| Objective:To identify the differential proteins related to insomnia and Yuanqi capsules in the treatment of insomnia,find the pathological changes of insomnia and provide experimental basis for the mechanism of action of Yuanqi Capsule in the treatment of insomnia,the molecular weight and protein content of differential proteins in normal mice and insomnia mice were identified by mass spectrometry with systemic biology methods.Methods:(1)180 SPF Kunming mice were randomly divided into six groups:blank control group,p-chlorophenylalanine(PCPA)model control group,Yuanqi capsule low,medium and high treatment group,and western medicine control group.Molding method A mouse model of insomnia was prepared by intraperitoneal injection of PCPA suspension.(2)The mice were decapitated and the parietal cortex proteins of each group were extracted and trypsin lysed to determine the protein concentration.The whole proteome expression difference analysis was performed on the brain tissue of each group by ion orbital trap(Orbi-trap)mass spectrometry.The differentially expressed proteome was analyzed by the knowledge mining method(Ingenuity EPA database).(3)The effects of Yuanqi capsule on the threshold latency of sleep and sleep time of mice with threshold dose of pentobarbital sodium were observed in the sleep experiment of mice with threshold dose of pentobarbital sodium.(4)The proteome group differentially expressed between the Chinese medicine group and the model group was analyzed by the knowledge mining method(Ingenuity IPA database)for the metabolic pathway network,and the mechanism of the difference between the normal group and the model histone was analyzed.(5)Western Blot was used to detect the contents of different proteins in the 6 groups of samples to verify the IPA metabolic network pathway analysis results.(6)The content of each monoamine neurotransmitter was measured by enzyme-linked immunosorbent assay(ELISA)and compared statistical analysis.Results:The sodium pentobarbital sleep test confirmed that Yuanqi Capsule can shorten the sleep latency of normal mice and insomnia mice caused by PCPA and prolong sleep time;A total of 2,206 proteins were detected by mass spectrometry,107 of which were up-regulated and 88 of which were down-regulated in the model group and the normal control group,28 kinds of differentially expressed proteins were obtained in each dose group of Yuanqi capsule and model group.Using IPA software to analyze the differentially expressed proteins in a network pathway and combing the literature search,we found five signaling pathways related to insomnia,namely oxidative phosphorylation,mitochondrial dysfunction,protein kinase A signaling pathway,melatonin signaling pathway,14-3-3 protein signaling and GABA resepter signaling pathway.After treatment with Yuanqi Capsule,the effect of GABA receptor signaling pathway was only found between Yuanqi Capsule-treated mice and insomnia model mice.Western Blot results showed that GABA receptor protein was significantly up-regulated in the model group,while it is decreased significantly after treatment with Yuanqi capsule.Therefore,we believe that the mechanism of action of sedative and hypnotic in Yuanqi Capsule is mainly related to the regulation of GABA receptor.The results of ELISA showed that the content of 5-HT and NE in serum decreased significantly after treatment with Yuanqi capsule,and the level of GABA was significantly increased.Conclusion:Using proteomics techniques,we found that oxidative phosphorylation,mitochondrial dysfunction,protein kinase A signaling pathway,melatonin signaling pathway,14-3-3 protein signaling pathway signaling and protein associated with insomnia.Yuanqi Capsule mainly plays a role in the treatment of insomnia by acting on the GABA receptor signaling pathway.The use of proteins related to insomnia can further study the disease markers and drug targets of insomnia,and further explore the molecular biological mechanism of Yuanqi Capsule in the treatment of insomnia. |
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