辐射诱导的大鼠放射性食管炎的蛋白质组学分析

Proteomic analysis of radiation⁃induced esophagitis in rats

目的利用定量蛋白组学研究电离辐射对大鼠食管组织中蛋白表达谱的影响,揭示放射性食管炎发生发展潜在的分子机制。方法将24只雄性SD大鼠按简单随机化方法分为对照组、25 Gy照射组和35 Gy照射组,每组8只。照射后7 d收集食管组织提取总蛋白。通过串联质谱标签(TMT)标记定量蛋白质组学技术和生物信息学分析方法,探究大鼠受照后食管组织蛋白表达谱的变化。利用免疫组织化学法和Western blot法对两个关键蛋白Hp和Ndufs4的表达进行验证。结果与对照组相比,25 Gy照射后共有847个差异表达蛋白(上调483个,下调364个),35 Gy照射后共有699个差异表达蛋白(上调443个,下调256个)。不同剂量照射后共同上调的蛋白有326个,主要涉及免疫反应和炎症反应相关的生物学过程和信号通路;共同下调的蛋白有210个,主要涉及能量产生和代谢相关的生物学过程和信号通路。进一步通过蛋白互作(PPI)网络构建,筛选出节点蛋白共155个,其中上调节点蛋白最相关的功能途径分别是先天性免疫应答、补体和凝血级联以及先天性免疫系统,下调节点蛋白最相关的功能途径分别是通过氧化有机化合物获得能量、氧化磷酸化以及三羧酸(TCA)循环和呼吸电子传递。这些功能分别富集到9个补体相关的上调节点蛋白和5个线粒体相关的下调节点蛋白。电离辐射后,Hp蛋白表达显著上调(t=27.94、10.96,P<0.001),Ndufs4蛋白表达显著下调(t=59.27、54.07,P<0.001),与蛋白测序结果一致。结论电离辐射可引起大鼠食管组织中蛋白表达谱的改变,这些差异表达的蛋白涉及到多种免疫过程、炎症反应和能量代谢异常等放射生物学相关的功能通路。筛选验证的关键蛋白可以为放射性食管炎提供潜在的生物标志物。

Objective To investigate the impacts of ionizing radiation on protein expression profiles in esophageal tissues of rats using quantitative proteomics,in order to reveal the molecular mechanisms underlying the onset and development of radiation-induced esophagitis(RIE).Methods A total of twenty-four male SD rats were divided by simple randomization into three groups:the control,25 Gy irradiation,and 35 Gy irradiation groups,and their esophageal tissues were collected at 7 d post-irradiation to extract total protein.Then,changes in the protein expression profiles of the esophageal tissues in irradiated rats were investigated using tandem mass tag(TMT)-labeled quantitative proteomics and bioinformatics analysis.Additionally,the expressions of two key proteins,Hp and Ndufs4,were validated using immunohistochemistry and Western blot.Results A comparison with the control group revealed a total of 847 differentially expressed proteins(DEPs;483 up-regulated and 364 down-regulated)following 25 Gy irradiation and 699 DEPs(443 up-regulated and 256 down-regulated)following 35 Gy irradiation.Different radiation doses led to common 326 up-regulated proteins,which were mainly involved in biological processes and signaling pathways related to immune and inflammatory responses,and 210 down-regulated proteins,which were primarily involved in biological processes and signaling pathways related to energy production and metabolism.Furthermore,a total of 155 proteins were screened using a constructed protein protein interaction(PPI)network.Of these proteins,the up-regulated ones were most associated with three functional pathways,namely innate immune responses,complement and coagulation cascades,and innate immune system,while the down-regulated ones were most associated with energy acquisition via oxidizing organic compounds,oxidative phosphorylation,and the tricarboxylic acid(TCA)cycle and respiratory electron transfer.These functions were enriched with nine complement-related up-regulated and five mitochondria-related down-regulated proteins,respectively.Ionizing radiation significantly up-regulated Hp(t=27.94,10.96,P<0.001)and down-regulated Ndufs4(t=59.27,54.07,P<0.001),consistent with the protein sequencing result.Conclusions Ionizing radiation can change the protein expression profiles in the esophageal tissues of rats,and these DEPs are involved in multiple radiobiology-related functional pathways such as immune processes,inflammatory responses,and abnormal energy metabolism.Screening and validation of key proteins are helpful for identifying potential biomarkers of radiation-induced esophagitis.