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 联合治疗导致胆汁淤积性肝损伤与原卟啉IX(PPIX)、a 血红素前体，以人孕烷X受体(HPXR)依赖的方式在小鼠。因此， 利福平激活hPXR可通过PPIX积聚导致DILI；因此，拮抗hPXR可以 预防帝力的临床后果。该项目旨在评估预防DILI的方法，并 增加对帝力作用机制的认识。在这个项目完成后，我将阐明一部小说 异烟肼在DILI发生发展中的作用及对一种新的治疗药物的评价 与DILI相关的病理标志物。这些信息最终可能被用来减少发病率。 以及在人类体内的抗结核作用范围。使用hPXR转基因小鼠模型和原代人类 肝细胞，我将评估我们开发的一种新型hPXR小分子抑制剂的潜力。 防止与利福平和异烟肼治疗相关的肝损伤的实验室。PPIX累积将是 用LC/MS/MS对小鼠进行鉴定；hPXR靶基因的表达将通过蛋白质印迹进行鉴定 人原代细胞和人源化小鼠的qRT-PCR分析。肝脏损伤将通过以下方式进行评估 分析小鼠血清中的肝酶活性，并进行组织病理学检查。ALAS1/ALAS1(速率限制 在两种hPXR中，利福平和异烟肼(hPXR的一种配体)都能诱导 转基因小鼠和原代人肝细胞。这种诱导是通过与一种 HPXR的拮抗剂。利福平和异烟肼同时治疗的hPXR小鼠肝脏中PPIX增加。 异烟肼可使FECH/FECH降解。这种降解可能是由一种铁络合物介导的。 异烟肼的代谢产物。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物在降解中的潜在作用 FECH的。早期发现肝损伤后用小分子拮抗hPXR可能是可行的 减少ALAS1诱导和随后导致肝脏的PPIX积聚的治疗策略 受伤。由1名具有3年以上工作经验的研究生成功完成此项目应 需要大约一年的时间来完成并提供细胞生物学、分子生物学、 和病理学。

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.