Protective Effect And Mechanism Of Genistein On Broiler Challenged With Escherichia Coli O78

Four experiments were conducted to investigate the regulation of genistein on immune function and intestinal barrier function of chicken challenged with Escherichia coli(E.coli)078 and its mechanism.Experment 1 was conducted to evaluated the effect of genistein supplementation on broiler challenged with E.coli 078.Ia a 3 × 2 factorial arrangement,432 one-day-old male broiler chicks(Arbor Acres)were assigned to 6 groups.Birds were fed a basal diet without or with 20,40 mg/Kg genistein and intraperitoneally injected with E.coli 078(1.0×108 CFU/mL)or saline at 19 old.Genistein had no effect on broiler performance.E.coli 078 infection reduced broiler performance and increased broiler mortality.Genistein(20 mg/Kg)addition decreased the mortality of birds during challenge.The results indicated that genistein(20 mg/Kg)addition reduced the mortality of colibacillosis.Experment 2 was conducted to investigate the inhibitory effects of genistein on E.coli 078.The effects of genitein and bacterial isolated from cecum with or without genitein on E.coli 078 was determinded by Oxford Cup.Genitein had no inhibitory effect on growth of E.coli 078.Genistein could increase proliferation of lactobacillus and bifidobacterium isolated from cecal contents,which could suppress E.coli 078 in co-culture assay.However genitein could not improve the effects of Lactobacillus and bifidobacterium on E.coli 078.The results indicated that genitein han no direct inhibitory effect on the growth of E.coli O78,nor by proliferation of lactobacillus and bifidobacterium.Experment 3 was conducted to investigate the effects of genistein on immune function of broiler challenged with E.coli 078.In a 3 X 2 factorial arrangement,432 one-day-old male broiler chicks were assigned to 6 groups.Birds were fed a basal diet without or with 20,40 mg/Kg genistein and intraperitoneally injected with E.coli 078(1.0×108 CFU/mL)or saline at 19 old.While reduced the thymus and bursa index,E.coli 078 infection increased the heart and liver index of broilers.While relieved the increase of total protein and globulin,genistein increased serum lysozyme and serum IgA levels during challenge.The proportion of neutrophils,basophils,monocytes and CD4+ T lymphocytes were reduced by genistein supplement.Genistein addition inhibitited the mRNA expression of TNF-a,IL-1β,IL-8,MyD88,and NF-κB.To further verified the effect of genistein on peripheral blood lymphocytes,peripheral blood lymphocytes,isolated from broiler whole blood,were chanllenged with E.coli 078 and LPS.The effects of genistein on subpopulation composition of peripheral blood lymphocytes were analyzed.The ratio of CD3+and CD4+ was increased when peripheral blood lymphocytes were exposed to E.coli O78 and LPS,but not CD4+/CD8+.The effect of E.coli 078 and LPS was inhibited by genistein.Peripheral blood lymphocytes pre-exposed to E.coli 078 had an negative effect on permeability of IPEC-1,which was attenuated by genstein.The results indicated that genistein may inhibit the inflammatory response of broilers challenged by E.coli 078 by regulating innate immunity and acquired immunity,thereby enhancing the immune function of broilers against E.coli 078 infection.Experment 4 was conducted to investigate the effects of genistein on intestinal epithelial barrier function of broiler challenged with E.coli 078.In a 3 × 2 factorial arrangement,432 one-day-old male broiler chicks were assigned to 6 groups.Birds were fed a basal diet without or with 20,40 mg/Kg genistein and intraperitoneally injected with E.coli 078(1.0×108 CFU/mL)or saline at 19 old.Genistein could reduce the depth of ileal crypts in broilers and increase the ratio of villus height to crypt depth.The intestinal permeability of broilers was increased during challenge,and genistein can reduced it.Genistein enhanced the expression of ZO-1 and Claudin-1.To further verified the effect of genistein on intestinal barrier function,IPEC-1 was pretreated with 10,25,50 μM genistein and then incubated with E.coli 078.Finally,TEER,permeability,and expression of tight jucton proteins were analyzed.Addition,the invasion of E.coli 078 and Salmonella-GFP of IPEC-1 and peripheral blood lymphocytes were analyzed.Genistein could increase the expression of ZO-1,Claudin-1,E-cadherin,PI3K and mTOR.The effect of genstein on barrier function was abolished by tyrosine phosphatase inhibitors and inhibitors of Akt.The invasion of E.coli 078 and Salmonella was inhibited by genstein,which was related to tyrosine kinase inhibition.The results indicated that genistein improyed intestinal barrier function via tyrosine kinase inhibition and expression of tight junction,thereby enhancing the intestinal epithelial barrier function of broilers against E.coli 078 infection..In conclution,in vivo,genistein can reduce the mortality of colibacillosis by regulating the innate immunity and acquired immunity and improving the intestinal barrier faction.In vitro,genitein han no direct inhibitory effect on the growth of E.coli 078.Genistein reversed the negative effects of peripheral blood lymphocytes on permeability of IPEC-1,when it was pre-expoxed to E.coli 078.Genistein improved the barrier fuction of IPEC-1,which was related to tyrosine kinase inhibition and Akt.