| Snakehead fish(Channa argus,C.argus)is very susceptible to hepatobiliary syndrome during the aquaculture.Cholestatic liver injury,a typical symptom of hepatobiliary syndrome,is usually accompanied by bile acid metabolism disorders and intestinal microbiota imbalance.Lactic acid bacteria(LAB)play an active role in regulating liver and gallbladder diseases and the balance of intestinal microbiota.In the study,the C.argus liver injury model was constructed in vivo and in vitro.The LAB isolated from the C.argus were used as the starting point,and the FXR and related signal pathways were the study focus to explore the molecular mechanism of homologous LAB on regulating cholestatic liver injury in C.argus.To reveal the protective mechanism of homologous LAB against cholestatic liver injury in C.argus and to further understand fish bile acid metabolism mode,provide insights for solving the liver and gallbladder syndrome of C.argus in actual production,and promote the healthy feeding of C.argus and the scientific applications of LAB in the aquaculture industry.This study consisted of six parts and the main results and conclusions are as follows:(1)Screening of excellent LAB from C.argus and study on its probiotic properties in vitroIn order to evaluate the probiotic properties of aquatic animal-derived lactic acid bacteria,a total of 178 gram-positive and catalase-negative LAB isolates were isolated from the intestines of healthy C.argus.Ten LAB isolates(W1,W16,W24,C4,C9,C11,L1,L3,L12 and L19)with high cholesterol-lowering ability were screened out in MRS-CHOL medium.Five LAB isolates(W1,W24,C11,L1 and L19)were screened by Oxford cup method that can inhibit common aquatic pathogens.Among them,W24 and L19 strains showed relatively strong broad-spectrum antibacterial activity,which could inhibit 5 species(A.veronii TH0426,A.veronii ATCC35624,A.veronii JL-8155,A.hydrophila ATCC7966 and A.salmonicida ATCC27013)or 6 species(A.veronii TH0426,A.veronii AV115,A.veronii JL-8155,A.hydrophila ATCC7966,A.caviae ATCC15468 and A.salmonicida ATCC27013)of aquatic pathogens.Through in vitro probiotic characteristics analysis,these five strains can survive under the conditions of low p H,high p H and high concentration of bile salts,have high surface hydrophobic activity and high self-aggregation ability,and are resistant to pepsin and trypsin environment conditions.In summary,two LAB isolates(W24 and L19)with excellent comprehensive performance were retained,16S r RNA sequencing and phylogenetic tree analysis were performed,and they were identified as Enterococcus faecalis W24(Genbank:MT102745.1)and Lactococcus lactis L19(Genbank: MT102746.1).Physiological and biochemical characteristics identification results show that W24and L19 isolates can ferment a variety of sugars,which are in line with the characteristics of LAB.The results of drug susceptibility test showed that W24 and L19 isolates were sensitive to most antibiotics;animal safety tests showed that W24 and L19 isolates were safe and non-pathogenic to C.argus.The above results indicate that W24 and L19 isolates may be candidate strains with development potential for the development of functional microecological feeds,and provide a theoretical basis for the selection of excellent LAB for aquatic animals.(2)Effect of homologous and excellent LAB on the growth,humoral immunity,antioxidant capacity and health of liver,intestine and gallbladderIn order to evaluate the effects of single or combined supplemented with L.lactis L19 or/and E.faecalis W24 in the feed on the growth,humoral immunity,antioxidant capacity and health of the liver,intestines and gallbladder.Fish were fed with a basal diet(CK)supplemented with L.lactis(L19),E.faecalis(W24),and L.lactis L19+E.faecalis W24(L+W)at 1.0×10~8 CFU/g basal diet for 56 days.The results showed that the single or combine supplemented with L.lactis L19 or/and E.faecalis W24 diets significantly increased the growth parameters FBW,WG,FER,SGR and PER(P<0.05),and significantly increased the digestive enzyme activities(protease,amylase and lipase)in the stomach,intestine and liver(P<0.05),effectively improved the growth performance and digestion and absorption of nutrients;Significantly increased the activity of LZM and the contents of Ig M,C3 and C4 in the serum(P<0.05),significantly increased the activities of SOD,CAT,GSH-Px and T-AOC in the liver and intestines(P<0.05),significantly reduced the MDA content(P<0.05),effectively enhanced the humoral immunity and improve the antioxidant capacity;Significantly reduce the levels of ALT,AST,TC,TG and TBA in serum(P<0.05),showed a strong ability to lower cholesterol,can effectively protect the health of liver and gallbladder,and has the potential to prevent and treat common diseases such as fatty liver and hepatobiliary syndrome;Significant increased the thickness,fold height and fold width of the midgut of C.argus(P<0.05)to protect the intestinal morphology;Enriched the intestinal microbial diversity,increase the beneficial bacteria such as Firmicutes and reduce the harmful bacteria such as Proteobacteria,and adjusted the intestinal microbiota,and showed effectively protection for the intestinal health of C.argus.The above results are based on the single supplemented with 1.0×10~8 CFU/g L.lactis L19 showed the best comprehensive effect,which is better than the single supplemented with E.faecalis W24 and the combination of these two strains.L.lactis L19 is a probiotic strain with important development prospects,which can effectively improve the growth performance,enhance immunity and antioxidant capacity,regulate the diversity of intestinal microbiota,and effectively protect the health of liver,intestine and gallbladder of C.argus.(3)Exploring the relationship between metabolites of homologous LAB and BA metabolism based on non-targeted metabolomics technologyIn order to explore the relationship between the metabolites of homologous L.lactis L19 and BA metabolism,this study used the UHPLC-Q-TOF MS analysis platform to conduct a non-targeted metabonomics study on the intestinal contents of the C.argus.After combining the positive and negative ion modes,a total of 404 metabolites(290 positive and 114 negative)were identified;all metabolites identified by the combined positive and negative ions were classified and counted according to their chemical classification attribution information.Among them,the proportion of metabolites of organic acids and their derivatives,organic oxygen compounds,lipids and lipidoid molecules are relatively high,respectively 17.327%,8.168%,6.931%.There were17 and 1 significantly different metabolites in positive and negative ion modes,mainly involving organic acids and their derivatives and organic nitrogen compounds,respectively.L.lactis L19can promote the transformation of the structure and quantity of the different metabolites in the intestine,and significantly affect the metabolic activity of C.argus.Significantly different metabolites,mainly N-palmitoylsphingosine and glutamate-lysine,were enriched in the control group,while significantly different metabolites,mainly sphingosine,L-palmitoylcarnitine,taurodeoxycholic acid and N-acetylmannosamine,were enriched in the L19 group.The KEGG pathway annotation of remarkably different metabolites is mainly enriched in amino sugar and nucleotide sugar metabolism.In addition,it is also significantly enriched in sphingolipid metabolism,riboflavin metabolism and sphingolipid signal pathways.L.lactis L19 is indeed involved in regulating BA metabolism,lowering cholesterol levels,helping to regulate the body’s metabolic level and protecting the health of C.argus.(4)A novel model construction of lithocholic acid-induced cholestasis and transcriptome analysis in C.argusIn order to construct a model of cholestatic liver injury in snakehead fish,this study explored the effects of supplementation with 0%,0.05%,0.1%,0.2%,0.4%and 0.6%lithocholic acid(LCA)on serum biomarkers,survival rate and liver histopathology of C.argus,and established LCA-induced cholestasis model of C.argus.The model was confirmed by the transcriptome analysis and the m RNA expression of genes related to bile acid(BA)metabolism.Survival rate results showed that the fish fed 0.4%and 0.6%LCA died from the first day.The Biochemical analysis results showed that the levels of ALT,AST,TBA and ALP in serum were markedly increased(P<0.05)in C.argus with cholestasis induced by 0.2%,0.4%and 0.6%LCA.The results of liver morphologic and histopathological section showed that 0.1%,0.2%,0.4%and 0.6%LCA treatment for 7 days all resulted in liver toxicity and cholestasis,while the liver color in 0.4%and0.6%LCA treatments showed large areas of green and large-scale focal necrosis.Thus,the optimal modeling time for LCA-induced cholestasis of C.argus is 7 days,and the optimal modeling concentration is 0.2%.In the study,101.09 GB of clean data a total of 83,513 unigenes and 107,325 transcripts was obtained by liver transcriptome sequencing of C.argus using the Illumina platforms.A total of 206 DEGs,i.e.,112 up-regulated DEGs and 94 down-regulated DEGs,were identified between the control and LCA treatment groups based on the liver transcriptome analysis.Notably,the key BA metabolism-related DEGs,such as bsep,shp,fxr,mrp2,mrp3,mrp4,cyp7a1,cyp8b1,cyp27a1 and hmgcr were found in the liver transcriptome.(5)Molecular mechanism verification of homologous LAB regulating cholestatic liver injury of C.argus in vivo.In order to clarify the molecular mechanism of the homologous LAB regulating the cholestatic liver injury of C.argus,this study determined the homologous L.lactis L19 against LCA-induced cholestatic liver injury in C.argus on serum BA metabolism biomarker,liver tissue section,BA metabolism-related genes and proteins,and intestinal microbiota diversity.Biochemical results illustrated that LCA feeding markedly increased the serum AST,ALT,ALP,TBA,TBIL and DBIL activities compared to control(P<0.05)and treatment with L19 diet reversed the increase of these BA biomarkers.Liver histology results identified the amelioration of LCA-induced cholestasis after L19 treatment.Gene analysis indicated that L19 activated fxr and regulated BA metabolism,including promoted BA transport from the liver into the intestine by inducing the expressions of fxr-target genes bsep,mrp2,mrp3,mrp4,and Ostβ,and reduced BA reabsorption by inhibiting the expression of ntcp.In addition,L19 reduced BA synthesis by inhibiting the expressions of fxr-target genes cyp7a1,cyp8b1,cyp27a1,and hmgcr via the Fxr-Shp signaling pathway,and promoted BA metabolism by inducing the expression of sult2a1.Protein analysis indicated that L19 promoted BA metabolism and transport through activating the Fxr-target proteins Bsep,Shp and Cyp7a1.Intestinal microbiota analysis demonstrated that the LCA group was dominated by Proteobacteria and Chlamydiae,while L19 treatment can reduce the abundance of Proteobacteria,increase the abundance of Firmicutes,and regulate the composition of intestinal microbiota.For the first time,we found that L.lactis L19 can activate Fxr,regulate BA metabolism and intestinal microbiota to exert protective effects against cholestatic liver injury.Therefore,L.lactis L19 might be a potent measure for the prevention against LCA-induced cholestatic liver injury of fish,possibly via Fxr-Bsep and Fxr-Shp signal pathway and intestinal microbiota alteration.(6)Liver cell injury model construction of C.argus and the molecular mechanism verification of homologous LAB regulating liver cell injury in vitro.In order to clarify the molecular mechanism of the homologous LAB regulating the liver cell injury of C.argus,this study used Fxr as the key target protein for the treatment of liver cell injury,used LCA to construct the liver cell injury model,and used the Fxr inhibitor GS to specifically block Fxr and its related signaling pathways will further explore the protective mechanism of homologous L.lactis L19 against liver injury in C.argus in vitro.The results showed that the LCA-induced liver cell injury model of C.argus was successfully constructed.The optimal concentration of LCA was 20μM,and the optimal duration of action was 24 h.1.0×10~8 CFU/m L L.lactis L19 ECP showed a protective effect on LCA-induced liver cell injury,remarkably decrease the levels of liver injury-related biochemical parameters AST,ALT,ALP and LDH(P<0.05),and regulate the expressions of BA metabolism-related genes fxr,bsep,mrp2,shp,cyp7a1 and cyp8b1 and BA metabolism-related proteins Fxr,Bsep,Shp and Cyp7a1.It is revealed that L.lactis L19 is a possible molecular mechanism that regulates the liver cell injury of C.argus by activating Fxr and its related signal pathways.In vitro test results show that L.lactis L19 can enhance the proliferation of hepatocytes,promote the efflux and metabolism of toxic BA,inhibit the BA synthesis and uptake,and play a protective role in LCA-induced liver cell injury in C.argus.At the gene and protein levels,it was verified that L.lactis L19 can activate Fxr and its related signal pathways,and proved that the protective effects of L.lactis L19 on LCA-induced liver cell injury was blocked by GS.In summary,the homologous L.lactis L19 is an ideal probiotic candidate strain,which can activate the key target Fxr and its related signaling pathways in vivo and in vitro to regulate the cholestatic liver injury of C.argus.L.lactis L19 shows a potential development in the treatment of liver and gallbladder related diseases. |
PDF Full Text Request