Lipid Metabolism And Proteomic Research Of Gypenosides On HDL₂-induced HepG2 Cells

Objective Observing the mechanism of Gypenosides(GPS)on cholesterol uptake and proteome in HDL2-induced HepG2 cell via Molecular biology,cell biology and proteomics.To lay a good foundation for Chinese Drug researching on cholesterol regulatory.Methods ①MTT assay was used to determine the optimal concentration of GPS for HepG2 cells in vitro.Than cells were treated with different concentrations of GPS toghter with 300mg/L HDL2(HDL-CE)for 24h.Intracellular lipid accumulation and content of cholesterol were tested respectively by oil red 0 staining and enzyme colorimetric.②Qualitative and quantitative analysis of lipid droplets in HepG2 cells were detected by fluorescence microscopy and flow cytometry.③The mRNA and protein expression of human scavenger receptor-B1 and HMGCR were detected through real-time PCR and Western immunoblotting.④Differential expression proteins were found via two-dimensional electrophoresis combined with mass spectrometric identification technology.Results ①0～120 mg/L concentration of GPS had no effect on HepG2-derived cholesterol-road cell.Oil red O staining showed,compared with normal group,the accumulation of intracellular lipid droplets,the content of total cholesterol in HepG2 cells were significantly increased after treatment with 300mg/L HDL2,as well as incubated with different concentrations of GPS and 300mg/L HDL2.②The increased lipid droplets from HepG2 cells were confirmed from HDL2 through fluorescence microscopy and flow cytometry.③The mRNA and protein expression of human scavenger receptor-B1 and HMGCR were significantly decreased after treated with GPS.④40 differential expression proteins had been identified via mass spectrometry,including heat shock protein 60,thioredoxin related protein,calumenin,EF-hand domain,calreticulin,nucleobindin serine/arginine-rich splicing factor,galectin-1,M2-pyruvate kinase,valosin-containing protein,3-hydroxyisobutyrate dehydrogenase precursor,endoplasmic reticulum protein 29,heterogeneous nuclear ribonucleoprotein K,heterogeneous nuclear ribonucleoprotein C,DEAD-box protein abstrakt,ZNF254 protein,Enoyl Coenzyme A hydratase,tropomodulin 3,stomatin-like protein,eukaryotic translation initiation factor 3,guanine nucleotide-binding protein,proteasome beta 3,ubiquinol-cytochrome c reductase core I protein,galactokinase 1,isocitrate dehydrogenase 3(NAD+),which had been down-regulated in HDL2 treated group and up-regulated in GPS treated group,along with those up-regulated in HDL2 treated group and down-regulated in GPS treated group proteins,such as peroxiredoxin 2,superoxide dismutase,heme binding protein 1,glutathione S-transferase P1,ssodium/hydrogen exchanger regulatory factor 1,retinoblastoma binding protein 7,heat shock protein 27,Stathmin1/oncoprotein18,WD-40 repeat protein,26S proteasome,Ran-binding protein 1,PSMA3,proteasome Y,cargo selection protein TIP47,pyrophosphate 1.Conclusion ①GPS can promote HDL2-derived cholesterol ester transport to the liver for metabolism,which reduced excessive accumulation of cholesterol in the plasma.In the consequence of excessive cholesterol intake,GPS can feedback regulate mRNA and protein expression of SR-B1(membrane receptor)and HMGCR(cholesterol synthesis rate-limiting enzyme),so as to protect the liver cells.②Gypenosides can maintain cellular redox homeostasis state,intracellular calcium homeostasis,cytoskeleton,cell metabolism,energy metabolism and stability through regulating cellular associated-proteins expression,which has provided a guarantee for the reverse cholesterol transport(RCT).