基于LncRNA介导的肝窦内皮细胞线粒体功能障碍探讨吡咯里西啶生物碱的肝毒性机制

Pyrrolizidine alkaloids (PAs) are a type of natural hepatotoxic compounds and new insights into the pathogenesis of PAs-induced hepatotoxicity are urgently needed. Increasing amounts of evidence have indicated that long noncoding RNAs (LncRNAs) have important regulatory potential in drug-induced liver injury. However, the contribution of LncRNAs to Chinese herbal medicines-induced liver injury remains unknown. Our previous studies showed that the expression level of LncRNA H19 and ASncmtRNA2, which play the key role in regulating mitochondrial functions, was higher in sinusoidal endothelial cells (SECs) than Kupffer cells and hepatocytes. Furthermore, PAs inhibited the expression of H19 and up-regulated ASncmtRNA2 expression. At the same time, PAs induced apoptosis by activation of mitochondrial-mediated apoptotic and ROS pathway in SECs. So we propose a novel hypothesis to elucidate the mechanism of PAs-induced hepatotoxicity by LncRNA regulation of mitochondrial activity in SECs. A co-culture system of SECs and hepatocytes, and PAs-induced toxicity model of mice will be used to describe the multifaceted effects of lncRNAs on mitochondrial function, more particularly on the balance between oxidative phosphorylation and glycolysis, and on the production of reactive oxygen species and apoptosis. At the same time, microarray expression profiles of lncRNAs and mRNAs will be determined in mice treated with PAs. Differential expression, pathway and gene network analyses will be developed to identify possible functional LncRNAs in PAs-induced toxicity. And the serum exosomal-LncRNAs relevant to mitochondrial activity will be investigated. The goal of this project is to contribute to deepen the understanding of PAs-induced hepatotoxicity and to represent a novel non-invasive potential biomarker of PAs-induced hepatotoxicity.

中药中吡咯里西啶生物碱(PAs)肝毒性机制尚未明了，尤其肝窦内皮细胞(SECs)毒性研究较少。LncRNA与疾病关系密切，但尚无LncRNA对中药肝毒性的调控研究。前期实验发现，LncRNA H19/ASncmtRNA2在SECs中表达远高于其它肝细胞，PAs引起SECs线粒体功能异常及H19/ASncmtRNA2表达变化，而H19/ASncmtRNA2能调控线粒体功能，因此本课题提出“LncRNA介导SECs线粒体功能障碍引起PAs肝毒性”假说，以LncRNA为研究主线，在SECs及小鼠PAs毒性模型中考察LncRNA对SECs线粒体功能调控；通过芯片检测、共表达网络分析，寻找SECs损伤特异性LncRNA调控网络；考察血浆外泌体中是否含PAs毒性预警分子的LncRNA。在阐明PAs对SECs更敏感的分子机制同时，为PAs肝毒性早期诊断和治疗提供突破点，为药源性肝损伤机制研究提供新视角。

中药中吡咯里西啶生物碱(PAs)肝毒性机制尚未明了，尤其是肝窦内皮细胞(LSECs)相关毒性研究较少。长链非编码RNA (LncRNA)与肝脏疾病关系密切，本课题开展了LncRNA介导LSECs线粒体功能障碍引起PAs肝毒性研究。本课题以LncRNA为研究主线，在LSECs及小鼠PAs毒性模型中考察LncRNA对LSECs功能调控；通过芯片检测、共表达网络分析，寻找LSECs损伤特异性LncRNA调控网络。进而寻找到下游效应分子脂质运载蛋白2（Lcn2），发现Lcn2可能通过其铁载体功能促进肝脏氧化损伤，通过增加肝脏铁蓄积，加重PAs所致肝损伤；而Lcn2的抗体能够减轻铁蓄积，上调Bmp2/Bmp6/Hamp通路，缓解肝脏凋亡，减轻LSEC损伤。Lcn2属于分泌蛋白，可作为潜在的血清生物标志物预警HSOS的早期病变，指导HSOS的临床诊断与治疗。本项目通过究，完成了项目预期的研究目标，针对PAs肝毒性的复杂性特点，从整体细胞、分子水平进行多层次的深入研究，建立PAs诱导的肝毒性模型，以此客观、全面的考察PAs对肝窦内皮细胞凋亡的影响。研究结果丰富了PAs毒性的传统研究，发现了其潜在治疗靶点和早期毒性预警标志物。课题目前已发表论文6篇，其中SCI收载5篇。培养研究生5名。