代谢组学测定对大鼠百草枯中毒程度评估的研究

Evaluation the degree of paraquat poisoning in rats by metabolomics

目的基于气相色谱-质谱联用(GC-MS)代谢组学技术筛选潜在性的用于评估急性百草枯(PQ)中毒大鼠严重程度的血浆代谢物生物标志物,为PQ中毒的早期诊断及病情进展提供依据。方法24只SD大鼠按随机数字表法分为生理盐水对照组、低剂量中毒组(40mg/kg)、中剂量中毒组(80mg/kg)、高剂量中毒组(120mg/kg),每组6只。观察大鼠一般情况,于灌胃后24h采血,采用GC-MS方法检测血浆中内源性小分子代谢物,并进行代谢轮廓及随机森林分析;生化检测大鼠肝肾功能,与筛选的潜在生物标志物进行典型相关分析。HE染色明确百草枯中毒大鼠肺损伤程度。结果PQ染毒剂量越大,SD大鼠出现食量减少、少动、气促等临床症状越明显,肺组织、肝、肾功能损伤越严重。相对于对照组,不同剂量百草枯中毒大鼠的血浆中检测出了30种差异性血浆代谢产物,随着百草枯中毒剂量的增加,与对照组大鼠血浆代谢轮廓特性的差异性变化越显著。乙酸、乙二酸、L-丙氨酸、D-果糖、葡萄糖肟、谷氨酰胺、丙酸、肌醇、苯乙酸等代谢物的改变对百草枯中毒大鼠血浆代谢轮廓特征变化贡献度最高,代谢通路分析提示在百草枯急性中毒大鼠体内的能量代谢、蛋白质代谢、脂肪酸代谢和糖代谢发生改变。采用典型相关对代谢物特征和临床特征进行分析,相关系数R为0.92,代谢物L-脯氨酸,L-苯丙氨酸,酪氨酸、肌醇和生化指标ALT、AST、BUN、CR对两组特征的相关性贡献最大。结论基于气相色谱-质谱(GC-MS)的代谢组学可以揭示百草枯中毒病情严重程度的潜在生物标志物,且代谢组学研究结果对于解析百草枯代谢通道机制、探讨百草枯中毒后的解毒途径具有重要价值。

Objective Screening of potential plasma metabolite biomarkers for the evaluation of the severity of acute paraquat(PQ)poisoning in rats based on GC-MS metabolomics technology,providing a basis for the early diagnosis and disease progression of PQ poisoning.Methods 24 SD rats were divided into normal saline control group,the low-dose intoxicati6 on group(40mg/Kg),the medium-dose intoxication group(80mg/kg)and the high-dose intoxication group(120mg/kg)according to the random number table method.Every group has 6 SD rats.Observed the general conditions of rats after gastrointestinal perfusion,then collected abdominal aorta blood of rats in 24 hours.Endogenous small molecule metabolites in plasma were detected by GC-MS method,and metabolic profile and random forest analysis were performed.Biochemical examination of rat liver and kidney function,and in combination with endogenous small molecule metabolites to execute canonical correlation analysis.The lung injury degree of paraquat poisoning rats was determined by HE staining.Results The higher the dose of PQ,the more obvious clinical symptoms such as decreased food intake,decreased movement and shortness of breath appeared in SD rats,and the more serious the lung tissue,liver and kidney function damage was.Compared with the control group,30 different plasma metabolites were detected in different doses of paraquat poisoning rats,and with the increase of paraquat poisoning dose,the plasma metabolic profile changed more significantly.The change of acetic acid,oxalic acid,L-alanine,D-fructose,glucose oxime,glutamine,propionic acid,inositol and phenylacetic acid have high contribution to PQ rats plasma metabolic profile change.The metabolic pathway analysis indicated that the energy metabolism,protein metabolism,fatty acid metabolism and sugar metabolism were disturbed in PQ rats.Using canonical correlation analysis to analyze metabolite characteristics and clinical features,and find the correlations,correlation coefficient R is 0.92,metabolites L-valine,L-phenylalanine,Tyrosine,Inositol and biochemistry indicator ALT、AST、BUN、CR contributed the most to the correlation of the two groups of features.Conclusions Our results indicate that metabolomics based on gas chromatography-mass spectrometry(GC-MS)can reveal potential biomarkers for assessing the severity of acute paraquat poisoning.In addition,the results of metabolomics study are of great value in analyzing the mechanism of paraquat metabolism pathway and exploring the detoxification pathway after paraquat poisoning.