分析中药黄药子水提取物诱导小鼠肝损伤后的基因表达谱，探讨黄药子中毒导致肝损伤的分子作用机制。

SPF级雌性昆明小鼠40只，按随机数字表法分为4组，每组10只。黄药子干预组小鼠分别以低(6 g/kg/d)、中(12 g/kg/d)、高(24 g/kg/d)剂量的黄药子水提取物连续灌胃21 d，正常对照组以同体积纯水灌胃。对比观察4组小鼠的体重增长值、肝脏指数、血清丙氨酸氨基转移酶(ALT)和天冬氨酸转移酶(AST)活性以及肝组织经苏木精-伊红(HE)染色后的病理变化，确定可引起小鼠明显肝损伤的给药剂量。利用RNA-Seq技术分别构建黄药子诱导组(DB组)和对照组(Normal组)小鼠肝脏的数字化基因表达谱(DGE)文库，对比两个DGE文库的差异表达基因，并进行基因本体论(GO)功能注释和京都基因与基因组百科全书(KEGG)通路分析。

与对照组相比，仅高剂量组小鼠出现明显的肝损伤，表现为同时存在体重增长值降低、肝脏指数增高、ALT及AST活性增高(P均<0.05)以及肝组织病理学变化(结构明显破坏、肝索受压紊乱、肝细胞明显肿胀且呈气球样变性、肝小叶内中性粒细胞浸润)。以高剂量组小鼠为DB诱导组，对照组为Normal组，分别测序并构建肝组织的DGE文库：两组分别检测出13 214 893和11 124 617条碱基序列，过滤并剔除带有接头、带有N碱基和测序质量低的reads后分别得到12 819 933和10 786 300条序列。根据所得的两组DEG文库，对比筛选出了312个显著差异表达的基因，其中上调基因148个，下调基因164个。对两组差异表达基因进行聚类分析：两组均表达的高质量序列有9537个，仅在DB组表达的有702个，仅在Nomal组表达的有539个。GO功能注释主要归类为生物学过程和分子功能两个条目。根据KEGG通路分析结果，差异表达的基因主要涉及8条通路：视黄醇代谢通路、花生四烯酸代谢通路、过氧化物酶体增殖物激活受体(PPARs)信号通路、细胞色素P450(CYP450)酶代谢通路、CYP450酶催化的外源物质代谢通路、甾类激素生物合成通路、谷胱甘肽代谢通路、不饱和脂肪酸的生物合成过程。

黄药子的诱导作用严重影响了小鼠肝脏的结构、功能以及相关功能基因的表达；其造成肝损伤的作用机制可能是黄药子在肝内经CYP450酶代谢产生了带有自由基的代谢产物，使胞质膜和细胞器脂质过氧化、机体抗氧化能力减弱，或通过直接影响肝内脂质代谢、增强肝脏脂肪酸氧化反应，从而导致肝损伤。

The present study was designed to detect the gene expression profiles associtaed with Dioscorea bulbifera L. (DB)-induced liver injury in mice by RNA-sequenceing (RNA-Seq), a next-generation sequencing technology, and analyzed its molecular mechanisms.

Forty SPF Kunming female mice (4-6 weeks old, 20±2 g)were purchased from Shanghai Slack Laboratory Animal Co. Ltd (animal certification number: 2015000533474). Animals were randomly divided into the control group, high, moderate and low dose groups (n=10 in each group). Mice in the latter three groups were administered intragastrically (i.g.) with DB decoction (24, 12 and 6 g/kg/d, respectively) for 21 consecutive days, while the control group were given equal volume of pure water.After experiment the mice were killed, and their livers were collected. The optimum dose to induce hepatotoxicity was chosed by compared the treated groups and the control group in terms of mice weight gain, liver index, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and liver tissue pathology after H&E staining. Two digital gene expression (DGE) libraries of mice liver in the control and the DB group were created by RNA-seq, following with data analysis and differential gene expression analysis. Finally, Gene ontology (GO) enrichment and kyoto encyclopedia of genes and genomes (KEGG) pathway analyses were implemented on all differentially expressed genes to elucidate their biological functions and processes. The analyses were carried out using the SPSS 20.0 software package, and the results were expressed as ±s. Comparison between two groups was done with independent sample T-test, with P<0.05 considered as statistically significant. Moreover, padj<0.05 was set as the threshold for significantly differential expression in DGE library analysis.

Firstly, obvious liver injury was observed only in the high-dose group, which mainly manifested as decreased weight gain, elevated liver index and serum ALT and AST levels (P<0.05). Histopathological changes were apparent in liver tissue, with destruction of structure, liver cell swelling and cord derangement and infiltration of neutrophilic granulocytes. A total of 13, 214, 693 and 11, 124, 617 raw reads were generated from the control and DB exposure DGE libraries respectively, with 12, 819, 933 and 10, 786, 300 clean reads left for assembly after removing adaptor related, containing N and low quality reads. According to the statistical result of the two DGE libraries, 302 genes were detected differentially expressed with 148 up-regulated and 164 down-regulated. And 9537 high-quality sequences were observed co-expression among two DGE libraries, with 702 in the DB group only and 539 in the normal group only, after clustering analysis. GO annotation there mainly had molecular function and biological process. All differentially expressed genes were mapped to 140 pathways of KEGG database, and the top 8 significantly enriched KEGG pathways were retinol metabolism, arachidonic acid metabolism, PPAR signaling pathway, drug metabolism-cytochrome P450, metabolism of xenobiotics by cytochrome P450, steroid hormone biosynthesis, glutathione metabolism and biosynthesis of unsaturated fatty acids.

The results showed that DB may induce hepatotoxicity by seriously damaging structures and functions of liver and influence related gene expressions. The DGE sequencing technology provides a better way to revealed that the molecular mechanisms of DB induced hepatotoxicity may include generation of oxygen free radical after DB metabolized by the CYP450 enzyme in liver, which resulted in lipid peroxidation of cellular membrane and organelle and decrease of anti-oxidation capacity, or DB may cause liver injury by increasing intrahepatic lipid metabolism and fatty acid oxidation.