外源性化学物修饰PDC-E2硫辛酰区在原发性胆汁性胆管炎发病中的作用机制研究

Primary biliary cirrhosis (PBC) was one of the common autoimmune liver diseases. The characteristic autoantibody (anti-mitochondrial antibody) mainly recognized the lipoyl domain within E2 subunit of pyruvate dehydrogenase complex (PDC-E2LD), which was vulnerable to external environmental factors as it was also the active center of the enzyme, especially the lipoic acid (LA) group on the side chain of the lysine residue (aa259K). Our preliminary investigation had found that the replacement of this LA with 2-octynic acid (2OA) might be one of the primary pathogenic mechanisms for PBC. In this study, this LA on aa259K will be only modified by 2OA or N-acetyl-p-benzoquinoneimine (NAPQI) with intein-mediated protein ligation. The conformational changes on peptide chain of PDC-E2LD induced by the modifications and replacements will be comparatively tested with electron paramagnetic resonance and the inhibitive ELISA will be employed to detect the antibodies in PBC sera against the new antigen epitopes due to the conformational changes. These antibodies will be purified and identified and their IgG/IgM types will be determined, and also, the possible cross immune reactions among them will be explored. These results, combined with the clinical documents including the distribution characters of the disease staging, will demonstrate that different external chemical xenobiotics (including some drug metabolites such as NAPQI) might lead to the break of immune tolerance to PDC-E2 by various modification ways, which might alter the backbone structure of PDC-E2LD in similar way to very likely produce the across immunity among them. This study will demonstrate the pathogenic roles and the mechanisms of the environmental chemical xenobiotics in the onset of PBC.

原发性胆汁性胆管炎(PBC)是常见自身免疫性肝病。特征性抗线粒体抗体主要识别PDC-E2硫辛酰区(PDC-E2LD),该区也是酶的活性中心,易受外环境因素影响,特别是该区aa259K侧链的硫辛酰基(LA)。我们初步研究发现2-辛炔酸(2OA)替代此LA可能诱导PBC发病。本研究利用intein介导蛋白连接使2OA及N-乙酰-对-苯醌亚胺(NAPQI)分别只修饰此LA,与2OA替代LA及正常比较,电子顺磁共振检测不同修饰后PDC-E2LD蛋白构象变化,PBC血清中探索识别这些构象变化诱导的特异抗体,纯化并鉴定这些抗体,检测它们的IgG/IgM类型及交叉免疫,结合它们临床病期分布特点分析,阐明不同化合物(包括药物代谢产物NAPQI)对aa259K的不同修饰方式,可能通过相似的蛋白构象改变产生交叉抗原新表位打破PDC-E2免疫耐受,从而揭示外环境中不同化合物在PBC发病中的可能作用及其机制。

原发性胆汁性胆管炎（PBC）特征性抗线粒体抗体（AMA）主要识别PDC-E2的内硫辛酰区（ILD），其aa259K侧链的硫辛酰基（LA）容易被外源性化合物修饰或替代，可能诱发PBC。我们之前的研究显示日用品中常用添加剂2-辛炔酸（2OA）替代LA可诱导PBC发病。N-乙酰-对-苯醌亚胺（NAPQI）是扑热息痛的中间代谢物，可能参与扑热息痛相关的PBC肝损。我们利用intein介导体外蛋白连接技术，获得aa259K被4个不同化合物修饰的ILD：LA-ILD（天然ILD）、2OA-ILD（2OA替代LA的ILD）、NAPQI-LA-ILD和2OA-LA-ILD（分别为NAPQI和2OA修饰LA后的ILD），确认它们仍保留被AMA识别的天然抗原性，电子顺磁共振（EPR）检测显示与天然LA-ILD比较，3种修饰的ILD蛋白主链构象发生相似改变，且在部分PBC的血中发现特异抗体只识别被修饰的ILD，提示修饰ILD导致构象改变可成为部分患者的新抗原表位。纯化3种特异抗体，它们不识别天然LA-ILD，但识别各自修饰的ILD，且均以IgM型为主，多存在于PBC早期，而识别ILD的自身抗体以IgG亚型为主，存在于PBC各期。结论：常用日用品添加剂2OA及扑热息痛代谢产生的亲电子化合物NAPQ作为外环境化合物，可通过修饰LA-ILD中aa259K侧链的LA，产生新的抗原构象表位，诱导自体免疫，再经表位扩展机制等导致对PDC-E2免疫失耐受，最终导致PBC发病。本结果对临床通过控制某些外源性化合物及药物防治PBC提供科学依据。利用本课题研究机会，我们初步研究还发现，2OA在结缔组织病伴发PBC及原发性PBC发病中作用的重要性是不同的；分子结构环化的ILD对AMA-E2阴性的PBC患者潜在临床诊断价值；JAK/STAT信号通路参与PBC发病，JAK1/2抑制剂潜在PBC治疗价值。