Studies Of G₀/G₁ Cell Cycle Arrest Caused By Aflatoxin B₁ And Protective Effect Of Se In Thymocytes Of Broilers

Aflatoxin B₁（AFB₁）is a toxic product of Aspergillus fungi.AFB₁ can cause immune damage,genotoxicity,and oxidative stress.Selenium（Se）,the essential trace elements,which has the effects of preventing cancer,preventing oxidative damage and preventing mutations.Previous studies in our laboratory showed that,AFB₁ intake can cause the thymus cell cycle arrest in G₀/G₁ phase,and sodium selenite can alleviate the above effects of AFB₁.However,there is no relevant research report on the molecular mechanism of AFB₁ causing thymic cell cycle arrest in chicks and the effect of selenium on its mitigation.This study uses sodium selenite as a selenium source to feed broiler chickens that ingest AFB₁.The pathological changes of thoracic tissue were observed by histopathology;flow cytometry was used to detect the thymus cell cycle,the quantitation PCR（q RT-PCR）was used to detect the transcriptional level of thymocyte cycle-related genes,and immunohistochemistry was used to detect PCNA protein expression level.In this experiment,288 chicks,one day old,were divided into the 4 group,which were fed with the control diet（0.332 mg/kg selenium）,AFB₁ diet（add with 0.6 mg/kg AFB₁）,+Se diet（add 0.4 mg/kg selenium）and AFB₁+Se diet（add 0.6 mg/kg AFB₁ and 0.4 mg/kg selenium）for 21 days.Thymus histopathological results showed that,the difference between the+Se group and the control group is not obvious;when compared with the control group,the AFB₁ group had more vacuoles and nuclear fragments in thoracic cortex,and congestion in the thymus medulla area.Compared with AFB₁ group,the AFB₁+Se group had clear thymus tissue structure without pathological injury.At the same time,the AFB₁+Se group had no significant difference compared with the control group.Flow cytometry showed the percentage of cell population distribution in G₀/G₁ phase.There was no significant difference between the+Se group and the control group at each age（P>0.05）.Compared with the control group,at each age,the AFB₁ group was significantly or extremely significantly elevated（P<0.01 or P<0.05）.Compared with the AFB₁ group,the AFB₁+Se group was significantly decreased at 14 and 21 days of age（P<0.05）;at the same time,the AFB₁+Se group had no significant difference compared with the control group at each age（P>0.05）.Immunohistochemistry showed that the integrated optical density（IOD）value of PCNA protein showed that.There was no significant difference between the+Se group and the control group at 7 and 14 days（P>0.05）.Compared with the control group,at each age,the AFB₁ group was significantly or extremely significantly lower（P<0.01 or P<0.05）.Compared with AFB₁ group,AFB₁+Se group was significantly increased at 7 and 14 days of age（P<0.05）;at the same time,there was no significant difference between AFB₁+Se group and control group at 7 and 14 days of age（P>0.05）.QRT-PCR detection results show that:Compared with the control group,p27,p53,p21,p16,Cyclin E,Cdk6,Cdk2 and PCNA genes at 7,14,and 21 days of age,p27 gene at 21 days of age,p15 and Cyclin D₁ genes in 7 and 14 days of age,the transcription levels of these genes in the+Se group were not significantly different from the control group（P>0.05）.Compared with the control group,ATM,p53,p15,and p16 genes at 7,14,and 21 days of age,the p21gene at 7 and 21 days of age,the p27 gene at 21 days of age,the transcription level of these genes in the AFB₁ group were significant or extremely significantly higher than the control group（P<0.01 or P<0.05）;Cyclin D₁,Cyclin E,Cdk6,and Cdk2 genes at 7 and 14 days of age,PCNA gene at 7,14 and 21 days of age,the transcription levels of these genes in the AFB₁group were significantly or extremely significantly lower than control group（P<0.01 or P<0.05）.AFB₁+Se group compared with the AFB₁ group,ATM and p21 genes at 7 and 21days of age,the p53,p15 and p16 genes at 7,14,and 21 days of age,the p27 gene at 21 days of age,the transcription levels of these genes were significantly or extremely significantly decreased（P<0.01 or P<0.05）;Cyclin D₁ and Cyclin E genes at 7 and 14 days of age,Cdk6 and Cdk2 genes at 7 days of age,PCNA gene at 7,14,and 21 days of age,the transcription levels of these genes were significant or extremely significantly increased（P<0.01 or P<0.05）.At the same time,compared with the control group,ATM,p53,p27,Cdk2,and PCNA genes at 7,14,and 21 days of age,the p21,p15,p16,and Cyclin D₁ genes at 7 and 14 days of age,Cyclin E gene at the 7 and 21 days of age,Cdk6 gene at 7 days of age,the transcription levels of these genes in the AFB₁+Se group were not significantly different from the control group（P>0.05）.Therefore,the effect of AFB₁ on the transcription level of these genes was alleviated in the AFB₁+Se group.Taken together,uptake of 0.6 mg/kg of AFB₁ could block cell cycle at G₀/G₁ phase by p15（p16）-Cyclin D₁/Cdk6,p27-Cyclin E/Cdk2,and ATM-p53-p21-Cyclin E/Cdk2（PCNA）routes in the thymus of broilers,and 0.4 mg/kg selenium could protect the broiler’s thymocytes from the G₀/G₁ arrest through those routes.This may be the molecular mechanism of AFB₁-induced G₀/G₁ cell cycle arrest,and protective role of Se on thymocytes cell cycle arrest caused by AFB₁.