| Objective: X.sorbifolia is a kind of edible plant with a long history of medicinal use.It is called the whole body is treasure.Its seeds are important oil crops,which can be used as raw materials for producing diesel oil,and its young leaves can be used as excellent raw materials for producing health tea.Long-term consumption of yellowhorn tea(YT)has the effects of diuresis,hemostasis,dispelling dampness,reducing blood lipid and cholesterol.But there is no experimental evidence yet.Therefore,in this study,the hypolipidemic effect of YT extract on hyperlipidemia mice was investigated by establishing a hyperlipidemia mouse model and using pharmacological methods and liver metabolomics analysis.The chemical constituents of YT extract were detected by HPLC-MS/MS method,and the main active constituents,targets and potential signaling pathways of YT extract were predicted by network pharmacological methods.Combined with in vivo cell experiments to verify the expression levels of core genes in related signaling pathways,it provides scientific evidence for the effect of YT extract on hyperlipidemia(HLP).Methods: 1)Thirty-six KM male mice were randomly divided into six groups(normal group,model group,YT-0.15g/kg,YT-0.3g/kg,YT-0.6g/kg,positive group).A hyperlipidemia mouse model was established by high fat diet induction.Body weight,food intake,perirenal and periepididymal fat index,organ index(liver,heart,kidney,spleen),serum TG,TC,LDL-C,HDL-C levels and liver TG and TC levels were detected.Pathological symptoms of liver,kidney and adipose tissue were observed by HE staining.The levels of liver inflammatory factors(IL-6,IL-1β,TNF-α),oxidative stress indicators(SOD,CAT,GSH-Px,MDA),adiponectin and leptin were determined by enzyme-linked immunosorbent assay(ELISA).2)LC-MS/MS method was used to detect the characteristics,classification,relative content of metabolites in mouse liver and the expression of HLP-related metabolic pathways,so as to reveal the metabolic profile of YT extract in HLP mouse liver and Spearman correlation analysis was used.To study the correlation analysis of liver metabolites,body weight,food intake,liver inflammatory factor indexes and oxidative stress indexes of HLP mice,and to provide scientific basis for the role of HLP pathological mechanism.3)The chemical constituents of YT extract were analyzed qualitatively and quantitatively by HPLC-MS/MS method.Chemical constituents with content greater than 1% were used as potential active constituents for HLP treatment.Combined with network pharmacology,the active constituents and targets of YT extract for HLP treatment were preliminatively predicted.4)Finally,the in vitro experiment of human hepatocellular carcinoma cell HepG2 steatosis model induced by oleic acid(OA)with YT extract was conducted to preliminatively verify the core targets and pathways.CCK-8 method was used to detect the effects of YT extract and OA on the survival rate of HepG2 cells.The contents of TG,TC,LDL-C and HDL-C in supernatant were detected by oil red O staining and biochemical analyzer.The expression levels of IL1 B,IL6,TNF,PPARA,PPARG and VEGFA genes were detected by q PCR.Results: 1)In vivo animal experiments showed that compared with normal group,body weight,fat index,liver index,serum and liver TG,TC and serum LDL-C levels of hyperlipidemia mice induced by high fat diet were significantly up-regulated(p <0.05),and YT extract significantly decreased serum and liver TG and TC levels.Levels of inflammatory and oxidative stress markers IL-6,IL-1β,TNF-α,MDA and leptin(p < 0.05).The levels of oxidative stress indexes(SOD,CAT,GSH-Px)and adiponectin were significantly increased by YT extract.Histopathological tests showed that YT extract significantly improved lipid deposition in liver,kidney and adipose tissue.2)Liver metabolomics studies showed that high fat diet induced significant changes in the liver endogenous molecules of hyperlipidemia mice.After YT extract ingestion,12 different metabolites were recovered significantly,including adenosine monophosphate,citicoline,d UMP,N(6)-(1,2-dicarboxyethyl)AMP,NAD,nicotinamide N-oxide,PC(22:5(4Z,7Z,10 Z,13Z,16Z)/18:2(9Z,12Z)),pyridoxine,ribose-1-phosphate,serylthreonine,SM(d18:1/18:1(9Z)),uridine-5’-monophosphate.Pathway analysis showed that YT extract improved HFD-induced hyperlipidemia in mice through three main metabolic pathways,namely glycerol phospholipid metabolism,vitamin B6 metabolism,niacin and niacinamide metabolism.3)A total of127 compounds were detected in the positive and negative ion mode in YT extract by HPLC-MS/MS method,among which 24 compounds with content higher than 1%were selected as potential chemical components for HLP treatment.Network pharmacological analysis predicts,The effective active ingredients of YT extract in treatment of HLP are quercetin,isamoltan,1,4-Phenylenebis(1-piperidinylmethanone),9-Benzyl-3,9-diazaspiro[5.5]undecan-2-one and N-(4-Hydroxy-3-nitrophenyl)-2-[(1-methyl-2,4,6-trioxohexahydr-o-5-pyrimidinyl)carbonyl]hydrazinecarboxamidecatechi n compounds such as;The first 10 core targets of YT extract in treatment of HLP were TNF,IL6,IL1 B,PPARA,PPARG,AKT1,VEGFA,CRP,ACE and CCL2.Pathway analysis showed that YT extract may affect a variety of signaling pathways related to hyperlipidemia,including AGE-RAGE signaling pathway,PPAR signaling pathway,TNF signaling pathway,insulin resistance and cholesterol metabolism in diabetes complications.4)In vitro HepG2 cell validation experiments showed that YT extract could inhibit OA induced steatosis of HepG2 cells.After YT extract intervention,the number of intracellular lipid droplets stained with oil red O gradually decreased,and the TG content of HepG2 cells significantly decreased.In addition,the expression levels of the core genes IL6,IL1 B,TNF,PPARA,PPARG and VEGFA in the critical pathway were detected by q PCR.Compared with the normal group,the m RNA expressions of IL6,IL1 B,TNF,PPARG and VEGFA in the OA group were significantly increased.The m RNA level of PPARA was significantly decreased(p <0.05)and the above results were significantly reversed after YT treatment.Conclusion:Based on animal experiments in vivo and metabonomics,this paper revealed the effect and mechanism of the YT extract on hyperlipidemia.Then HPLC-MS/MS method was used to detect the chemical constituents of YT extract and network pharmacology was used to predict that the key pathways for reducing blood lipids of yellowhorn tea were AGE-RAGE signaling pathway,PPAR signaling pathway and TNF signaling pathway in diabetes complications.Validation network analysis of in vitro HepG2 cell assay.This study has a preliminary understanding of the mechanism of lowering blood lipid of the extract of yellowhorn tea,which lays a theoretical and scientific basis for further discussion. |
