Effects Of Bacillus Amyloliquefaciens TL Regulating IGF1/PI3K Signaling Pathway On Liver Lipid Metabolism Of Broilers

Bacillus amyloliquefaciens is a gram-positive bacterium,which is widely used as a probiotic for feeding because of its wide antibacterial spectrum,high enzyme production,good stability and strong stress resistance.Previous studies in vivo have shown that B.A-TL can promote the fat deposition of Cobb broilers by regulating sugar/lipid/protein metabolism.Some studies have pointed out that 95%of lipid in poultry are synthesized in the liver.In this study,Bacillus amyloliquefaciens TL was added to the basic diet of Cobb broilers,gene expression in liver tissue of broiler was analyzed by transcriptome technology,and the effect of Bacillus amyloliquefaciens TL secondary metabolites on IGF signaling pathways were investigated by cell testing and signal pathway blocking test.Two groups of Cobb broilers were fed to 35 days of age,one group was fed normally and the other group was fed with B.A-TL.1.Serum biochemical indexes:At 21 days of age,compared with the control group,serum glucose in B.A-TL group increased by 35.90%（p<0.05）;IGF-1 increased by15.5%（p<0.05）;TG increased by 31.19%（p<0.05）.At 35 days of age,GLU increased by17.80%（p<0.05）;IGF-1 decreased by 19.76%（p<0.05）.There were no difference in GH and INS content throughout the whole process.The results showed that Bacillus amyloliquefaciens TL could accelerate gluconeogenesis,inhibit glycolysis and lipid oxidation.Without affecting the secretion of GH,it can promote the synthesis of IGF-1 in the liver of broilers at the early growth stage（1-21 days）,and a large amount of IGF-1 can be secreted into the circulation system to promote the rapid development of muscle and other tissues,thus achieving significant weight gain effect.2.Liver tissue morphology:HE staining of liver tissue at 21 days and 35 days showed normal liver morphology without lesions.Oil red staining showed that the number of lipid droplets in the liver of B.A-TL group was more than that of the control group at the same time,indicating that B.A-TL had no damage to the liver tissue and could promote the synthesis of lipid substances in the liver.3.Screening differentially expression genes:Transcriptome analysis was carried out on the 21 days liver tissue,DESeq2 with variance analysis software to|log₂FC|≥1 or higher and p-value<0.05 for screening criteria to determine the differentially expression genes,358 differentially expression genes were detected in liver tissue,including 145up-regulated genes and 213 down-regulated genes,pyruvate carboxykinase 1,fatty acid synthase and other genes were significantly up-regulated in B.A-TL group.4.Functional annotation analysis of differentially expression genes:GO analysis found that the function of differentially expression genes was mainly related to protein and lipid metabolism,ion transport,stimulus response,immune response and cell growth and differentiation.KEGG enrichment analysis showed that the DEGs had high common enrichment degree of calcium/Ras/MAPK/PI3K/signaling pathway.The results showed that after feeding B.A-TL,the metabolism and lipid/protein synthesis activities of the body were enhanced,the immune stimulation was reduced,and finally the metabolic activities were accelerated,the protein fat deposition activity was enhanced and the anti-stress ability was enhanced.5.Secondary metabolites analysis:The metabolites of the B.A-TL supernatant was detected by LC-MS/MS.Tyrosine accounted for 29.02%of the supernatant.The exopolysaccharides components of B.A-TL were analyzed and detected by electrochemical detector.Galactose,glucose and mannose accounted for 98.81%of the exopolysaccharides.6.In vitro test:LMH cells were stimulated with 10^-4 fermentation supernatant,100μg/m L exopolysaccharides and 0.25 mmol/L tyrosine for 4 h.The results showed that fermentation supernatant and tyrosine could increase the IGF-1 secretion of LMH cells（p<0.05）.Oil red staining showed that lipid droplets in the fermentation supernatant and tyrosin groups were more than those in the control group.Western blot test showed that fermentation supernatant and tyrosin could stimulate the expression of FASN in LMH cells.In summary,B.A-TL activates IGF signaling pathway by stimulating hepatocyte synthesis of IGF-1 and plays a regulatory role in the expression of FASN,thus affecting lipid metabolism.7.Signal pathway blocking test:IGF-1R inhibitors（Picropodophyllin）,PI3K inhibitors（LY294002）and MEK inhibitors（PD98059）were used to block the corresponding pathway in vitro.RT-q PCR and Western blot were used to verify the effects of fermentation supernatant,exopolysaccharides and tyrosine on IGF signaling pathway in LMH cells.The results showed that secondary metabolites of B.A-TL mainly regulated the expression of FASN by affecting the downstream PI3K pathway of IGF pathway.8.In summary,B.A-TL activates the IGF signaling pathway by stimulating liver cells to synthesize IGF-1,activates the downstream PI3K pathway to regulate the expression of FASN,and regulates the expression of liver fat production genes and fat oxidation genes to affect lipid metabolism.A large amount of IGF-1 is secreted into the circulatory system to reach muscle tissue,promoting rapid muscle growth,achieving weight gain effect.