The Improvement Of Non-Alcoholic Fatty Liver Disease In Mice Through The Use Of Alpha-Lactalbumin Peptide And Its Mechanism

Non-alcoholic fatty liver disease(NAFLD)is a common chronic disease that often accompanies complications such as hyperlipidemia and insulin resistance,posing a serious threat to human health.Dietary interventions have positive effects on the prevention and treatment of NAFLD.α-lactalbumin(α-La)is the main component of whey protein which has the effects of reducing body weight,lowering blood lipids,and maintaining insulin homeostasis,however the effect and mechanism of α-La on NAFLD is unknown.Many studies demonstrated that oligopeptides have positive effects in weight loss,improvement of hepatic lipid degeneration and insulin resistance.In this study,α-La hydrolysate were obtained by simulating gastrointestinal digestion,and screened out the bioactive peptide with improvement effect on NAFLD from α-La hydrolysate using cell and animal experiments and clarify its regulatory mechanism.NAFLD mouse model was established by high-fat diet and the ameliorative effects of α-La and whey protein isolate(WPI)on NAFLD in mice was investigated.Both α-La and WPI treatment significantly reduced body weight,organ index,blood lipid and glucose levels in NAFLD mice,with α-La being more effective than WPI.Compared with the model group,the levels of serum triglycerides(TG),high-density lipoprotein(HDL-C),aspartate aminotransferase(AST),alanine aminotransferase(ALT),and insulin in α-La group were reduced to the normal physiological range,and serum total cholesterol(TC)content reduced by 35.80% and levels of liver TG and TC reduced by 32.35% and 31.29%,respectively.α-La activated hepatic Ppara m RNA and protein expression,subsequently upregulated m RNA expression of Cpt1 a,Acox1 and Atgl and protein expression of CPT-1a to accelerate fatty acid oxidation,downregulated m RNA expression of Pparg1,Acc1 and Scd1 and protein expression of SCD-1 to inhibit fatty acid synthesis.The α-La hydrolysate was obtained by simulating gastrointestinal digestion,with the oligopeptide(molecular weight <1 k D)content accounting for 59.38% of the total peptide.The different hydrolysate fractions were obtained by ultrafiltration and dextran gel G-15 column chromatography,and combined with cell experiments to screen the bioactive fraction F3 with the ability inhibiting lipid accumulation in Hep G2 cells.Mass spectrometry results indicated that the F3 contains 59 peptides with molecular weights between 400 and 1700 Da.The oligopeptide GINY(Gly-IleAsn-Tyr)and the DQW(Asp-Gln-Trp)with inhibitory effect on cellular lipid accumulation were screened out from the highly abundant oligopeptides of F3 through molecular docking and cell experiments.Cell proteomics results demonstrated that the oligopeptides GINY and DQW inhibited cellular lipid accumulation by mediating the PPAR signaling pathway,oxidative stress signaling pathway and oxidative phosphorylation signaling pathway.Further research found that GINY and DQW significantly reduced the content of reactive oxygen species(ROS)in lipid accumulation Hep G2 cells,increased the activity of superoxide dismutase(SOD)and catalase(CAT)as well as mitochondrial membrane potential and ATP content.GINY and DQW targeted as PPARα to promote fatty acid oxidation by upregulating m RNA and protein expression of fatty acid oxidation-related genes(Cpt1a and Fabp1)and inhibited fatty acid synthesis by downregulating m RNA and protein expression of fatty acid synthesis-related genes Fasn and enhancing ACC1 protein phosphorylation.Moreover,GINY and DQW also alleviated oxidative stress and mitochondrial damage by upregulating m RNA and protein expression of oxidative stress-related genes(Ppargc1a,Nrf2,and Ho-1),thereby inhibiting lipid accumulation.Finally,the NAFLD mouse model was used to further investigate the ameliorative effects and mechanisms of oligopeptides DQW and GINY on NAFLD in mice.DQW and GINY significantly reduced body weight,organ index,Lee’s index,and blood lipid levels in mice.Compared with the model group,serum TG,HDL-C,AST and insulin levels of mice in the high-dose DQW and GINY intervention groups were restored to the normal physiological range.Meanwhile,the DQW and GINY intervention groups showed significantly lower liver TG and malondialdehyde(MDA),higher CAT and SOD activities,reduced hepatic lipid droplets and increased mitochondrial numbers.DQW and GINY activated the PPARα pathway by upregulating the m RNA and protein expression of Ppara,subsequently promoted liver fatty acid oxidation and reduced oxidative stress damage by up-regulating the m RNA and protein expression of fatty acid oxidation-related genes(Cpt1a and Fabp1)and oxidative stress-related genes(Nrf2 and Ho1),inhibited lipid synthesis by downregulating the m RNA and protein expression of lipid synthesis-related genes(Srebf1c,Acc1,and Scd1)and promoting ACC1 protein phosphorylation.Furthermore,DQW and GINY activated IRS1/PI3K/AKT pathway,subsequently accelerated glucose uptake and glycogen synthesis in hepatocytes,thereby reducing blood glucose levels.Hence,DQW and GINY both improved NAFLD in mice and improvement effect of high-dose group was significantly better than that of low-dose group.In summary,the high-abundance oligopeptides DQW and GINY released fromα-La by simulated gastrointestinal digestion could promote fatty acid oxidation,inhibit fatty acid synthesis,alleviate oxidative stress injure,and improve glucose metabolism,thereby alleviating NAFLD.The revelation of the mechanism of DQW and GINY improving NAFLD not only provides theoretical support for the screening of bioactive peptides with inhibiting effect on lipid accumulation,but also provides scientific basis for the high value utilization of α-La and its functional food development.