补骨脂和淫羊藿是临床常用药对,既往研究显示两药可能引起特异质肝损伤,然而其物质基础以及发生机制尚不清楚.该研究基于肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)介导的免疫应激小鼠模型,以补骨脂及淫羊藿中 2 个主要化学成分补骨脂甲素和朝藿定B为研究对象,通过检测细胞上清液中乳酸脱氢酶(lactate dehydrogenase,LDH)释放量和小鼠血浆中丙氨酸氨基转氨酶(alanine aminotransferase,ALT)、天冬氨酸氨基转氨酶(aspartate transaminase,AST)含量,苏木精-伊红(hematoxylin-eosin,HE)染色观察肝脏病理学变化情况,分别在细胞及动物水平评估补骨脂甲素联合朝藿定B引起肝损伤的程度.借助拟靶向代谢组学技术和多元统计分析方法,考察给药前后小鼠血浆中内源性代谢物的影响,筛选差异代谢标志物,并进行京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集通路分析.结果表明,在细胞水平,使用TNF-α刺激2 h后,相较于正常组及单独给药组,补骨脂甲素可以使HepG2 细胞中LDH释放量显著增加;补骨脂甲素联合朝藿定B后,LDH释放量在原来基础上进一步显著增加.在动物水平,对于正常小鼠,单独或联合补骨脂甲素均不引起肝损伤;对于免疫应激模型小鼠,单独给药补骨脂甲素或者朝藿定B均可引起肝损伤,补骨脂甲素或者朝霍定B联合给药时肝损伤进一步增强,表现为小鼠血浆中ALT、AST含量显著升高,并伴随肝脏组织大量炎性免疫细胞浸润.基于拟靶向代谢组学技术,筛选得到 7 个共有差异代谢物,根据受试者操作特性(receiver operating characteristic,ROC)曲线分析结果,其中曲线下面积(area under curve,AUC)大于 0.9 的包括D-氨基葡萄糖 6-磷酸、N1-甲基-2-吡咯-5-甲酰胺、17β-硝基-5a-雄甾烷、葛花苷元-7-O-葡萄糖醛酸苷、N-(1-脱氧-1-果糖基)缬氨酸.因此,这 5 个差异代谢物可能是潜在的生物标志物.此外,烟酸盐和烟酰胺代谢及亚油酸代谢通路可能是补骨脂甲素联合朝藿定B诱导特异质肝损伤的关键代谢通路.综上,在TNF-α介导免疫应激的条件下,朝藿定B联合补骨脂甲素导致机体代谢扰动可能是二者协同导致特异质性药物性肝损伤(idiosyncratic drug-induced liver injury,IDILI)的重要机制.
Pseudo-targeted metabolomics study of immune stress-mediated idiosyncratic liver injury induced by synergistic effects of bavachin and epimedin B
Chinese patent medicine preparations containing Epimedii Folium and Psoraleae Fructus have been associated with the occurrence of idiosyncratic drug-induced liver injury(IDILI).However,the specific toxic biomarkers and mechanisms underlying these effects remain unclear.This study aimed to comprehensively assess the impact of bavachin and epimedin B,two principal consti-tuents found in Psoraleae Fructus and Epimedii Folium,on an IDILI model induced by tumor necrosis factor-α(TNF-α)treatment,both in vitro and in vivo.To evaluate the extent of liver injury,various parameters were assessed.Lactate dehydrogenase(LDH)re-lease in the cell culture supernatant,as well as the levels of alanine aminotransferase(ALT)and aspartate transaminase(AST)in mouse plasma were measured.Additionally,histological analysis employing hematoxylin-eosin staining was performed to observe liver tissue changes indicative of the severity of liver injury.Furthermore,a pseudo-targeted metabolomics approach was employed,followed by multivariate analysis,to identify differential metabolites.These identified metabolites were subsequently subjected to Kyoto Encyclo-pedia of Genes and Genomes(KEGG)pathway enrichment analysis.The results showed that at the cellular level,after 2 hours of TNF-α stimulation,bavachin significantly increased the release of LDH in HepG2 cells compared to the normal group and the group treated alone;after the combination of bavachin and epimedin B,the release of LDH further significantly increased on the original ba-sis.Similarly,although the individual or combination treatments of bavachin and epimedin B did not induce liver injury in normal mice,the combination of both drugs induced marked liver injury in TNF-α treated mice,leading to a significant elevation in plasma AST and ALT levels and substantial infiltration of inflammatory immune cells in the liver tissue.Pseudo-targeted metabolomics analysis identi-fied seven common differential metabolites.Among these,D-glucosamine-6-phosphate,N1-methyl-2-pyridone-5-carboxamide,17beta-ni-tro-5a-androstane,irisolidone-7-O-glucuronide,and N-(1-deoxy-1-fructosyl)valine emerged as potential biomarkers,with an area under the curve(AUC)exceeding 0.9.Furthermore,our results suggest that the metabolism of nicotinic acid and nicotinamide,as well as the linoleic acid metabolic pathway,may play pivotal roles in bavachin and epimedin B-induced IDILI.In conclusion,within an immune-stressed environment mediated by TNF-α,bavachin and epimedin B appear to induce IDILI through disruptions in metabolic processes.
Chinese patent medicinebavachinepimedin Bidiosyncratic liver injurytumor necrosis factor-αpseudo-targeted metabolomics
NETL
NSTL
万方数据
维普
