急性百草枯中毒小鼠肺损伤机制的蛋白组学分析

Proteomic analysis of lung injuries induced by paraquat in mice

目的探索百草枯(paraquat,PQ)肺损伤的分子机制。方法6至8周龄的C57BL/6雄鼠按照完全随机化分组的方法分为四组,实验组(共3组,每组9只)经腹腔注射40 mg/kg PQ建立染毒模型,对照组(9只)小鼠腹腔注射同等剂量生理盐水。PQ注射后2 d、7 d和14 d,分别麻醉后处死小鼠取出肺组织,苏木精-伊红染色法(HE)观察肺组织的病理变化,并应用串联质谱标记技术(tandem mass spectrometry tag technology,TMT)分析各时间点实验组与对照组比较,肺组织中差异蛋白表达情况,并进行功能性分析。结果与对照组比较,PQ-2 d、7 d、14 d分别有91个(69升高,22降低)、160个(103升高,57降低)和78个(45升高,33降低)蛋白的差异表达有统计学意义;亚细胞定位结果显示,与对照组相比,PQ-2 d和PQ-7 d组差异蛋白均以细胞外分布最多,而PQ-14d组中差异蛋白位于细胞核内最多;GO分析显示,与对照组相比,PQ-2d和7 d以体液免疫和凝血相关的反应为主,PQ-14d以中性粒细胞聚集等趋化和调控反应为主;KEGG分析显示,补体-凝血级联在PQ-2 d和7 d组中占据首要地位,而PQ-14 d组中以细胞色素P450对异生物的代谢途径为首。结论本研究应用TMT技术对PQ中毒小鼠不同时间点的肺脏进行蛋白组学分析,从蛋白分子角度揭示了PQ肺损伤的分子机制,发现并提出体液免疫和补体-凝血途径是PQ肺损伤的关键,为后续临床研究和治疗方向提供了重要的理论基础。

Objective To explore the molecular mechanism of paraquat(PQ)-induced lung injuries.Methods Male C57BL/6 mice aged 6 to 8 weeks were randomly divided into four groups.Mice in the experimental groups(three groups,nine rats in each group)were intraperitoneally injected with 40 mg/kg PQ to establish an infection model,and mice in the control group(n=9)were intraperitoneally injected with the same dose of saline.Mice were sacrificed at day 2,7 and 14 after PQ administration.Pathological changes of lung tissues from mice model were observed by Hematoxylin-eosin staining.The expression of different proteins in the lung tissues at different time points were detected and identified by tandem mass spectrometry tag technology(TMT),and the functional analysis was performed.Results Compared with the control group,there were 91(69 up and 22 down),160(103 up and 57 down)and 78(45 up and 33 down)proteins in the PQ-2 d,7 d,and 14 d groups,respectively,and there was significant difference of protein expression.The subcellular localization analysis showed that compared with the control group,the differentially-expressed proteins in the PQ-2 d and-7 d groups were mainly distributed in the extracellular space,while in the PQ-14 d group were mainly distributed in the nuclear.GO analysis showed that compared with the control group,the differentially-expressed proteins in the PQ-2 d and PQ-7 d groups were mainly involved in humoral immunity and coagulation-related reactions,while in the PQ-14 d group were mainly involved in chemotactic and regulatory responses such as neutrophil aggregation.The KEGG signaling pathway analysis showed that the complement and coagulation cascades was the most important pathway in the PQ-2d and PQ-7 d groups,while metabolism of xenobiotics by cytochrome P450 was the most important pathway in the PQ-14 d group.Conclusions It is the first time that TMT was used to analyze PQ-induced lung injuries in mice model at different time points.This study demonstrates the molecular mechanism of PQ-induced lung injuries at protein levels,and elucidates that humoral immunity and complement-coagulation pathways charge the main role of PQ-induced lung injuries.This study may provide an important theoretical basis for further research and clinical treatment.