Mechanism Of Ziziphi Spinosae Semen For Nourishing Heart And Tranquilizing Mind Based On The Whole Regulation Of Brain And Intestine

The research content of this paper is part of the National Natural Science Foundation Project of China“Study on the Synergistic Mechanism of Ziziphi Spinosae Semen（ZSS）Multicomponent for Nourishing Heart and Tranquilizing Mind Based on the Brain-Gut-Microbiota Axis".On the basis of preliminary chemical research,mainly from the perspective of overall regulation of the brain and intestine,the research is carried out on the mechanism and material basis of ZSS to nourish heart and tranquilize mind.This dissertation is divided into three chapters,the main research content and results are as follows.Chapter one,Literature reviewOn the basis of consulting and summarizing the relevant literatures,this chapter systematically reviewed the previous research progress on ZSS,and made a more systematic summary of the chemical constituents and pharmacological effects of ZSS.The characteristics and research progress of insomnia animal models were also reviewed,which provides an objective and mature animal model for revealing the ZSS’s nourishing heart and tranquilizing mind effect.At the same time,from the angle of "brain-gut-microbiota axis",this paper studied the internal relationship between the axis and the occurrence and development of insomnia,which provides reference for the study of the molecular mechanism and material basis of ZSS in the treatment of insomnia from the perspective of the brain and intestine overall regulation.Chapter two,Study on the mechanism of ZZS for nourishing heart and tranquilizing mind based on the whole regulation of brain and intestineSection 1,Research on the intervention effect of ZSS on insomnia rats based on "brain-gutmicrobiota axis"The insomnia rat model was established with p-chlorophenylalanine（PCPA）.After 10 days of intervention with ZSS,the urine,blood and tissues were collected to evaluate the effect of ZSS on insomnia rats.The results showed that the learning and memory ability of insomnia rats decreased significantly.Pathological changes appeared in the hypothalamic-pituitary-adrenal axis（HPA axis）related tissues and thymus.The results of ELISA analysis showed that the levels of HPA axis hormones,neurotrophic factors,inflammatory factors and brain gut peptide in insomnia rats were significantly different from those in control group.Administration of ZSS could improve the pathological changes of brain and intestinal tissue caused by insomnia to a certain extent,adjust the level of neurotrophic factors,inflammatory factors and so on.The structure and diversity of intestinal flora in insomnia rats were studied by 16S rRNA gene sequencing technology,and the regulatory effect of ZSS on intestinal flora in insomnia rats was explored.The result showed that compared with the control group,there were several different floras in the feces of insomnia model group from phylum level to genus level,among which 27 different genera were found at genus level.After the intervention of ZSS extract,the relative abundance changes of intestinal flora in insomnia model rats could be recalled,including Corynebacterium₁,Coriobacteriaceae_UCG-002,unclassified_fErysipelotrichaceae,norank_fErysipelotrichaceae and Allobaculum,which suggested that ZSS could improve the imbalance of intestinal flora in insomnia rats.In addition,the analysis results showed that the contents of short-chain fatty acids（SCFAs）in feces and cecal contents of insomnia rats were significantly lower than that of control group,and the SCFA content in insomnia rats returned to normal level in varying degrees after administration of ZSS extract.To sum up,ZSS extract can regulate the changes of brain,gut and intestinal flora diversity,and then regulate the "brain-gut-microbiota axis"as a whole.Section 2,Study on the regulation of the metabolic network of ZSS on insomnia rats based on metabolomicsBased on UPLC QTOF/MS technology,the plasma and urine of insomnia rats were studied by metabolomics to determine the changes of metabolites related to insomnia model and the intervention effect of ZSS.A total of 24 potential biomarkers were identified in the model group,including 7 in plasma and 17 in urine,which were different from those in control group.After given different doses of ZSS extract,18 different metabolites tended to normal level（P<0.05）,which were mainly involved in Taurine and hypotaurine metabolism,D-glutamine and Dglutamate metabolism,phenylalanine metabolism,pentose and glucuronate interconversions,arachidonic acid metabolism and other pathways.On this basis,the Pearson correlation coefficient between the related bacteria and biomarkers of insomnia was calculated by R language,and the heatmap of the relationship between intestinal microorganisms and metabolic differences in insomnia rats were drawn.The results showed that the five intestinal bacteria with significant callback after administration of ZSS were related to a variety of metabolites.The metabolic pathway related to insomnia is mainly amino acid metabolism,which is consistent with the amino acid transport and metabolism in the prediction of intestinal flora function,suggesting that insomnia is the result of synergism between metabolism and intestinal flora.The insomnia symptom can be improved by ZSS extract to adjust the metabolic spectrum and intestinal flora composition.Section 3,Study on the mechanism of ZSS on the basis of "pseudo-germfree" modelThe "pseudo-germfree" model was constructed by the combination of enema and mixed antibiotics to verify the role of intestinal flora in the treatment of insomnia with ZSS.Results showed that the community composition of intestinal flora in "pseudo-germfree" rat was single,and the absolute abundance was significantly lower than that of control group,mainly Klebsiella under Proteobacteria.Based on the construction of "pseudo-germfree" rat,PCPA was injected intraperitoneally to establish insomnia rat model.Ten days after administration of ZSS,the histopathological,biochemical and structural sequencing of intestinal flora were analyzed to evaluate the effects of ZSS on "pseudo-germfree" and normal insomnia rats.The results showed that indexes of insomnia rats were changed compared with the control group.The indexes of the insomnia rats based on the control rats tended to be normal after the administration of ZSS,while the insomnia rats based on the "pseudo-germfree" model had poor callback effect after given the same dose of ZSS,suggesting that the intestinal flora plays an important role in the prevention and treatment of insomnia with ZSS.Chapter three,Study on the material basis of ZZS nourishing heart and tranquilizing mind based on the whole regulation of brain and intestineSection 1,Evaluation of regulating effect of ZSS different components on insomnia ratsBased on the insomnia model induced by PCPA,the fatty oil,saponin and flavonoid enrichment parts of ZSS were reflux extracted with petroleum ether,70%ethanol and separated and purified by D101 macroporous resin to analyse the intervention effect of behavior,organ indexes,histopathology and biochemical indexes in insomnia rats.The results showed that the different components of ZZS had different degree of callback effect on various indexes of the insomnia model rats,among which ZSS saponin enrichment part and the combination of ZSS saponin and fatty oil components had better callback effect.The above results suggestd that ZSS saponins were the main material basis for regulating pathological changes and biochemical indexes,while there was no significant difference between the combination with fatty oil parts and ZSS saponins alone.Section 2,Evaluation on the regulatory effect of ZSS multi components on intestinal flora structure in insomnia model ratsBased on 16S rRNA gene sequencing technology,the regulatory effects of different components of ZSS on intestinal flora of insomnia rats were studied.LEfSe analysis showed that there were 41 differential genera in the feces of insomnia rats compared with the control group.After the intervention of different components of ZSS,the relative abundance of Bacteroides,Barnesiella,Dubosiella,Monoglobus and norank_fOscillospiraceae in rat intestine caused by insomnia could be reversed.Different components can regulate the above changes in different degrees.Among them,ZSS flavonoid enrichment part and the combination of ZSS flavonoid and fatty oil components had better effects on the first three flora genera,while ZSS saponin enrichment part could regulate most of the different flora in rat intestine.The results suggested that ZSS saponins had a general regulatory effect on the different flora of insomnia.From the perspective of regulating the structure of intestinal flora,it was suggested that ZSS saponins were closely related to the effect of ZSS on nourishing heart and tranquilizing mind.