1、食品化学 食品科学 2023,Vol.44,No.04 9活性氧激活缺氧诱导因子-1加速宰后成熟 初期牛肉能量代谢郭雨轩,陈 骋*,师希雄,郭兆斌,马国源,马纪兵,余群力(甘肃农业大学食品科学与工程学院,甘肃 兰州 730070)摘 要:为探究活性氧(reactive oxygen species,ROS)在宰后成熟初期调控牛肉缺氧诱导因子-1(hypoxia-inducible factor-1,HIF-1)对能量代谢的影响。牛肉样品分别用H2O2、N-乙酰半胱氨酸(N-acetyl-L-cysteine,NAC)和生理盐水(对照)处理。在低温成熟0.5、6、12、24、48 h分别测定RO
2、S含量、脯氨酰羟化酶(proline hydroxylase,PHD)活力、磷酸肌醇-3激酶(phosphatidylinositol 3-kinase,PI3K)/蛋白激酶B(protein kinase B,AKT)和HIF-1蛋白质表达水平、能量代谢水平以及pH值的变化。结果发现:H2O2处理组初始(0.5 h)ROS含量显著高于其他2 组(P0.05),且在48 h内始终处于显著较高水平(P0.05),NAC处理能够在短期内(0.512 h)抑制ROS积累;H2O2处理组PHD活力在0.548 h内显著低于其他2 组(P0.05),同时,在0.512 h和0.56 h内PI3K和磷酸化
3、AKT表达水平显著高于其他2 组(P0.05)。上述差异使H2O2处理组HIF-1表达水平在648 h内显著高于其他2 组(P0.05)。能量代谢方面,较高的HIF-1表达水平使H2O2处理组糖原分解和R值升高速率在624 h内显著快于其他2 组(P0.05),并且pH值在第12小时提前达到极限。综上所述,牛肉中ROS通过抑制PHD活力和激活PI3K/AKT信号通路调控HIF-1稳定表达,进而提升宰后成熟初期以糖酵解为主的能量代谢水平以及pH值降低速率,可能对牛肉品质的形成产生重要影响。关键词:牛肉;活性氧;缺氧诱导因子-1;能量代谢;宰后成熟Reactive Oxygen Species A
4、ccelerates Energy Metabolism by Activating Hypoxia-Inducible Factor-1 in Bovine Muscle at the Initial Stage of Postmortem AgingGUO Yuxuan,CHEN Cheng*,SHI Xixiong,GUO Zhaobin,MA Guoyuan,MA Jibing,YU Qunli(College of Food Science and Engineering,Gansu Agricultural University,Lanzhou 730070,China)Abstr
5、act:This study aimed to investigate the effect of reactive oxygen species(ROS)on early postmortem energy metabolism in bovine muscle via regulating the expression of hypoxia-inducible factor-1(HIF-1).The muscle samples were treated with hydrogen peroxide(H2O2),N-acetyl-L-cysteine(NAC),and normal sal
6、ine(control),separately.The ROS content,proline hydroxylase(PHD)activity,phosphoinositide-3 kinase/protein kinase B(PI3K/AKT)and HIF-1 expression levels,energy metabolism,and pH were determined at 0.5,6,12,24 and 48 h postmortem.The results revealed that the H2O2-treated group showed a significantly
7、 higher ROS content than the other two groups at 0.5 h postmortem(P 0.05)and remained at a high level within 48 h,while NAC inhibited the accumulation of ROS for a short period of time(during 0.512 h)postmortem.The PHD activity in the H2O2-treated group was significantly lower than that in the other
8、 two groups during 0.548 h(P 0.05).Meanwhile,the expression levels of PI3K from 0.5 to 12 h and phosphorylated AKT(p-AKT)from 0.5 to 6 h were significantly higher than those in the other groups(P 0.05).Consequently,the expression level of HIF-1 in the H2O2-treated group was significantly higher than
9、 that of the other two groups during 648 h(P 0.05),the rate of glycogenolysis and the rate of increase in R value were significantly faster in the H2O2-treated group during 624 h(P 0.05),and the pH of the H2O2-treated group reached the limit value earlier(at 12 h).In conclusion,ROS can activate the
10、stable 收稿日期:2022-04-07基金项目:国家自然科学基金地区科学基金项目(31860426);甘肃省教育厅青年博士基金项目(2021QB-36);甘肃省自然科学基金项目(21JR7RA818)第一作者简介:郭雨轩(1997)(ORCID:0000-0002-9646-893X),男,硕士研究生,研究方向为畜产品加工。E-mail:*通信作者简介:陈骋(1988)(ORCID:0000-0001-7870-2836),男,助理研究员,博士,研究方向为畜产品加工。E-mail:10 2023,Vol.44,No.04 食品科学 食品化学expression of HIF-1 by r
11、educing the activity of PHD and up-regulating the PI3K/AKT signaling pathway and consequently accelerate energy metabolism dominated by glycolysis and the decrease in pH in bovine muscle at the initial stage of postmortem aging,which may have a crucial impact on meat quality.Keywords:bovine muscle;r
12、eactive oxygen species;hypoxia-inducible factor-1;energy metabolism;postmortem agingDOI:10.7506/spkx1002-6630-20220407-072中图分类号:TS251.52 文献标志码:A 文章编号:1002-6630(2023)04-0009-08引文格式:郭雨轩,陈骋,师希雄,等.活性氧激活缺氧诱导因子-1加速宰后成熟初期牛肉能量代谢J.食品科学,2023,44(4):9-16.DOI:10.7506/spkx1002-6630-20220407-072.http:/GUO Yuxuan,C
13、HEN Cheng,SHI Xixiong,et al.Reactive oxygen species accelerates energy metabolism by activating hypoxia-inducible factor-1 in bovine muscle at the initial stage of postmortem agingJ.Food Science,2023,44(4):9-16.(in Chinese with English abstract)DOI:10.7506/spkx1002-6630-20220407-072.http:/宰后成熟过程中以糖酵
14、解为主要途径的能量代谢反应对牛肉品质的形成起至关重要的作用1。屠宰后缺氧迫使肌肉细胞启动缺氧应答反应,通过糖酵解维持细胞能量供应,该过程引起肌肉pH值变化进而对嫩度、保水性、色泽、风味等关键品质产生重要影响2。目前,大量学者基于饲喂管理、屠宰工艺以及宰后成熟等不同角度,对调控糖酵解反应的因素进行了全面研究。例如,采用电刺激处理提升宰后成熟过程中牦牛肉能量代谢水平,加速pH值降低继而促进牦牛肉骨架蛋白质降解,有利于牦牛肉嫩度的改善3。饲料中添加直链淀粉以提升宰后能量代谢底物浓度和关键酶活性,间接调节pH值变化速率从而影响肌肉的保水性4。但是,宰后成熟过程中牛肉能量代谢受到宰前、屠宰及成熟环节众多
15、因素的影响,现有研究尚不能完全解释宰后能量代谢的调控机理。缺氧状态下,肌肉细胞能量代谢方式向糖酵解的转变依赖于蛋白质的表达。缺氧诱导因子-1(hypoxia-inducible factor-1,HIF-1)作为细胞缺氧条件下能量代谢的重要调控因子,其氧敏感的亚基HIF-1直接调控大多数糖酵解酶的表达或激活5-6。供氧正常时,HIF-1被脯氨酰羟化酶(prolineamide hydroxylase,PHD)降解,反之HIF-1则正常表达并上调糖酵解酶表达,促使细胞能量代谢转变为糖酵解7。近期研究认为,细胞缺氧应激过程中ROS通过信号传导功能对糖酵解反应的启动表现出重要调控作用。缺氧导致肌肉细
16、胞线粒体的电子传递受阻而产生大量活性氧(reactive oxygen species,ROS),部分ROS扩散至细胞质中作为信号传导分子调控多种细胞生理反应,其中就包括细胞能量代谢8。目前,ROS介导HIF-1调控细胞能量代谢主要有2 种途径:一是ROS通过细胞氧化修饰作用引起PHD活力降低,抑制PHD对HIF-1的降解作用,促进HIF-1稳定积累9;二是ROS能够激活磷酸肌醇-3激酶(phosphatidylinositol 3-kinase,PI3K)并促进蛋白激酶B(protein kinase B,AKT)磷酸化,磷酸化AKT(p-AKT)调控HIF-1稳定表达并与细胞缺氧反应元件(hypoxia response elements,HRE)结合,进而启动糖酵解反应10。但是,宰后成熟过程中牛肉细胞糖酵解反应的调控是否依赖于上述机制还有待研究。因此,本研究选取牛背最长肌,采用ROS激活剂H2O2、清除剂N-乙酰半胱氨酸(N-acetyl-L-cysteine,NAC)和生理盐水处理后,在温度为4、相对湿度为85%条件下成熟,依次在第0.5、6、12、24、48小时测定ROS细