Glutathione Peroxidase 3 Regulates The Mechanism Of Selenium-Deficient Pulmonary Fibrosis In Chicken Through Mediating Macrophage Extracellular Traps

Selenium（Se）is considered an important non-metallic trace element that plays a crucial role in animal health,the proper functioning of the immune system,and the improvement of reproductive ability.Se ingested by animals mainly functions by synthesizing selenoprotein,among which glutathione peroxidase 3（Gpx3）is one of the important antioxidant enzymes in the glutathione family and the only secretory enzyme in the glutathione family.Gpx3 is mainly synthesized by the kidney and acts with blood flow to target organs,and has been shown to have important roles in antioxidant,anti-inflammatory,anti-cancer and anti-obesity.Macrophage extracellular traps（METs）are a newly discovered suicidal macrophage immune mechanism that acts by releasing DNA reticulums containing MPO,NE,and cit H3,among others,and are able to damage epithelial cells and thus play a role in the fibrotic process.In addition to kidney,lung is also a tissue rich in Gpx3expression,and Se deficiency can significantly reduce Gpx3 expression in lung,however,whether Se deficiency can regulate the release of METs through selenoprotein and the mechanism of action in lung fibrosis is unclear.Therefore,in this thesis,1-day-old AA broilers were fed a normal diet with selenium content of 0.278 mg/kg and a low-selenium diet with selenium content of 0.039 mg/kg in the control and selenium-deficient groups,respectively.After 42 days of feeding,a selenium deficient broiler model was obtained.In addition,extract chicken embryos primary type II alveolar epithelial cells（AEC-II）and construct a Gpx3 knockdown AEC-II cell model or a co-culture model of AEC-II cells and chicken macrophages（HD11）in vitro.Through H&E staining,Sirius red staining,Masson trichrome staining,immunofluorescence staining,antioxidant index detection,q RT PCR,Western Blot,scanning electron microscope,Hcecht Sytox Green staining,ER-Tracker Red-Fluo-4AM endoplasmic reticulum calcium staining,DCFH-DA probe method and other methods,the pathological changes of broiler lung tissue,the expression of 25 selenoproteins,chemokines and inflammatory factors,as well as the level of oxidative stress,the degree of fibrosis and the release of METs were detected,aiming to clarify the effects of selenium deficiency on pulmonary fibrosis in broiler lungs,the relationship between METs and selenium-deficient pulmonary fibrosis,and the molecular mechanism of Gpx3’s role in it.The main findings were as follows:（1）The Se content in the lung tissues of broilers in the Se-deficient group was significantly reduced（p<0.001）.The histomorphological observations showed that compared with the control group,the lung tissues of the Se-deficient group were disorganized and had obvious solid changes,the alveolar wall,bronchial wall and vascular wall were significantly thickened,the alveoli of the reticular structure were destroyed,and a large number of inflammatory cells and red blood cells were exuded.Masson staining results showed that the alveoli of lung tissue in the Se-deficient group were severely damaged,loss of alveoli and solid changes in some tissues,thickened bronchial and vascular walls,and there were large amounts of blue collagen fibers deposited around the vessels and bronchioles and even fibrous bundles formed in some areas.Sirius red staining results also showed that the alveoli in the Se-deficient group were unevenly distributed with some alveolar walls broken,the interlobular spaces of lung were widened,and some solid areas were visible,and there were large amounts of red-stained collagen fiber bundles between the vessels,bronchioles and lung lobules.The above results indicate that selenium deficiency promotes lung tissue damage in broilers and can lead to pulmonary fibrosis.（2）The results of m RNA levels of 25 selenoproteins in broiler lungs showed that all selenoproteins in the Se-deficient group were significantly reduced,except for the m RNA levels of Dio2 and Dio3（p>0.05）,in which Gpx3 and Sel I changed most significantly（p<0.001）,suggesting that Gpx3 may play an important role in the process of Se-deficient pulmonary fibrosis.（3）The assay results of tissue oxidative stress showed that the content of oxidation products H₂O₂ and MDA increased and the activities of antioxidant enzymes CAT,T-SOD and T-AOC weakened in the lung tissue of broilers in the Se-deficient group.The results of antioxidant enzyme expression level assay revealed that the expression of antioxidant enzymes HO-1,NQO1,SOD1,SOD2,SOD3 and CAT were significantly decreased in the Se-deficient group（p<0.05）.Knockdown of Gpx3 in AEC-II cells significantly increased the level of ROS production and was significantly inhibited by NAC.The above results indicate that the decrease in Gpx3 expression caused by selenium deficiency leads to oxidative stress in the lung tissue of broilers.（4）The results of chemokine detection showed that the expression of chemokines CCL1,CCL4,CCL5,CCL17,CXCL12,CXCL13,and CXCL14 in the lung tissue of selenium deficient broilers was significantly increased（p<0.05）.After knocking down Gpx3 in AEC-II cells in vitro,the transcription levels of chemokines CCL1,CCL4,CCL5,CCL17,CXCL12,CXCL13,and CXCL14 were significantly higher than those in the si NC group（p<0.05）.The above results indicate that the decrease in Gpx3 expression caused by selenium deficiency can promote macrophage recruitment to damaged lung tissue.（5）The results of macrophage extracellular trap assay showed that the expression of MPO and cit H3,the main markers of METs,and CD68,a macrophage marker,were significantly increased in the lung tissue of the selenium-deficient group（p<0.05）,the expression of METs formation-related proteins PKCα,PKCβ,PKCε,PLCγ,MPO,NE and cit H3 was also significantly elevated（p<0.05）.Hochest-Sytox Green staining showed an increase in the formation of METs in HD11 cells cultured in selenium deficient medium.In the co-culture system of si Gpx3-AEC-II cells and HD11 cells,the release of METs in HD11 cells was significantly increased（p<0.05）,and the expression of METs forming related proteins PKCα,PKCβ,PKCε,PLCγ,MPO,NE and cit H3 was also significantly increased（p<0.05）,the level of endoplasmic reticulum calcium was significantly reduced（p<0.05）,and the production of ROS was significantly increased（p<0.05）.When the ROS inhibitor NAC was added,it not only inhibited the generation of ROS in the co-culture system,but also effectively inhibited the release of METs.The above results indicate that the decrease in Gpx3 expression caused by selenium deficiency leads to oxidative stress in lung tissue which in turn promotes the release of METs.（6）The results of inflammatory response assay showed that the expression of inflammatory factors COX-2,IL-1β,IL-6,IL-8,TNF-αand i NOS in lung tissue of broilers in Se-deficient group was significantly higher than that in control group（p<0.05）,and the expression of IFN-γwas significantly lower than that in control group（p<0.05）,and the immunofluorescence results showed that the expression of IL-1βand TNF-αin lung tissue of Se-deficient group was significantly higher（p<0.001）.After knockdown of Gpx3 in AEC-II cells in vitro,the expression of intracellular inflammatory factors COX-2,IL-1β,IL-6,IL-8,TNF-αand i NOS was significantly higher and the expression of IFN-γwas significantly lower,and the immunofluorescence results showed that the expression of IL-1βand TNF-αwas significantly higher in the cells of si Gpx3 group.After co-culture of PMA-stimulated HD11 cells with AEC-II cells,a significant increase in the expression of the above pro-inflammatory factors was also measured in AEC-II cells（p<0.05）.The above results indicate that the decrease in Gpx3 expression caused by selenium deficiency not only directly causes inflammation in the lungs,but also promotes the generation of METs,leading to inflammation in the lungs.（7）The results of tissue fibrosis detection showed that the expression levels of fibrosis-related genes MMP2,MMP9,TIMP2,COL1,COL3,FN,and Vimentin in the lung tissue of broiler chickens with selenium deficiency were significantly increased（p<0.05）.Immunofluorescence detection of epithelial marker genes E-cadherin and mesenchymal marker genes N-cadherin showed that the expression of E-cadherin was decreased and N-cadherin was increased in Se-deficient lung tissues,and the expression of TGF-β/Smad pathway-related genes TGF-β,Smad2,Smad3 and mesenchymal marker genes N-cadherin and ACTA2 were significantly increased,while epithelial marker genes E-cadherin and ZO-1 were significantly decreased（p<0.05）.After knocking down Gpx3 in AEC-II cells in vitro,the expression of TGF-β/Smad pathway-related genes and interstitial marker genes in cells significantly increased,while the expression of epithelial marker genes significantly decreased（p<0.05）.After co-culture of PMA-stimulated HD11 cells with AEC-II cells,METs significantly stimulated the expression of TGF-β/Smad pathway-related genes and mesenchymal marker genes in AEC-II cells,while significantly suppressing the expression of epithelial marker genes（p<0.05）.The above results indicate that the decrease in Gpx3 expression caused by selenium deficiency can not only promote EMT in AEC-II cells directly,but also promote EMT in AEC-II cells by promoting the release of METs,which ultimately causes lung fibrosis.In summary,selenium deficiency caused fibrosis in broiler lung tissues,while Gpx3 expression decreased,inducing oxidative stress and inflammatory reactions,promoting the release of chemokines and macrophage recruitment to the lungs,promoting the release of METs and subsequently inducing EMT.The above results suggest that selenium deficiency regulates METs through Gpx3/ROS axis to induce EMT causing chicken lung fibrosis.This study is the first to investigate the regulatory mechanism of Gpx3 on METs release and the role of METs in pulmonary fibrosis,and to provide a new idea for the prevention and treatment of pulmonary fibrosis,as well as a reference for further exploration of the biological function of Gpx3.