Study On The Material Foundation And Mechanism Of Efficacy Of Tiansiyin Decoction For The Treatment Of Senile Dementia Based On Key Technologies Of System Biology

Objective:In this paper,the network pharmacology method was used to explore the mechanism and material basis of Tiansiyin decoction（TSYD）in the treatment of Alzheimer’s disease,and to predict the main components,targets and pathways.It lays a foundation for further research of TSYD.Ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry（UHPLC-QQQ-MS）was used to quantify the potential active components in TSYD and determine the main components.Using Caenorhabditis elegans wild type（N2）and transgenic AD model（CL4176）as model organisms,through oxidative stress resistance experiment,lifespan experiment,and paralysis mitigation experiment,etc.,the mechanisms of TSYD on preventing and treating dementia and delaying aging was further explained.Method:1.Network pharmacological study of TSYD in treating AD:In the TCMSP database,AD is used as the research object,and all targets and components related to AD in Morinda officinalis and Cuscuta are reversed,and the ADME parameter differences between the components are compared.The network pharmacology method established TSYD’s"component-target"network,"target-pathway"network and protein-protein interaction（PPI）network.Finally,Gene Ontilogy and KEGG enrichment analysis was performed on candidate targets to explore the mechanisms and material basis of TSYD in the treatment of AD.2.Medicinal chemistry research of TSYD:First,the total flavonoids content of TSYD and its medicinal materials was determined.Then acetonitrile-0.2%phosphoric acid aqueous solution was used as the mobile phase,and the chemical composition of TSYD was qualitatively analyzed by HPLC by comparing the retention time and the maximum absorption wavelength of the reference substances.Then UHPLC-QQQ-MS was applied to establish the standard curve of each reference substance and their content in TSYD were measured.3.Study on the drug uptake of TSYD:Caenorhabditis elegans administered TSYD at an optimal dose.After 24 hours,the worms were collected,ground and processed by protein precipitation.UPLC-MS-QQQ analysis technology was applied to determine the drug uptake values of main components in TSYD.Qualitative analysis and quantification results of in vivo uptaken components were obtained by applying setting mass spectrometry conditions and comparing retention times.4.Pharmacological effects of TSYD:Through paraquat oxidative stress resistance experiment,the optimal concentration of TSYD with antioxidant effect was obtained.Based on the result,life-span experiment was conducted to further confirm the pharmacological effects of TSYD.The paralysis relief experiment was performed using CL4176 Caenorhabditis elegans,and the optimal drug concentration for alleviating Aβtoxicity was obtained.5.Proteomics study of Caenorhabditis elegans induced by TSYD:Transfer the Caenorhabditis elegans in L1 phase to NGM plates coated with TSYD solution,about 8000 Caenorhabditis elegans were seeded in each plate.The S.medium added NGM plate was used as an empty control.When the nematodes were cultured to the oviposition period,the residual E.coli and drugs in the epidermis were washed away and stored in a 1.5 mL centrifuge tube.Two groups of Caenorhabditis elegans samples were labeled with TMT peptides,identified by mass spectrometry,and the bioinformatics of the two groups of differentially expressed proteins were analyzed using KOBAS software.Results:1.Using network pharmacological analysis,13 types of AD-related components in TSYD were obtained.8 components belong to Morinda officinalis and 5 components belong to Cuscuta.Combine the targets related to AD,inflammation,and aging obtained from the CTD,Innate,and GenAge databases with the targets corresponding to the TSYD components predicted by Swiss Targetprediction to obtain 8 overlapping targets,namely ABL1,PTGS2,AKT1,PIK3R1,PIK3CA,IGF1R,HIF1A and PTK2.Through KEGG pathway enrichment analysis,candidate targets were mainly enriched in the endocrine resistance pathway.2.Eight reference materials were selected and quantified by UHPLC-QQQ-MS.Their contents were:hypericin 0.2963%;chlorogenic acid0.2352%;crystal blue glycoside 0.1688%;muscarin glycoside 0.0891%;kaempferol 0.0390%;deacetylated trifoliate 0.0059%;quercetin 0.0010%;isorhamnetin 0.0001%.The content of the flavonoids in TSYD was 1.83%.Different concentrations of TSYD-H₂O solution have different scavenging effects on DPPH free radicals.The concentration was between 0.2 mg/mL²5 mg/mL,the dose-dependent effects were more obvious;25 mg/mL⁵0mg/mL,the change tended to be gentle.3.By using UHPLC-QQQ-MS method,the drug uptake results of 7potential active components from TSYD by C.elegans elegans were ontained.Ingredients and contents were 0.375μg/mL of hypericum,0.5831μg/mL of chlorogenic acid,0.691μg/mL of crystal blue glycoside,0.1131μg/mL of myricin,0.0995μg/mL of kaempferol,and 0.0556μg/mL of isorhamnetin.4.To establish the oxidative stress resistant experiment using paraquat pre-treated C.elegans,8 concentrations of TSYD were selected:4 mg/mL,8mg/mL,16 mg/mL,24 mg/mL,32 mg/mL,40 mg/mL,80 mg/mL and 400mg/mL.The results showed that 8 mg/mL was the optimal concentration.The results of paralysis relief experiment showed that the TSYD solution with concentrations from 0.8 mg/mL to 32 mg/mL can delay the paralysis of CL4176 C.elegans,with a better paralysis relief efficacy of 3.2 mg/mL.At160 mg/mL and 320 mg/mL,toxic effects may occur due to too high drug concentrations.Finally,TSYD（16 mg/mL）and hypericin can significantly extend the lifespan of wild-type C.elegans N2.5.A total of 3349 key protein spots were identified between the 2 groups of C.elegans samples.Compared with the blank group,there were 563differentially expressed proteins in the TSYD group,including 277 positive regulatory proteins and 286 negative regulatory proteins.Gene ontology analysis showed that these differentially expressed proteins are mainly involved in biological processes such as cellular stratum corneum formation,fatty acid production,ATP binding,and DNA binding.KEGG pathway analysis further showed that these proteins were mainly concentrated in drug metabolism-cytochrome P450,biosynthesis of unsaturated fatty acids,fatty acid metabolism,glutathione metabolism,systemic lupus erythematosus,and cytochrome P450 metabolism of xenobiotics.Conclusion:This article uses network pharmacology technology to evaluate the efficacy of TSYD for the treatment of AD.In addition,the use of TMT proteomics technology to explore the mainly regulated proteins after TSYD was taken by Caenorhabditis elegans revealed that TSYD mainly regulates vit-5,vit-1,vit-3,vit-4,Acdh-1,gst-31,gst-27,gst-24,gst-5,gst-13,gst-12,gst-38,gst-10,scl-2 and other protein expression activation or inhibition,regulate drug metabolism-Cytochrome P450,biosynthesis of unsaturated fatty acids,fatty acid metabolism pathways,thereby exerting the effect of extending lifespan.