Effects Of Toll-Like Receptor Signaling Pathway On Splenic Inflammatory Injury In Selenium-Deficient Chickens

Selenium is an essential trace element for animals to maintain normal physiological activities.Toll-like receptors(TLRs)is an important pattern recognition receptors(PRRs).Among them,TLR4 signal pathway is an important regulation pathway of inflammatory factors,which participates in the occurrence and regulation of many diseases.At present,there are a few researches on pathogenesis of selenium deficiency in in chicken spleen inflammatory injury.In particular,it is unclear how ChTLR participates in the splenic inflammatory damage mechanism of seleniumdeficient chickens.Therefore,in this experiment,the mechanism of spleen inflammatory injury in selenium deficient chickens was studied from the point of view of TLR and its signal transduction.First of all,a total of 200 one-day-old healthy broilers were randomly divided into the experimental group and the control group.The experimental group was fed with self-made lowselenium diet and the control group was fed with complete formula feed.The spleens were taken in 15,25,35,45 and 55 days,respectively.Using histopathological and ultrapathological methods to observe the pathological morphological change.The expression of 8 kinds of ChTLRs(ChTLR,ChTLR2,ChTLR3,ChTLR4,ChTLR5,ChTLR7,ChTLR5 and ChTLR21),My D88,TRIF,NF-κB and cytokines(IL-6,TNF-α,IL-2 and IFN-γ)mRNA in spleen were detected by quantitative realtime PCR,western blot method to detect ChTLR4 protein expression changes,and principal component analysis(PCA)to analyze the correlation of 8 kinds of ChTLRs and connective proteins My D88,TRIF.Pathological morphology results showed that the red pulp and white pulp of the spleen tissue of the experimental group were not clearly defined,the number of lymphocytes decreased,the nucleus shape of the lymphocytes was irregular,and the distribution of chromatin in the nucleus was uneven,the nuclear membrane was ruptured;the mitochondria were swollen,the crest was disordered and broken;and the endoplasmic reticulum was swollen.The results of realtime fluorescence quantitative PCR showed that 8 kinds of ChTLRs were expressed in the spleens of chicks at 15,25,35,45 and 55 days old,but the expression levels were different.Compared with the control group,the relative expression of ChTLR,ChTLR2,ChTLR3 mRNA in the experimental group increased first and then decreased,the relative expression of ChTLR5,ChTLR7,ChTLR5 mRNA decreased,and the relative expression of ChTLR21 mRNA first decreased and then increased.All ChTLRs were expressed in the spleen of chickens of different ages,and there were differences.Among them,the gene level expression and protein level of ChTLR4 were higher than those in the control group at 15-45 days,and the difference was very significant on the 15 th day(P<0.01).The relative expression of TRIF mRNA in the experimental group was higher than those of the control group at 25-55 days,and the relative expression of NF-κB and IL-6 mRNA in the experimental group was higher than that in the control group at 15-45 days.Western blot results showed that the expression of ChTLR4 protein at 15-45 d was significantly higher than that of the control group(P<0.01).The results of principal component analysis showed that ChTLR4 and TRIF had a strong correlation.After that,we studied chicken macrophage HD11.After the ChTLR4 gene of HD11 cells was silenced,the mRNA expression of ChTLR4 and its pathway molecules in HD11 cells was observed.The results showed that after ChTLR4 siRNA was transfected into HD11 cells,the expression levels of ChTLR4,IL-6 and NF-κB mRNA in the cells were significantly down-regulated compared with the experimental group(P<0.05).After TRIF siRNA was transfected into HD11 cells,the expression levels of IL-6,TRIF and TNF-α mRNA in the cells were significantly reduced compared with the experimental group(P<0.05).In summary,ChTLR4 plays an important role in regulating the expression of inflammatory cytokines in the spleen of selenium-deficient chickens.Selenium deficiency can cause inflammatory damage to the spleen of chickens through the ChTLR4 / TRIF / NF-κB signaling pathway.This study elaborated the pathogenesis of selenium-deficient chicken spleen immune injury at the molecular level,enriched the theoretical research content of chicken selenium-deficient immune injury,and provided an important scientific basis for the effective prevention and treatment of avian metabolic diseases.