短期血清饥饿胁迫对猪骨骼肌卫星细胞代谢及自噬发生的影响

doi:10.11843/j.issn.0366-6964.2024.08.014

摘要/Abstract

摘要：

旨在研究短期饥饿胁迫对猪骨骼肌卫星细胞(skeletal muscle satellite cells，SMSCs)代谢及自噬发生的影响，解析自噬机制在骨骼肌发育调控网络中的地位和作用，为改良家猪产肉性状提供理论依据。将本实验室分离并保存的SMSCs细胞系复苏，按培养体系中血清浓度将细胞分为20%血清组(对照组)、15%血清组、10%血清组、5%血清组和0%血清组，以形成不同程度的饥饿胁迫。当细胞融合度达到70%~80%后再培养24 h进行检测，每组4个重复。流式细胞术检测各组细胞凋亡率、膜电位和活性氧水平；试剂盒测定ATP水平；WB检测自噬标志蛋白LC3B-Ⅱ、p62和通路相关蛋白AMPK、mTOR的表达；透射电镜检测细胞线粒体形态和细胞器的变化。结果显示，随着细胞中血清浓度降低，细胞活力、p62蛋白量、p-mTOR/mTOR比值逐渐降低；细胞凋亡率、ROS水平、ATP水平、LC3B-Ⅱ蛋白水平、p-AMPK/AMPK比值、细胞内自噬溶酶体数量、细胞核及线粒体异常率则逐渐升高；与对照组相比，15%组膜电位显著升高，5%和0%组极显著降低。这表明，短期血清饥饿胁迫能诱导基于AMPK/mTOR信号通路的自噬发生，加快细胞代谢，但同时存在加快细胞凋亡、抑制细胞增殖等毒副作用。

关键词: 猪, 自噬, SMSCs, 血清饥饿胁迫, AMPK/mTOR通路

Abstract:

This study aimed to investigate the effects and mechanisms of transient starvation on the metabolism and autophagy of porcine skeletal muscle satellite cells (SMSCs), to analyze the role of autophagy in skeletal muscle development, and to help design strategies for improving meat production traits in domestic pigs. Resuscitate the SMSCs cell lines isolated and preserved in the laboratory, and divide the cells into 5 groups based on the serum concentration: 20% serum group (control group), 15% serum group, 10% serum group, 5% serum group, and 0% serum group, to form varying degrees of hunger stress. When the cells fuse to 70%-80%, culture for 24 hours for detection, with 4 replicates in each group. Cell apoptosis, membrane potential, reactive oxygen species(ROS) levels were detected by flow cytometry; ATP levels were measured using a reagent kit, and the expression of autophagy marker proteins(LC3B-Ⅱ, p62) and pathway-related proteins(AMPK, mTOR) was detected by WB; transmission electron microscopy was used to detect changes in mitochondrial morphology and organelles in cells. The cell viability, level of p62 protein, and the p-mTOR/mTOR ratio decreased with the decrease of serum concentration; apoptosis rate, level of ROS, level of ATP, the level of LC3B-Ⅱ protein, the p-AMPK/AMPK ratio, the number of autophagy lysosomes and the abnormal rate of nucleus and mitochondria increased; Compared with the 20% serum concentration group, the 15% group showed a significant increase in membrane potential, while the 5% and 0% groups showed a highly significant decrease. This indicates that short-term serum starvation can induce autophagy induced by the AMPK/mTOR signaling pathway and accelerate cell metabolism, but at the cost of some toxic effects such as accelerating cell apoptosis and inhibiting cell proliferation.

Key words: porcine, autophagy, SMSCs, serum starvation, AMPK/mTOR pathway

中图分类号:

S828.2

王怡, 高娟, 胡悦旻, 杨跃飞, 范博钧, 鞠辉明. 短期血清饥饿胁迫对猪骨骼肌卫星细胞代谢及自噬发生的影响[J]. 畜牧兽医学报, 2024, 55(8): 3408-3417.

Yi WANG, Juan GAO, Yuemin HU, Yuefei YANG, Bojun FAN, Huiming JU. Effect of Transient Serum Starvation on Metabolism and Autophagy of Porcine Skeletal Muscle Satellite Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3408-3417.

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