The Research Of The Roles Of Selenoprotein W In Selenium-deficiency Induced Chicken Muscular Injuries

Selenium(Se) is one of the essential elements for animals, however, the diet Se deficiency induces skeletal muscle damage in a variety of animals. A lot of previous studies discussed the Se-deficiency-related skeletal muscle damages from the angle of oxidative stress, apoptosis, inflammation, selenoproteins, et al, but the exact mechanism of Se-deficiency-related skeletal muscle damages was still unclear. Although the information about Se-deficiency-related skeletal muscle damages in livestocks have been reported, the complete and thorough research on its mechanism in poultry is still less. With the advent of the post genome era, multiple omics analysis with a large amount of data was widely used in disease diagnosis and research. Based on the characteristics of its high efficiency, systematicness and deep-going, omics analysis has greatly promoted the understanding and research of human and animal diseases related mechanism. So the usage of transcriptome, proteomics and other omics analysis has become more important for the study of livestock and poultry disease.Selenoprotein W(Selw) is one of highly expressed selenoprotein in chicken skeletal muscle. Selw plays important roles in the maintenance of skeletal muscle normal function, in the response to the diet Se levels, and in the regulation of redox homeostasis. However, as an important selenoprotein in skeletal muscle, the role and biological function of Selw in Se-deficiency-related skeletal muscle injury was known. Therefore: Se-deficiency-related chicken skeletal muscle damage model was established, and the antioxidative enzymes activities, apoptosis levels, expression of selenoproteins, HE staining, ultrastructure, and ion levels and distribution in skeletal muscle were detected. In addition, the transcriptome analysis, proteomics analysis and ionomic analysis were used to screen out and validate the differentially expressed genes and the related signaling pathways in Se-deficiency related skeletal muscle injury. The aim was to reveal the possible mechanism and the signal pathways that were related to Se-deficiency skeletal muscle damage. Selw-deficiency primary chicken myoblasts were established, and the antioxidative enzymes activities, apoptosis genes levels, mitochondrial membrane potential, expression of selenoproteins, ultrastructure, and Ca levels and Ca channels expression were examined in myoblasts. In addition, the Realtime PCR and Western blot were used to validate the signal pathways that were screened out by omic analysis. It was to discuss the biological function of Selw, and its role in the Se-deficiency-related muscles injuries. Moreover, Selw knockout chicken embryo models were established by using(CRISPR)/CRJSPR-associated 9(Cas9) technology. Then the antioxidative enzymes activities, expression of selenoproteins, HE staining, ultrastructure, Ca levels and Ca channels expressions were detected in chicken embryo. Moreover, the signal pathways that were screened out by multi-omic analysis were validated to reveal the biological function of Selw and the relation of Selw with Se-deficiency related skeletal muscle injuries.The results showed that:(1) In skeletal muscle, 5 different microRNAs, 725 different transcripts(mRNA and lncRNA), and 450 different proteins were detected by transcriptome and proteomics analysis(P < 0.05). By multiple omics analysis, several signaling pathways were screened out and validated including: PI3K-AKT signaling pathways, calcium signaling pathways, FoxO signaling pathways, and endoplasmic reticulum protein processing signal pathways, autophagy signaling pathways, sugar metabolism signal pathways, energy metabolism signaling pathways, the ubiquitin-proteasome signaling pathway and other signaling pathways. It indicated that a variety of signaling pathways were involved in Se-deficiency induced skeletal muscle injuries.(2) The levels and distributions of ions in skeletal muscles were detected. The results showed that the Ca levels showed a decreasing trend, and V, Cr, Mn, Cu, Cd and Hg were increased(P < 0.05), indicating that the imbalances of Ca, V, Cr, Mn, Cu, Cd and Hg were related with Se-deficiency skeletal muscle injuries.(3) In the Se deficiency chicken skeletal muscles models, Selw deficiency myoblast and e mbryo modesl, we verified the antioxidative enzymes activities, ROS levels, ultrastructure, PI3K-AKT pathways, calcium signaling pathways, FoxO signaling pathway, apoptosis pathway, autop hagy pathway, methanism pathway, endoplasmic reticulum protein processing, ubiquitin-proteasom e signaling pathway, and analyzed omics data and enrichment. The results indicated that Selw i nduced the Se-deficiency related skeletal muscle injuries by activating ROS-JNK-FoxO-Ubi-prote asome/autophagy-lysosome-apoptosis/necrosis, and by inbihiting the ROS-JNK-PI3K-AKT-mTORprotein synthesis/energy/glycometabolism signal pathway.(4) by detecting the expression of selenoproteins in Se deficiency chicken muscels, and Sel w deficiency myoblast and embryo, we found that Selw involved in Se deficiency related skelet al oxidative injuries by influenced the epression of Gpx1, Gpx2, Gpx3, Gpx4, Dio1, Dio2, Dio3, Txnrd1, Txnrd3, Selt, Selu, Seln, Sel15, Sels.(5) In the Selw deficiency primary chicken myoblast and embryo, we detected the Ca leve ls, the expression of Ca channels and the response of Ca channels to inhibitor. The results sho wed that Selw deficiency induced Ca leakage and linked Se deficiency related Ca leakage in c hicken muscles by influencing the levels and sensitivity of SERCA, CACNA1 S, TRPC1, and T RPC3. Combing with the omic analysis, enzymes activities, ultrastructure, we analyzed the corre lation between Ca signal and oxidative, and showed that Ca signal plays important role in the Ca/ROS-JNK-FoxO-Ubi-proteasome/autophagy-lysosome-apoptosis/necrosis pathway, and Ca/ROS-J NK-PI3K-AKT-mTOR-protein synthesis/energy/glycometabolism signal pathway.