大鼠过劳死模型的建立及基于模型的能量代谢和氧化应激的蛋白质组学和代谢组学分析

Establishment of Rat Karoshi Model and Modelbased Proteomic and Metabolomic Analyses of Energy Metabolism and Oxidative Stress

目的过劳死已成为一个日益严重的问题,然而由于对其机制理解不足,其识别标准并不清楚。本文旨在建立大鼠过劳死模型,并用于探索急性身心疲劳对心脏功能的损伤以及相关机制。方法本研究采用健康雄性Sprague-Dawley(SD)大鼠作为实验动物,随机分为对照组和实验组,对照组大鼠给予正常饲养和休息;实验组大鼠进行了7天的负重游泳和睡眠剥夺。过劳建模过程中死亡的为猝死组(D),存活的为存活组(S)。期间监测大鼠生理功能指标的变化,包括心电图和呼吸。存活组大鼠于第7天时断颈处死。取各组大鼠的心尖组织,进行蛋白质消化、iTRAQ标记和定量数据分析以确定差异表达蛋白(DEP)。此外,还进行GC-MS分析以鉴定差异代谢物。对疲劳组和过劳死组共同的差异代谢物和蛋白质进行整合分析,构建相关代谢通路网络。结果线粒体氧化磷酸化、支链氨基酸分解、溶酶体自噬等模块功能增强,这些变化为过劳状态下的心脏提供了更多的ATP。此外,过氧化物酶体代谢和高铁血红素转移到血红素结合蛋白途径被发现上调,突显了氧化应激和血红蛋白代谢在过劳死模型中的潜在影响。蛋白质组学结果还提示,在急性过劳死的不同时期,可能发生了代谢的重编程。结论代谢重编程可能为心脏提供足够的能量,缓解过度劳累和疲劳对心脏细胞的氧化应激和损伤。这些发现揭示了心脏对过劳死的反应机制,为进一步研究过劳死奠定了基础。

Objective Karoshi,death from overwork,is a serious problem with unclear identification standards and mechanisms.This study aims to establish a karoshi rat model by integrating weight-bearing swimming and sleep deprivation.This model will enable us to investigate the adverse effects of acute physical and mental fatigue on cardiac functions and explore the response mechanisms to overwork using integrated omics approaches,specifically metabonomics and proteomics.Methods The experimental design involved healthy male sprague-dawley(SD)rats subjected to weight-bearing swimming and sleep deprivation for 7 d.The rats were monitored for changes in physiological function indexes,including electrocardiogram and respiration.Protein digestion,iTRAQ labeling,and quantitative data analyses were performed to determine differentially expressed proteins(DEPs).Additionally,GC-MS analysis was conducted to identify differential metabolites.The integration analysis of differential metabolites and proteins shared by the fatigue group and the overwork group was performed to construct a relevant metabolic pathway network and integrate the proteomics and metabolomics data.Statistical analysis was carried out using one-way ANOVA and Duncan’s multiple range t-tests.Results The rats subjected to weight-bearing swimming and sleep deprivation showed various physical and behavioral changes associated with fatigue,including hair disorder,decreased muscle tension,reduced food intake,and weight loss.Analysis of cardiac functions revealed cardiac hypertrophy and heart failure in the fatigue and karoshi groups,as evidenced by changes in heart color,myocardial fiber structure,heart rate,respiratory rate,and cardiac ultrasound measurements.Metabolomics analysis using GC-MS identified several differential metabolites in response to overwork,including amino acids involved in various metabolic pathways.Proteomic analysis using iTRAQ technology identified DEPs in the fatigue and karoshi groups,with a subset of DEPs shared by both groups.The GO analysis revealed that the up-regulated DEPs were primarily associated with mitochondria and peroxisomes in the cellular component category.The Reactome analysis further highlighted the enrichment of DEPs in the transfer of ferriheme from methemoglobin to hemopexin pathway.Integration analysis of the DEPs and differential metabolites revealed the activation of autophagy,increased mitochondrial oxidative phosphorylation,enhanced branched-chain amino acid degradation,and altered peroxisomalβ-oxidation.These findings suggested complex metabolic adaptations to meet the increased energy demands during overwork while also dealing with oxidative stress.Furthermore,the reprogramming of energy metabolism was observed,with upregulation of fatty acidβ-oxidation enzymes and glycolysis-related enzymes in the fatigue group,indicating a shift towards glucose metabolism.In contrast,the karoshi group showed a decreased dependence on fatty acids as an energy source and increased utilization of glucose.The model proposed in this study highlights the interconnected metabolic changes involving mitochondria,peroxisomes,and lysosomes in response to overwork.The findings contribute to our understanding of the mechanisms involved in overwork-related pathologies and provide a basis for further research in the field of karoshi.Conclusion Overall,metabolic reprogramming might provide sufficient energy to the heart,alleviate oxidative stress and damage to cardiac cells in response to excessive exertion and fatigue.Our findings provide an insight into response mechanism to overwork death and lay a foundation for further research on overwork death.