Mechanism Of Selenium Deficiency Regulating Pneumonia Induced By Necroptosis Of Chicken Lung Epithelial Cells Through ROS/TLR4/NF-κB Signaling Pathway

Selenium（Se）plays an essential role in monitoring animal and human health.Selenium deficiency in vivo will make dysfunction and damage of many organs and tissue,such as inflammation,apoptosis,necroptosis and autophagy.Lung is one of the target organs of selenium deficiency.Many studies showed that inflammatory damage will occur in the lungs of animals when selenium deficiency occurs,leading to pneumonia.However,the specific mechanism of these lung injuries caused by selenium deficiency is still insufficient.As one of the main receptors of the innate immunity,TLR4 plays a significant role in the regulation of pneumonia.At the same time,due to the membrane rupture characteristics of necroptosis,the substances liberated are closely related to the growth of pneumonia.In order to explore the relationship between the pathogenesis of pneumonia,TLR4 and necroptosis in broilers with selenium deficiency,this experiment was carried out by establishing chicken selenium deficiency pneumonia model and chicken embryo primary alveolar epithelial cell type II selenium deficiency model.The morphological varieties of chicken lung tissue were witnessed by H&E staining,and the necroptosis of alveolar type II epithelial cells was observed by flow cytometry and AO/EB fluorescence staining.Oxidative stress related indicators were detected using an oxidative stress kit and ROS staining.The expressions of TLR4/NF-κB signaling pathway,necroptosis,heat shock protein,inflammation genes,selenoprotein and energy metabolism genes were discovered by q RT-PCR and Western Blot.In vitro,two concentrations of TLR4 inhibitor（TAK242）were used to observe the changes of alveolar type II epithelial cell injury.This experiment explored the mechanism of lung injury in selenium-deficient broilers from molecular biology,and the results were as follows:（1）The results of H&E staining exhibited that the alveolar structure in NC group was obvious,alveolar cells had clear boundaries,the staining was regular,and there was no apparent congestion in the interstitial lung.In-Se group,lung tissue was congested,alveoli and interstitial spaces were obviously bleeding and filled with a large number of blood cells,inflammatory cells were obviously infiltrated,cells were necroptotic,nuclei were condensed or dissolved,and alveolar cells were unclear.These results indicated that selenium deficiency could cause inflammatory damage to chicken lung tissue.（2）The expression levels of 20 selenoproteins DIOl,DIO2,DIO3,TXRNDl,TXRND2,TXRND3,GPXl,GPX2,GPX3,GPX7,SELH,SELO,SELM,SELK,SELS,SEP15,SPS2,SEPP1,SEPXL and SELU in chicken lung tissue and alveolar type II epithelial cells were detected.The expression of 17 kinds of selenoproteins in lung tissue of selenium-deficient chickens and 18 kinds of selenoproteins in alveolar type II epithelial cells of selenium-deficient chickens were significantly down-regulated（P<0.05）,which indicated that selenium deficiency significantly affected the expression of selenoproteins in lung tissue and alveolar type II epithelial cells of chickens.（3）The results of oxidative stress tests on chicken lung tissue and alveolar type II epithelial cells displayed that contrasted with NC group,the activities of T-AOC,CAT,SOD and GSH-Px in-Se group decreased,whereas the contents of MDA and H₂O₂increased.At the same time,the results of ROS staining of alveolar type II epithelial cells exhibited that the amount of green fluorescent spots in the-Se group raised compared with that in the NC group,and the fluorescence intensity increased significantly,indicating that selenium deficiency would cause oxidative stress in chicken lung tissue.（4）Expressions of genes related to TLR4/NF-κB pathway in chicken lung tissue and alveolar type II epithelial cells exhibited that contrasted with NC group,the-Se group of m RNA and protein expressions in TLR4,My D88,TRAF6 and NF-κB were significantly additional（P<0.05）.However,contrasted with the-Se group,the expression of these above genes in the-Se+10 n M TAK242 group was obviously reversed,and when the inhibitor concentration additted to 100 n M,the above tested genes were further reduced（P<0.05）.These results represent that selenium deficiency will be able to trigger the expression of My D88/TRAF6/NF-κB pathway through TLR4.（5）Expressions of genes related to necroptosis in chicken lung tissue and alveolar type II epithelial cells exhibited that contrasted with NC group,the-Se group of m RNA and protein expressions in RIPK1,RIPK3 and MLKL were significantly increased.Flow cytometry and AO/EB staining were used to evaluate the occurrence of necroptosis of alveolar type II epithelial cells.Compared with NC group,the cell necroptosis rate in-Se group increased,but after adding TLR4 inhibitor,the cell necroptosis rate decreased,and the inhibitory impact increased with the addition of inhibitor concentration.These results represented that selenium deficiency caused necroptosis of chicken lung tissue through TLR4 signaling pathway.（6）The expressions of heat shock proteins and energy metabolism related genes in chicken lung tissue and alveolar type II epithelial cells demonstrated that the m RNA and protein expression levels of HSP60,HSP70,and HSP90 in-Se group were significantly increased contrasted with those in the NC group.After adding TAK242,the expression of the above indexes decreased significantly（P<0.05）.Compared with NC group,the m RNA expressions of ACO2,HK1,HK2,PFK,PK,SDHB and LDHA in-Se group were dramatically decreased.However,after TAK242was added,the expression of above indexes increased（P<0.05）.These results indicated that selenium deficiency leads to the increase of heat shock protein expression and energy metabolism disorder through TLR4 signaling pathway.（7）The expressions of inflammation interrelated genes in chicken lung tissue and alveolar type II epithelial cells exhibited that compared with NC group,the expression of IFN-γ,Pt GEs,COX-2,TNF-α,IL-1βand IL-6 in-Se group increased dramatically（P<0.05）.The addition of TAK242 in the cell test dramatically decreased the expression of the above genes（P<0.05）.The results showed that selenium deficiency induced inflammatory damage in chicken lung tissue.In summary,selenium deficiency can cause oxidative stress in chicken lung tissue and activate TLR4/NF-κB pathway,heat shock protein activation,and energy metabolism disorders can induce necroptosis and inflammation.We can conclude that selenium deficiency is mediated by ROS/TLR4/NF-κB signal pathway regulates necroptosis of chicken lung epithelial cells to induce pneumonia.This study is the first to elaborate the molecular mechanism of selenium deficiency causing chicken pneumonia,providing a new direction for drug research and development of pneumonia.