Study On The Hypolipidemic Mechanism Of Stilbene Glycosides Based On LC-MS Technology And Network Pharmacology

Objective: To establish an UHPLC-Q-TOF-MS/MS method to analyze the metabolites and metabolic pathways of stilbene glycosides in healthy rats and hyperlipidemia model rats in vivo and in vitro,which provide a theoretical basis for the further study of stilbene glycosides,and to use metabolomics and network pharmacology to study the target and mechanism of stilbene glycosides in the treatment of hyperlipidemia.Methods: The fresh feces of healthy rats and hyperlipidemia model rats were collected to establish an intestinal bacteria incubation system.Plasma,bile,urine and fecal samples were collected from healthy rats and hyperlipidemia model rats after intragastric and tail intravenous injection of stilbene glycoside,respectively.The incubation solution of intestinal bacteria and the biological samples in vivo were pre-treated by methanol precipitation of protein.UHPLC-Q-TOF-MS/MS online acquisition technology,ESI source,negative ion mode,and full scan acquisition mode were used to collect data,and the results were imported into Metabolite Pilot 2.0.2 software to infer the possible metabolites according to the cleavage rules of the parent drug.Metabolomics research based on liquid mass spectrometry technology and network pharmacology research methods based on systems biology theory,were established to expound the possible mechanism of stilbene glycosides in the treatment of hyperlipidemia and investigate whether it produces hepatotoxicity.Male SD rats were randomly divided into five groups: healthy group,hyperlipidemia model group,high-dose administration group,medium-dose administration group and low-dose administration group and serum samples of each group were collected.LC/MS was used to collect data from samples of each group.The raw data were imported into Progenesis QI and SIMCA-P 14.1 software for data post-processing.The different metabolites between different groups were screened and related metabolic pathways were analyzed.Meanwhile,the efficacy of stilbene glycosides in lowering blood lipid and whether it produces hepatotoxicity were evaluated by combining with biochemical.Multiple databases were searched for the corresponding targets of stilbene glycosides and hyperlipidemia by network pharmacological methods,and the core targets were obtained by constructing a protein-protein interaction network screening.GO enrichment analysis and KEGG pathway analysis were performed using Metascape database.Results: A total of 47 metabolites were found in both healthy group and hyperlipidemia model group of rats.Among them,a total of 43 metabolites were found in the healthy group of rats,including 39 kinds after intragastric administration,18 kinds after intravenous administration,and 5 kinds of intestinal bacteria incubation solution.A total of 24 metabolites were found in the hyperlipidemia model group,including 17 for intragastric administration,6for intravenous administration and 5 for intestinal bacteria incubation solution.The main metabolic pathways involved glucuronic acid binding,sulfuric acid binding,sugar removal reaction,reduction reaction and so on.The results showed that there were differences in the metabolism of stilbene glycosides in the two different models.A total of 89 different metabolites were identified between the healthy group and the hyperlipidemia model group of rats,and a total of 94 differences were identified between the stilbene glycoside administration group and the hyperlipidemia model group.Metabolites mainly involved changes in glycerophospholipid metabolism,sphingolipid metabolism,primary bile acid biosynthesis and purine metabolism pathways.And 7β,12α-dihydroxycholic acid,L-acetylcarnitine,glycolic acid,(3α,5β,12β,17xi)-12,24-dihydroxy-24-oxacholine-3-yl-D-Glucopyranoside and N-[(3α,5β,7β,12α)-3,7,12-trihydroxy-24-oxycholine-24-yl]glycine were selected as the potential biomarker for the treatment of hyperlipidemia with stilbene glycosides.Subsequently,using network pharmacology methods,a total of 8 stilbene glycosides related targets were screened for the treatment of hyperlipidemia and 39 core targets were screened after PPI network analysis,involving related pathway such as HIF-1signaling pathway.Conclusions: The difference in the metabolites of stilbene glycosides in healthy rats and hyperlipidemia model rats indicated that the changes in the physiological state of hyperlipidemia rats had an impact on the processes of stilbene glycosides in vivo,suggesting that model animals should be used in the study of the pharmacodynamic form of stilbene glycosides in vivo;the differences in metabolism under different administration forms indicated that the intestinal flora had an influence on the metabolic process of stilbene glycosides,which is worthy of further study.Through metabolomics and network pharmacology studies,it was found that the treatment of hyperlipidemia with stilbene glycosides mainly involved signaling pathways such as glycerophospholipid metabolism,purine metabolism and phosphatidylinositol.This study provided a theoretical basis for elucidating the mechanism of stilbene glycosides in the treatment of hyperlipidemia.