炎症状态TLR4/IL-1R1介导伏立康唑致肝损伤机制研究

Drug-induced liver injury (DILI) has become a serious issue in the practice of clinical medicine. Voriconazole (VCZ) is the preferred drug for prevention and treatment of fungal infections by invasive aspergillus. Clinical application of VCZ is limited severely due to the high incidence of DILI. However, the mechanism underlying VCZ-associated hepatotoxicity remains unknown. Preliminary experimental results showed that VCZ induced DILI was significantly affected by inflammation, and disrupted bile acid homeostasis together with upregulated mRNA levels of TLR4 were observed. Based on highly homologous structural domain between TLR4 and IL-1R1, VCZ may induce DILI through TLR4/IL-1R1 activation and relevant signal pathway. In the present study, sandwich-cultured human and rat hepatocyte and animal models will be employed to assess the liver injury of VCZ under inflammation state. Then, TLR4/IL-1R1 pathway mediated regulation of bile acid synthesis enzymes and transporters will be evaluated. Molecular pharmacokinetics of VCZ under inflammation state will also be investigated. Finally, molecular mechanism of VCZ induced liver injury will be studied by silence or knockout of TLR4 or/and IL-1R1 using shRNA and lentivirus infection technology. In summary, the current research will not only provide a practical interpretation for clinical use of VCZ, but also offer new thinking and strategy for therapeutic regimen optimization in clinic.

药物性肝损伤（DILI）已成为临床用药安全不容忽视的重要问题。伏立康唑（VCZ）是侵袭性真菌感染防治首选药物，而DILI高发生率严重限制了临床应用。目前，VCZ致DILI的机制仍不清楚。我们前期研究发现VCZ致DILI受炎症影响、胆汁酸平衡紊乱且TLR4 mRNA表达显著上调。基于TLR4与IL-1R1结构域高度同源，推测VCZ很可能通过炎症诱导激活TLR4/IL-1R1相关通路导致DILI。本项目拟采用“三明治”培养人与大鼠肝细胞、整体动物炎症模型考察VCZ肝毒性，评价炎症时VCZ介导TLR4/IL-1R1通路调控胆汁酸合成关键酶与转运体的作用，明确VCZ致DILI的分子药动学机制，进一步采用shRNA等技术沉默/敲除TLR4或/和IL-1R1，阐明VCZ致DILI的分子生物学机制。本研究将为临床安全应用VCZ提供理论依据，为优化VCZ临床治疗方案提供新思路和新策略，具有重要的临床意义。

药物性肝损伤（DILI）是临床用药安全不可忽视的重要问题。伏立康唑（VCZ）是侵袭性真菌感染的一线治疗药物，因其较高的DILI发生率严重限制了其临床应用。但VCZ致DILI的机制仍不清楚。本研究采用大鼠原代肝细胞模型考察了VCZ的肝毒性，证实其在正常状态下，肝毒性不明显。采用无毒剂量的LPS炎症大鼠模型，探讨了炎症状态下VCZ的分子药动学变化机制及致肝损伤的分子机制。研究结果提示，炎症状态下，VCZ的代谢及消除明显减慢，相关代谢酶Cyp2c11、Cyp3a1/2、Cyp2c6的基因及蛋白表达水平显著降低；可见明显的肝损伤，大鼠血浆中AST、ALP、γ-GGT与非炎症状态比较均有显著性差异，肝脏组织中ALP与AST也具有显著性差异；HE染色可见肝脏组织明显损伤，细胞间隙变大，炎性浸润，细胞骨架模糊，边缘呈不规则状态。炎症状态下，VCZ显著上调了TLR4及NF-κB的mRNA表达，显著下调了FXR、CAR和PXR的基因水平；胆汁酸外排转运体BSEP、MRP2的基因表达水平均显著下调，而NTCP及胆汁酸合成关键酶CYP7A1的基因表达水平显著上调。采用LC-MS/MS测定实验大鼠肝脏及血浆中的胆汁酸。结果表明，炎症状态下，VCZ可使肝脏组织中TDCA、GCDCA、CDCA等多种胆汁酸含量显著升高，使血浆中TDCA、TMCA、GCDCA、MCA、CDCA、DCA等的含量显著升高。上述结果提示炎症状态下，VCZ可能通过激活TLR4-NF-κB，介导FXR调控胆汁酸代谢相关通路导致了胆汁淤积性肝损伤。进一步采用TLR4基因敲除小鼠验证了炎症对VZC所致肝损伤的调控机制。临床标本研究证实炎症对VCZ代谢及肝损伤产生了明显影响。本项目研究结果将为临床安全应用VCZ提供理论依据，为优化VCZ临床治疗方案提供新思路和新策略，具有重要的临床意义。