Selenium Deficiency Induces Mitochondrial Redox Oxidative Stress Through MiR-365-3p/SelT Axis,Causing Myoblasts Proliferation Disorders In Broilers

MicroRNA（miRNA）plays an important biological role in maintaining skeletal muscle homeostasis and other aspects.Mi RNA affects the expression of many genes and participates in the regulation of organ development and function by inhibiting the post transcriptional translation of target m RNA.Selenium（Se）is an essential trace element for humans and animals,which mainly functions in the form of selenoprotein.Adequate Se can ensure the healthy development of animal skeletal muscles.Selenoprotein T（SelT）is an important member of the selenoprotein family,which has a strong redox regulation function.miR-365-3p has the function of targeted regulation of SelT.During the formation of muscle fibers,myoblasts first enter the cell cycle for rapid proliferation.However,there is still a lack of research on the role and regulatory mechanism of SelT in maintaining the redox balance of myoblasts and promoting cell proliferation.Based on this,here,we established Se deficient broiler models,SelT knockdown/overexpression and miR-365-3p knockdown/overexpression myoblasts and chicken embryo models,as well as H₂O₂ or mitochondrial oxidative stress inhibitor Mito-TEMPO treated myoblast models.We applied H&E staining,immunohistochemistry staining,immunofluorescence staining,TUNEL staining,flow cytometry,Ed U staining,real-time quantitative PCR,protein immunoblotting,and kit testing methods,By detecting the levels of oxidative stress,mitochondrial function,mitochondrial biogenic ability,ATP content,cell cycle,apoptosis,proliferation potential,MHC content,etc.in myoblasts,the aim is to elucidate the mechanism of Se deficiency inducing mitochondrial oxidative stress through the miR-365-3p/SelT axis,inhibiting mitochondrial biogenesis,and ultimately leading to dysproliferation of myoblasts.The results indicate that:（1）Se deficiency significantly upregulated the expression of miR-365-3p in broiler skeletal muscle tissue and downregulated the level of SelT（p<0.05）.Se deficiency led to fracture of skeletal muscle fibers in chickens,disordered and loose fiber arrangement,abundant adipocytes in the stroma,and inflammatory cell infiltration and fiber necrosis.The cross-sectional area of muscle fibers decreases,skeletal muscle atrophy,and a significant decrease in overall body weight of broilers（p<0.05）.The skeletal muscle changes in miR-365-3p overexpression or SelT knockdown chicken embryos were similar to those in vivo.On the contrary,knocking down miR-365-3p or overexpressing SelT significantly increased the cross-sectional area of skeletal muscle fibers in chicken embryos,and the muscle fibers were plump and filled（p<0.05）.The above results indicate that Se deficiency can induce damage to chicken skeletal muscles through the miR-365-3p/SelT axis.（2）The detection results of oxidative stress,DNA oxidative damage,and apoptosis levels showed that Se deficiency significantly increased the content of H₂O₂ and malondialdehyde（MDA）and activity of T-NOS in muscle tissue and decreased the activity of antioxidant enzymes CAT,Gpx,GSH,SOD and T-AOC,increased the levels ofγH2AX and 8-OH DG and the number of TUNEL positive cells（p<0.05）.Results consistent with in vivo experimental trends were obtained in chicken embryo tissues and myoblasts overexpressing miR-365-3p or knocking down SelT.Targeted inhibition of Mito SOX using Mito-TEMPO could effectively alleviate the changes in the aforementioned indicators caused by the decrease in SelT levels in myoblasts.In addition,overexpression of SelT effectively inhibited oxidative stress induced by H₂O₂ treatment（p<0.05）.However,when miR-365-3p mimic was co transfected with the SelT plasmid,the protective effect of SelT on cells disappeared.The above results indicate that Se deficiency inhibits SelT through miR-365-3p,induces mitochondrial oxidative stress,DNA damage,and promotes cell apoptosis.（3）The detection results of mitochondrial dynamics genes showed that Se deficiency caused the expression of fusion and division genes MFN1,MFN2,OPA1,DRP1,CLPP,PGC-1a,TRAM,and PPAR-γin skeletal muscle mitochondria were downregulated.Overexpression of miR-365-3p or knockdown of SelT resulted in similar changes in the indicators in myoblasts.Targeted inhibition of Mito SOX using Mito-TEMPO could significantly alleviate mitochondrial homeostasis disorders caused by decreased SelT levels（p<0.05）.H₂O₂ treatment did not cause significant changes in the expression of kinetic genes,but when miR-365-3p mimic was co transfected with the SelT plasmid,H₂O₂ treatment significantly downregulated the expression of the genes（p<0.05）.The above results indicate that Se deficiency inhibits SelT through miR-365-3p and induces mitochondrial homeostasis disorders in myoblasts.（4）The results of mitochondrial oxidative phosphorylation function test showed that Se deficiency significantly reduced the levels of oxidative phosphorylation（OXPHOS）enzymes NDFB8,SDHB,VQCRC2,MTCO1 and ATP5A1,and ATP production in skeletal muscle tissues（p<0.05）.Overexpression of miR-365-3p or knockdown of SelT also caused the loss of mitochondrial JC-1 membrane potential of myoblasts,blocked the process of oxidative phosphorylation of cells,and finally led to a significant reduction of ATP synthesis in chicken embryo muscle tissue and myoblasts（p<0.05）.Mito-TEMPO treatment could effectively alleviate the oxidative phosphorylation dysfunction caused by the reduction of SelT level.After H₂O₂treatment,compared with myoblast cells transfected with SelT plasmid alone,co transfection of miR-365-3p mimic with SelT plasmid significantly downregulated the expression of the kinetic genes（p<0.05）.The above results indicate that Se deficiency inhibits SelT through miR-365-3p,resulting in dysfunction of oxidative phosphorylation in myoblasts.（5）By detecting MHC levels in tissues and cells,as well as indicators related to myoblast cell cycle and proliferation,the damage and proliferation ability of myoblasts were evaluated.The results showed that Se deficiency significantly downregulated the level of MHC in skeletal muscle tissue.Overexpression of miR-365-3p or knockdown of SelT also caused significant downregulation of ki67 and MHC levels in chicken embryo muscle tissue and myoblasts.The percentage of myoblasts in G0/G1 and G2/M phases increased,the percentage of myoblasts in S phase decreased,and the number of Ed U positive cells decreased,accompanied by activation of p53/p21 signaling pathway and significant reduction of Cyclin A,Cyclin B,Cyclin D,and Cyclin E levels（p<0.05）.Targeted inhibition of Mito SOX using Mito-TEMPO could significantly alleviate the myoblast cell cycle arrest caused by decreased SelT levels（p<0.05）.Similarly,H₂O₂ treatment of miR-365-3p mimic and SelT plasmids co transfected cells yielded similar results,while H₂O₂treatment of SelT overexpressing cells did not cause significant changes in these indicators.The above results indicate that Se deficiency inhibits SelT,induces myoblast cell cycle arrest,and inhibits its proliferation through miR-365-3p.In summary,Se deficiency inhibits SelT through miR-365-3p targeting,disrupts mitochondrial redox homeostasis,hinders ATP generation,induces myoblast cell cycle arrest,promotes cell apoptosis,and ultimately leads to damage to broiler skeletal muscles.Selenium deficiency induces mitochondrial redox oxidative stress through miR-365-3p/SelT axis,causing myoblasts proliferation disorders.This study provides a new direction for further exploring the function of SelT and also provides reference for comparative medicine.