基于非靶向代谢组学的吡虫啉暴露致人肝细胞HepG2毒性效应研究
The toxic effects of imidacloprid exposure on HepG2 cell based on non-targeted metabolomics
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摘要:
背景 吡虫啉是一种被广泛使用于农业生产中的新烟碱类杀虫剂，在人群生物样本中检出率高。已有研究显示吡虫啉暴露与肝损伤相关性较高，但具体机制不明。
目的 观察吡虫啉暴露对人肝细胞HepG2毒性效应及其非靶向代谢谱的扰动影响，分析其主要参与的生物学过程和信号通路，探讨吡虫啉肝毒性可能的分子机制。
方法 制备人肝细胞HepG2细胞悬液接种于96孔板中，分为空白对照组、二甲基亚砜（DMSO）溶剂对照组和多种浓度的吡虫啉染毒组，每组设5个平行样。MTT法测定不同浓度吡虫啉（1、2.5、5、7.5、10 mmol·L−1）染毒8 h后的HepG2细胞存活率，分析其剂量-效应关系。选用细胞存活率为80%对应的吡虫啉暴露浓度（3 mmol·L−1）对HepG2细胞进行染毒，应用UHPLC-Q-TOF/MS技术进行非靶向代谢组学分析，并与空白对照组和DMSO溶剂对照组进行比较分析，筛选各组间差异代谢物，利用京都基因与基因组百科全书（KEGG）数据库进行注释，分析其富集的生物过程和相关信号通路。
结果 与空白对照组和DMSO溶剂对照组比较，吡虫啉暴露组HepG2细胞存活率降低，差异有统计学意义（
P ＜0.001）。暴露于2.5mmol·L−1吡虫啉8 h后，HepG2细胞存活率约为86%。非靶向代谢组学研究发现，暴露3 mmol·L−1吡虫啉可引起HepG2细胞包括肌酸（变量权重值VIP=1.11,P ＜0.001）、精氨酸（VIP=1.47,P =0.048）、牛磺酸（VIP=4.28,P =0.001）、α-D-葡萄糖（VIP=1.90,P =0.006）等在内的61种差异代谢产物水平紊乱，主要涉及哺乳动物雷帕霉素靶蛋白（mTOR）信号通路（P ＜0.001）、精氨酸和脯氨酸代谢（P =0.002）及半乳糖代谢（P =0.015）等。结论 吡虫啉暴露能够显著抑制HepG2细胞存活率，干扰mTOR信号通路、精氨酸和脯氨酸代谢、半乳糖代谢等通路。
Abstract:Background Imidacloprid is a neonicotinoid insecticide that is widely used in agricultural production, with a high detection rate in human biological samples. Previous studies have shown a high correlation between imidacloprid exposure and liver injury, but the specific mechanism is still unknown.
Objective To observe potential toxic effects of HepG2 cells and its perturbation of non-targeted metabolic profile after imidacloprid exposure, and to explore possible molecular mechanisms of hepatotoxicity of imidacloprid by analyzing invovlved biological processes and signaling pathways.
Methods HepG2 cell suspension was prepared and seeded in a 96-well plate, which was divided into blank control group, dimethyl sulfoxide (DMSO) solvent control group and imidacloprid exposure groups with multiple concentrations. Each group was set with 5 parallel samples. The viability of HepG2 cells viability were determined after 8 h of exposure to different concentrationsof imidacloprid (1, 2.5, 5, 7.5, 10 mmol·L−1), and the dose-effect relationship was analyzed. A proper concentration (3 mmol·L−1 with 80% viability) was chosen for imidacloprid exposure, non-targeted metabolomic analysis was applied to the cultivated HepG2 cells using UHPLC-Q-TOF/MS technology, the differential metabolites between groups were screened, and the bioprocess and related signaling pathways of their enrichment were annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.
Results Compared to the other two groups, the survival rates of HepG2 cells in the imidacloprid exposure groups decreased. A survival rate of about 86% of HepG2 cells was found in HepG2 cells exposed to 2.5 mmol·L−1 imidacloprid exposure. The non-targeted metabolomics studies showed that 61 metabolites were significantly affected in HepG2 cells after 3 mmol·L−1 imidacloprid exposure, including creatine (variable importance in projection VIP=1.11,
P <0.001), arginine (VIP=1.47,P =0.048), taurine (VIP=4.28,P =0.001), and α-D-glucose (VIP=1.90,P =0.006). The differential metabolites enriched in bioprocess and related signaling pathways were mainly directed to mTOR signaling pathways (P <0.001), arginine and proline metabolism (P =0.002), and galactose metabolism (P =0.015).Conclusion Imidacloprid exposure can significantly inhibit the survival rate of HepG2 cells, and interfere with the mTOR signaling pathway, arginine and proline metabolism, galactose metabolism, and so on.
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