Study On The Protective Mechanism Of Lactobacillus Rhamnosus LV108 Fermented Milk Against Non-alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease(NAFLD)is one of the most common chronic liver disease.The pathogenesis of non-alcoholic fatty liver disease is complex,and there is a lack of ideal treatment.Probiotics have extensive physiological functions,including anti-oxidation,regulation of intestinal flora and enhance immunity function make probiotics the focus of research.In this study,the NAFLD modle was used to study the protective effect of the L.rhamnosus LV108 fermented milk in NAFLD.The present research adopted the method of proteomics to carry out differential analysis of liver proteome on NAFLD model rats treated with probiotic.In addition,bioinformatics analysis was carried out to screen out the differential proteins and important pathways which played a key role in the treatment of NAFLD by L.rhamnosus LV108 fermented milk and provide valuable clues for deepening our understanding of its mechanism at the molecular level.The main results were as follows:1.The protective effect of the L.rhamnosus LV108 fermented milk in NAFLD.Forty male SD rats were randomly divided into normal group,model group,LV108 fermented milk group and mixed fermented milk group.The normal group was treated with normal feed,and the rest groups were treated with high fat diet.At the same time,LV108 fermented milk group and mixed fermented milk group were given LV108 fermented milk and mixed fermented milk(1.0 mL/100 g,109 CFU/mL)respectively.The results showed that there was no significant difference in food intake between the groups.After the fourth week,the weight gain of the LV108 fermented milk group and mixed fermented milk group was significantly lower than that of the model group.Serum indicators show that LV108 fermented milk and mixed fermented milk could reduce TG,TC and LDL-C,increase HDL-C.It can be seen from the rats liver oxidation index that the LV108 fermented milk and mixed fermented milk could increase the activity of antioxidant enzymes SOD,GSH-Px,CAT and the content of non-enzymatic antioxidant GSH,and eliminate the superoxide anion free radical,hydroxyl free radical,NO free radical and other free radicals,reduce lipid peroxide MDA.The protective effect by LV108 fermented milk and mixed fermented milk in NAFLD may be achieved by reducing the food-effect ratio of high-fat diet,relieve lipid accumulation,regulate lipid and liver lipid oxidation damage.2.Regulatory effect of L.rhamnosus LV108 fermented milk on NAFLD in rats.Tandem Mass Tag(TMT)relative quantitative proteomics was used in this research,which displays changes in liver protein expression in rats among normal group(N),modle group(M),and L.rhamnosus LV108 fermented milk group(A).Furthermore,2 differentially expressed proteins were selected for parallel reaction monitoring(PRM)analysis to further verify the results of MS.In addition,bioinformatics analysis was carried out to screen out the differential proteins which played a key role in the treatment of NAFLD by LV108 fermented milk and provide valuable clues for deepening our understanding of its mechanism at the molecular level.A total of 4155 corresponding proteins were identified by MS.A total of 26 differentially expressed proteins were found between A and M group of which 16 were up-regulated and 10 down-regulated.Most of the differentially expressed proteins were involved in apoptosis and lipid metabolism.The key differentially expressed proteins(BFAR and Cyt-C)were verified by parallel reaction monitoring.The results indicated that the expression quantities of BFAR in the M group were markedly up-regulated compared with those in the N group,whereas the expression quantity of it in the A group was down-regulated compared with that in the M group.Furthermore,expression quantities of Cyt-C in the A group were was markedly down-regulated compared with that in the M group.The expression quantities of Cyt-C in the M group were not significantly changed compared with that in the N group.It was consistent with the results of MS,which further validates the accuracy of MS.The differentially expressed proteins screened in this study can effectively separate the M from the A and the N from M.The hierarchical clustering analysis thus supported that the differentially expressed proteins screened out were reasonable.GO functional annotation analysis categorized the 26 differentially expressed proteins in A/M into biological processes,cellular components,and molecular functions according to their annotation.The most prevalent biological processes were single-organism process.The most prevalent cellular components were located in the cell part,cell and intracellular.The most predominant molecular functions were binding.In addition,activation of cysteine-type endopeptidase activity involved in apoptotic process by cytochrome c and other important biological processes change significantly.Caspase binding and other molecular function change significantly.BRCA1-A complex and other positioning proteins change significantly.Using KEGG pathway analysis,we enriched 143 KEGG pathways by the differentially expressed proteins in M/N.The 40 KEGG pathways had changed significantly.These pathways may be involved in the formation of NAFLD.We enriched 37 KEGG pathways by the differentially expressed proteins in A/M.Phenylalanine,tyrosine and tryptophan biosynthesis,and Sulfur metabolism pathways had changed significantly.A total of 17 KEGG pathways had be enriched by the differentially expressed proteins in A/M and N/M.LV108 fermented milk inhibits hepatocyte apoptosis and relieves hepatic injury by regulating key proteins in NAFLD pathway,apoptosis and other important pathways.LV108 fermented milk also improves fat metabolism induced by high-fat diet and restores lipid metabolism balance by regulating multiple key proteins in signal pathways such as synthesis and metabolism of various amino acids,glycolysis/gluconeogenesis,peroxisomes and P53 signaling pathway.In addition,PPI revealed the interaction between phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase,Protein SDA1 homolog and Surfeit 6 in A/M.LV108 fermented milk has a promoting effect on the synthesis of ribosomes and proteins,and its influence on the glycolytic pathway will also become the focus of subsequent research.We hypothesize that LV108 fermented milk attenuates NAFLD by inhibiting apoptosis and alleviating hepatocellular injury.