Study On The Intestinal Transport Of Lactobacillus Acidophilus S-layer Protein Modified Cholesterol-lowering Active Peptides Liposome

Morbidity and mortality of cardiovascular disease increased year by year as a big factor of human death,serum cholesterol is considered to induce coronary heart disease,atherosclerosis and other important risk factor for cardiovascular disease.Statistics show that the total cholesterol in the serum increases by 0.6mmol/L,and the risk of coronary heart disease increases by 34%,thus regulating the level of cholesterol in the serum is conducive to human health.The cholesterol-lowering peptides can reduce the cholesterol content in serum by regulating the expression of cholesterol absorption genes,but the major problems of peptides are easily degraded by enzymes and low bioavailability.Liposomes have a class cell structure,which is mainly consumed by the reticuloendothelial system in the body and change the absorption and distribution of the encapsulated drugs in the body,with targeted,slow release and low toxicity.The self-assembly and the good gastrointestinal tolerance of Lactobacillus acidophilus S-layer protein can improve the stability of liposomes.In this experiment,the effects of three casein-derived cholesterol peptides Leu-Gln-Pro-Glu(LQPE),Val-Ala-Pro-Phe-Pro-Glu(VAPFPE)and Val-Leu-Pro-Val-Pro-Gln(VLPVPQ)on cholesterol absorption was researched through Caco-2 cell experiment,and the key gene expression which influences cholesterol metabolism was also studied.The L.acidophilus S-layer protein coated positive liposome was prepared to embed cholesterol peptide LQPE.Caco 2 cell model was established to study intestinal transportation of embedding LQPE.(1)The cholesterol-lowering activity of milk source peptide was studied.The effects of LQPE,VAPFPE and VLPVPQ on the activity of caco-2 cells were determined by MTT assay.The influence of Caco-2 cells on cholesterol absorption was determined by incubation of caco-2 cells with LQPE,VAPFPE and VLPVPQ respectively.Experimental results showed that the three kinds of different concentrations of milk source peptides have the cholesterol decreasing function.The concentration on 0.4 mg/mL LQPE had the highest cholesterol absorption effect,with high inhibition rate reached to 87.66± 1.76%.The inhibition rate of VAPFPE was 42.25±3.46%,even lower than that of 0.05 mg/mL LQPE and VLPVPQ.The absorption of cholesterol in Caco-2 cells was decreasing as concentration of peptide increasing.(2)The effect of three peptides on the cholesterol absorption mechanism of caco-2 cell model was researched.The effect of peptides on key protein NPC1L1 expression in cholesterol absorption pathway was researched;Effects on transcription levels of key genes ABCG8,ACAT2,MTP,ABCA1,SREBP1 and SREBP2 were also investigated.The results showed that the amount of NPC1L1 protein in the cells decreased significantly after incubation with caco-2 cell model.RT-qPCR was used to detect the transcriptional level of regulatory genes in cells.The mRNA expressions of ABCG8 and ABCA1 were increased significantly by adding LQPE,and VLPVPQ down-regulated the transcription level of MTP.(3)Positive liposome coated with L.acidophilus CICC 6074 S-layer protein.The S-layer protein was purified and co-incubated with liposomes.Observed through transmission electron microscopy and fluorescence transmission electron microscopy,the coated liposome size increased,and the coated liposome surface was fluorescence.The particle size of liposomes increased from 166.2 nm to 540.7 nm.Zeta potential was changed from+69.1 mV to+22.9 mV,demonstrating that S-layer protein was successfully assembled on liposome surface.(4)The intestinal transportation of L.acidophilus S-layer protein coated LQPE peptide liposomes was studed.The intestinal transportation of LQPE,liposome embedding LQPE and L.acidophilus S-layer protein-coated liposome embedding LQPE were studied using Caco-2 cell monolayer.The results showed that the liposome embedding LQPE encapsulation rate was 89.02%±0.92%.The concentration of LQPE in the BL side of Caco-2 cells was increased after embedded with liposomes,and the concetraion of tranported LQPE embedded with S-layer protein-coated liposome was highest.