Mechanism Of Lasctobacillus Reliving Channa Argus Fatty Liver Induced By High-fat Diet

Fatty liver disease of fish is the most common nutritional disease in aquaculture all over the world,especially in freshwater aquaculture species in China.Liver fat deposition is the main cause of inducing fatty liver.As a typical probiotic,the research in the field of aquatic products proves that it can regulate the immunity of the body.However,the research on alleviating fatty liver of fish by lactic acid bacteria is relatively few,and the molecular mechanism of lipid-lowering is still unclear.Therefore,in this study,Channa argus was used as the experimental animal,and Lactobacillus casei,Lactobacillus plantarum and Lactobacillus rhamnosus were used to intervene C.argus fatty liver.The molecular mechanism of three strains of lactic acid bacteria relieving C.argus fatty liver was explored through in vivo and in vitro experiments.There are five parts in this study,and the results are as follows:(1)Establishment of C.argus fatty liver modelIn order to explore the molecular mechanism of C.argus fatty liver formation,a high-fat diet with 9%(Control),16%(L16)and 22%(L22)fat content was set up on the basis of basic diet,and C.argus was fed continuously for 90 days,and samples were taken at 30 days,60 days and 90 days.Through tissue section observation,the results showed that after 90 days,the cells in the Control group were compact and free of vacuoles.Compared with Control group and L16group,the content of TG and TC in serum and liver in L22 group increased significantly,while the activity levels of antioxidant enzymes SOD,CAT and GSH-Px in serum decreased significantly.Further study showed that the expression level of NF-?B in liver of L22 group was significantly higher than that of L16 group and Control group,and the expression levels of apoptosis-related genes Bax,Cas3,Cas8 and Cas9 in L22 group were also significantly higher than those of L16 group and Control group,and the levels of GOT and GPT in serum of L22 group were significantly higher than those of L16 group and L22 group,which proved that feeding C.argus with diet supplement 22%fat level could successfully induce C.argus fatty liver.(2)Exploring the key genes of C.argus fatty liver induced by high fat diet based on transcriptome technologyOn the basis of the establishment of C.argus fatty liver model,the differential genes between high-fat group and control group were analyzed by transcriptome sequencing.the results showed that the enrichment of differential genes between high-fat group and control group involved 158pathways,and KEGG enriched up to 312 signal pathways,among which immune regulation,stress regulation and fat metabolism were mainly related.Equivalent to the control group,high-fat diet can significantly up-regulate the expression of inflammatory factors and apoptosis-related genes,inhibit the expression of fat degradation-related genes such as PPAR?,ACOX1 and LPL in C.argus liver,and inhibit the expression of cholesterol metabolism-related genes such as LXR?and SREBP-1c,which are all related to fat degradation.Therefore,it is speculated that these are the key genes that high-fat diet leads to C.argus fatty liver.(3)Effects of three lactic acid bacteria on growth,immunity,antioxidation and intestinal flora of C.argusTo study the effects of L.casei,L.plantarum and L.rhamnosus on the growth,immunity,antioxidant function and intestinal flora of C.argus,three strains of lactic acid bacteria were fed with 1×10~9CFU/m L coated feed for 56 days,and then the samples were taken to test the growth performance,feed efficiency,antioxidant index,immune index and intestinal flora abundance.The results showed that the addition of L.casei and L.rhamnosus in the diet decreased the relative abundance of Proteobacteria in the intestine of C.argus and improved the structure of intestinal flora.Compared with the Control group,adding L.rhamnosus in the diet can improve the growth performance and feed efficiency of C.argus,and L.casei,L.plantarum and L.rhamnosus can increase the activity levels of SOD,CAT and GSH-Px in serum and tissues of C.argus,and up-regulate the expression of SOD and GST genes in tissues and the expression of immune-related genes.The antioxidant capacity and immunity of C.argus were improved,and it was resistant to A.hydrophila infection.The protective effect of L.rhamnosus was higher than the other two groups,and the protective rate was as high as 50%,while the protective rates of L.casei and L.plantarum were 37.5%and 40%,respectively.(4)Effects of three lactic acid bacteria on fatty liver induced by high fat diet in C.argusOn the basis of C.argus fatty liver model,the high fat feed was coated with L.casei,L.plantarum and L.rhamnosus.after feeding for 90 days,the liver lipid level,inflammatory factors,antioxidant enzyme activity and intestinal flora were measured.With H&E staining and oil red o staining,it can be observed that L.casei,L.plantarum and L.rhamnosus could improve the fatty liver symptoms of C.argus induced by high fat diet.L.casei and L. rhamnosus could improve the intestinal flora structure and inhibit the relative abundance of Proteobacteria,and L.rhamnosus regulates the expression of downstream ACOX1 and LPL genes by activating PPAR?,thus alleviating the symptoms of C.argus fatty liver.L.casei participates in regulating lipid metabolism by activating LXR?and LPL,and alleviates liver fat deposition of C.argus.L.plantarum has no significant effect on intestinal flora abundance.unlike L.casei and L.rhamnosus,L.plantarum regulates SREBP-1c gene expression by activating LXR?,and alleviates fatty liver symptoms of C.argus.(5)Based on PPAR?/LXR?,the mechanism of three lactic acid bacteria alleviating fatty liver induced by high fat diet in C.argus was explored in vitroThe fat deposition model of C.argus primary hepatocytes was established by oleic acid.The results showed that OA concentration of 2.5 mmol/L could significantly increase the contents of TG,LDL and FFA,and inhibit the expression of PPAR?and LXR?,which led to fat deposition.On this basis,GW6471 and GSK2033 were used to inhibit PPAR?and LXR?,respectively,to explore the molecular mechanism of L.casei,L.plantarum and L.rhamnosus relieving fat deposition.The results showed that L.rhamnosus activated the transcription level of ACOX1 and LPL by activating PPAR?.After inhibiting PPAR?activity,the expression level of these genes decreased significantly and the intracellular TG level increased significantly.However,when inhibiting LXR?activity,the intracellular TG level had no difference with L.rhamnosus and OA groups.L.plantarum reduced LDL and FFA content and inhibited PPAR?activity by activating LXR?expression,but the intracellular TC and FFA levels were no different from those of L.plantarum and OA groups,while the intracellular lipid level increased significantly when LXR?activity was inhibited.L.casei regulates the up-regulation of PPAR?,LXR?and LPL,and inhibiting PPAR?and LXR?will lead to the increase of intracellular lipid level.To sum up,in vivo experiments have proved that L.casei,L.plantarum and L.rhamnosus could effectively alleviate the fatty liver symptoms of C.argus induced by high-fat diet,and the lipid-lowering effect of L.rhamnosus is slightly better than the other two strains.In vitro experiments further prove that the molecular mechanisms of relieving fatty liver symptoms of L.casei,L.plantarum and L.rhamnosus are different.the results of this study provide scientific reference for the research on the pathogenesis of fatty liver of C.argus,and also provide theoretical basis for solving fatty liver of current aquaculture economic varieties.