The Protective Effect Of Se On The Apoptosis Pathways Of Chicken’s Spleen Caused By Aflatoxin B₁

Aflatoxin B₁（AFB₁）is widely found in mouldy raw materials and feedstuffs,as the most toxic secondary mycotoxins produced by different strains of Aspergillus flavus and Aspergillus parasiticus,and has strong immunotoxicity and carcinogenicity.Selenium（Se）,an important component of some antioxidant enzymes and selenium-P protein in animals and humans,is an essential trace element for the growth of humans and animals and has the functions of anti-cancer,anti-oxidation and enhancing immune functions and antagonizing certain toxins.Studies have shown that excessive apoptosis of immune cells is an important cause of immunosuppression,and reports about selenium-antagonizing AFB₁ harm to the body have gradually attracted people’s attention.At present,there have been some reports on AFB₁-induced apoptosis in the immune organs of chicks,but the study of AFB₁ signaling pathways leading to apoptosis of immune organs is still lacking.In this study,288 one-day-old Cobb chicks were randomly divided into four groups and fed control diets（AFB₁=0 mg/kg,Se=0.3mg/Kg）and the+Se diet（Se=0.7mg）./Kg),AFB₁diet（AFB₁=0.6 mg/kg）and AFB₁+Se diet（AFB₁=0.6 mg/kg,Se=0.7 mg/Kg）for 21days.histopathology,flow cytometry,TUNEL staining and qRT-PCR were used to study the effect of AFB₁ on apoptosis signal pathway of chicken spleen and the antagonistic effect of selenium on it.The result is as follows:At 7 days of age,there was no significant difference in absolute and relative weight of spleens between the AFB₁ group and the AFB₁+Se group,compared with the control group and+Se group（p>0.05）.At the age of 14 days,the absolute weight of spleens in the AFB₁group were significant lower than the control group,+Se group,and AFB₁+Se group（p<0.05or 0.01）;at the age of 21 days,the absolute weight of the spleens in the AFB₁ group were significant or extremely significant lower than the control and+Se groups（p<0.05 or 0.01）.At the 14 and 21 days of age,spleen index in the AFB₁ group was significantly lower than the control group（p<0.05）.The results of histopathological showed that no obvious pathological changes were obversed in four groups at 7 days of age.At 14 and 21 days of age,there were congestion in the red pulp region of the spleen in the AFB₁ group,a large amount of irregular blank areas appeared in the ellipsoid,and decreased of lymphocytes in the splenic nodules.The morphological structure of spleen in the+Se group was similar to that in the control group,and the histological structure was normal.In AFB₁+Se groups,the spleen tissue structure of the chicks returned to normal.The results of flow cytometry showed that at 7,14,and 21 days of age,the apoptotic rate of spleen cells in AFB₁ group was significantly higher than that of control and+Se groups（p<0.01）;AFB₁+Se group spleen cell apoptosis The rate was significantly lower than that in the AFB₁ group（p<0.05）.There was no significant difference in splenocytes of chicks between the+Se group and the control group（p>0.05）.The results of TUNEL staining showed that the change of TUNEL positive cells in each group was consistent with that of flow cytometry.The results of Real-time PCR indicated that the mRNA expression levels of Caspase-3,Caspase-8,Caspase-10,Fas,FasL,TNF-R1,TNF-a,Fas,GRP78 and GRP94 in the AFB₁group were obviously increased at 7,14 and 21 days of age（p<0.05 or 0.01）,when compared with the control group.In comparison to the AFB₁ group,these values in the AFB₁+Se group were also obviously decreased（p<0.05 or 0.01）.In addition,no significant differences in these values were found between the+Se group and control group（p>0.05）.There was no significant difference between the+Se group and the control group.In summary,the results showed that 0.6mg/kg AFB₁ in the diet could lead to reduced spleen index,lymphocyte decrease,and increased apoptosis in the chicks.Death receptor pathway related molecules Caspase-3,Caspase-8,Caspase-10,TNF-α,TNF-R1,FasL and Fas,as well as mRNA expression levels of the endoplasmic reticulum pathway-associated molecules GRP78 and GRP94 were up-regulated.The addition of 0.4 mg/kg selenium（using sodium selenite as the selenium source）in the diet slowed these changes.This indicates that AFB₁ may induce apoptosis of spleen cells through activation of death receptors and apoptotic molecules in the endoplasmic reticulum pathway,while selenium may play a protective role by antagonizing the effects of AFB₁ on these two pathways.This study provided a new theoretical basis for the detoxification mechanism of selenium to AFB₁.