Study On The Mechanism Of Selenoprotein S Regulating The Occurrence Of Chicken Selenium Deficient Arteritis By Mediating Neutrophil Extracellular Traps Formation

Selenium(Se)is an important essential micronutrient that plays an important role in various physiological processes such as organismal development and immune response.Se ingestion by the body plays a role in the body mainly in the form of selenoproteins,among which selenoprotein S(Sel S)is widely distributed in arterial blood vessels and has various functions such as antioxidant and anti-inflammatory.Se deficiency in poultry causes significant pathological damage to vascular tissues,including increased permeability,inflammatory infiltration of vascular endothelial cells,and necrosis of endothelial cells.Neutrophil external traps(NETs)are a newly identified mechanism of neutrophil activation that activates vascular endothelial cells,thus becoming a key link in vascular inflammatory injury.Previous studies have found that Se deficiency can lead to vascular injury and decreased neutrophil function in chickens,accompanied by oxidative stress and decreased selenoprotein expression.However,whether Se deficiency affects the formation of nets through selenoprotein and the mechanism of NETs in the occurrence of Se deficient arteritis are not clear.Therefore,180 1-day-old Ross 308 male broilers were randomly divided into two groups with 90 in each group.The dietary Se level of the control group(C group)was 0.2 mg/kg and that of the Se deficient group(-Se group)was0.03 mg/kg.After feeding for 42 days,the Se deficient chicken model was obtained,and then 45 broilers were randomly selected from the C group and the-Se group.Lipopolysaccharide(LPS)was injected intraperitoneally at the dose of 200 mg/kg·bw,which was called C+LPS group and-Se+LPS group.The peripheral blood and aortic tissues of broilers in each group were collected after LPS treatment for 2 h.Histopathological changes,expression of chemokines,expression of inflammatory factors,expression and colocalization of genes involved in the formation of NETs,expression of oxidative stress-related indicators,expression of 25 selenoproteins in the aortic tissue of chickens were evaluated by Hematoxylin&Eosin(H&E)staining,proteomic detection techniques,real-time PCR(qRT-PCR),Western Blot(WB),immunofluorescence(IF),bioinformatics,to determine the effect of Se deficiency induced-aortic injury in chickens.The peripheral blood neutrophils of broilers were isolated.The effects of Se deficiency on lactate dehydrogenase(LDH)release,reactive oxygen species(ROS)production and NETs formation in neutrophils were detected by scanning electron microscope(SEM),Sytox Green staining and DCFH-DA staining.The coculture system of neutrophils and PAECs was established,and the formation of NETs induced by si Sel S-PAECs,si Sel S-PAECs and inflammatory response induced by NET-DNA were detected respectively,in order to clarify the correlation between NETs and chicken Se deficient arteritis,the mechanism of Sel S regulating the formation of NETs,and the mechanism of NETs in the occurrence of chicken Se deficient arteritis.The results are as follows:(1)The plasma Se content in the-Se group was decreased significantly.Histopathological observation showed that there was infiltration of lymphocytes,plasma cells and monocytes,proliferation of collagen fibers in intima and adventitia,partial rupture of elastic fibers in media and slight uplift of intima in the broilers aorta of the-Se group.In the broilers aorta of the C+LPS group,the destruction and interruption of aortic elastic plate,alternating scar tissue,uneven intimal surface,a large number of plaque uplift,a large number of collagen fiber hyperplasia in adventitia,vascular wall thickening and inflammatory cell infiltration can be seen.In the broilers aorta of the-Se+LPS group,there were degeneration and necrosis of smooth muscle cells,destruction of elastic fibers,inflammatory cell infiltration,rupture of internal and external elastic membrane,granulomatous inflammatory reaction,including infiltration of multinucleated giant cells,epithelioid cells,lymphocytes and monocytes.Using qRT-PCR,WB,and IF,we found that the expression levels of inflammatory cytokines tumor necrosis factor-α(TNF-α),interferon-α(IFN-α),interleukin-1β(IL-1β),IL-6,IL-8,IL-17 and granulocyte macrophage colony-stimulating factor(GM-CSF)were significantly increased in the broilers aorta of the-Se group,which indicated that Se deficiency could cause inflammatory injury in broilers aorta.(2)Proteomic analysis showed that 179 proteins were upregulated and 173 proteins were downregulated in the aorta of Se deficient chickens.GO and KEGG enrichment analysis of differentially expressed proteins(DEPs)showed that DEPs were mainly involved in redox pathway,energy metabolism pathway,myocardial contraction pathway and peroxisome proliferator activated receptor(PPAR)pathway.The DEPs related to inflammatory response,oxidative stress and PPAR pathway were screened and verified by qRT-PCR,and the accuracy of sequencing was confirmed.(3)Using qRT-PCR,WB,and IF analyses,we found that the expression levels of neutrophil chemokine C-C motif chemokine 1(CCL1),CCl4,CCL17,chemokine ligand 12(CXCL12),CXCL13,and CXCL14),NETs formation related proteins myeloperoxidase(MPO),neutrophil elastase(NE),NADPH oxidase 2(NOX2),protein kinase Cα(PKCα),PKCβ,PKCζ and phosphoinositide specific phospholipase Cγ(PLCγ)were increased in the broilers aorta of the-Se group,and these indicators were more significantly increased in the-Se+LPS group.Using SEM and Sytox Green staining,we found that NETs formation was increased in broilers peripheral blood neutrophils of the-Se group,which indicated that Se deficiency could recruit neutrophils around inflamed aorta and promote the release of NETs from neutrophils in broilers aorta and peripheral blood.(4)The m RNA expression levels of 25 selenoproteins in broilers aorta were detected by qRT-PCR.It was found that compared with the C group,the expression of selenoproteins in the-Se group and the-Se+LPS group were decreased,among which the m RNA expression level of Sel S decreased most significantly.The other selenoproteins significantly affected by Se deficiency were iodothyronine deiodinase 1(DIO1),DIO2,DIO3,Sel K,glutathione peroxidase 4(GPX4)Sel M,Sel P2,Sel I and GPX1,indicate that Se deficiency reduces the expression of selenoprotein in broilers aorta,and suggest that Sel S may play an important role in Se deficient arteritis injury.After silencing Sel S in chicken arterial endothelial cells(PAECs)by cell transfection in vitro,the expression of TNF-α and IL-1β was significantly increased by IF detection,indicating that Sel S plays an important regulatory role in arteritis.Further,si Sel S-PAECs were cocultured with broilers peripheral blood neutrophils.It was found that the formation of NETs increased significantly,indicating that the decrease of Sel S expression can cause the release of inflammatory factors from arterial endothelium and promote the formation of NETs.(5)qRT-PCR was used to detect the m RNA expression levels of antioxidant genes catalase(CAT),heme oxygenase 1(HO-1),superoxide dismutase 1(SOD1)and SOD2 in broilers aorta.It was found that the m RNA expression levels of the above antioxidant genes were significantly lower in the-Se group than in the C group;The levels of ROS in broilers peripheral blood neutrophils in the-Se group and the C+LPS group were significantly increased by fluorescence staining,indicating that Se deficiency can cause oxidative stress in broilers aorta and peripheral blood neutrophils.After stimulating the broilers peripheral blood neutrophils to produce NETs with phorbol-12-myristic acid-13-acetate(PMA)in vitro,it was cocultured with PAECs.It was found that the expression of TNF-α and IL-1β in PAECs was increased significantly,indicating that NET-DNA can induce inflammatory response in PAECs.ROS bursts were detected in si Sel S-PAECs,si Sel S-PAECs induced-NETs formation and NET-DNA induced-PAECs inflammatory response.However,in si Sel S-PAECs pretreated with ROS inhibitor N-acetyl-L-cysteine(NAC),NETs almost did not form compared with NC-PAECs pretreated with NAC.Combined with the results of in vivo experiments,it shows that the reduced expression of Sel S can induce the formation of ROS dependent NETs and cause the inflammatory response of PAECs.(6)KEGG enrichment analysis of proteomics showed that PPAR pathway was significantly enriched in Se deficient broilers aorta.qRT-PCR was used to detect the activation of PPAR pathway in broilers aorta.It was found that Se deficiency significantly decreased the expression level of PPARα,PPARβ and PPARγ,indicating that Se deficiency can inhibit PPAR pathway in broilers aorta.Further,in the tests of si Sel S-PAECs,si Sel S-PAECs induced-NETs formation,and NET-DNA induced-PAECs inflammatory response,qRT-PCR and WB were used to detect the expression level of PPAR pathway related proteins.It was found that compared with the corresponding C groups,the m RNA and protein expression levels of PPARα,PPARβ and PPARγ in the coculture medium of each experimental group were decreased significantly,indicating that the decreased expression of Sel S can inhibit the PPAR pathway.In addition,si Sel S-PAECs pretreated with PPAR agonist bezafibrate(BZF)lost the ability to induce the formation of NETs,and alleviated the inflammatory process of PAECs.Combined with the results of in vivo experiments,it shows that the activation of PPAR pathway can reverse the formation of NETs induced by decreased expression of Sel S,and then alleviate arteritis.In conclusion,Se deficiency leads to the decrease of Sel S expression in broilers aorta,the occurrence of oxidative stress,the increase of neutrophil chemokine release,the recruitment of neutrophils to aorta,and then induces the occurrence of Se deficient arteritis by promoting the increase of NETs release.This study provides the important theoretical basis for the study of the mechanism of Se deficiency induced vasculitis in chickens,and further enriches the experimental data of Se deficiency induced vasculitis in chickens.