溶酶体组织蛋白酶参与宰后牛肉AIF介导细胞凋亡研究

Lysosomal Cathepsins in Apoptosis-inducing-factor-mediate Apoptosis of Bovine Muscle during Postmortem Aging

为研究牛肉宰后成熟过程中凋亡诱导因子(AIF)介导细胞凋亡及溶酶体组织蛋白酶参与AIF活化机制,选取6头西门塔尔杂交牛进行屠宰,取其背最长肌于4℃条件下成熟,在不同成熟时间点(0、6、12、24、72、120、168 h)进行活性氧(ROS)相对含量、溶酶体膜稳定性、组织蛋白酶活性、线粒体膜通透性、AIF表达及细胞核形态的测定。结果表明,宰后成熟过程中,牛肉ROS相对含量呈先降低后升高的趋势(P<0.05);溶酶体膜稳定性逐渐降低(P<0.05);组织蛋白酶B、D和L的活性逐渐升高(P<0.05);线粒体膜通透性增大(P<0.05);AIF在线粒体中的表达量于宰后12 h显著升高,在之后的成熟时间降低(P<0.05),AIF在细胞核中的表达呈上升的趋势(P<0.05),与线粒体中的下降趋势相一致;细胞核体积逐渐增大、部分核溶解,并伴随细胞间隙变大的现象。表明牛肉在宰后成熟过程中生成ROS,ROS的累积导致溶酶体膜稳定性失衡,使组织蛋白酶被释放,作用于线粒体,之后线粒体膜通透性发生变化,释放AIF,活化的AIF进入细胞核内诱导凋亡发生。

With the aim to investigate apoptosis-inducing-factor（ AIF）-mediate apoptosis and mechanism of lysosomal cathepsins involved in AIF activation of bovine muscle during postmortem aging,six healthy Simmental crossbreeding local yellow cattle were slaughtered and longissimus thoracic muscles were removed from the carcasses then aged at 4℃ for 6 h,12 h,24 h,72 h,120 h and 168 h. At the end of each storage period,ROS content,lysosomal membrane stability,cathepsins activities,mitochondrial membrane permeability,the expression of AIF and nuclear morphology were measured. The results suggested that ROS content was decreased from 0 h to 6 h,but it was significantly increased from 6 h to168 h during postmortem aging（ P 〈0. 05）. Lysosomal membrane stability was significantly decreased from 0 h to 168 h（ P〈 0. 05）. Cathepsin B,D and L activities were increased as the postmortem aging was increased（ P 〈0. 05）. Mitochondrial membrane permeability was significantly increased during postmortem aging（ P〈 0. 05）. The AIF level in mitochondria was significantly increased from 0 h to 12 h and decreased as the postmortem aging was increased,the amount of AIF in the nucleus was increased during postmortem aging（ P 〈0. 05）. The volume of cell nucleus was gradually increased and some nucleus were dissolved and broken during bovine postmortem aging. It was concluded that the generation of ROS may lead to imbalance of lysosomal membrane stability,and then cathepsins were released to cytoplasm. Subsequently, the mitochondrial outer membrane was permeabilized, and AIF was translocated to the cytosol and nucleus,where it induced apoptosis of nucleus.