| Exosome is an extracellular vesicle secreted by cells,which can deliver its contents to target cells to play a specific role.As an important bioactive ingredient in milk,exosomes have been proved to have many physiological functions,such as regulating autoimmunity,preventing inflammation,and promoting intestinal development.Among them,micro RNAs(mi RNAs)are considered to be important functional performers in milk derived exosomes.Previous studies focused on the differences in the composition and functional components of different dairy products,but so far there have been few studies on the biological effects of exosomes in different dairy products.Therefore,in this study,using breast milk and fresh milk as controls,the morphological characteristics and surface marker protein of exosomes from different milk products(ultra-high temperature sterilization(UHT)milk,freezed-dried powder and formula powder)were firstly compared.Furthermore,normal and Lipopolysaccharide(LPS)induced RAW264.7macrophage model was constructed to observe the effect of different milk products exosomes stimulating the expression of mi RNAs,secreting cytokines and mediating signaling pathways of immune cells,in order to provide a theoretical basis for the research and development of milk exosomes in China.1.Characterization of exosomes in different dairy productsFirstly,the characterization differences of exosomes in different milk products were compared by Malvin particle size analyzer,transmission electron microscope and western blot.The results showed that the peak value of exosomes in each extraction group was only one,which was between 100-150 nm.The exosomes in each group were round or oval with lipid bilayer structure.It can be seen that the standard exosomes can be extracted from breast milk,fresh cow milk,UHT milk,freeze-dried powder and formula powder.Among them,the breast milk exosome mixture had the cleanest background,and the exosome mixture in the milk group contained relatively more background impurities,and exosome particles were easy to aggregate,especially the electron microscope images of formula powder exosome showed more impurity protein fusion.In addition,fresh milk exosome surface marker protein CD63,TSG101 and CD9 bands were slightly deeper and thicker,UHT milk and lyophilized powder group exosome surface marker protein bands were slightly shallower,formula powder group exosome surface marker protein bands were the shallowest.2.Studies on the regulation of mi RNA expression in macrophages by exosomes from different milk productsNormal and LPS-induced macrophage models were established to analyze the effects of exosomes extracted from human milk,fresh cow milk,UHT milk,freeze-dried powder and formula powder on the expression of mi RNAs in macrophages.The results showed that each exosome group significantly promoted the expression of immune-related mi RNAs,such as mi RNA-223,mi R-181 a,and mi R-155,in normal macrophages,but had no effect on mi R-30 d.The expressions of mi R-155 and mi R-223 in the breast milk group were higher than those in the fresh milk group,and the level of mi R-181 a was slightly lower than that in the fresh milk group,but there was no significant difference.The expression of mi R-181 a in fresh milk group was not significantly different from that in lyophilized powder group,but was significantly higher than that in UHT milk group and formula powder group(P<0.05).There was no significant difference in the expression of mi R-223 and mi R-155 between fresh milk group and milk product group(P>0.05).However,after co-culture with LPS-induced macrophages,each exosome group showed the role of promoting mi R-223 and mi R-181 a,and inhibiting the overexpression of mi R-155.Mi R-223 and mi R-155 expression were increased and decreased significantly in the breast-feeding group compared with the other groups(P<0.05);the expression of mi R-181 a in fresh milk group was significantly higher than that in other groups(P<0.05),inhibiting mi R-155 overexpression was similar to that of the lyophilized powder group,and significantly higher than that of the UHT milk group and formula powder group(P<0.05).3.Studies on the activation of immunity by exosomes from different dairy productsExosomes in breast milk,fresh milk,UHT milk,freeze-dried powder and formula powder were used to stimulate normal macrophages to investigate the effects of exosomes in different milk products on macrophage activation.The results showed that all exosome groups could activate macrophages,including promoting cell proliferation and increasing the secretion of NO,IL-1?,TNF-? and IL-6.Among them,exosomes in fresh milk and breast milk showed obvious immune-activating effects.The effect of fresh milk group on IL-1? was significantly higher than that of milk products group(P<0.05);there was no significant difference between the lyophilized powder group and the freeze-dried powder group in promoting TNF-? and IL-6,which was better than the UHT milk group and formula powder group.4.Study on inhibition of LPS-mediated inflammation by exosomes from different milk productsLPS-induced macrophage inflammation model was established to explore the effects of different milk products exosomes on the inhibition of inflammatory response.The results showed that the breast milk exosome group had the best effect on inhibiting LPS-induced ROS accumulation and pro-inflammatory factor level increase in macrophages.Exosomes in fresh milk group inhibited the production of NO and IL-6,and down-regulated the expression of IL-1?m RNA significantly higher than those in milk products group.Fresh milk group and freeze-dried powder group had better ability to reduce IL-6 and TNF-? transcription levels than UHT milk group and formula powder group.The ROS production of fresh milk group was not significantly different from that of freeze-dried powder group and UHT milk group,but was significantly lower than that of formula powder group(P<0.05).In addition,exosomes in each group can decrease the expression of TLR4/NF-?B signaling pathway induced by LPS to different degrees.The protein expression of TLR4 receptor in breast milk,fresh milk,freeze-dried powder group and UHT milk group had no statistical difference,which was significantly higher than that in formula powder group.The inhibitory effect of fresh milk group on the phosphorylation of p65 and I?B? was significantly lower than that of breast milk group.The protein expression levels of p-p65 and p-I?B? in fresh milk group were similar to those in freeze-dried powder group,and significantly higher than those in UHT milk group.The inhibition of phosphorylation of p65 and I?B? protein in formula group was the worst.These results suggest that exosomes extracted from breast milk,fresh cow milk,pasteurized milk,freeze-dried powder and formula powder all have a bidirectionally regulated effect on the immune system,namely,up-regulating the immune activity of normal macrophages and down-regulating the inflammatory response of macrophages induced by LPS.The immunoregulatory effect of fresh milk group was similar to that of freeze-dried powder group,and higher than that of UHT milk group.The immunoregulatory effect of formula powder group was the lowest.This may be related to different levels of exosomes regulating the expression of immune-related mi RNAs in macrophages in each group. |
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