Study On The Anti-insomnia Mechanism And The Change Rule Of Anti-insomnia Core Components Of Faeces Bombycis Based On The Network Pharmacology

Objective:To clarify the mechanism of of Faeces Bombycis(FB)on “ anti-insomnia ” from the perspective of network pharmacology,and on that basis,to study the dynamic changes of the core components of “ anti-insomnia ” in the process of “ Morus alba L.to Faeces Bombycis ”.And to clarify the scientific nature of the fecal traditional Chinese medicine Faeces Bombycis as a medicine from the perspective of ingredients,and promote the inheritance and development of traditional Chinese medicine of feces.Methods:1.Study on the mechanism of Faeces Bombycis in the treatment of insomnia based on network pharmacologyTo collect the chemical components of FB through databases such as TCMSP;to collect component targets through databases such as Pharm Mapper;to verify the efficacy of FB on insomnia through disease enrichment analysis;and to collect disease targets related to insomnia through databases such as CTD.Then,the intersection of the component targets and disease targets was mapped;used STRING database to perform PPI analysis,GO analysis and KEGG pathway analysis on the intersection target;and visualize the results through Cytoscape software;finally used Auto Dock Vina to verify the molecular docking of anti-insomnia core components and hub targets.2.Verification of the “ anti-insomnia ” effect and mechanism of Faeces BombycisThe insomnia animal model was established by intraperitoneal injection of PCPA to investigate the effects of Faeces Bombycis on the falling asleep rate,sleep latency and sleep duration of mice induced by sodium pentobarbital.The Morris water maze experiment and the open field test were used to investigate the effect of FB on the behavior of insomnia mice.The influence of anxiety and other behaviors related to insomnia further verified the“ anti-insomnia ” effect of FB.On this basis,the effects of FB on the content of Glu,GABA and the expression of GABAARα1 and GABAARγ2 were studied,to clarified the mechanism of FB on “ anti-insomnia ”.3.Study on the change rule of anti-insomnia core componentsFaeces Bombycis is the feces of Bombyx mori L,and its composition includes undigested Morus alba L.and related substances in the Bombyx mori L’s gastrointestinal tract.The gastrointestinal tract of Bombyx mori L is generally in an anaerobic environment.Therefore,the GC-MS,HPLC and other methods were used to study the changes in the “ quality ” and“ quantity ” of volatile components,flavonoids isoquercitrin,and GABA from Morus alba L.to FB,which in order to clarifies the scientific nature of fecal traditional Chinese medicine Faeces Bombycis as a medicine from the perspective of ingredients.Results:1.Study on the mechanism of FB in the treatment of insomnia based on network pharmacologyWith OB ≥ 20% and BBB >-0.3 as the screening threshold,a total of 7 components that meet the requirements were obtained.Combined with the literature,4 components related to insomnia were added.Therefore,there are a total of 11 components related to the anti-insomnia effect of FB.There were 366 targets for component action;608 targets for insomnia had been collected through OMIM and other databases.The intersection of component targets and disease targets were mapped.129 common targets were analyzed by PPI,GO and KEGG.PPI results showed that FB had 15 core targets and 8 core components in the treatment of insomnia;GO analysis results showed that FB treatment of insomnia mainly involves biological processes such as chemical synaptic transmission and GABA signaling pathway;KEGG analysis results showed that FB mainly treated insomnia by regulating the pathways of the GABAergic system;the results of molecular docking were further verified that the core target and core components have a good combination.2.Verification of the “ anti-insomnia ” effect and mechanism of Faeces BombycisThe results of the pentobarbital sodium synergy experiment showed that compared with the control group,the falling asleep rate and the sleep duration were significantly reduced(P < 0.05),the sleep latency was significantly increased(P < 0.05)of mice in the model group.After giving FB,compared with the model group,the M-FB and H-FB groups significantly increased the fall asleep rate and the sleep duration,and significantly shortened the sleep latency(P < 0.05)of insomnia mice.The results of the OFT showed that,compared with the control group,the total movement distance,average speed,frequency of entering the central area and staying time in the central area of the model group increased significantly(P <0.05),while the resting time was significantly decreased(P <0.05).Compared with the model group,he total movement distance,average speed,frequency of entering the central area and staying time in the central area of the mice in the M-FB and H-FB groups were significantly reduced(P < 0.05),while the resting time was significantly prolonged(P < 0.05).The results of the Morris water maze experiment showed that,on the first day,the time to search for platform in the Morris water maze training,which had no statistical difference of each group;and from the second day,the time for the the model group mice to find the platform significantly prolonged(P < 0.05)while comparing with the control group.After giving FB,compared with the model group,the time to find the platform of the H-FB group was shortened(P < 0.05)from the second day,while the same index of the M-FB group was significantly shortened(P < 0.05)from the fourth day.The effect of FB on the content of Glu and GABA showed that,compared with the control group,the Glu content in the hypothalamus and colon of the model group was significantly increased(P < 0.05),the GABA content was significantly decreased(P < 0.05),and which was significantly increased(P < 0.05)in the M-FB and H-FB groups while after treatment,and when comparing with the model group,the Glu content of the M-FB and H-FB groups was significantly reduced(P < 0.05).The results of immunohistochemical experiments showed that,compared with the control group,the expression of GABAARα1 and γ2 in the hypothalamus of the model group was significantly reduced(P < 0.05);after administration of FB,compared with the model group,those indexs of mice in the M-FB and H-FB group was significantly increased(P < 0.05).3.Study on the change rule of anti-insomnia core componentsThe GC-MS results show that,compared with Morus alba L.and “ simulated-FB ”,the unique components in FB are Di-epi-.alpha.-cedrene,2-Ethyl-1-hexene,3-methylaminopropylamine,geranylacetone,3,5-dihydroxypentylbenzene,isocar-boxene epoxide,farnesyl acetone.Compared with Morus alba L.and “ simulated-FB ”,the increased content of FB included octamethylcyclotetrasiloxane,cyclopentadimethylsiloxane,β-ionone,cedar,phytone,and isophytol.The results of changes in the content of flavonoids isoquercitrin in FB showed that compared with Morus alba L.,with the increase of “ vacuum ” time,the content of isoquercitrin in “ simulated-FB ” increased sharply(P <0.05);compared with “ simulated-FB ”(8 h),the content of isoquercitrin in FB was significantly increased(P <0.05).The results of changes in the GABA content of the amino acid component of FB showed that compared with Morus alba L.,as the “ vacuum ” time increased,the GABA content in“ simulated-FB ” increased sharply(P <0.05);while the GABA content in FB was compared with that in Morus alba L.significantly reduced(P <0.05).Conclusion:Faeces Bombycis acts on related targets through volatile oil,isoquercitrin,GABA and other core components,to regulates the pathways of the GABAergic system which play an“ anti-insomnia ” effect.During the formation of Faeces Bombycis,the gastrointestinal environment of Bombyx mori increased the content of volatile components,flavonoids and other medicinal components.The fecal Chinese medicine Faeces Bombycis has a certain degree of scientificity as a medicine.