马兜铃酸Ⅰ致小鼠急性肝毒性的转录组学分析

朱哿瑞; 王静; 黄恺; 彭渊; 刘成海; 陶艳艳

doi:10.3969/j.issn.1001-5256.2021.10.026

马兜铃酸Ⅰ致小鼠急性肝毒性的转录组学分析

DOI: 10.3969/j.issn.1001-5256.2021.10.026

朱哿瑞¹,
王静¹,
黄恺²,
彭渊¹,
刘成海^{1, 2},
陶艳艳^1, ,

1.
上海中医药大学附属曙光医院肝病研究所，上海 201203
2.
上海市中医临床重点实验室，上海 201203

基金项目:

“重大新药创制”科技重大专项 (2019ZX09201001);

上海市科委科技支撑项目 (19401901500);

上海市中医药三年行动计划 (ZY-[2018-2020]-CCCX-5001)

详细信息

通信作者:
陶艳艳，taoyanyan1023@126.com

中图分类号: R575
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出版历程
- 收稿日期: 2021-03-04
- 录用日期: 2021-05-13
- 出版日期: 2021-10-20

A transcriptomic analysis of acute hepatotoxicity induced by aristolochic acid Ⅰ in mice

1.
Institute of Liver Diseases, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
2.
Shanghai Key Laboratory of Traditional Chinese Medicine, Shanghai 201203, China

Research funding:

The Special Project for Significant New Drug Research and Development in the Major National Science and Technology Projects of China (2019ZX09201001);

The Science and Technology Innovation Action Plan of the Shanghai Municipal Science and Technology Commission (19401901500);

The Three-Year Action Plan of for the Development of TCM in Shanghai (ZY-[2018-2020]-CCCX-5001)

摘要

摘要: 目的探讨马兜铃酸Ⅰ(AAⅠ)致小鼠急性肝损伤的分子机制。方法雄性C57BL/6小鼠15只，随机分为正常组(n=6)和处理组(n=9)。处理组小鼠以AAⅠ 20 mg/kg剂量腹腔注射，连续5 d，第6天处死、取材。检测血清ALT、AST，HE染色观察肝组织学变化；两组各随机选择3例肝组织样本提取RNA进行高通量转录组测序。通过生物信息学分析和功能预测筛选出表达显著的差异基因；再采用实时荧光定量PCR (qRT-PCR) 对部分差异基因进行验证。计量资料两组间差异比较采用t检验。结果与正常组比较，处理组ALT、AST水平明显升高(t值分别为4.331、4.947，P值均＜0.01)；处理组肝组织HE染色可见肝小叶结构紊乱，小叶内见点状坏死。通过筛选条件[logFC＞2倍且P＜0.05]获得差异基因共计1352个，其中上调基因703个，下调基因649个。对这些差异性表达的基因进行GO和KEGG富集分析(P＜0.05)，小分子分解代谢、中性粒细胞参与的免疫应答、分泌颗粒细胞质囊泡腔、细胞质囊泡腔、细胞外结构组织、细胞外基质组织等相关的GO分类呈显著富集；化学致癌、视黄醇代谢、花生四烯酸代谢、类固醇激素合成、癌症中的转录失调、蛋白质消化和吸收、炎症介质对TRP通道的调节、药物代谢、补体和凝血级联、谷胱甘肽代谢、PPAR信号通路等在KEGG通路中显著富集。聚类分析发现明显下调基因包括Srd5a1、Lipc、Aqp8、Hba-a1、Slco1a1、Pklr等，采用qRT-PCR得以证实(P值均＜0.05)。结论 AAⅠ有明显急性肝毒性，其毒性主要涉及化学致癌、视黄醇代谢、花生四烯酸代谢、类固醇激素合成、癌症中的转录失调等环节。
- 马兜铃酸 /
- 转录组 /
- 化学性与药物性肝损伤 /
- 小鼠, 近交C57BL
Abstract: Objective To investigate the molecular mechanism of aristolochic acid Ⅰ (AAⅠ) inducing acute hepatotoxicity in mice. Methods A total of 15 male C57BL/6 mice were randomly divided into normal group with 6 mice and treatment group with 9 mice. The mice in the treatment group were given intraperitoneal injection of AAⅠ at a dose of 20 mg/kg for 5 consecutive days and were sacrificed to collect samples on day 6. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured, and HE staining was used to observe liver histological changes; three liver tissue samples were randomly selected from each group, and RNA was extracted for high-throughput transcriptome sequencing. Bioinformatics analysis and functional prediction were used to screen out differentially expressed genes, and quantitative real-time PCR (qRT-PCR) was used for validation. The t-test was used for comparison of continuous data between two groups. Results Compared with the normal group, the treatment group had significant increases in the activities of ALT and AST (t=4.331 and 4.947, both P < 0.01), as well as disordered structure of hepatic lobules and spotted necrosis in hepatic lobules shown by HE staining. A total of 1352 differentially expressed genes were obtained based on the screening conditions of logFC > 2 and P < 0.05, among which there were 703 upregulated genes and 649 downregulated genes. The GO and KEGG enrichment analyses of these differentially expressed genes showed significant enrichment in GO terms (such as small molecular catabolism, immune response involving neutrophils, cytoplasmic vesicle lumen in secretory granules, cytoplasmic vesicle lumen, extracellular structural organization, and extracellular matrix) and KEGG pathways (such as chemical carcinogenesis, retinol metabolism, arachidonic acid metabolism, steroid hormone biosynthesis, transcriptional dysregulation in cancer, protein digestion and absorption, regulation of TRP channel by inflammatory mediators, drug metabolism, complement and coagulation cascade, glutathione metabolism, and the PPAR signaling pathway). A cluster analysis (P < 0.05) showed that significantly downregulated genes included Srd5a1, Lipc, Aqp8, Hba-a1, Slco1a1, and Pklr, which were validated by qRT-PCR (all P < 0.05). Conclusion AA Ⅰ can lead to significant acute hepatotoxicity, which mainly involves the processes such as chemical carcinogenesis, retinol metabolism, arachidonic acid metabolism, steroid hormone biosynthesis, and transcriptional dysregulation in cancer.
- ARISTOLOCHIC ACID /
- Transcriptome /
- Chemical and Drug Induced Liver Injury /
- Mice, Inbred C57BL

HTML全文

图 1 两组小鼠体质量变化

下载: 全尺寸图片幻灯片

图 2 正常组与处理组小鼠肝组织切片(HE染色，×100)

下载: 全尺寸图片幻灯片

图 3 正常组与处理组差异表达基因qRT-PCR分析

注：与正常组比较，*P＜0.05，**P＜0.01。

下载: 全尺寸图片幻灯片

表 1 基因引物序列

基因	正向引物(5′-3′)	反向引物(5′-3′)
β-actin	TGACGAGGCCCAGAGCAAGA	ATG GGCACAGTGTGGGTGAC
Srd5a1	CTACTGGATGCGCTAGTCTAC	TCAGAAACATAGCTAGCAGGAC
Lipc	CTACAAACACATTGCAGAGCAT	GTTGCAAGGAGTCAATGAAGAG
Aqp8	GCTCCGCTCTCTTCATCTTCATCG	GAAGTGTCCACCGCTGATGTTCC
Hba-a1	ACCACCAAGACCTACTTCCCTCAC	CAGAGCATCGGCGACCTTCTTG
Slco1a1	TGGATTTATCACTGGGAGCTTT	AGGAAATGAGGTGATGCCATTA
Pklr	GGCTCAGAAGATGATGATTGGA	ATCGCATGTTGCATCTTTACAG

下载: 导出CSV

表 2 两组小鼠血清ALT、AST水平的比较

组别	动物数(只)	ALT(U/L)	AST(U/L)
正常组	6	13.95±1.56	13.44±2.05
处理组	9	102.15±34.10	53.62±22.73
t值		4.331	4.947
P值		0.002	0.001

下载: 导出CSV

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