目的 基于16S rRNA测序技术和非靶向代谢组学技术初步探讨白鲜皮Dictamni Cortex对斑马鱼幼鱼中的肝毒性机制。方法 将斑马鱼幼鱼置于0、100、200、300、400、500、600、700、800、1 000 μg/mL白鲜皮药液中24 h，统计死亡个数及致死率，计算10%致死浓度（sublethal concentration，LC₁₀），据此设置白鲜皮低、中、高给药剂量；LC₁₀下暴露24 h，检测斑马鱼幼鱼中丙氨酸氨基转移酶（alanine aminotransferase，ALT）、天冬氨酸氨基转移酶（aspartate aminotransferase，AST）、白蛋白（albumin，ALB）、超氧化物歧化酶（superoxide Dismutase，SOD）、丙二醛（malondialdehyde，MDA）、层黏连蛋白（laminin，LN）、谷胱甘肽（glutamate，GLU）活性，采用16S rRNA测序技术分析白鲜皮对斑马鱼幼鱼肠道菌群的分布影响；基于非靶向代谢组学技术探讨其生物标志物的变化和影响的代谢通路，结合Spearman分析法对肠道门属水平优势菌群和差异代谢物进行相关性分析。结果 白鲜皮在斑马鱼幼鱼中LC₁₀为572.43μg/mL。与对照组比较，白鲜皮100、300、500 μg/mL均能升高斑马鱼幼鱼的ALT、AST、MDA、LN、GLU活性（P＜0.05、0.01），降低SOD、ALB活性（P＜0.05、0.01）；16S rRNA测序结果表明白鲜皮能升高变形菌门、衣原体门等菌群的丰度（P＜0.05、0.001），降低拟杆菌门等菌群的丰度（P＜0.01）；代谢组学分析鉴定出32个关键差异代谢物，通路分析表明白鲜皮可通过参与鞘脂代谢、嘌呤代谢、花生四烯酸代谢、不饱和脂肪酸的生物合成、药物代谢-细胞色素P450、半乳糖代谢、甘油磷脂代谢、氨基糖和核苷酸糖代谢产生肝毒性。结论 白鲜皮可以导致斑马鱼幼鱼肝毒性，其机制可能是调节肠道菌群结构和鞘脂代谢、嘌呤代谢、花生四烯酸等代谢途径进而促进炎症反应、氧化应激、细胞凋亡和代谢激活。

Objective To investigate the mechanism of hepatotoxicity of Baixianpi (Dictamni Cortex) in larvae zebrafish using 16S rRNA equencing technology combined with non-targeted metabolomics techniques. Methods The zebrafish were placed in 0, 100, 200, 300, 400, 500, 600, 700, 800, 1 000 μg/mL concentration gradients for 24 h, the number of deaths and the lethality rate were counted, and the sublethal concentration (LC₁₀) was calculated, and the low-, medium- and high-doses of Dictamni Cortex were set according to this, and the exposure was 24h under LC10. The activities of ALT, AST, ALB, SOD, MDA, LN and GLU in larvae zebrafish were detected. To analyze the effect of Dictamni Cortex on the distribution of larvae zebrafish by 16S rRNA sequencing, to explore the metabolic pathways of the biomarkers based on non-targeted metabolomics technology. Combining Spearman analysis for correlation between differential intestinal flora and differential metabolites. Results The LC₁₀ of Dictamni Cortex on larvae zebrafish is 572.43 μg/mL. Compared with the control group, 100, 300, 500 μg/mLgroups of Dictamni Cortex can increase the content of ALT, AST, MDA, LN and GLU (P < 0.05, 0.01), and decreased the content of SOD and ALB (P < 0.05, 0.01). Results of 16S rRNA sequencing revealed that Dictamni Cortex can increase the abundance of Proteobacteria, Chlamydiae, Deinococcota, Verrucomicrobiota (P < 0.05, 0.001), decreasing the abundance of Bacteroidota (P < 0.01). Metabolomic analysis identified 32 key differential metabolites, and pathway analysis showed that Dictamni Cortex could produce toxicity by participating in sphingolipid metabolism, purine metabolism, arachidonic acid metabolism, galactose metabolism, glutathione metabolism, amino sugar and nucleotide sugar metabolism, biosynthesis of unsaturated fatty acids, drug metabolism-cytochrome P450. Conclusion Dictamni Cortex can cause hepatotoxicity in larval zebrafish and change intestinal flora structure and influence sphingolipid metabolism, purine metabolism, arachidonic acid metabolism and then promote inflammatory response, oxidative stress, apoptosis and metabolic activation.

R285.5

国家重点研发计划(2022YFC3502104)