Toll-like Receptor Signal Transduction Mechanism Of Quercetin On Beta-defensin And Microflora In Ileum Of Broilers

As one of ideal antibiotic substitutes,quercetin has many biological activities,such as antioxidant,anti-inflammatory,antibacterial,immunoregulatory,etc.The purpose of this experiment was to study the effect of quercetin on the intestinal microflora of AA broilers and mechanism of action by measuring m RNA expression of avian ?-defensin(Av BD),Toll-like receptors(TLR),genes relating to TLR signaling pathways and ileal microflora,thereby provide a theoretical basis for the antibiotic substitution s.480 healthy Arbor Acre(AA)broilers with similar weight(1 day old)were randomly assigned to 4 treatments,with 6 replicates in each batch and 20 broilers in each.These broilers were reassigned 4 groups 7 days later according to the principle of similar weight.Control treatment was fed on a basic diet,and treatments were fed on a basal diet supplemented with 0.02%,0.04%,and 0.06% quercetin,respectively.The experimental period was six weeks.Results showed that:(1)Effects of quercetin on growth performance of AA broilers: Compared with control,there was no significant difference in body weight at the end of trial,average daily gain,average daily feed intake and feed/gain ratio in AA broilers of three quercetin groups(P>0.05).(2)Effects of quercetin on mortality of AA broilers: Compared with control,the mortality of AA broilers in three quercetin groups was decreased.(3)Effect of quercetin on ileal microflora in AA broilers: Compared with control,1)at the class level,quercetin decreased relative abundance of Clostridia in ileum of AA broilers;2)at the order level,0.02% and 0.06% quercetin increased relative abundance of Bifidobacteriales,whereas 0.04% quercetin decreased that of Bifidobacteriales;The relative abundance of Clostridiales in three quercetin groups was decreased;3)at the family level,0.02% quercetin increased relative abundance of Bifidobacteriaceae,whereas decreased relative abundance of Enterobacteriaceae,Lachnospiraceae,Coriobacteriaceae and Desulfovibrionaceae in ileum of AA broilers;0.04% quercetin decreased relative abundance of Bifidobacteriaceae,Enterobacteriaceae,Lachnospiraceae,Coriobacteriaceae and Desulfovibrionaceae in ileum of AA broilers;0.06% quercetin increased relative abundance of Bifidobacteriaceae and Enterobacteriaceae,whereas decreased relative abundance of Lachnospiraceae,Coriobacteriaceae and Desulfovibrionaceae in ileum of AA broilers;4)at the genus level,0.02% quercetin increased relative abundance of Bifidobacterium,whereas decreased relative abundance of Enterobacter,Salmonella,Escherichia and Staphylococcus in ileum of AA broilers;0.04% quercetin decreased relative abundance of Bifidobacterium,Enterobacter,Salmonella,Escherichia and Staphylococcus in ileum of AA broilers;0.06% quercetin increased relative abundance of Bifidobacterium,Enterobacter and Salmonella,whereas decreased relative abundance of Escherichia and Staphylococcus in ileum of AA broilers;5)at the species level,0.02% quercetin increased relative abundance of Bifidobacterium bohemicum,Lactobacillus crispatus,Lactobacillus reuteri,Lactobacillus salivarius,Lactobacillus ingluviei,Lactobacillus vaginalis,Lactobacillus oris,Lactobacillus kitasatonis,Lactobacillus johnsonii,Lactobacillus gasseri and Lactobacillus agilis,whereas decreased relative abundance of Salmonella enterica,Staphylococcus aureus,Escherichia coli,Helicobacter pylori,Clostridium perfringens,Lactobacillus aviaries and Lactobacillus gallinarum in ileum of AA broilers;0.04% quercetin increased relative abundance of Bifidobacterium bohemicum,Lactobacillus aviaries,Lactobacillus reuteri,Lactobacillus gallinarum,Lactobacillus salivarius,Lactobacillus ingluviei,Lactobacillus johnsonii and Lactobacillus gasseri,whereas decreased relative abundance of Helicobacter pylori,Salmonella enterica,Lactobacillus crispatus,Escherichia coli,Lactobacillus vaginalis,Lactobacillus oris,Staphylococcus aureus,Lactobacillus kitasatonis,Clostridium perfringens and Lactobacillus agilis in ileum of AA broilers;0.06% quercetin increased relative abundance of Bifidobacterium bohemicum,Salmonella enterica,Clostridium perfringens,Lactobacillus crispatus,Lactobacillus gallinarum,Lactobacillus salivarius,Lactobacillus ingluviei and Lactobacillus gasseri,whereas decreased relative abundance of Escherichia coli,Staphylococcus aureus,Helicobacter pylori,Lactobacillus aviaries,Lactobacillus reuteri,Lactobacillus vaginalis,Lactobacillus oris,Lactobacillus kitasatonis,Lactobacillus johnsonii and Lactobacillus agilis in ileum of AA broilers;6)0.02% and 0.06% quercetin increased Shannon index in ileum of AA broilers.(4)Effects of quercetin on m RNA expression of Av BD in ileum of AA broilers: 0.02% quercetin up-regulated m RNA expression of Av BD4,Av BD6(P<0.05),Av BD3 and Av BD11(P<0.01)in ileum of AA broilers,while down-regulated that of Av BD12 and Av BD13(P<0.05);0.04% quercetin up-regulated m RNA expression Av BD9(P<0.05),Av BD7 and Av BD10(P<0.01)in ileum of AA broilers;0.06% quercetin significantly increased m RNA expression of Av BD5,Av BD8(P<0.05),Av BD10,Av BD11 and Av BD13(P<0.01)in ileum of AA broilers;There was no significant difference in m RNA expression of Av BD1,Av BD2 and Av BD14 in three quercetin groups(P>0.05);Moreover,0.04% and 0.06% quercetin significantly increased m RNA expression of total Av BD(P<0.05;P<0.01)as compared to control.(5)Effect of quercetin on m RNA expression of TLRs in ileum of AA broilers: Compared with control,0.02% quercetin significantly decreased m RNA expression of TLR21(P<0.05),TLR1 A,TLR1B,TLR2 A and TLR4(P<0.01)in ileum of AA broilers;0.04% quercetin significantly decreased m RNA expression of TLR1 A,TLR1B and TLR2A(P<0.01),whereas increased m RNA expression of TLR7(P<0.05)and TLR4(P<0.01)in ileum of AA broilers;0.06% quercetin significantly increased m RNA expression of TLR1 A,TLR1B,TLR2 A and TLR3(P<0.01)in ileum of AA broilers;There was no significant difference in m RNA expression of TLR2 B,TLR5 and TLR15 in three quercetin groups(P>0.05).Moreover,0.02% and 0.06% quercetin significantly decreased m RNA expression of total Av BD(P<0.01).(6)Effects of quercetin on TLR signaling pathway in ileum of AA broilers: Compared with control,in ileum of AA broilers,0.02% quercetin up-regulated m RNA expression of interferon-regulatory factor 7(IRF7)(P<0.01),while down-regulated m RNA expression of I?B kinase ?(IKK?),I?B kinase ?(IKK?),inhibitor ? of NF-?B(I?B?),TNF receptor associated factor 3(TRAF3),nuclear factor kappa-B(NF-?B)(P<0.05),I?B kinase ?(IKK?),inhibitor ? of NF-?B(I?B?),myeloid differentiation factor 88(My D88),Toll-interleukin 1 receptor domain containing adaptor protein(TIRAP/MAL),TANK binding kinase 1(TBK1)(P<0.01);0.04% quercetin significantly increased m RNA expression of tumor necrosis factor receptor associated factor 6(TRAF6)(P<0.01),while down-regulated m RNA expression of I?B? and NF-?B(P<0.05),IKK?,IKK?,I?B?,My D88,TIRAP/MAL,TRAF3(P<0.01);0.06% quercetin significantly decreased m RNA expression of interleukin 1?(IL-1?),IKK?(P<0.05),My D88,TIRAP/MAL,TBK1(P<0.01);There was no significant difference in m RNA expression of interferon-regulatory factor 3(IRF3),interferon ?(IFN-?),interferon ?(IFN-?),interleukin 6(IL-6),interleukin 8(IL-8),tumor necrosis factor ?(TNF?),transforming growth factor ?(TGF?),Toll-interleukin 1 receptor domain containing adaptor inducing IFN-? protein(TRIF),TRIF-related adaptor molecule(TRAM)and transforming growth factor ?-activated kinase(TAK1)in three quercetin groups(P>0.05).In conclusion,appropriate dosage of quercetin regulated ileal microflora in AA broilers via regulating m RNA expression of Av BDs and TLR signaling pathway,thereby maintaining the ileal microecology balance,decreased mortality.