Study On The Protective Effect And Mechanism Of Blueberry Anthocyanins Against Non-alcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease(NAFLD),relating to a series of metabolic syndromes such as obesity and diabetes,has become a universal health issue.Liver lipid deposition and oxidative stress play important roles in NAFLD pathogenesis.Until now,there still lacks widely accepted drugs for NAFLD treatment.Thus,it is of great significance to find potentially effective bioactive compounds from fruits and vegetables for the amelioration of NAFLD.Blueberry is a kind of popular fruit all over the world,with high edible value and health benefits due to its good taste and nutrition abundance.Clinical researches have shown that blueberry can effectively regulate lipid metabolism,therefore preventing and improving metabolic syndromes such as obesity,diabetes,and cardiovascular disease.However,the specific active substances and mechanisms have not been fully clarified.It was evidenced that anthocyanin,a compound rich in blueberry,has various bioactivities such as antioxidative,hypolipidemic,anti-inflammatory and anti-cancer,etc.Therefore,this research focused on the major anthocyanins of blueberry and investigated the effects and mechanisms of active blueberry anthocyanins on NAFLD.The main results are summarized as follows:(1)The effects of blueberry anthocyanins on lipid accumulation were estimated using an in vitro NAFLD cell model,and malvidin-3-O-glucoside(M3G)and malvidin-3-O-galactoside(M3Ga)presented main effects on ameliorating lipid accumulation,the effects of which on oxidative stress of NAFLD were further investigated.Normal human hepatocyte cell line L02 was cultured with a mixture of free fatty acids(FFA)with a 1:2 ratio of palmitic acid to oleic acid,using 0.2 m M as the experimental concentration.It was found that blueberry anthocyanins M3G and M3Ga attenuated lipid deposition in the NAFLD cell model significantly,the lipid accumulation of which decreased 27.89% and 24.17% at the anthocyanin dose of 400 ?M(p < 0.05),respectively,compared with FFA group.The other 8 kinds of anthocyanins(malvidin-3-O-arabinoside,delphinidin-3-O-galactoside,delphinidin-3-O-arabinoside,petunidin-3-O-galactoside,petunidin-3-O-arabinoside,delphinidin-3-O-glucoside,petunidin-3-O-glucoside,cyanidin-3-O-glucoside)had no positive effect on lipid accumulation in liver cells.Considering the important role of oxidative stress in NAFLD pathogenesis,the effects of M3G and M3Ga on oxidative stress in the NAFLD cell model were evaluated.Results showed that the reactive oxygen species(ROS)level in M3G and M3Ga treated group was significantly decreased versus FFA-alone group.Moreover,M3G and M3Ga could effectively scavenge excessive superoxide anions,hinder the decrease of mitochondrial membrane potential and alleviate intracellular glutathione declination induced by FFA.It was worth noting that M3G decreased cell superoxide anions and restored glutathione levels in a dose-dependent manner while M3Ga not.The cellular superoxide anions in M3Ga(100 and 200 ?M)treated group(129.84% and127.84%)had no difference with the FFA-alone group(139.23%).The glutathione levels of M3G with 100,200,and 400 ?M treatments(84.45%,89.37%,and 91.15%,respectively)behaved better than M3Ga of the equal doses(83.71%,83.26%,and80.35%,respectively).(2)It was unveiled that M3G,which showed an optimal activity on NAFLD,attenuated hepatic liver deposition and oxidative stress through regulating TFEB-mediated lysosomal function and activating Nrf2/ARE.Lysosome plays an important role in lipid metabolism,while the results showed that M3G could significantly enhance the number of lysosomes and protein expression of LAMP1(a marker protein of lysosome),cathepsin B,and cathepsin D in NAFLD cell model,indicating the improvement of lysosomal function.Transcriptional factor EB(TFEB)is a master regulator of the lysosomal pathway.Further investigation showed that M3G promoted TFEB nuclear translocation and upregulated the expression of TFEB target genes(TFEB,LAMP1).Results of molecular docking indicated that M3G could form hydrogen bonds at the glutamine site(Gln10,Gln17,Gln18)and glutamate site(Glu14)of TFEB,as well as forming hydrophobic interaction at the alanine site(Ala16)of TFEB,which might explain the above results.Considering that M3G significantly attenuated oxidative stress in the NAFLD cell model,the Nrf2/ARE signaling pathway,an important cellular antioxidant defense system,was studied.The results revealed that M3G upregulated the gene expression of Nrf2,phase II detoxification enzymes(HO-1,NQO1,?-GCL)and antioxidant enzymes(SOD,GPx)in NAFLD cell model.Moreover,the enzyme activities of SOD,GPx,CAT was strengthened by M3G.In summary,this research evaluated the efficacy of blueberry anthocyanins on NAFLD and revealed that M3G regulated TFEB-mediated lysosomal function and redox state via Nrf2/ARE activation,thereby alleviating NAFLD.This research helped to expand the understanding of the blueberry anthocyanins bioactivity,and provided ideas for the development of functional foods that benefits NAFLD as well as deep-processing products of blueberry anthocyanins.