宰后成熟初期线粒体活性氧（ROS）调控牛肉糖酵解反应机理研究

Glycolysis is a crucial biochemical pathway in postmortem muscles that directly influences meat quality. Hypoxia-driven cells trigger glycolysis to maintain postmortem energy supply. Researches have confirmed that the expression of rate-limiting enzymes activated by the adenosine monophosphate-activated protein kinase (AMPK) plays an important role in glycolysis activation. Moreover, recent studies found that reactive oxygen species (ROS) generated in mitochondria of the muscles are directly involved in the regulation of glycolysis, but the specific mechanism remains unclear. Therefore, this project intends to investigate the substrate and product concentration, glycolysis potential, rate-limiting enzymes activity, and ROS concentration during early postmortem aging. This project will also analyze the relations between glycolysis and ROS, confirm the primary generation site of ROS in mitochondria, reveal the influencing mechanism of ROS on expression levels or enzyme activities of the key signaling molecules and rate-limiting enzymes in glycolysis activation signaling pathway, and explore the oxidation mechanism of ROS on glycolysis related proteins. The mechanism will be investigated through high-performance liquid chromatographs, gel electrophoresis, proteomics, western blot, chromatographic-tandem mass spectrometry, and bioinformatics techniques. Based on above studies, this project attempts to reveal regulatory mechanism of ROS on bovine muscle glycolysis through signal transduction and protein oxidation pathways. Finally, the results will enrich the theoretical basis for the overall understanding of the regulation mechanism of glycolysis in postmortem muscles and promote the development of new technologies for prediction and improvement of meat quality.

糖酵解是肌肉成熟过程中的重要生化途径，直接影响肌肉最终品质，宰后缺氧应激调控肌肉细胞糖酵解启动以维持能量供应。目前已经证实腺苷酸活化蛋白激酶激活限速酶表达是启动糖酵解反应的重要途径，而近期研究发现肌肉线粒体产生的活性氧（ROS）也直接参与糖酵解的调控，但具体机理尚不清楚。本项目采用高效液相色谱、蛋白质电泳、蛋白质组学、免疫印迹、液相色谱串联质谱以及生物信息学等技术，分析宰后成熟初期肌肉糖酵解底物和产物浓度、糖酵解潜力、限速酶活性以及ROS浓度的变化规律；阐明糖酵解反应与ROS的关系，并确证线粒体产生ROS的主要位点；揭示ROS对糖酵解激活通路中关键信号分子和限速酶表达水平及其活性的影响机理；探究ROS对糖酵解进程中相关蛋白质的氧化作用机制。从信号传导和蛋白质氧化的角度解析ROS调控牛肉糖酵解反应的机理，为丰富宰后肌肉糖酵解反应调控机制、发展肌肉品质预测和改善新技术提供理论参考。

糖酵解是肌肉成熟过程中的重要生化途径，直接影响肌肉最终品质，宰后缺氧应激调控肌肉细胞糖酵解启动以维持能量供应。近期研究发现肌肉线粒体产生的ROS也直接参与糖酵解的调控，但具体机理尚不清楚。本项目采用激光扫描共聚焦、蛋白质电泳、免疫印迹、TMT-蛋白质组学以及生物信息学等技术全面分析了宰后成熟初期牛肉中ROS影响糖酵解反应的机理及其对肉嫩化进程的影响。主结果如下：（1）宰后成熟初期牛肉中ROS快速积累，糖酵解酶活力短期内显著升高，糖酵解底物糖原转化为乳酸，引起牛肉pH值降低。动力学分析发现ROS含量、糖酵解指标符合线性变化规律，相关性分析表明ROS含量可以正向调节糖酵解水平；（2）体内结合体外孵化研究确定线粒体复合物Ⅰ和复合物Ⅲ是宰后成熟过程中牛肉细胞ROS的主要产生位点。外源ROS处理能够调控糖酵解限速酶活力升高，显著提升肌肉糖酵解潜力；（3）ROS一方面抑制PHD酶活力，削弱其对HIF-1α的降解作用，另一方面迅速激活PI3K/AKT信号通路，加速HIF-1α的稳定积累，为激活糖酵解反应提供必要条件；（4）宰后成熟初期HIF-1α在细胞中迅速表达积累并向细胞核转移，一方面调控细胞中葡萄糖转运关键蛋白质GLUT-1表达上调，保障糖酵解底物的供应，另一方面诱导糖酵解限速酶HK和PFK酶活力升高，从而提升糖酵解潜力，促使牛肉pH值在短期内下降到pHu值；（5）ROS能够引起8种糖酵解相关蛋白质表达上调，并且以PGAM、ENO、PDHB为主要节点，共同促进宰后初期牛肉糖酵解潜力。（6）提升宰后成熟初期牛肉中的ROS含量可以明显缩短宰后僵直期，促进肌原纤维结构破坏，加速肌肉剪切力值的下降和MFI值升高，表明宰后成熟初期牛肉中的ROS可通过调控糖酵解水平提升，间接促进肉的嫩化进程。综上所述，宰后成熟初期牛肉不可避免产生ROS，后者通过激活HIF-1α信号通路和诱导糖酵解关键酶表达的途径提升牛肉糖酵解水平，从而促进了牛肉嫩度的改善。上述机制为基于能量代谢的肉品质预测和调控提供了理论依据。