| Zexie decoction(AA)was first recorded in the “Synopsis of Prescriptions of The Golden Chamber” by Zhang Zhongjing in the Eastern Han Dynasty and has been in clinical use for a long time.It is extracted with water in the ratio of 5:2 from Alismatis Rhizoma(AR)and Atractylodis Macrocephalae Rhizoma(AMR),and is included in the “Catalogue of Ancient Classic Recipes(First Batch)” issued by the State Administration of Traditional Chinese Medicine.Modern pharmacological studies have shown the efficacy of AA on vertigo and metabolism-related diseases such as hyperlipidemia,hypertension and non-alcoholic fatty liver disease(NAFLD).However,the material basis of the chemical composition of its aqueous decoction is weak,and the therapeutic mechanism for NAFLD remains unclear.To address these issues,this thesis systematically characterized the chemical composition of AA based on high performance liquid chromatography(HPLC)and liquid chromatography-mass spectrometry(LC-MS).A C57BL/6 mouse model of NAFLD was established and two animal experiments were conducted to validate the therapeutic effects of AA.Mass spectrometry imaging,targeted metabolomics,untargeted metabolomics and transcriptomics were further utilized to search for biomarkers related to pharmacological efficacy and explore the scientific connotation of AA for the treatment of NAFLD.Firstly,the extraction and full chemical characterization of AA was carried out.The preparation of AA was carried out according to a process documented in ancient texts,and the average yield of AA lyophilised powder was 29.99%.A comprehensive quantitative characterization of its chemical composition was further carried out using four analytical methods.(1)The monosaccharides and oligosaccharides were quantified using high performance liquid chromatography with diode array detection(HPLCDAD).(2)The content of polysaccharide was quantified using a phenol-sulphuric acid method.(3)The content of 14 amino acids was quantified using a pre-column derivatization method.(4)The content of five nucleosides was quantified using high performance liquid chromatography with ultraviolet spectrophotometry(HPLC-UV).In addition,the content of moisture was quantified using a drying method.Finally,the composition of 93.41% of AA was clarified.For the unknown and other types of constituents in AA,they were analysed using a LC-MS method and 33 chemical components,mainly fat-soluble,were identified.Secondly,the therapeutic effect of AA on a dyhomeostasis mouse model of nonalcoholic fatty liver was confirmed.The Gubra-Amylin NASH(GAN)diet induced NAFLD model in C57BL/6 mice was selected and two animal experiments were conducted sequentially to verify the therapeutic effect of AA on NAFLD.The results showed that AA significantly reduced body weight and tissue weight(especially liver and white adipose tissue weight)in NAFLD mice,with little effect on food intake,while increasing the amount of urine in mice.Blood biochemical indices and pathological sections indicated that AA could protect the liver,alleviate steatosis and lobular inflammation,while lowering blood lipids and regulating glucose tolerance.Thirdly,the modulatory effects of AA on known biomarkers associated with NAFLD were investigated.Using desorption electrospray ionization mass spectrometry imaging(DESI-MSI),eight lipid types and a total of 56 lipids in mouse liver were analyzed and identified,visually represented their variation between normal and model groups as well as between drug administration groups.Using PCR technology,the metabolic modulation of intestinal flora by AA was analysed and it was found that AA improved intestinal disorders and significantly increased the abundance of four probiotic species,especially promoting the growth of Akkermansia muciniphila.Using targeted metabolomics,a method for the quantification of 31 bile acids was established and a method for the quantification of 8 short-chain fatty acids was previously established in the laboratory.It was found that AA could reduce bile acid accumulation and increase the abundance of short-chain fatty acids.Using transcriptomics,the mechanism of action of AA on epididymal adiposity in NAFLD mice was investigated.Differential gene enrichment analysis and pathway analysis were performed to demonstrate that AA affected epididymal fat in model mice through PPAR signaling pathway,regulation of lipolytic pathway in adipocytes pathway,thermogenic pathway,insulin resistance pathway,AMPK signaling pathway and citric acid cycle pathway,and thus treated NAFLD.Finally,non-targeted metabolomics techniques were used to explore the therapeutic effects of AA on NAFLD and to search for biomarkers associated with the efficacy.Three animal experiments with a combined total of ten mouse urine collections were conducted to demonstrate the dynamic changes of endogenous metabolites in urine at different time points of drug administration.The reversal of the metabolic profile by AA administration was demonstrated and differential ions were sought for the normal and model groups,and for the model and AA administration groups.Ultimately,a total of 14 potential biomarkers were identified and could be used for follow-up monitoring,including 2 stable significantly different ions,7 stable dose-dependent ions and 5 stable time-dependent ions.In addition,60 potential biomarkers were identified,and pathway analysis revealed fatty acid β-oxidation pathway was the most significant pathway,suggesting that the mechanism of action of AA may be closely related to this.In conclusion,we have clarified the chemical composition of AA and confirmed its therapeutic effects on non-alcoholic fatty liver liver disease,identified potential biomarkers associated with its efficacy,and explored the possible mechanisms underlying the onset of AA’s effects.This has laid a foundation for the subsequent application and bio-quality evaluation of AA. |
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