■药物诱导的肝损伤(DILI)是临床试验和上市后引入期间药物开发失败的主要原因。目前的生物标志物,如ALT和AST,缺乏准确检测所需的特异性和灵敏度。外泌体,保护LncRNAs免受RNase降解,可以为生物标志物提供可靠和容易获得的选择。
■RNA测序用于鉴定差异表达的LncRNAs(DE-LncRNAs),然后在这项研究中从血浆外泌体中分离LncRNAs。通过透射电子显微镜(TEM)进行外泌体表征,纳米粒子跟踪分析(NTA),和蛋白质印迹(WB)。生物信息学分析包括功能富集和共表达网络分析。建立5个大鼠模型,并采用实时定量PCR方法验证两种外泌体LncRNAs的特异性和敏感性。
■成功建立了APAP诱导的肝细胞损伤模型,用于RNA测序,导致几个差异表达的外泌体LncRNAs的鉴定。选择8个上调的外泌体DE-LncRNA用于验证。其中,NONRATT018001.2(p<0.05)和MSCRG.73954.4(p<0.05)显示表达水平增加超过2倍。在肝细胞损伤和肝内胆汁淤积模型中,与血清生物标志物ALT和AST相比,NONRATT018001.2和MSCRG.73954.4均显示出更早的增加。然而,直到最后的时间点才观察到组织学变化.在脂肪肝模型中,NONRATT018001.2和MSTRG.73954.4在21天比ALT和AST更早增加。到第七天,肝脏组织有轻微的脂肪变性,而两个候选外泌体LncRNAs的表达水平超过2和4倍,分别。在肝纤维化模型中,NONRATT018001.2和MSTRG.73954.4在每个时间点都显示出增加。到了第49天,在肝组织中观察到肝细胞坏死和纤维化,与NONRATT018001.2显示增加8倍以上。使用心肌损伤模型验证了鉴定的外泌体DE-LncRNA的特异性,并且它们在病例组和对照组之间没有显着差异。
■NONRATT018001.2和MSTRG.73954.4具有作为生物标志物的潜力,用于区分药物引起的不同类型的器官损伤,特别是能够早期预测肝损伤。进一步的实验,如siRNA干扰或基因敲除,有必要探索这些LncRNAs的潜在机制。
UNASSIGNED: Drug-induced liver injury (DILI) is a leading cause of drug development failures during clinical trials and post-market introduction. Current biomarkers, such as ALT and AST, lack the necessary specificity and sensitivity needed for accurate detection. Exosomes, which protect LncRNAs from RNase degradation, could provide reliable and easily accessible options for biomarkers.
UNASSIGNED: RNA-sequencing was used to identify differentially expressed LncRNAs (DE-LncRNAs), followed by isolation of LncRNAs from plasma exosomes in this study. Exosome characterization was conducted by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot (WB). Bioinformatics analysis included functional enrichment and co-expression network analysis. Five rat models were established, and quantitative real-time
PCR was used to verify the specificity and sensitivity of two candidate exosomal LncRNAs.
UNASSIGNED: The APAP-induced hepatocellular injury model was successfully established for RNA-sequencing, leading to the identification of several differentially expressed exosomal LncRNAs. Eight upregulated exosomal DE-LncRNAs were selected for validation. Among them, NONRATT018001.2 (p < 0.05) and MSTRG.73954.4 (p < 0.05) exhibited a more than 2-fold increase in expression levels. In hepatocellular injury and intrahepatic cholestasis models, both NONRATT018001.2 and MSTRG.73954.4 showed earlier increases compared to serum biomarkers ALT and AST. However, no histological changes were observed until the final time point. In the fatty liver model, NONRATT018001.2 and MSTRG.73954.4 increased earlier than ALT and AST at 21 days. By the 7th day, minor steatosis was evident in liver tissue, while the expression levels of the two candidate exosomal LncRNAs exceeded 2 and 4 times, respectively. In the hepatic fibrosis model, NONRATT018001.2 and MSTRG.73954.4 showed increases at every time point. By the 49th day, hepatocellular necrosis and fibrosis were observed in the liver tissue, with NONRATT018001.2 showing an increase of more than 8 times. The specificity of the identified exosomal DE-LncRNAs was verified using a myocardial injury model and they showed no significant differences between the case and control groups.
UNASSIGNED: NONRATT018001.2 and MSTRG.73954.4 hold potential as biomarkers for distinguishing different types of organ injury induced by drugs, particularly enabling early prediction of liver injury. Further experiments, such as siRNA interference or gene knockout, are warranted to explore the underlying mechanisms of these LncRNAs.