| Selenium is an essential trace element for humans and animals to maintain normal physiological activities.Selenium deficiency not only endangers human health but also endangers the health of many animals,which brings great economic losses to the aquaculture industry.Selenium deficiency can also induce inflammatory injury of tissues and organs and autophagy.Toll-like receptors（TLRs）are important pattern recognition receptors in the body.Among them,the TLR4 pathway can regulate the occurrence of many diseases.At present,there are few studies on autophagy of chicken bursa of Fabricius induced by selenium deficiency,especially the mechanism of TLR involved in autophagy of chicken bursa of Fabricius induced by selenium deficiency is not known.How does the TLR4 pathway mediate selenium deficiency-induced autophagy in the bursa of Fabricius?This is the main objective of our study.Firstly,200 one-day-old healthy broilers were randomly divided into the selenium deficiency group and control group.Chickens in the selenium deficiency group（L group）were fed with a self-made selenium deficiency diet,and the selenium content was determined as 0.004 mg/kg.Chickens in the control group（C group）were fed a normal diet supplemented with selenium（i.e.,Na2Se O3was added to the selenium deficiency diet）,and the selenium content was determined to be 0.2 mg/kg.The bursa tissues were collected at 15,25,35,45 and 55 days.The histopathological changes and autophagy of the bursa of Fabricius were observed by histopathology and transmission electron microscopy.Real-time fluorescent quantitative PCR was used to detect the mRNA expression changes of ChTLR4 and its signaling pathway conducting molecules,inflammatory factors,and autophagy-related factors in the bursa of Fabricius.Western blot was used to detect the changes of ChTLR4,MyD88,Beclin1,and LC3 proteins in the bursa of Fabricius.The results showed that the cortex and medulla of the bursa of Fabricius in the selenium deficiency group were unclear,the volume of lymphoid follicles became smaller,and the number of cells in lymphoid follicles decreased.Mitochondria swelled,vacuolated,cristae arranged disorderly,and fractured.Autophagosomes were formed in the cytoplasm.The results of real-time fluorescence quantitative PCR showed that compared with the control group,the expression level of ChTLR4 mRNA in the chicken bursa of Fabricius in the selenium deficiency group was increased at 15～55 days,and the difference was significant at 15 days（P<0.05）,and the difference was extremely significant at 25,35,45 and 55 days（P<0.01）.The mRNA expression levels of signal transduction molecules MyD88,inflammatory factors NF-κB,IL-8,TNF-α,and autophagy-related factors ATG5,Beclin1,LC3-I,and LC3-II were up-regulated.The protein expression levels of MyD88 and Beclin1 and the ratio of LC3-II/LC3-I in the selenium deficiency group were significantly up-regulated（P<0.01）,and the protein expression level of ChTLR4 was significantly up-regulated at 25～55 days（P<0.01）.The results showed that a low selenium diet could activate ChTLR4 and up-regulate the expression level of inflammatory factors through the TLR signaling pathway,resulting in the inflammatory injury of the bursa of Fabricius.Selenium deficiency also induces autophagy in the chicken bursa of Fabricius.To explore the correlation between TLR signaling pathway and autophagy in the chicken bursa of Fabricius,HD11 cells were used as the research object,and the selenium deficiency model in vitro was established.Three groups were established:the control group,selenium deficiency group,and selenium deficiency+TAK242（TLR4 inhibitor）group.The control group was cultured in DMEM/F12 medium containing 10%FBS.The selenium deficiency group was treated with DMEM/F12 medium containing 1%FBS,10μg/m L insulin and 5μg/m L transferrin for 5days.The selenium deficiency+TAK242 group was added 1μmol/L TAK242 based on selenium deficiency culture medium,and the other conditions were the same as the selenium deficiency group.CCK8 method was used to detect the viability of HD11 cells.The dynamic changes of the mRNA expression of ChTLR4 and its signal transduction molecules,inflammatory factors,and autophagy-related factors in HD11 cells were detected by fluorescence quantitative PCR.The changes of ChTLR4,MyD88,Beclin1,and LC3 proteins in HD11 cells were detected by Western blot.In order to further verify the integrity of autophagocyte flow in the chicken bursa of Fabricius induced by selenium deficiency,the integrity of autophagocyte flow in HD11 cells was observed by using autophagocyte double-label adenovirus.The results showed that compared with the control group,selenium deficiency HD11 cell viability decreased.The mRNA expression levels of ChTLR4,MyD88,NF-κB,IL-1β,IL-8,TNF-α,ATG5,Beclin1,LC3-I,and LC3-II in HD11 cells in the selenium deficiency group were significantly up-regulated（P<0.01）.Compared with the selenium deficiency group,the mRNA expression levels of ChTLR4,MyD88,NF-κB,ATG5,LC3-I,and LC3-II in HD11 cells in the selenium deficiency+TAK242 group were significantly decreased（P<0.01）,and the mRNA expression levels of IL-1β,IL-8,TNF-α,and Beclin1 were significantly decreased（P<0.05）.Compared with the control group,the protein expression levels of ChTLR4,MyD88,Beclin1,and the ratio of LC3-II/LC3-I in HD11 cells in the selenium deficiency group were significantly increased（P<0.01）.Compared with the selenium deficiency group,the protein expression levels of ChTLR4,MyD88,Beclin1,and the ratio of LC3-II/LC3-I in HD11 cells in selenium deficiency+TAK242 group were significantly decreased（P<0.01）.It was found that selenium deficiency could form a complete autophagy flow,and TAK242 was added to block the formation of autophagy lysosomes,and the autophagy flow was blocked.The results showed that the TLR signaling pathway was correlated with autophagy and showed a positive correlation.In summary,selenium deficiency regulates the expression of autophagy-related genes in bursa of Fabricius by activating ChTLR4/MyD88/NF-κB signal pathway,and induces autophagy in bursa of Fabricius,which leads to inflammatory injury of bursa of Fabricius in chickens. |
