免疫应激介导的淫羊藿苷协同补骨脂甲素致特异质肝损伤的拟靶向代谢组学研究

Investigation of synergistic induction of idiosyncratic drug-induced liver injury by Bavachin and Icariin under immunological stress conditions using pseudotargeted metabonomics

既往研究显示,仙灵骨葆(Xianling Gubao capsule,XLGB)肝损伤具有特异质属性,但是XLGB中免疫促进成分和肝损伤易感成分如何协同导致特异质肝损伤(idiosyncratic drug-induced liver injury,IDILI)机制尚不清楚,本研究采用拟靶向代谢组学探讨其可能机制.建立了肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)介导的体内外免疫应激模型,检测了细胞上清液中乳酸脱氢酶(lactate dehydrogenase,LDH)释放量和小鼠血浆中天冬氨酸氨基转氨酶(aspartate aminotransferase,AST)及丙氨酸氨基转氨酶(alanine aminotransferase,ALT)含量,并用HE染色观察了肝脏病理学变化情况,对比考查了补骨脂甲素(bavachin,Bav)和淫羊藿苷(icariin,Ica)单用或联合TNF-α的毒性差异.采用超高效液相色谱-四级杆飞行时间串联质谱(ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry,UPLC-QTOF/MS)技术分析不同组别小鼠血浆样品的代谢轮廓谱,结合多元统计分析方法研究了补骨脂甲素和淫羊藿苷对特异质肝损伤小鼠血浆中内源性代谢物的影响,筛选代谢标志物,并借助全基因组及代谢途径数据库(Kyoto Encyclopedia of Genes and Genomes,KEGG)进行代谢富集通路分析.细胞和动物表型实验结果初步表明,TNF-α可成功诱导HepG2细胞和小鼠免疫应激,此时补骨脂甲素和淫羊藿苷配伍给药对肝细胞和肝脏的损伤程度显著增加(P<0.01),肝脏生化和组织病理学分析也显示了类似的结果.拟靶向代谢组学结果显示,TNF-α组和对照组筛选并鉴定了26个与免疫激活相关的差异代谢物,Bav/TNF-α组和TNF-α组以及Ica/TNF-α组和TNF-α组分别筛选并鉴定了24和32个与肝损伤相关的差异代谢物,Bav/Ica/TNF-α组和Bav/TNF-α组以及Bav/Ica/TNF-α组和Ica/TNF-α组分别筛选并鉴定了12和23个与肝损伤相关的差异代谢物,Venn图和受试者工作特征曲线(receiver operating characteristic curve,Roc curve)分析得到3个由补骨脂甲素和淫羊藿苷协同影响的肝损伤代谢标志物,分别是磷脂酰乙醇胺36:5、磷脂酰乙醇胺38:6和S-硫代-L-半胱氨酸(area under the curve(AUC)>0.9).进一步将Bav/Ica/TNF-α组和Bav/TNF-α组以及Bav/Ica/TNF-α组和Ica/TNF-α组间的差异代谢物分别进行KEGG通路富集分析,共同富集的通路是甘油磷脂、花生四烯酸和亚油酸代谢.综上,在TNF-α介导免疫应激的条件下,细胞和机体内代谢网络的改变可能是淫羊藿苷协同补骨脂甲素导致IDILI的潜在机制.

Identifying the mechanisms underlying the immune-stimulating and susceptibility-related aspects of Xianling Gubao(XLGB)that lead to idiosyncratic drug-induced liver injury(IDILI)poses a significant challenge due to the heterogeneous nature of XLGB.In this study,we employed a pseudotargeted metabonomics approach to investigate the potential mechanisms.Utilizing a TNF-α-mediated HepG2 cell and mouse model,the release of lactate dehydrogenase(LDH)in cell supernatant was assessed,the levels of aspartate aminotransferase(AST)and alanine aminotransferase(ALT)in mouse plasma were measured,and the liver pathology changes were observed using HE staining.These evaluations aimed to compare the hepatotoxicity induced by bavachin(Bav)and icariin(Ica)individually and in combination with TNF-α.The UPLC-QTOF/MS technique was employed to analyze the metabolic profiles of mouse plasma samples.Multivariate statistical analysis was then used to investigate the impact of Bav and Ica on endogenous metabolites in mouse plasma,identify metabolite markers,and conduct KEGG enrichment pathway analysis.Phenotypic experiments demonstrated that TNF-α successfully induced immune stress in both cells and mice.Furthermore,co-exposure to Bav,Ica,and a non-toxic dose of TNF-α prestimulation resulted in more pronounced hepatotoxicity and more significant liver injury compared to Bav and Ica administered individually(P<0.01).These findings were corroborated by liver biochemistry and histopathology analyses.Moreover,pseudotargeted metabonomics identified 26 differential metabolites associated with differences in immune activation between the TNF-αgroup and the control group.Additionally,24 differential metabolites were linked to differences in liver injury between the Bav/TNF-α group and the TNF-α group,32 differential metabolites were associated with differences in liver injury between the Ica/TNF-α group and the TNF-α group,and 12 differential metabolites were related to differences in liver injury between the Bav/Ica/TNF-α group and the Bav/TNF-α group.Another 23 differential metabolites were identified in relation to differences in liver injury between the Bav/Ica/TNF-α group and the Ica/TNF-α group.Three potential metabolite markers(PE 36:5,PE 38:6,and s-Sulfo-l-cysteine)associated with IDILI were identified in the Bav/Ica/TNF-α group compared to either the Ica/TNF-α or Bav/TNF-α groups through Venn diagram and ROC curve analysis(AUC>0.9).Furthermore,pathway enrichment analysis revealed that glycerophospholipid,arachidonic acid,and linoleic acid metabolic pathways were significantly enriched in the Bav/Ica/TNF-α group compared to the Ica/TNF-α or Bav/TNF-α groups.In conclusion,these results suggest that alterations in intracellular metabolism may contribute to the synergistic induction of IDILI by the incompatible combination of Bav and Ica in the presence of TNF-α.