目的 基于16S rRNA測(cè)序技術(shù)和非靶向代謝組學(xué)技術(shù)初步探討白鮮皮Dictamni Cortex對(duì)斑馬魚幼魚中的肝毒性機(jī)制。方法 將斑馬魚幼魚置于0、100、200、300、400、500、600、700、800、1 000 μg/mL白鮮皮藥液中24 h，統(tǒng)計(jì)死亡個(gè)數(shù)及致死率，計(jì)算10%致死濃度（sublethal concentration，LC₁₀），據(jù)此設(shè)置白鮮皮低、中、高給藥劑量；LC₁₀下暴露24 h，檢測(cè)斑馬魚幼魚中丙氨酸氨基轉(zhuǎn)移酶（alanine aminotransferase，ALT）、天冬氨酸氨基轉(zhuǎn)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）、層黏連蛋白（laminin，LN）、谷胱甘肽（glutamate，GLU）活性，采用16S rRNA測(cè)序技術(shù)分析白鮮皮對(duì)斑馬魚幼魚腸道菌群的分布影響；基于非靶向代謝組學(xué)技術(shù)探討其生物標(biāo)志物的變化和影響的代謝通路，結(jié)合Spearman分析法對(duì)腸道門屬水平優(yōu)勢(shì)菌群和差異代謝物進(jìn)行相關(guān)性分析。結(jié)果 白鮮皮在斑馬魚幼魚中LC₁₀為572.43μg/mL。與對(duì)照組比較，白鮮皮100、300、500 μg/mL均能升高斑馬魚幼魚的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測(cè)序結(jié)果表明白鮮皮能升高變形菌門、衣原體門等菌群的豐度（P＜0.05、0.001），降低擬桿菌門等菌群的豐度（P＜0.01）；代謝組學(xué)分析鑒定出32個(gè)關(guān)鍵差異代謝物，通路分析表明白鮮皮可通過參與鞘脂代謝、嘌呤代謝、花生四烯酸代謝、不飽和脂肪酸的生物合成、藥物代謝-細(xì)胞色素P450、半乳糖代謝、甘油磷脂代謝、氨基糖和核苷酸糖代謝產(chǎn)生肝毒性。結(jié)論 白鮮皮可以導(dǎo)致斑馬魚幼魚肝毒性，其機(jī)制可能是調(diào)節(jié)腸道菌群結(jié)構(gòu)和鞘脂代謝、嘌呤代謝、花生四烯酸等代謝途徑進(jìn)而促進(jìn)炎癥反應(yīng)、氧化應(yīng)激、細(xì)胞凋亡和代謝激活。

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

國家重點(diǎn)研發(fā)計(jì)劃(2022YFC3502104)