Selenium Deficiency Caused Oxidative Stress-induced Necroptosis Through MiR-138-5p/SelM Axis To Regulate The Mechanism Of Hepatitis In Chickens

Selenium（Se）as an essential trace element for humans and animals,its lack of content is a serious threat to human and animal health.Selenium deficiency can lead to Keshan disease in humans,exudative qualities in chickens,nutritional myotonic dystrophy and other pathological changes.The liver is one of the important target organs for selenium,and selenium deficiency can lead to liver damage.Selenium mainly functions through selenoprotein,and currently there are 25types of selenoprotein gene sequences identified in chickens.Selenoprotein M（SelM）is an important member of the selenoprotein family.As a key thioredoxin like enzyme in the endoplasmic reticulum,SelM is closely related to hepatocyte degeneration and is presumed to be related to Ca²⁺pathway and redox regulation.Micro RNA（miRNA）is a small non coding RNA that mediates post transcriptional gene silencing of target genes by targeting the 3’untranslated region of m RNA,and mediates different biological reactions in vivo.As an important member of the miR-138 family,miR-138-5p plays an important role in liver diseases.However,at present,the role of miR-138-5p/SelM and necroptosis in chicken selenium deficient hepatitis,whether SelM deficiency causes liver inflammation,and the specific biological mechanism have not been clarified.This study proposes the scientific hypothesis that selenium deficiency triggers hepatic oxidative stress through the miR-138-5p/SelM axis causing cellular Ca²⁺overload and energy metabolism disorders inducing necroptosis leading to hepatitis.Based on this,we established an in vivo chicken liver selenium deficiency model and divided into selenium-deficient and control groups of 60 animals each,which were sampled at 15 d,25 d and 35 d,respectively.In vitro assays were performed to construct miR-138-5p knockdown and overexpression models and SelM knockdown LMH cell models,respectively,and validated using the oxidative stress inhibitor NAC and Ca²⁺antagonist BAPTA-AM.According to different treatment methods,we divided LMH cells into the following 14 groups:NC,M,and I groups;si NC,si SelM-50,si SelM-75,and si SelM-100groups;si NC,si SelM,and si SelM+I groups;si NC,si SelM,si+NAC,and si+BAPTA-AM groups.Transmission electron microscopy,H&E staining,oxidative stress detection kit,ROS staining,Fluo4-AM/ER staining,AO/EB staining,flow cytometry,Western blot and q RT-PCR were used to detect morphological changes in chicken liver tissues and miR-138-5p,SelM,oxidative stress indicators（CAT,SOD,GSH,MDA,H₂O₂,TNOS,and i NOS）,Ca²⁺pathway-related genes（CAMKIIαand SERCA1）,energy metabolism-related genes（SDHB,LDHB,HK1,HK2,PK,PFK,and PDHX）,necroptosis-related genes（RIPK1,RIPK3,and MLKL）and inflammation-related genes（NFκB,NLRP3 TNFα,COX2,IL6,IL8,IL10,IL1β,PTGES,PTGER2 and PTGER4）,and changes in ROS expression in LMH cells.To reveal the molecular mechanism of chicken liver injury caused by miR-138-5p/SelM,the research results are as follows:（1）The morphological examination results showed that after feeding a selenium deficient diet,chickens gradually developed typical symptoms of selenium deficiency and exudative qualities,characterized by the presence of pale green jelly like substances under the skin of the chest and abdomen,which represented the successful establishment of a chicken selenium deficiency model.The results of H&E staining revealed that the hepatocytes in the control group were well-arranged and morphologically normal.In the selenium deficiency group,liver cells were disorderly arranged and accompanied by a large number of inflammatory cell infiltration.Ultrastructural observation of liver tissues by transmission electron microscopy showed that liver cells in the control group were neatly arranged,with intact cell membrane and nuclear membrane,rich in mitochondria and normal mitochondrial morphology,and the rest of organelles were not abnormal.In the selenium deficiency group,partial nuclear fragmentation,membrane integrity destruction,organelles outflow,mitochondrial damage and breakage,accompanied by mitochondrial vacuolation,were observed.The above results indicate that selenium deficiency leads to necroptosis of liver cells and inflammatory damage to liver tissue.（2）The results of miR-138-5p knockdown and overexpression model and SelM knockdown model construction showed that the optimal transfection concentration of miR-138-5p mimic was50 n M,the optimal transfection concentration of miR-138-5p repressor was 100 n M,and the optimal transfection concentration of SelM siRNA was 100 n M.Validation with q RT-PCR and Western blot revealed that miR-138-5p upregulation leads to SelM downregulation（p<0.05）.（3）The results of oxidative stress assay showed that selenium deficiency caused a significant decrease in CAT and SOD activities and GSH content in chicken liver tissues,while MDA and H₂O₂contents as well as TNOS and i NOS activities were significantly increased（p<0.05）.Overexpression of miR-138-5p and knockdown of SelM similarly caused changes in oxidative stress indicators and caused an increase in ROS levels（p<0.05）.This suggests that miR-138-5p/SelM is involved in the oxidative stress in the liver caused by selenium deficiency.（4）The results of calcium homeostasis assay showed that the expression of Ca²⁺channel-related protein SERCA1 was significantly down-regulated and p-Ca MK2α/Ca MK2αwas significantly up-regulated after feeding selenium-deficient diets（p<0.05）.The addition of the oxidative stress inhibitor NAC significantly suppressed the downregulation of SERCA1 and upregulation of p-Ca MK2α/Ca MK2αcaused by overexpression of miR-138-5p and knockdown of SelM in in vitro assays（p<0.05）.The results suggest that miR-138-5p/SelM is involved in hepatic calcium overload induced by selenium deficiency.（5）The results of energy metabolism assay showed that selenium deficiency caused down-regulation of energy metabolism-related genes SDHB,LDHB,HK1,HK2,PK and PFK expression and up-regulation of PDHX expression in chicken liver tissue（p<0.05）.The addition of NAC and Ca²⁺inhibitor BAPTA-AM in in vitro assays significantly suppressed the down-regulation of SDHB,LDHB,HK1,HK2,PK and PFK expression and up-regulation of PDHX expression caused by overexpression of miR-138-5p and knockdown of SelM（p<0.05）.The results suggest that miR-138-5p/SelM is involved in the disturbance of liver energy metabolism induced by selenium deficiency.（6）The results of the necroptosis assay showed that selenium deficiency caused a significant increase in the expression of RIPK1,RIPK3 and MLKL,genes associated with necroptosis in chicken liver tissue（p<0.05）.The addition of NAC and BAPTA-AM in in vitro assays significantly suppressed the elevated expression of RIPK1,RIPK3 and MLKL caused by overexpression of miR-138-5p and knockdown of SelM（p<0.05）.The results suggest that miR-138-5p/SelM is involved in liver necroptosis induced by selenium deficiency.（7）The results of inflammatory factor assay showed that selenium deficiency caused elevated expression of inflammatory factors NFκB,NLRP3,TNFα,COX2,IL6,IL8,IL1β,PTGES,PTGER2 and PTGER4 and decreased expression of IL10 in chicken liver tissues（p<0.05）.The addition of NAC and BAPTA-AM significantly suppressed the elevated expression of NLRP3 and TNFαcaused by overexpression of miR-138-5p and knockdown of SelM in in vitro assays（p<0.05）.The results suggest that miR-138-5p/SelM is involved in liver inflammation caused by selenium deficiency.In conclusion,selenium deficiency causes inflammatory and necroptosis in chicken liver tissue.miR-138-5p targeting SelM triggers oxidative damage in hepatocytes,resulting in intracellular Ca²⁺overload,disturbed energy metabolism,and necroptosis ultimately leading to liver inflammation.Selenium deficiency induces oxidative stress through miR-138-5p/SelM axis inducing necroptosis regulating chicken hepatitis.This study enriches the mechanism of liver disease caused by selenium deficiency and provides a theoretical basis for the study of avian selenium deficiency,as well as a reference for comparative medicine.