Study On The Mechanism Of Inhibitory Effect Of Extracts And Active Compounds Of Pomegranate Flowers Against Hyperlipidaemia Based On Cell Metabonomics

Pomegranate flowers(PF)are the dried petals of pomegranate(Punica granatum L.),known as "gulina" in Uyghur medicine.The main chemical components are flavonoids,polyphenols,triterpenoids,volatile oils,etc.Compounds include betulinic acid,luteolin,apigenin,biochanin A,etc.Which have anti-inflammatory,antioxidant,hepatoprotective and anti-tumour effects against type 2 diabetes and complications,circulatory system protection,etc.In traditional medicine,pomegranate flowers are often used for the treatment of diabetes and its complications,etc.Fewer studies have been conducted on the anti-hyperlipidaemic properties of pomegranate flowers.Therefore,in this paper,palmitic acid-induced Hep G2 cells were used as an in vitro hyperlipidemic model in order to investigate the efficacy and mechanism of action of pomegranate flower and its active compounds against hyperlipidemia.Objectives:To combine network pharmacology and molecular docking techniques to screen compounds and possible targets of pomegranate flower against hyperlipidaemia,to explore its pharmacodynamic effects and its mechanism of action in combination with Hep G2 cell assays,to identify potential biomarkers of pomegranate flower for the treatment of hyperlipidaemia by cellular metabolomics and to enrich for relevant metabolic pathways.Methods:1.To construct a "drug-component-target-disease" diagram of pomegranate flower and hyperlipidaemia using network pharmacology and molecular docking methods,and to explore the targets,enrichment pathways and active components of pomegranate flower against hyperlipidaemia.Molecular docking of pomegranate flower compounds with key targets was carried out to screen the active ingredients.2.Based on the results of network pharmacology and molecular docking analysis,Hep G2 cells were cultured and the effects of palmitic acid,pomegranate flower extract and the active monomer compounds betulinic acid,lignan,apigenin and biochanin A on the viability of Hep G2 cells were determined by the Cell Counting Kit-8(CCK-8)method.A model of lipid deposition in Hep G2 cells was established using palmitic acid,and the pharmacological effects were investigated by quantifying intracellular lipid content based on absorbance values by oil red O staining and intracellular total cholesterol(TC)and triacylglyceride(TG)content by GPO-PAP method.Annexin VFITC/PI double-staining flow cytometry assay to detect the number of early and late apoptotic cells after intervention with pomegranate flower extract and its four active compounds in Hep G2 cells.Western blotting assay and q RT-PCR assay to determine the expression of PI3K/AKT,JAK2/STAT3 pathway-related targets and apoptotic targets in Hep G2 hyperlipidemic cells.3.The intracellular metabolites of pomegranate flower extract,the active compounds betulinic acid,luteolin,apigenin and biochanin A after intervention with Hep G2 cells were subjected to information collection by UPLC-LTQ-Orbitrap-MS method,principal component analysis of the differential metabolites,identification of the differential metabolites and enrichment analysis of the associated metabolic pathways.Result:1.A total of 15 active compound components in pomegranate flowers,475 targets associated with the 15 active compounds in pomegranate flowers,2420 targets associated with hyperlipidaemia,and 240 intersecting targets of pomegranate flowers for the treatment of hyperlipidaemia were collected.240 intersecting targets were imported into cytoscape 3.7.2 software for topological analysis to screen the core targets of pomegranate flowers against hyperlipidaemia.The top 10 targets in terms of degree are SRC,MAPK1,MAPK3,AKT1,HSP90AA1,STAT3,TP53,RXRA,PIK3 CA,PIK3R1,which are mainly involved in molecular functions such as protein phosphorylation,ATP binding,and negative regulation of apoptosis.Related pathways are mainly involved in PI3K/AKT signalling pathway,MAPK signalling pathway,AGE-RAGE signalling pathway in diabetic complications,etc.2.Palmitic acid-induced Hep G2 cells showed different degrees of reduction in the number of intracellular lipid droplets and intracellular TC and TG contents after each group of drug interventions,with pomegranate flower extract and the active monomer compound betulinic acid showing better hypolipidemic effects.The pomegranate flower extract and the active compounds in its 4 compounds could improve the effect of hyperlipidemic disease by regulating the expression of PI3K/AKT and JAK2/STAT3 related genes and MAPK.Pomegranate flower extract and betulinic acid,luteolin,apigenin and biochanin A could inhibit early and late apoptosis of Hep G2 cells caused by palmitate lipotoxicity to achieve anti-hyperlipidemia.3.Samples were significantly separated by principal component analysis in the control,model and each dosing group and each metabolite moved towards the blank group after pharmacological intervention,indicating that pomegranate flower and its active monomeric compounds had a significant ameliorative effect on lipid deposition in Hep G2 cells.Pathway enrichment of the differential metabolites showed that PFE,betulinic acid,apigenin and biochanin A administration mainly affected the glycerophospholipid metabolic pathway,and luteolin mainly affected the taurine and subtaurine metabolic and glycerophospholipid metabolic pathways.Conclusion:In palmitic acid-induced Hep G2 hyperlipidemic cells,pomegranate flower extract,betulinic acid,luteolin,apigenin and biochanin A all improved lipid deposition to varying degrees,with pomegranate flower extract and betulinic acid showing better hypolipidemic effects.Based on the results of network pharmacology and molecular docking studies,it was hypothesized that the anti-hyperlipidemic effect of pomegranate flower was achieved through multi-component,multi-target and multi-pathway.Hep G2 cell experiments showed that its mechanism of action was achieved by inhibiting free fatty acid-induced apoptosis and regulating the expression of genes related to PI3K/AKT and JAK2/STAT3 pathways.This study is the first to investigate the hypolipidemic effect and mechanism of action of Garnet in terms of improving lipid deposition in human hepatocellular carcinoma Hep G2 cells,which is important for the maintenance of hepatic lipid homeostasis and the prevention and treatment of cardiovascular diseases.