| Hen egg, as a cholesterol-rich food, nutritional advantages and disadvantages of the current status quo is still controversial. So the object of this study were hen eggs and its active ingredients, and the study was carried out to evaluate the egg cholesterol nutritional pros and cons though animal experiment, screen the active ingredients from eggs with regulation role of cholesterol absorption, investigate cholesterol absorption and metabolism regulation mechanism of egg active lipids. The main research contents and results are as follows:In order to investigate the influence of egg-enriched diets and cholesterol diet on SD rats’plasma, hepatic and fecal lipid levels, and on gene expression levels of transporters, receptors and enzymes involved in cholesterol metabolism after90d dietary interventions. Sprague-Dawley rats fed an egg-enriched diet had lower plasma triglycerides, total cholesterol, low density lipoprotein (LDL)-cholesterol, hepatic triglyceride, and cholesterol concentrations, and greater plasma high-density lipoprotein cholesterol concentration, fecal neutral sterol and bile acid concentrations than those fed a plain cholesterol diet. Hen egg yolk had no effect on sterol12a-hydroxylase (CYP8B1) and sterol27α-hydroxylase (CYP27A1); but upregulated mRNA levels of hepatic LDL-receptor, cholesterol7α-hydroxylase (CYP7A1) and lecithin cholesterol acyltransferase, and downregulated hepatic hydroxymethylglutaryl-(HMG)-CoA reductase and acyl-CoA:cholesterol acyltransferase (ACAT) after90days. The present study has demonstrated that an egg-enriched diet has no significant impact on the plasma lipid levels of healthy SD rats, could restrain de novo cholesterol synthesis and deposits in the form of cholesterol esters, and activate bile acid synthesis and excretion in feces and deposition.Caco-2monolayers model was established and assessed with morphology, enzymology, transepithelial electrical resistance (TEER) and the apparent permeation rate (Papp)-The transmission electron micrograph showed well-differentiated cells with microvilli and a tight junction between the cells, the alkaline phosphatase activity in the apical side was significantly increased and higher than that in the basolateral side with the time, the transepithelial electrical resistance reached a relatively stable value within the scope of220Ω·cm2-600Ω·cm2, and the Lucifer Yellow apparent permeability coefficient was lower than1.0×10-6cm/s. Therefore, the Caco-2cell monolayers were appropriate for use in the transport study, and the best in vitro models to study cholesterol absorption and transport study.Caco-2monolayers model was used to study bidirectional transport of cholesterol in a series of concentrations with different pH values, and investigated impact of ezetimibe on cellular transport of cholesterol. The permeation rate of cholesterol across Caco-2cell monolayers was examined in both direction of A-B and B-A with micellar solutions (100μmol/L of cholesterol) in donor media at pH7.4and at pH7.4in receiving media, which were the best conditions according the preliminary experiments. The uptake ratio value was calculated as4.73, which show that the mechanism of cholesterol permeation was suggested as active uptake in absorptive transport with carrier proteins mediated-transport. In the presence of ezetimibe (10μmol/L), a significant decrease of the uptake ratio was observed compared to the data in the absence of the NPC1L1-inhibitor. It was found that the absorptive transport was competitively inhibited by ezetimibe.The lipids and proteins were extracted from hen eggs and digested with the help of in vitro artificial gastrointestinal simulation system. Then these functional factors were studied to compare the impact of cholesterol uptake and transport in Caco-2monolayers model by using liquid scintillation counter. It was found that egg proteins (egg white protein, egg yolk protein and egg ovomucin), phospholipids (phosphatidylcholine and sphingomyelin) and n-3series polyunsaturated fatty acids (EPA and DHA) significantly inhibited cholesterol uptake and transport of cholesterol in the Caco-2monolayers model, palmitic acid and lysophosphatidylcholine significantly promoted cholesterol uptake and transport of cholesterol in the Caco-2monolayers model. However, the effect of mono-unsaturated fatty acids and phosphatidyl ethanolamine on cholesterol uptake and transport of cholesterol in the Caco-2monolayers model were no significant impact. These results indicate that egg contain the regulation of cholesterol absorption transport ingredients, which provide a theoretical basis for further study on the regulation of cholesterol metabolism network and mechanism.Determination of physicochemical characteristic of cholesterol micelles with egg-yolk phospholipids were evaluated by detecting the binding capacity with taurocholate, micellar solubility of cholesterol and taurocholate, and molecular weight changes of micelles after adding phospholipids. The present study indicated that egg-yolk SM and PC interfered with sodium taurocholate micelles, decreased the micellar solubility of cholesterol and taurocholate, and increased the bile acid-binding capacity and size of the taurocholatelecithin-cholesterol micelles. Since cholesterol has a higher affinity for bile salt micelles containing SM and PC, release from the micelles is reduced as a monomer and less absorbed to brush border membranes. Consequently, it is plausible that excess phospholipids especially SM could suppress Caco-2monolayers absorption of cholesterol by modulating the physicochemical properties of mixed micelles.Cholesterol micellar solutions with egg-yolk PC and SM in cultural medium were added in Caco-2cell, total RNA and protein were isolated from the cells and detected expression of transcriptional and protein levels of key proteins in the pathway of cholesterol absorption in Caco-2cells by RT-qPCR and Western-blot. Egg-yolk sphingomyelin and phosphatidylcholine could significantly inhibit Niemann-Pick Cl-like1protein (NPC1L1) levels and mRNA levels, which suggest that the cholesterol-inhibitory effect be mediated in part by their action at the NPC1L1gene and protein level, while NPC1L1gene might be relative with Sterol regulatory element binding protein (SREBP) activation was inhibited. High concentrations of egg-yolk PC increased ATP-binding cassette transporter (ABCA1) mRNA levels, suggesting that PC promoted ABCA1activity and accelerated the removal of cholesterol from Caco-2cell by reverse cholesterol transport process. Egg-yolk SM might inhibit cholesterol absorption by down-regulating mRNA expression of caveolin1.Cholesterol micellar solutions with egg-yolk fatty acids (PA, OA, LA, AA, EPA, DHA) cultural medium were added in Caco-2cell, total RNA and protein were isolated from the cells and detected expression of transcriptional and protein levels of key proteins in the pathway of cholesterol absorption in Caco-2cells by RT-qPCR and Western-blot. EPA and DHA could significantly inhibit NPC1L1levels and mRNA levels, which suggest that the cholesterol-inhibitory effect be mediated in part by their action at the NPC1L1gene and protein level, while NPC1L1gene might be relative with SREBP activation was inhibited. However, high concentrations of PA and OA could increase NPC1L1levels and mRNA levels, which were consistent with the increase of cholesterol uptake and transport in Caco-2monolayers model. High concentrations of EPA and DHA decreased ABCA1mRNA levels, suggesting that PC inhibited ABCA1activity and weakened the removal of cholesterol from Caco-2cell by reverse cholesterol transport process, while high concentrations of PA and OA increased acyl-CoA:cholesterol acyltransifying enzyme2(ACAT2) levels and mRNA levels to promote the conversion of cholesterol ester and eventually involved in the formation of chylomicrons. EPA and DHA might inhibit cholesterol absorption by down-regulating mRNA expression of caveolin1. |
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