生长猪骨骼肌热应激损伤中硒蛋白表达调控及其抗应激机制分析

The heat stress (HS) induced the changes of energy metabolism of skeletal muscle from growth to maintenance, which is the main reason that HS jeopardizes growth performance and carcass traits of growing pigs. Previous studies indicate that supplementation of dietary Se can help alleviate the negative consequences of HS. Preliminary research of our laboratory revealed HS up-regulated several selenoproteins in C2C12 cells, which indicated those selenoproteins played important roles in cells under heat stress. However, whether selenoproteins act as the target genes for heat stress prevention, and whether they play roles in alleviating skeletal muscle heat stress injury by impacting on cell energy metabolism through AMPK/mTOR signaling pathways are still unclear. In this project growing DLY (Duroc×Landrace×Yorkshire) pigs and pig skeletal muscle satellite cells will be taken as research materials to develop animal and cell heat stress models and following experiments will be conducted: 1) Investigate the effects of different levels of dietary selenium supplementation on expression of selenoproteins, AMPK/mTOR signal, energy metabolism related genes or proteins and heat stress response related indicators in skeletal muscle of growing pigs under heat stress conditions; 2) Compare gene or protein expressions of selenoproteins and some related indicators in pig skeletal muscle satellite cells (in vitro）under the heat stress condition; 3) Screen and analyze the target selenoprotein genes associated with the heat stress response in in vivo and in vitro models; 4) Further investigate the effects of key selenoprotein gene silence or overexpression on skeletal cell energy metabolism and heat stress response by adopting gene knockdown and overexpression technology; 5) Based on above experiments the protective roles of key selenoproteins in skeletal muscle of growing pigs during heat stress injury will be evaluated and analyzed. The results of the project may provide target genes and theory evidence for adequate nutrition application for growing pigs and alleviate the negative consequences of heat stress in hot summer day.

热应激影响肌肉组织能量代谢是导致生长猪生长性能下降、肉品品质变差的主要原因。日粮中添加硒可缓解热应激对畜禽健康和生产性能的影响；申请人前期研究发现，热应激显著上调小鼠成肌细胞部分硒蛋白表达，预示这些硒蛋白在细胞热应激调控中起重要作用。但硒蛋白是否是热应激调控靶标基因，是否通过AMPK/mTOR信号通路影响细胞能量代谢而调控肌肉组织热应激效应尚不清楚。本项目拟构建DLY生长猪和猪骨骼肌卫星细胞热应激模型；考察日粮硒水平对热应激猪肌肉组织硒蛋白、AMPK/mTOR信号、能量代谢相关基因蛋白表达及相关指标的影响；在细胞水平考察热应激对硒蛋白和相关考察指标的影响；分析筛选重要硒蛋白；进一步采用基因干扰和超表达技术探讨重要硒蛋白表达调控对骨骼肌细胞能量代谢及热应激效应的影响；综合分析重要硒蛋白在骨骼肌热应激调控中可能的分子机制。研究结果可望为生长猪热应激的预防和有效营养调控提供分子靶标和理论基础。

本研究旨在考察日粮硒水平对热应激（HS）生长猪骨骼肌和肝脏组织硒蛋白、能量代谢相关基因蛋白表达及相关指标的影响。通过人工构建生长猪热应激模型，诱导了生长猪生长性能显著下降，直肠温度和呼吸频率显著升高，并造成了生长猪胴体组成，肉品质和内分泌的负面影响。并考察了不同剂量的硒代蛋氨酸羟基类似物（HMSeBA）添加（0.2, 0.4和0.6mg Se/kg）对生长猪生产性能，胴体组织，肉品质，内分泌和骨骼肌和肝脏组织代谢和硒蛋白相关基因和蛋白的影响。结果表明：1）HS对生长猪生长性能、胴体性状和肉质均有显著影响，并伴随骨骼肌中4个代谢相关基因和7个硒蛋白基因异常表达。2）日粮添加HMSeBA部分缓解了HS对胴体性状的负面影响，改善了肉质。这些改善伴随血清和肌肉的抗氧化能力以及GPX1、GPX3、GPX4和SELENOP蛋白表达的增加，0.4和0.6 mg Se/kg HMSeBA可恢复所有异常表达的代谢相关基因和硒蛋白基因。3）HS显著降低肝脏重量、糖原含量。4）HS诱导生长猪肝脏代谢紊乱，导致6个代谢相关基因和11个硒蛋白基因的异常表达。HMSeBA添加可提高肝脏抗氧化能力。0.4或0.6 mg/kg HMSeBA可恢复肝脏重量、糖原含量。补充HMSeBA恢复了生长猪肝脏中 15个硒蛋白基因的表达。并上调了肝脏中p-AMPK的蛋白水平。进一步在体外构建了C2C12肌管热应激模型，并考察了亚硒酸钠（SS）和硒代蛋氨酸（SeMet）对HS的缓解效应。结果表明：4）HS可下调C2C12肌管MYOGENIN和MYOD基因和蛋白水平。5）HS导致肌管细胞15个和17个硒蛋白基因的表达分别在4天和8天发生显著变化。补充SS和SeMet可不同程度地下调HSP70的基因和蛋白表达，并在第4、8天部分恢复MYOGENIN和MYOD的基因和蛋白表达。其中，SS和SeMet分别改变了HS肌管细胞中16个和10个硒蛋白基因的表达。上述研究结果表明，热应激显著影响生长猪的生长性能、胴体性状和肉质，并诱导肝脏代谢紊乱和C2C12肌管细胞损伤，添加HMSeBA形式的硒可以部分缓解热应激引起的负面影响，主要是通过恢复骨骼肌，肝脏和C2C12肌管细胞硒蛋白基因表达模式和代谢相关基因和蛋白的表达实现。