The Mechanism Of Intestinal Microbiota Mediated Liver Damage Induced By Ochratoxin A In Ducks

The wide occurrence of ochratoxin A(OTA)and its high thermal stability makes the eradication of OTA from the food chain very difficult.OTA has been shown to be nephrotoxic,hepatotoxic,teratogenic and immunotoxic to several species of animals.More and more studies have confirmed that intestinal microorganisms are related to many diseases.Mycotoxins could alter the composition and diversity of intestinal microbome.This study hypothesizes that OTA could induce liver injury by regulating intestinal microbiota.The aim of this study was to investigate the effects of OTA on growth performance,OTA residues in different organs,cecum microbiota composition and function,intestinal permeability and liver inflammation at the overall level,molecular biological level(transcription level and protein level)and microbiological level to reveal the mechanism of intestinal microbiota mediated OTA-induced liver injury of ducks.The experiment is divided into the following six parts:Part 1: Effect of OTA on growth performance and OTA residues in different organs of Peking ducks.A total of 30 one day old healthy male Peking ducklings were randomly assigned into two groups,each consisting of 3 replicates of 5 ducklings.At the first week,ducklings received a basal diet,and normal drinking water.On day 8,ducklings were given OTA in doses of 0(CON group)or 235 μg/kg(OTA group)body weight in 0.1 M Sodium bicarbonate by oral gavage once a day for two weeks.The results showed that:(1)OTA had no effect on final weight,average daily feed intake(ADFI),average daily gain(ADG)and feed/gain ratio(F/G)of ducks(P>0.05).(2)OTA oral gavage had no effect on the relative weights of organs except increased relative weight of liver(P<0.05).(3)There were no difference of intestinal relative length and weight between CON and OTA groups(P<0.05).(4)OTA residue amount in different tissues from high to low were kidney,cecum,liver,breast,duodenum,jejunum,ileum,thigh and serum,respectively.(5)Histological analysis indicated that OTA resulted in the infiltration of inflammatory cells in the liver and increased the proportion of inflammatory cells(P<0.0001).Together,ducks treated with 235 μg/kg body weight OTA had no effect on growth performance,but increased relative weight of liver,and extensive inflammatory cell infiltration in OTA treated liver.The OTA residue amount in cecum was next to kidney.Part 2: Effect of OTA on composition and function of cecum microbiota.Animal grouping and treatment was the same to part 1.Cecum digesta was collected on d 21 and sequenced by16 Sr DNA.The results showed that:(1)OTA treatment decreased cecum microbiota richness and diversity(P<0.05).(2)The dominant phylum of duck were Bacteroidetes,Firmicutes,Actinobacterin,Fusobacteria,proteobacteria and Deferriabacteres.OTA increased the relative abundance of Bacteroidetes(phylum),Bacterides(Genus)and Bacterides plebius(species)(P<0.05).(3)OTA treatment significantly decreased cellular processes(quorum sensing)(P<0.05),increased KEGG pathways associated with inflammation and exposure to oxidative stress: LPS biosynthesis and citrate cycle(P<0.05).(4)The contribution of different strains to LPS biosynthesis was different,and Bacterides contribute most.(5)Cecum LPS content was higher in OTA group compared with CON group(P=0.09).Part 3: Effect of OTA on cecum tight junction.Animal grouping and treatment was the same to part 1.Mucus was collected to measured gene and protein expression related to intestinal tight junction.Liver was collected to 16 S sequencing and measured LPS level.The results showed that:(1)Relative expression of TJP-1m RNA,OCLN m RNA,TJP1 and Occludin protein abundance were up-regulated in OTA oral gavage group(P<0.05).(2)Serum LPS level was higher in OTA group than CON group(P<0.05).(3)OTA treatment group had higher serum pro-inflammatory cytokines L-1β(P<0.05)and IL-6(P=0.01)levels and had no effect on anti-inflammatory IL-10 and IFN-γ(P>0.05)levels.(4)The relative abundance of Bacteroidetes and Bacterides of liver were higher in OTA group than CON group(P<0.05),and this change was similar with cecum microbiota.(5)Compared with CON group,OTA increased liver LPS level significantly(P=0.0006).Part 4: OTA oral gavage promote liver inflammation.Animal grouping and treatment was the same to part 1.Liver sample was collected to analysis gene and protein expression related to TLR4 signaling pathway,inflammatory cytokines and liver histology.The results showed that:(1)OTA oral gavage increased TLR4(P<0.05),My D88(P<0.01),IKB-α(P<0.05),IL-6(P<0.01)and TNF-α(P<0.05)gene expression.At the protein level,TLR4(P<0.01),My D88(P<0.05),p-IKBα(P<0.05)and p-IKBα / IKBα(P<0.05)were increased in OTA group.(2)Liver pro-inflammatory cytokine IL-1β was extremely increased(P<0.0001),IL-6 was increased(P<0.05)after OTA treatment,and OTA oral gavage could decrease liver anti-inflammatory cytokines IL-10 and IFN-γ level(P<0.05).Part 4: OTA oral gavage for antibiotics treated ducks.One day old Peking male ducklings were divided randomly into 2 groups with 3 replicates(n = 5/replicates):(i)Antibiotics group: ducklings received a basal diet and drinking fluid containing streptomycin(1 g/L,Sigma),ampicillin(1 g/L,Sigma),and neomycin(1 g/L,Sigma)to clear intestinal bacteria.On day 8,ducks were given 0.1 M Sodium bicarbonate.(ii)Antibiotics + OTA group: ducklings received a basal diet and drinking fluid containing streptomycin(1 g/L),ampicillin(1 g/L),neomycin(0.5 g/L).On day 8,ducklings were given OTA in doses of 0 or 235 μg/kg body weight in 0.1 M Sodium bicarbonate by oral gavage once a day for two weeks.The results showed that:(1)OTA had no effect on growth performance,organ index,relative length and weight of intestinal of antibiotics treated ducks(P>0.05).(2)OTA residue amount from high to low were kidney,cecum,liver,breast,duodenum,jejunum,ileum,thigh and serum,respectively.(3)OTA had no effect on the cecum relative abundance of Bacteridetes and Bacteroides and LPS level of antibiotics treated ducks(P>0.05).(4)There were no difference between Antibiotics and Antibiotics + OTA on intestinal tight junction(TJP1 and Occludin)m RNA and TJP1 protein abundance(P>0.05).(5)There was on effect of OTA on serum LPS level and inflammatory cytokines in antibiotics treated ducks(P>0.05).(6)OTA had no effect on liver LPS level,gene expression and protein abundance related to TLR4 signaling pathway,liver inflammatory cytokines and liver morphology(P>0.05).Part 6: Fecal microbiota transplantation(FMT)experiment.One old Peking male ducklings were divided randomly into 4 groups(15 ducklings per group).(i)Control group was fed with basal diet and normal drinking fluid for 4 weeks.(ii)OTA group,at the first week ducklings received a basal diet,and normal drinking water,on day 8,ducklings were given OTA in doses of 0 or 235 μg/kg body weight in 0.1 M sodium bicarbonate by gavage every day for three weeks.(iii)FMT(CON)group.One day ducklings received a basal diet and drinking fluid containing streptomycin(1 g/L),ampicillin(1 g/L),neomycin(0.5 g/L)for two weeks to remove indigenous gut microorganisms.After this treatment,10 g of fresh stool was collected from the control group immediately upon defecation and was resuspended in 50 m L of normal saline,vortexed for 3 min and allowed to settle by gravity for 2 min,and the fresh stool was collected every day.Transplant into recipient ducklings was achieved by gavage with 1 m L/100 g body weight of the supernatant from the fecal sample once a day for 2 weeks.(iv)FMT(OTA)group.One day old ducklings received a basal diet and antibiotics in water for two weeks.After this treatment,10 g of fresh stool was collected from the OTA group,the collection and transplantation method with the same to FMT(CON)group.The results showed that:(1)Final weight in FMT(OTA)group was lower than FMT(CON)group(P=0.06),and the relative weight of liver were higher in FMT(OTA)group than FMT(CON)group(P<0.05).(2)The change trend of cecum microorganisms in FMT(CON)and FMT(OTA)ducks were the same with that in CON and OTA group,respectively.The relative abundance of Bacteridetes and Bacterides in FMT(OTA)group was significantly higher than that in FMT(CON)(P<0.05),but there was no significant difference between FMT(OTA)group and OTA group(P>0.05),and FMT(OTA)increased cecum LPS level significantly compared with FMT(CON)group(P=0.005).(3)Compared with FMT(CON)group,FMT(OTA)down regulated gene expression and protein abundance(TJP1 and Occludin)related to tight junction(P<0.05),and increased serum LPS level(P=0.07).(4)FMT(OTA)increase serum pro-inflammatory cytokine TNF-α level(P=0.005),and had no effect on serum liver function indexes(P>0.05).(4)Compared with FMT(CON),FMT(OTA)significantly increased the liver LPS level(P<0.05),significantly increased the relative expression of TLR4 m RNA,IL-6m RNA and TNF-α m RNA in duck liver(P<0.05),significantly increased TLR4 and My D88 protein abundance(P<0.05),and extremely increased the protein abundance of pIKBα and p-p65(P<0.01).In FMT(OTA)group,there was obvious infiltration of inflammatory cells in liver slices,and significantly increased the proportion of inflammatory cells(P<0.0001).From the above results,we concluded that OTA could change the composition of cecum microbiota,destroy the tight intestinal junction,result in bacterial translocation to the liver,and ultimately lead to liver injury.OTA fails to promote liver inflammation in antibiotic treated ducks After transplantation of fecal microorganisms in OTA group,the phenotypic and microbial changes are similar with OTA group.These indicating that intestinal microbiota participate in OTA-induced liver injury.