Epidemiological Investigation Of Pig Selenium Deficiency Disease In Longjiang County And Effect On Necroptosis Of Pig Brain Tissue

Selenium（Se）,one of the indispensable trace elements in the life activities of vertebrates,plays an important role in the pathophysiological pro cess of various diseases,such as neurodegenerative diseases,cancer,diabetes and cardiovascular diseases.Se deficiency diseases,as one of the common nutritional metabolic diseases that harm the pig industry,have attracted much attention in recent years.Se deficiency is one of the crucial factors related to nervous s ystem disease and necroptosis.MicroRNA（miRNA）is a short non-coding RNA composed of about 21-23nucleotides,which is widely found in animals,plants and microorganisms.It is a key facto r in the regulation of gene expression and plays an important role in the regulation of necroptosis.In order to clarify the prevalence of selenium deficiency disease in pigs in longjiang county and elucidate the mechanism of necroptosis of pig brain tissue,a preliminary investigation was conducted on the incidence of Se deficiency disease in pigs in 2019 in longjiang county.On the basis of replication of the model of Se deficient pigs（in vivo）and the model of Se deficiency cells（in vitro）,relevant indicators were detected by electron microscopy,qRT-PCR and Western blot（WB）.The results showed that:（1）The results of epidemiological investigation showed that the results of epidemiological investigation showed that in 2019,the percentage of cases wi th Se deficiency disease in pigs in longjiang county increased sharply（21.7%）from September.The percentage peaked at 27.4%in December and began to decline in January,dropping to 9.3%.In addition,the incidence of fattening pigs and weaned pigs is generally higher than that of sows.（2）We detected 24 selenoproteins（GPX1,GPX2,GPX3,GPX4,DIO1,DIO2,DIO3,TXNRD1,TXNRD2,TXNRD3,SELM,SELN,SELT,SELW,SELK,SELV,SEPX1,SEP15,SELH,SELI,SEPP1,SEPHS2,SELO,SELS）,and the mRNA levels in the cont rol and Se-deficient pig brain tissues.The gene expressions of 10 selenoproteins was downregulated under Se-deficient condition.Compared with the control brain tissue,the mRNA expressions of GPX1,SELM,SELN,SEPP1,SELV,SELT,SELW,SEPHS2,SEP15 and TXNRD2 in the Se-deficient group was reduced to 35%,60%,45%,55%,35%,65%,35%,30%,40%and 20%,respectively（p<0.05）.However,the gene expression of DIO1,DIO2,SELO,SELS,SELH and SELI were upregulated by approximately 0.2 to 3-fold under Se-deficient conditions compared with the levels in the control group（p<0.05）.In addition,the gene expression levels of 8 selenoproteins（GPX2,GPX3,GPX4,DIO3,SELK,SEPX1,TXNRD1 and TXNRD3）were not affected significantly by Se deficiency（p>0.05）.（3）The antioxidant kit detected that the activities of CAT,SOD,GPX,TXNRD and the contents of H₂O₂,OH^-and MDA were decreased in the brain tissues of pigs deficient in selenium,indicating that selenium deficiency would cause oxidative stress in the br ain tissues of pigs.（4）The expression of mir-29a-3p was down-regulated and the expression of TNFRSF1A（TNFR1）was increased in selenium-deficient pig brain tissues.Using bioinformatics prediction software Targetscane and Mi RDB detection results,it was found that mir-29a-3p may target TNFR1.Based on the establishment of in vitro cultured pig intestinal epithelial cell model,the mimic/inhibitor model of mir-29a-3p was successfully constructed,and the targeting relationship between mir-29a-3p and TNFR1 was verified by qRT-PCR and WB.（5）The changes of typical necroptosis in pig brain cells were observed by electron microscopy.The expression of the necroptosis markers（RIPK1,RIPK3,MLKL）was significantly increased and the expression of caspase 8 was significant ly decreased in the brain tissues of Se deficient pigs detected by qRT-PCR and WB.In vitro experiments showed that the expression of mir-29a-3p decreased with the decrease of Se content in the medium.mir-29a-3p inhibitor increased the number of necrotic cells and the accumulation of ROS,which were inhibited by Nec-1 and NAC,respectively.The above results indicated that low selenium caused necroptosis of pig brain cells through the targeted regulation of TNFR1 by mir-29a-3p.