Proteomics Analysis Of Buffalo Milk Exosomes And Its Effect On Liver Lipid Metabolism

The liver is the main metabolic organ of the animal body and plays an important role in regulating the lipid homeostasis of livestock and poultry.When the liver lipid metabolism is disrupted,livestock and poultry will be accompanied by fatty liver,which will then affect the production performance and ultimately hinder the normal development of animal husbandry.Therefore,it is urgent to find safe and effective measures to prevent and treat fatty liver disease.Milk exosome is a type of membranous nanocyvesicles with a diameter of approximately 30 nm to 150 nm,widely present in various animal milk.Milk exosomes encapsulate various functional components,which can mediate intercellular information transmission and regulate intestinal health and immune function.However,the impact and regulatory mechanism of buffalo milk exosomes(BME)on liver lipid metabolism are still unclear.In this study,BME was isolated and identified by ultrahigh speed centrifugation,the protein active components BME were analyzed,and the effect of BME on liver lipid metabolism was preliminarily explored through in vivo and in vitro experiments.In vitro experiments,Hep G2 cells were used as models,which were divided into oleic acid palmitic acid control group and BME treatment group(400 ng/μL);In vivo experiments were conducted on C57BL/6J mice,which were randomly divided into(1)normal control group(ND),(2)high fat model group(HFD),(3)BME low dose group(5 mg/kg,HFD-L),and(4)BME high dose group(20 mg/kg,HFD-H).The normal control group was fed with kcal control diet,while the rest were fed with high-fat diet for 8 weeks.According to the grouping,PBS or BME was gavaged every 48 hours for 28 days.The results showed that complete BME can be obtained through ultra high speed centrifugation.Tandem mass tags(TMT)Proteomics technology was used to analyze and identify the protein components of the BME.The results showed that 991 proteins were identified in BME.Compared with the milk exosomes from Holstein cows,there were 156 differentially expressed proteins with 103 upregulated and 53 downregulated.The highest differential protein enrichment was invloved in the taurine and subtaurine metabolic pathways which related to lipid metabolism,suggesting that BME may participate in the regulation of lipid metabolism.The in vitro results showed that BME significantly reduced the levels of triglycerides(TG)and total cholesterol(TC)in liver cells;significantly downregulated the expression levels of SREBP1,ACC,and HMGCR m RNA related to lipid metabolism synthesis,upregulated the expression levels of HSL and LIPC m RNA related to lipid metabolism regulation,and reduced liver cell lipid deposition;significantly increased the number of mitochondria,increased ATP production,and promoted lipid consumption.In addition,by increasing the activities of glutathione(GSH)and superoxide dismutase(SOD)in the liver as well as reducing the activity of malondialdehyde(MDA),the antioxidant capacity of the liver is improved,and the level of oxidative stress in the liver is alleviated.The in vivo results indicated that the BME could alleviate the weight gain induced by high-fat diet to a certain extent.At the same time,it significantly reduced the levels of TG,TC,and low-density lipoprotein cholesterol(LDLC)in the liver and serum of high-fat mice in a dose-dependent manner,increased the levels of high-density lipoprotein cholesterol(HDLC)in the liver and serum of high-fat mice,and reduced the levels of serum glutamic oxaloacetic transaminase(AST),alanine transaminase(ALT),and reduced the liver injury of high-fat mice.In addition,it could improve glucose tolerance and insulin resistance in mice,and increase energy consumption in mice.In summary,this study analysed the protein active components in buffalo milk exosomes and found that they could inhibit high-fat induced lipid deposition and alleviate glucose and lipid metabolism disorders in liver.This study provides new ideas for studying the nutritional value of buffalo milk and theoretical basis for improving lipid metabolism by milk derived exosomes.