Establishment Of Syndrome Cell Model And Its Application In The Biological Basis Research Of Chuanxiong In Promoting Blood Circulation And Removing Blood Stasi

Syndromes are the basis of TCM treatment according to syndrome differentiation,and the syndrome model is indispensable for the study of medicinal properties and efficacy,which is an important part in the of traditional Chinese medicine.At present,the syndrome animal model is more commonly used,and there are a series of problems,such as large difference of species,the complicated modeling methods,the lack of effective verification methods,and difficult to define the syndrome attribute.The in-depth study of the syndrome cell model is of great significance to explain the pathogenesis of syndromes and the mechanism of drug action.Therefore,this paper intends to establish the cell model of syndrome,and apply it to the biological basic research on the efficacy of Ligusticum Chuanxiong of blood activating and stasis dissolving.First of all,HepG2 cells were used as the research vectors,to establish a syndrome cell model by administering serum from a patient with a syndrome,and the modeling method is optimized in the process of modeling.Then,metabolomics was used to evaluate the model,the difference metabolites in the syndrome cell model and corresponding syndrome serum were compared,obtain the biological targets associated with the syndrome to verify the reliability and applicability of the model,construction of a model assessment method based on cell metabolomics.Finally,based on the modeling method and model evaluation method,the biological basic research on the efficacy of Ligusticum Chuanxiong on promoting blood circulation and removing blood stasis.Firstly,the comprehensive analysis of the chemical constituents of Ligusticum Chuanxiong in serum was carried out,and definite the blood components of Ligusticum Chuanxiong.Then,the blood stasis syndrome cell model was given by Chuanxiong drug-containing serum,to explore the differential metabolites in the cell model of blood stasis after administration of Chuanxiong-containing serum,using cell metabolomics methods.,discover potential biomarkers for the efficacy of Ligusticum Chuanxiong on promoting blood circulation and removing blood stasis,and exploring the Biological Basis of the Effects of Ligusticum Chuanxiong on promoting blood circulation and removing blood stasis at the cellular level.The main research contents and results are as follows:1.Study on the establishment of serum induced syndrome cell model.This chapter explores the establishment method of cell syndrome model.HepG2 cells were induced by human serum of liver-depression and spleen-deficiency syndrome(LDSDS)to establish a cell model of LDSDS in this research.The concentration of cells,the content of human serum in culture medium and the growth characteristics of model-cell(cell growth curve,the survival rate and apparent morphology were investigated by MTT assay and microscopy.The results showed that HepG2 cells were in an exponential growth phase within 2×10-3～1×104(cell/hole)concentration and 24 to 72 h,and it has a good linear relationship with absorbance value,3×103(cell/hole)concentration was selected for subsequent experiments.The results showed that,model cells have the characteristics of normal growth,slow proliferation and stable morphological structure inducted by 10%serum in 24-72 h.2.Verification of cell model based on metabolomics.In this study,the methods of cell metabolomics extraction and extraction analysis were optimized.The extraction methods of single-phase extraction and two-phase extraction cell metabolism were optimized.,at the same time,HILIC,Generic-RP and lipidomic-rp LC-MS were optimized for different extracts,and different extraction solvent and solvent ratio.The experimental results showed that 2:2:1 methanol/ethanol/water was used as the extraction solvent and Generic-RP was used as the analytical condition,which show the best extraction and analysis capabilities for single-phase cell extracts;1:1:1 methanol/water/chloroform as the extraction solvent provides the strongest ability to extract and separate two-phase cell extracts,the HILIC has the best performance for the extraction of aqueous extracts and Lipidomic-RP has the best performance for the extraction of organic phases from two phases.Based on the results,single-phase extraction combined with Generic-RP analysis was used to verify the syndrome cell model based on metabolomics.Metabolomics was used to analyze the human serum of normal individuals and patients with LDSDS,and cell models induced by these serums,respectively.We obtained the difference metabolites from serums and cell models of LDSDS,respectively;then compared the biomarkers from two metabolomics and their metabolic pathways,to verify that the reliability and applicability of the model.Metabolomics data were collected by UPLC Q-TOF-MS,and then all data were analyzed by multivariate statistical(PCA,OPLS-DA).Differential metabolites were selected according to VIP>1 and P<0.05,a total of 79 serum differential metabolites and 49 cell differential metabolites in the syndrome were identified,of which 19 were differential metabolites,including 9 metabolic pathways that play an important role in maintaining normal physiological activities of the human body,such as lipids,amino acids,nucleotides,and energy metabolism etc.It was shown that the established syndrome cell model could reflect the biological basis of LDSDS to some extent.Therefore,the syndrome cell model is judged successfully at this level.This study provides a reference for the establishment of TCM syndrome cell model3.Study on the biological basis of the efficacy of Rhizomo Chuanxiong in blood activating and stasis dissolving based on the syndrome cell model of qi stagnation and blood stasis syndrome(QSBSS).This chapter is based on the cell model of QSBSS,and discusses the biological basis of the efficacy of Rhizomo Chuanxiong in blood activating and stasis dissolving.First,establishment of an analytical method for the determination of components of chuanxiong in blood of rats by UPLC-Q-TOF/MS and a comprehensive analysis of the chemical constituents of Chuanxiong in serum.Finally,14 components of Chuanxiong into the blood were identified or inferred from Chuanxiong drug-containing serum,including 6 prototype components and 8 drug-derived metabolites.To obtain serum of QSBSS,the rat model of QSBSS was established by using high fat feed coupled with chronic stress stimulation,and the success of the model confirmed by examining behavioral and blood lipid biochemical indicators.The cell model of QSBSS was established using the method established in this paper.Investigating the effect of Chuanxiong drug-containing serum on the growth of cell model of QSBSS by measuring the survival rate of cells,the results show that the Chuanxiong drug-containing serum has a callback effect on the inhibition of cell proliferation caused by the model serum.The serum(Control and Model Group)and the cell model(Control,Model and Model given Chuanxiong drug-containing serum Group)of QSBSS were analyzed by metabolomic based on UPLC Q-TOF/MS technology,respectively.Then the difference metabolites of QSBSS be obtained from serums and cell models of QSBSS,respectively.There are 70 differential metabolites(45 in serums and 25 in cell models)were screened and identified through multivariate statistical analysis and including 8 identical differential metabolites.It is confirmed the success of the model of QSBSS.In addition,the identical metabolites showed a significant callback in the cells of Model given Chuanxiong drug-containing serum Group,suggesting that the efficacy of Rhizomo Chuanxiong in blood activating and stasis dissolving might be related to these identical differential metabolites.The results of principal component analysis showed that the Control,Model and Model given Chuanxiong drug-containing serum Group showed a clear clustering of the overall metabolic profile,it is demonstrating that the intracellular metabolic profile of the cell model of QSBSS significantly changed after administration of Chuanxiong drug-containing serum.The results of MetPA analysis showed that the differential metabolites found in the syndrome serum and the cell model of QSBSS involves Ether lipid metabolism,Sphingolipid metabolism,Pantothenate and CoA biosynthesis,Glycerophospholipid metabolism,Purine metabolism,Fatty acid metabolism and beta-Alanine metabolism pathway.It is suggested that the occurrence of the of QSBSS may be related to the disturbance of these pathways.The differential metabolites of callback in the cells of Model given Chuanxiong drug-containing serum Group were mainly involved in the metabolic pathway of Ether lipid metabolism and Glycerophospholipid metabolism.It is concluded that the effective components of Ligusticum Chuanxiong may play a role in blood activating and stasis dissolving involve in the regulation of lipid metabolism at the cell level.