hPXR antagonism in Anti-TB hepatotoxicity

药物性肝损伤（DILI）是抗结核治疗失败的重要原因。利福平和异烟肼 共同治疗导致胆汁淤积性肝损伤与原卟啉IX（PPIX）的积累， 血红素前体，在小鼠中以人胆甾烷X受体（hPXR）依赖性方式。因此 利福平对hPXR的激活可通过PPIX积累导致DILI;因此，hPXR的拮抗作用可 预防DILI的临床后果。该项目旨在评估预防DILI的方法， 增加对DILI机制的了解。在完成这个项目后，我将阐释一部小说 异烟肼在DILI发展中的作用，并提供了一种新的治疗药物的评价， 与DILI相关的病理标志物。这些信息最终可能会被用来降低发病率 和人类抗结核DILI的程度。使用hPXR转基因小鼠模型和原代人 肝细胞，我将评估一种新的hPXR小分子抑制剂的潜力，这是在我们的研究中开发的。 实验室，以防止与利福平和异烟肼治疗相关的肝损伤。PPIX累积将是 在小鼠中通过LC/MS/MS评估;通过进行蛋白质印迹评估hPXR靶基因表达 以及人原代细胞和人源化小鼠的qRT-PCR分析。将通过以下方式评价肝损伤： 通过血清和组织病理学检查分析小鼠肝酶活性。ALAS 1/Alas 1（限速 血红素合成酶）在hPXR和hPXR中均被利福平和异烟肼（hPXR的配体）诱导 转基因小鼠和原代人肝细胞。这种诱导通过与一种抗肿瘤药物共处理来调节。 hPXR的拮抗剂。PPIX在用利福平和异烟肼两者处理的hPXR小鼠的肝脏中增加。 FECH/Fech在异烟肼作用下降解。这种降解可能是由铁螯合的一个介导的。 异烟肼的代谢物。使用原代人肝细胞和人癌细胞系的蛋白质印迹分析 异位过表达FECH，我将评估异烟肼代谢产物在降解中的潜在作用。 的FECH。在早期检测到肝损伤后，用小分子拮抗hPXR可能是一种可行的方法。 减少ALAS 1诱导和随后PPIX积累导致肝损害的治疗策略 损伤由1名具有3年以上经验的研究生成功完成该项目应 需要大约1年的时间来完成并提供细胞生物学，分子生物学， 和病理

Drug-induced liver injury (DILI) is a significant cause of anti-tubercular therapy failure. Rifampicin and isoniazid co-treatment lead to cholestatic liver injury associated with the accumulation of protoporphyrin IX (PPIX), a heme precursor, in a human pregnane X receptor (hPXR)–dependent manner in mice. Therefore, the activation of hPXR by rifampicin can lead to DILI via PPIX accumulation; thus, antagonism of hPXR can prevent the clinical consequences of DILI. This project aims to evaluate a method of preventing DILI and to increase the understanding of the mechanisms of DILI. Upon completion of this project, I will elucidate a novel role of isoniazid in the development of DILI and provide an evaluation of a novel therapeutic agent in reducing the pathologic markers associated with DILI. This information may eventually be used to reduce the incidence and extent of anti-tubercular DILI in humans. Using an hPXR transgenic mouse model and primary human hepatocytes, I will evaluate the potential of a novel small-molecule inhibitor of hPXR that was developed in our laboratory to prevent liver injury associated with rifampicin and isoniazid treatment. PPIX accumulation will be assessed by LC/MS/MS in mice; hPXR target gene expression will be evaluated by performing western blot and qRT-PCR analyses of human primary cells and humanized mice. Liver injury will be evaluated by analyzing mouse liver enzyme activity in sera and via histopathologic examination. ALAS1/Alas1 (rate-limiting enzyme of heme synthesis) is induced in response to rifampicin and isoniazid (a ligand of hPXR) in both hPXR transgenic mice and primary human hepatocytes. This induction is modulated by co-treatment with an antagonist of hPXR. PPIX is increased in the liver of hPXR mice treated with both rifampicin and isoniazid. FECH/Fech is degraded in response to isoniazid. This degradation may be mediated by iron chelation of a metabolite of isoniazid. Using western blot analysis of primary human hepatocytes and human cancer cell lines ectopically overexpressing FECH, I will evaluate the potential role of an isoniazid metabolite in the degradation of FECH. Antagonism of hPXR with a small molecule after early detection of liver injury may be a viable therapeutic strategy to reduce the induction of ALAS1 and subsequent accumulation of PPIX resulting in liver injury. The successful completion of this project by 1 graduate student with 3+ years of experience should require approximately 1 year to complete and provide experience in the fields of cell biology, molecular biology, and pathology.