The Protective Effects Of Se On AFB₁-induced Tissue Damage And Cell Cycle Arrest In Chick’s Bursa Of Fabricius

Aflatoxin B₁（AFB₁）is the most toxic form of aflatoxins that is a naturally occurring secondary metabolites of Aspergillus parasiticus and Aspergillus flavus.AFB₁ presents potent hepatotoxic,carcinogenic,genotoxic,immunotoxic potential.Selenium（Se）with antioxidant and detoxification functions is one of the essential trace elements for human beings and animals.Our previous study showed that aflatoxin-contaminated corn induced G₂M phase block of bursal cells.Particularly,the supplement Se in diet could improve humoral immune function by alleviating AFB₁-induced histopathological lesions in BF.However,there are no reports about the molecular regulation of how Se protects against AFB₁-induced tissue damage and cell cycle arrest in BF.In our work,we studied the protective effects of Se on AFB₁-induced tissue damage and cell cycle arrest of chick’s BF through histological observation,flow cytometry,Real time fluorescent quantitative PCR（RT-qPCR）and biochemistry assay.A total of 288,one-day-old healthy Cobb chickens were randomly divided into four groups,which were control group（basal diet）,+Se group(0.4 mg·kg^-1 Se),AFB₁ group(0.6 mg·kg^-1 AFB₁)and AFB₁+Se group(0.4 mg·kg^-1 Se+0.6 mg·kg^-1 AFB₁).Each group consisted of three replicates with 24birds per replicate.Histopathological examination showed that the relative weight of BF was decreased.Compared with the control group,many obvious nuclear debris and a few vacuoles were noted in cortex and medulla of bursal follicles in the AFB₁ group at 7 and 14 days of age,while less nuclear debris and more vacuoles were observed at 21 days of age.No obvious histological changes were found in the control group,+Se group and AFB₁+Se group from 7to 21 days of age.Antioxidation parameters biochemistry analysis showed that,compared with the control group,AFB₁ significantly reduced（p<0.01 or 0.05）the content of GSH and the activities of GR,GSH-Px,CAT,SOD and·OH scavenging,but increased（p<0.05 or 0.01）MDA levels from 7 to 21 days of age.Treatment with dietary Se caused a significant increase（p<0.05 or0.01）in the content of GSH,the activities of GR,GSH-Px,CAT,SOD and·OH scavenging,and an obvious decrease（p<0.01）in MDA levels as compared to the AFB₁ group during the experiment.Moreover,these antioxidant markers had no significant difference（p>0.05）among the control group,+Se group and AFB₁+Se group.Therefore,we speculated that supplement 0.4 mg·kg^-1 Se could effectively protect the chicken’s BF against the AFB₁-induced histopathological lesions by reducing oxidative stress.Compared to the control group,the AFB₁ group at 7 days of age displayed a significant decrease（p<0.01）in the percentage of G₀G₁ phase cells,but obvious increase（p<0.01）at14 and 21 days of age.Furthermore,the percentage of bursal cells in G₂M phase was obviously higher（p<0.01）at 7 days of age,but was significantly lower（p<0.01）at 14 days of age in the AFB₁ group than those in the control group.Moreover,these cell phase percentages among the control group,+Se group and AFB₁+Se group did not show significant changes from 7 to 21 days of age（p>0.05）.By flow cytometry assay,the G₂M phase arrest at 7 days and G₀G₁ phase blockage at 14 and 21 days were noted in the AFB₁group.However,the arrest phenomenon was alleviated in the AFB₁+Se group.RT-qPCR results showed that AFB₁ caused an increase（p<0.05 or 0.01）in mRNA levels of ATM and Chk2,and a decrease（p<0.05 or 0.01）in mRNA exprssions of cdc25 and cyclin B₃/cdc2 complexes at 7 days.In addition,we found the increased（p<0.05 or 0.01）mRNA expressions in ATM,Chk2 and p21 in AFB₁ groups when compared with those of the control group,along with a decrease（p<0.05 or 0.01）in p53 and cyclin D₁/CDK6 complexes at 14 and 21 days.Notably,the mRNA expression levels of cdc25 were also decreased at 14and 21 days（p<0.05 or 0.01）.Moreover,in the AFB₁+Se group,supplement Se could restore these parameters to be close to those in the control group.These results indicated that Se ameliorated AFB₁-induced cell arrest through ATM-Chk2-cdc25-cyclin B₃/cdc2 signaling pathway at G₂M phase,via ATM-Chk2-cdc25-cyclin D₁/CDK6 route and ATM-Chk2-p21-cyclin D₁/CDK6 pathway at G₀G₁ phase.In summary,0.4 mg·kg^-1 Se supplied in diet could protect BF from AFB₁-induced histopathological lesions by diminishing oxidative damage,and ameliorated AFB₁-induced cell cycle arrest through ATM-Chk2-cdc25-cyclin B/cdc2 route in G₂M phase at 7 days,and via ATM-Chk2-cdc25-cyclin D/CDK6 route and ATM-Chk2-p21-cyclin D/CDK6 pathway in G₀G₁ phase at 14 and days.Our founds suggested that supplementation of sodium selenite could be a potential antioxidant to protect poultry from toxicity caused by AFB₁.