ABSTRACT
Background: 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. Materials and methods: 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. Results: 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. Conclusion: 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.
ABSTRACT
The thymus, the central immune organ in mammals, plays an important role in immune defense. Porcine reproductive and respiratory syndrome virus (PRRSV) infection in piglets can cause thymus injury and immunosuppression. However, the mechanisms of thymus injury remain unknown. This study was aimed at investigating the specific manifestations of thymus injury through the construction of a PRRSV-infected piglet model and histopathological observation. In this study, fourteen 40-day-old PRRSV-free piglets were randomly divided into two groups, eleven of which were intramuscularly injected with 3 mL of PRRSV WUH3 virus suspension (106 PFU /mL) in the infection group, and three of which were sham-inoculated with 3 mL of RPMI-1640 medium in the control group. Clinical necropsy and samples collection were performed on day 8 after artificial infection. With the Illumina platform, the transcriptomes of piglet thymus tissues from infected and control piglets were sequenced to explore the relationships of differentially expressed genes (DEGs) and signaling pathways with thymus injury. The immune organs of PRRSV-infected piglets were severely damaged. The histopathological findings in the thymus indicated that PRRSV infection was associated with a large decrease in lymphocytes, cell necrosis and cell apoptosis; an increase in blood vessels and macrophages; thymic corpuscle hyperplasia; and interstitial widening of the thymic lobules. The transcriptomic analysis results revealed that the Gene Ontology functions of DEGs were enriched primarily in biological processes such as angiogenesis, regulation of angiogenesis and positive regulation of cell migration. Moreover, greater numbers of blood vessels and macrophages were observed in the thymus in PRRSV-infected than control piglets. KEGG pathway enrichment analysis revealed that the DEGs were significantly enriched in the Toll-like receptor signaling pathway, chemokine signaling pathway, IL-17 signaling pathway and TNF signaling pathway. The expression of TLR8, IRF5, the chemokines CCL2, CCL3L1 and CCL5; and their receptors CCR1, CCR2 and CCR5 was significantly up-regulated in PRRSV infection, thus suggesting that these cytokines were associated with the pathological processes of thymus injury.
