Ameliorative Effects Of Selenium On Excess Apoptosis In Jejunum Of Broilers Caused By AFB₁ Through Death Receptor And Endoplasmic Reticulum Pathways

Aflatoxin B₁ is one kind of difuranocoumarin derivatives produced by a polyketide pathway by the fungus Aspergillus flavus and Aspergillus parasiticus,which can induce primary liver cancer,immunosuppression and oxidative stress in the body.However,the study of the digestive tract,which was first exposed to AFB₁,has hardly been reported.Selenium,as a necessary trace element in human and animal body,has antioxidant,anti-cancer and immunity enhancing effects in a certain dose range.At present,more and more attention has been paid to the study of the protective function of selenium on the toxic effects of AFB₁ in the body.However,the specific molecular mechanism of AFB₁ induced excessive apoptosis in broilers’jejunum mucosa cells and the antagonism of selenium have not been fully understood.In this study,sodium selenite was as the selenium source to feed the AFB₁-exposured broilers.Through histopathology,TUNEL assay,flow cytometry and real-time quantitative PCR,the cell apoptosis of jejunum,morphology structure changes,and the dynamic changes of death receptor and endoplasmic reticulum pathways related regulatory factors were detected.In this study,288 1-day-old Cobb broilers were selected and randomly divided into4 groups.There was 72 in each group and were fed with the control group diet（0 mg/kg AFB₁,0.3 mg/Kg Se）,Se group diet（0 mg/kg AFB₁,0.7 mg/Kg Se）,AFB₁ group diet（0.6mg/kg AFB₁,0.3 mg/Kg Se）and AFB₁+Se group diet（0.6 mg/kg AFB₁,0.7 mg/Kg）for 21days.The results are as follows:Histopathological examination showed that,only in the AFB₁ group,the epithelial cells were obviously shedding from the apical region of villi of jejunum.However,there was no obvious histological change in intestinal villus of control group,Se group and AFB₁+Se group.At 7,14 and 21 days of age,the villus height,villus/crypt ratio and villus area of the AFB₁ group were significantly lower than those of the control group,Se group and AFB₁+Se group（p<0.01 or p<0.05）.Compared with the control group,there were no significant differences in morphology index between Se group and AFB₁+Se group.The results of TUNEL assay showed that,in comparison with the control group,the number of TUNEL positive cells in AFB₁ group was significantly increased at 7,14 and 21days of age（p<0.01）.When compared with the AFB₁ group,the number of TUNEL positive cells in the AFB₁+Se group was decreased significantly at the age of 14 and 21 days（p<0.05）.In addition,in the 21-day test period,there were no significant differences of TUNEL positive cells between the control group and the Se group（p>0.05）.The results of flow cytometry showed that,when compared to the control group,the apoptotic rate of jejunal cells in AFB₁ group was significantly increased at 7,14 and 21 days of age（p<0.01）.When compared with AFB₁ group,the apoptotic rate of jejunal cells in AFB₁+Se group at the 14 and 21 days of age was significantly decreased（p<0.01）.In addition,in the whole test period,the apoptotic rate of jejunal cells had no significant changes between the Se group and the control group（p>0.05）.Real time fluorescent quantitative PCR results showed that,in the death receptor pathway,compared with the control group,the mRNA expressions of FAS,TNF-α,TNF-R1,CASPASE-3 and CASPASE-8 in AFB₁ group at 7,14 and 21 days of age were increased significantly（p<0.05 or p<0.01）.In comparison with the control group,the mRNA expressions of FASL and CASPASE-10 at the age of 14 and 21 days were increased significantly（p<0.05 or p<0.01）.Compared with AFB₁ group,the mRNA expressions of FAS,FASL,TNF-α,CASPASE-3 and CASPASE-10 in AFB₁+Se group were decreased significantly at 14 and 21 days of age（p<0.05 or p<0.01）,while the mRNA expressions of TNF-R1 and CASPASE-8 were decreased significantly at 7,14 and 21 days of age（p<0.05）.In the endoplasmic reticulum pathway,the mRNA expressions of GRP78 and GRP94 in the AFB₁ group were increased significantly at 7,14 and 21 days of age when compared with the control group（p<0.05 or p<0.01）.The mRNA expressions of GRP78 and GRP94 in the AFB₁+Se group were significantly lower than those of the AFB₁ group at 14 and 21 days of age（p<0.05 or p<0.01）.In addition,during the whole test period,there were no significant differences in the mRNA expressions of FAS,FASL,TNF-α,TNF-R1,CASPASE-3,CASPASE-8,CASPASE-10,GRP78 and GRP94 between the Se group and the control group（p>0.05）.In conclusion,0.6 mg/kg AFB₁ in the diet can inhibit the development of broilers’jejunal mucosa by up-regulating the mRNA expressions of FAS,FASL,TNF-R1,TNF-α,CASPASE-3,CASPASE-8,CASPASE-10,GRP78 and GRP94,resulting in excessive apoptosis of jejunal mucosal cells,and 0.4 mg/kg selenium supplement can alleviate these changes.This indicated that AFB₁ could induce death receptor FAS/FASL or TNF-α/TNF-R1to activate the upstream CASPASE-8 or CASPASE-10,and further activate downstream CASPASE-3,finally cell apoptosis was caused by the death receptor pathway.At the same time,AFB₁ can also stimulate endoplasmic reticulum pathway to induce the endoplasmic reticulum stress,and then the activation of the UPR reaction.With the release of GRP78 and GRP94,their mRNA expressions were up-regulated.A long term ER stress will induce apoptosis.Selenium supplementation can inhibit the activation of AFB₁ on these two pathways,which may be one of the molecular mechanisms of AFB₁ induced apoptosis in the jejunal mucosal cells and the antagonistic effect of selenium.