Extraction Of Polar Lipids From Bovine Milk And Their Immunomodulatory Activity On Macrophages

Milk polar lipids,containing hydrophilic groups and long-chain hydrophobic fatty acids,mainly existed in milk fat globule membrane.Polar lipids are mainly composed of phospholipids,glycolipids and its derivatives.Phospholipids can be divided into glycerol phospholipid and sphingosine phospholipid.Polar lipids have many physiological functions for human,such as promoting the development of infant nervous system and the maturation of intestinal.At the same time,the milk polar lipids can be prepared into a more stable liposome,which can be used as a drug carrier with good biological targeting and immunoregulation activity.The research extracts the polar lipids from diary products to prepare milk liposomes,and studies the immunomodulatory effect of milk liposomes on LPS induced macrophage.This study provides a new train of thought for the preparation and functional research of milk liposomes.In this study,extraction process of milk polar lipids from buttermilk and preparation conditions of milk liposomes were studied.The immunoregulatory effects of milk polar lipids on lipopolysaccharides(LPS)induced macrophages were investigated.The effects of liposomes on the immune regulation of macrophages induced by LPS were analyzed from the nuclear factor-kappa B(NF-?B)signaling pathway and the two receptor levels of TLR4 and SRA1.The main research methods,contents and conclusions are as follows.1.The extraction process of milk polar lipids was optimized by single factor and response surface method.The extracted polar lipids was also identified.The optimum extraction condition was determined by the content of phosphorus in polar lipid.The optimum extraction conditions were:1.5 g of buttermilk,4 mL of chloroform,2 mL of methanol,20? of extraction temperature,300 rpm of stirring speed and 10 min of extraction time.Under such condition,the final extraction of phospholipid is 67.35 mg.Phosphatidylcholine,phosphatidyl ethanolamine,phosphatidyl inositol,phosphatidyl serine and sphingomyelin were identified by thin layer chromatography(TLC),fourier transform infrared spectroscopy(FTIR)and liquid phase tandem time of flight mass spectrometry(HPLC-MS),and 33 different molecular species in polar lipids were identified.2.The liposomes were prepared with extracting milk polar lipids.The size of obtained liposomes was about 300nm,the average potential was-67.3mV.The liposomes had good stability.Through ELISA and RT-qPCR techniques,the inflammatory factors secreted by macrophages in the blank control group,the LPS induction group and the LPS control group and the mRNA content were measured respectively.The experiment was divided into five groups including blank control group,LPS control group,100?g/mL liposomes with LPS group,200?g/mL liposomes with LPS group and 400?g/mL liposomes with LPS group respectively.(1)TNF-a,IL-1?,IL-6,IL-8 and IL-10 inflammatory cytokines contents of cell suspension were measured by ELISA method.The content of four inflammatory cytokines,except IL-10,in the LPS-induced groups adding milk liposome was significantly lower than that of the LPS control group.The results further proved that milk liposome can inhibit macrophage excessive inflammatory reaction,and regulate macrophage immune activity;(2)The LPS-induced groups plus milk liposomes showed significantly lower mRNA expression of TNF-a,IL-1?,IL-6 and IL-8 inflammatory factor than LPS-induced group,and higher mRNA expression than blank group.The mRNA expression of IL-10 in the LPS-induced group added with liposome was significantly higher than that in the LPS control group.These results indicates that the milk liposomes could inhibit the excessive inflammatory reaction of macrophages,regulate macrophage immune activity.And the inhibitory effect of the liposomes on macrophage inflammatory factors was dose-dependent.3.To research the signaling pathways and function receptors of milk liposomes on inhibiting macrophage inflammatory response,western bloting technique was used to determine the content of macrophage intranuclear NF-?B p50,p65 and TLR4 signaling receptors and SRA1 phagotrophic receptor of the different groups including:control group,LPS-induced group plus milk liposomes and the LPS control group.The results were as follows:(1)the content of p50 and p65 in the LPS-induced group was significantly lower than that in the LPS control group,indicating that the milk liposomes were inhibited by the inhibition of the transcription of p50,p65 and the transcription of the inflammatory factors in NF-?B;(2)In addition,milk liposomes did not affect the expression of TLR4 signaling receptors,but inhibited the expression of SRA1 phagocytic receptors,thereby inhibiting the inflammatory response induced by LPS.