The Effect And Mechanism Of DHA-PC On Triglyceride Accumulation In HepG2 Cells

Large yellow croaker roe is highly edible due to its high content of phospholipids(PLs)with n-3 polyunsaturated fatty acid.Previously,our research found that PLs from large yellow croaker roe had physiological functions that it would regulate triglyceride(TG)aggregation in mice significantly.However,the function of its main component,DHA-Phosphatidylcholine(DHA-PC)on TG regulation,has not yet been clarified.Therefore,DHA-PC was used as experimental material to study the effect and mechanism of DHA-PC on triglyceride deposition based on HepG2 cell model in this study.The main results are as follows:1?DHA-PC liposomes were prepared by membrane dispersion using high-purity DHA-PC as raw material.And its morphology and particle size distribution were observed.Taking HepG2 cells as the research object,HepG2 cells were cultured with different concentrations of DHA-PC liposomes for 24,48 and 72 h.The cell viability was measured by MTT assay,and the cell integrity was determined.Then the safe concentration range of DHA-PC liposome on HepG2 cells was selected.The results show that the prepared DHA-PC liposomes have good morphology and uniform distribution,and the average particle size is 234.4 nm.The safe concentration range of DHA-PC liposomes is 0?g/mL and 40 ?g/mL.At this time,the cell survival rate is higher and the integrity is better.2.HepG2 cells were cultured for 24 h with different concentrations of oleic acid(OA)and high glucose DMEM containing 10%fetal bovine serum.The high-fat model in vitro was established by measuring cell activity,oil red O staining and intracellular lipid levels.After the model was established,fenofibrate drugs and DHA-PC liposomes of different concentrations were administered to HepG2 cells for 24,48 and 72 h.After the intervention,the content of intracellular TG was detected,andthe accumulation of lipid droplets in the cell was observed by oil red O staining.The results showed that the selected OA model concentration was 0.75 mM.At this concentration,HepG2 cells had no obvious proliferative damage.The cells contained a lot of lipid droplets,and TG accumulation reached a high level.Compared with the model group,different doses of DHA-PC liposomes(10 ?g/mL,25 ?g/mL and 40?g/mL)at different time(24 h,48 h and 72 h)can significantly reduce the triglyceride content in HepG2 cells and reduce the accumulation of intracellular lipid droplets.3?In order to further study the hypolipidemic mechanism of DHA-PC,we used RT-qPCR to detect the mRNA expression of PPARa,CPT1A,and FAS in HepG2 cells.Western blot was used to detect the protein expression levels of PPAR?,CPT1A,and FAS in HepG2 cells.The results showed that compared with the model group,the expression of FAS mRNA and protein could be down-regulated in different time periods(24 h,48 h and 72 h),the expression of PPARa mRNA and protein was up-regulated.The expression of CPT1A mRNA was significantly up-regulated after treatment for 72 h,and the expression of CPT1A protein could be up-regulated in different time periods(24 h,48 h,72 h).It is suggested that the hypolipidemic mechanism of DHA-PC may be related to the activation of PPARa,up-regulation of CPT1A and down-regulation of the expression of FAS.4?The iTRAQ proteomics technique was used to investigate the uptake mechanism of DHA-PC liposomes in HepG2 cells.The differentially expressed proteins between the blank group and DHA-PC liposome were screened,and the differentially expressed proteins were analyzed by Gene Ontology(GO)classification annotation,KEGG Pathway and protein interaction.The results showed that there were 232 differential proteins among the two groups of cells,of which 19 were up-regulated and 213 were down-regulated.The GO and KEGG Pathway analysis of differential proteins revealed that the metabolic pathways involved in these differential proteins include:fatty acid metabolism,RNA transport,fatty acid biosynthesis,AMPK signaling pathways and so on.By constructing the protein interaction network between the differential proteins and KEGG pathways,it was found that proteins that interact most at protein interaction sites include IKBKG,NUP93,STAT3,ACSL4 and so on.Presumably these proteins may play an important role in the euptake of DHA-PC liposomes by HepG2 cells.In summary,this study systematically studied the effect of DHA-PC from the large yellow croaker roe on the TG deposition in HepG2 cells,and elucidated the mechanism of its influence on TG deposition from the molecular level.On this basis,the mechanism of absorption of DHA-PC liposomes in HepG2 cells was initially explored.The results of this study provide theoretical basis for the development of DHA-PC as a functional food ingredient.