Effect On Lipid Metabolism Of High-density Lipoprotein From Egg Yolk And Its Regulation Mechanism

High-density lipoprotein from egg yolk（EYHDL）,also known as lipovitellin,is the second largest lipoproteins in egg yolk（EY）.We speculate that it can be used as a potential regulator of lipid metabolism for its anti-oxidation and blood pressure lowering properties.In this study,highly purified EYHDL was firstly prepared,and its structure and lipid composition were further characterized.Then,the effects of EYHDL on lipid metabolism in mice were systematically studied through nutritional intervention experiments.The lipidomic analysis in serum and liver was conducted to explore the mechanism of EYHDL regulation on lipid metabolism;In vitro cell experiments,systematic lipidomic and metabonomic analysis of cellular lipids and metabolites were conducted to reveal involved metabolic regulation pathways;The main research contents and results are as follows:（1）A two aqueous phase method was established to extracte EYHDL.The effects of NaCl and（NH₄）₂SO₄ on the purity and extraction rate of EYHDL were compared.The results indicated the extraction ratio of HDL is 73.7%with the highest purity of 83.0%under optimal extraction condition of pH 4.5 and 4%PEG6000 precipitation using NaCl.This method simplifies the separation procession,and is also environmentally friendly and efficient（p<0.05）.At the same time,high-efficiency purification of EYHDL was achieved by using DEAE-650M chromatography column.The particle size and secondary structure of purified EYHDL were further analyzed.The results confirmed that its particle size was about 10-20 nm,andα-helix andβ-turn angle were the most dominant secondary structure of its apoprotein,which indicats that highly stable structure of obtained EYHDL.（2）The lipid profile of EYHDL was analyzed by ultra performance liquid chromatography tandem mass spectrometry（UPLC-MS/MS）and gas chromatography mass spectrometry（GC-MS）.The results showed that phosphatidylcholine（PC）was the highest lipid component in EYHDL.The triglycerides（TG）contents of EYHDL was about1/4 of that of EY,while the higher levels of PC,phosphatidylinositol（PI）and diglyceride（DG）were found in EYHDL（p<0.05）.The comparison of differential lipids showed that22 TG differential lipids were detected in ES+mode,which was significantly lower in EYHDL than EY（p<0.01）.In the ES-mode,a total of 30 differential lipids were detected,including 10 P-choline（9 PCs,1 lysophosphatidylcholine（LPC））,and 15 P-ethanolamines(3 phosphatidylethanolamine（PE）,1 lysophosphatidylethanolamine（LPE）,4 PI and 1sphingomyelin（SM）.Compared with EY,most of the PC and PE were significantly increased in EYHDL（p<0.01）.In addition,the results of GC-MS showed that EYHDL contained higher levels of C18:1 and C22:6,while lower levels of C16:0 than EY（p<0.05）.（3）A metabolic syndrome（MetS）mice model was established using high fat to study the effect of EYHDL on blood lipid profile and gene expressions related to lipid metabolism for a 100-day intervention.The results showed that EYHDL intake significantly inhibited weight gain and the accumulation of abdominal adipose tissue,decreased serum low-density lipoprotein cholesterol（LDL-C）,TG,and hepatic TG and total cholesterol（TC）compared with high-fat diet（HFD）group（p<0.05）.At the same time,EYHDL intake also increased TC excretion,reduced the formation of fatty liver,and improved glucose and insulin resistance.The up-regulation of involved genes such as sterol regulatory element binding protein 1c（SREBP-1c）,acetyl-CoA carboxylase 1（ACACA）,fatty acid synthase（Fasn）,Glycerol-3-phosphate acyltransferase（GPAT）and stearoyl-CoA desaturase（SCD1）in HFD group was inhibited by EYHDL.（4）The fatty acids（FA）profile in the serum,liver and feces were analyzed by GC-MS.EYHDL significantly reduced total FA and saturated fatty acids（SFA such as C18:0and C16:0）in serum and liver,and SFA（C14:0 and C12:0）in the liver,unsaturated fatty acids（MUFA（C16:1,C16:0 and C18:1））and polyunsaturated fatty acids（PUFA（C18:3and C18:2））levels and promoted the efflux of polyunsaturated fatty acids（PUFA）such as C18:2 and C20:3（p<0.05）.Lipid profiles in serum and liver on 60 d and 100 d were further analyzed using UPLC-MS/MS.Compared with HFD,EYHDL significantly increased serum PC,FA and TG,and PI and SM in the liver（p<0.05）.Subsequently,the differential lipids were selected using OPLS-DA.A total of 10 lipid markers were detected in serum,and 5 of them,such as PI（16:0/18:2）,PI（16:0/20:4）,PI（18:0/20:3）,PI（18:0/20:4）,and PI（18:1/20:4）,were significantly down-regulated by EYHDL（p<0.01）.A total of 8 DG,DG（16:0/18:2）,DG（18:1/18:2）,DG（18:1/20:4）,DG（18:2/18:2）,DG（18:2/20:4）,DG（18:2/22:6）,DG（18:3/18:2）,and DG（22:5/18:2）,2 TG（TG（18:1/20:3/22:6）,TG（18:1/22:5/22:6））,and 3 FA（C20:4,C20:5 and C22:6）were down-regulated in liver（p<0.01）.Glycerophospholipid metabolism is the most relevant regulatory metabolic pathway through path analysis.（5）A fatty liver model in vitro was established by oleic acid（OA）-induced HepG2cell to explore the regulation mechanism of EYHDL on the formation of fatty liver.EYHDL significantly inhibited the accumulation of TG and oil droplets in hepatocytes,and significantly increased intracellular LPE and phosphatidylserine（PS）（p<0.05）.Further analysis of differential lipid metabolites revealed that EYHDL significantly reduced TG and increased LPC,acylcarnitine（CAR）and ceramide（Cer）（p<0.05）,which were inversely related to the formation of fatty liver.The metabolic pathway analysis also confirmed that glycerophospholipid metabolism is the most relevant metabolic pathway.In addition,ESI-QTRAP-MS/MS was used to analyze downstream metabolites,and 297 and230 metabolites were detected in positive and negative ion modes,respectively.A total of9 differential metabolites were identified compared to OA control group,7 lecithins（LPE16:0,LPA 16:0,LPC 16:0,LPC 18:0,LPC 20:2,LPC17:0 and LPC 18:3）,1 Glycerol 3-phosphate and 1 lipid（PAF C-16）,all of which detected significant metabolites were significantly down-regulated in the OA+EYHDL group（p<0.01）.The glycerophospholipid metabolism and choline metabolism are the most relevant metabolism.Altogether,the regulation of EYHDL is closely related to glycerophospholipid metabolism,which is consistent with animal experiments.（6）Further verification of glycerophospholipids metabolism was conducted.Long-chain fatty acids（LCFA）,the raw material for the glycerophospholipids metabolism,can cause abnormal metabolism of glycerophospholipids due to its abnormal transport and absorption.Therefore,a Caco-2 and HepG2 co-culture system was established to evaluate the effect of EYHDL on the dynamic transport and absorption of six C14:0,C16:0,C16:1,C18:0,C18:1,C18:2.The results indicated that EYHDL inhibited the transport of C16:1and C18:2 from AP to BL of Caco-2 monolayer and the accumulation of C16:0,C18:0 and C18:1 in HepG2 cells,which leading to the reduction of TG.Western-blot and RT-qPCR analysis of LCFA transport-related proteins showed that EYHDL inhibited the transport of C16:1 and C18:2 by inhibiting fatty acid transporter 2（FATP2）and fatty acid binding protein pm（FABPpm）in Caco-2.It also reduced the expression of SREBP-1c and its downstream lipogenic genes ACACA and SCD1,leading to the inhibition the synthesis of TG in HepG2 cells.