肾上腺皮质激素释放激素(CRH)、肾上腺皮质激素(ACTH)水平;采用UPLC-QE-MS检测血清中的差异代谢物信息。行为学测得FH组体质量、肾上腺指数较PCPA组均显著降低(P<0.05);ELISA测得FH组较PCPA组ACTH,CRH显著升高;血清样本采集的数据统计经分析鉴定出9种潜在生物标志物,心肾不交失眠主要有4条代谢通路,分别是磷酸戊糖代谢,丙氨酸、天冬氨酸和谷氨酸代谢,组氨酸代谢,牛磺酸和亚牛磺酸代谢。PCPA和多因素共同作用方法,均能成功复制出失眠模型,但多因素造模法与临床中医病证更为相似。采用动物行为学、酶联免疫、代谢组学能够从体外到体内、从宏观到微观、从个体到整体对心肾不交失眠大鼠模型进行综合评价。

Enzyme-linked immunosorbent assay(ELISA)and metabolomics were used to analyze and compare two animal models of heart-kidney insomnia,in order to explore a more ideal animal model and preliminarily explore the essence of heart-kidney insomnia.Based on the clinical symptoms and disease characteristics of heart-kidney insomnia,the animal model of heart-kidney insomnia was reproduced through intraperitoneal injection with p-chlorophenylalanine(PCPA)and multi-factor interaction.The animal model of disease-syndrome combination was evaluated by behavioral observation,ELISA and metabolomics.Wistar rats were randomly divided into normal group,PCPA group and compound model group(FH).The rats′behavior,body weight,adrenal index and spleen index were recorded.The levels of corticotropin releasing hormone(CRH)and adrenocorticotropin(ACTH)in serum were detected by ELISA,and the differential metabolites in serum were detected by UPLC-QE-MS.The body weight and adrenal index in FH group were significantly lower than those in PCPA group(P<0.05);whereas ACTH and CRH in FH group were significantly higher than those in PCPA group by ELISA;nine potential biomarkers were identified by serum sample statistics.There were four main metabolic pathways in cardiorenal insomnia:pentose phosphate metabolism,alanine,aspartic acid and glutamic acid metabolism,histidine metabolism,and taurine and subtaurine metabolism.PCPA and multi-factor interaction method can successfully replicate the insomnia model,but multi-factor modeling method is more similar to clinical traditional Chinese medicine syndrome.Animal behavior,ELISA and metabolomics were used to evaluate the rat model of cardiorenal insomnia from in vitro to in vivo,from macro to micro,and from individual to the whole.