[關鍵詞]
[摘要]
目的 基于16S rRNA測序技術和非靶向代謝組學技術初步探討白鮮皮Dictamni Cortex對斑馬魚幼魚中的肝毒性機制。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)計死亡個數(shù)及致死率,計算10%致死濃度(sublethal concentration,LC10),據(jù)此設置白鮮皮低、中、高給藥劑量;LC10下暴露24 h,檢測斑馬魚幼魚中丙氨酸氨基轉移酶(alanine aminotransferase,ALT)、天冬氨酸氨基轉移酶(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測序技術分析白鮮皮對斑馬魚幼魚腸道菌群的分布影響;基于非靶向代謝組學技術探討其生物標志物的變化和影響的代謝通路,結合Spearman分析法對腸道門屬水平優(yōu)勢菌群和差異代謝物進行相關性分析。結果 白鮮皮在斑馬魚幼魚中LC10為572.43μg/mL。與對照組比較,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測序結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);代謝組學分析鑒定出32個關鍵差異代謝物,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。結論 白鮮皮可以導致斑馬魚幼魚肝毒性,其機制可能是調節(jié)腸道菌群結構和鞘脂代謝、嘌呤代謝、花生四烯酸等代謝途徑進而促進炎癥反應、氧化應激、細胞凋亡和代謝激活。
[Key word]
[Abstract]
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.
[中圖分類號]
R285.5
[基金項目]
國家重點研發(fā)計劃(2022YFC3502104)