Abstract
BACKGROUND: Clonorchiasis, a neglected tropical zoonosis, is caused by chronic infection with Clonorchis sinensis (C. sinensis). This infection can lead to cholangitis, bile duct epithelial hyperplasia, periductal fibrosis, and cholangiocarcinoma (CCA). However, the underlying carcinogenic mechanisms of CCA remain poorly understood, and there is not a well-developed model for C. sinensis CCA. METHODS: A C. sinensis-infected Sprague-Dawley rat model, co-treated with N-nitrosodimethylamine, was established. The study comprised four groups: negative control (NC), C. sinensis infection (CS), N-nitrosodimethylamine induction (NDMA), and combined C. sinensis infection and N-nitrosodimethylamine induction (CSNDMA). Pathological damage to the hepatic ducts was evaluated at 10, 17, and 20 weeks after infection. The expression levels of the relevant genes and proteins were detected using quantitative real-time polymerase chain reaction (qPCR) and immunohistochemistry, respectively. In addition, transcriptome sequencing was carried out on hepatic tissues infected for 20 weeks. RESULTS: Histopathological analysis using hematoxylin and eosin (H&E) and Masson staining revealed bile duct dilation, inflammatory infiltration, and collagen deposition in the liver tissue of both CS and CSNDMA groups, with the most severe manifestations observed in the CSNDMA group. The CSNDMA group exhibited the earliest onset of CCA, occurring at 10 weeks post infection, with an overall incidence of 63%, peaking at 71% by 20 weeks. The CS group showed a 37% incidence of CCA, while only one case was noted in the NDMA group at 20 weeks. Quantitative PCR demonstrated that C. sinensis infection induced upregulation of tumor-related markers in the liver, including CK19, PCNA, TP53, ITGB1, and MMP2, particularly when co-exposed to N-nitrosodimethylamine. Immunohistochemistry detected the significant overexpression of CK19, CK7, and PCNA along bile ducts. Transcriptome sequencing further indicated that C. sinensis significantly affected circadian rhythm and metabolic reprogramming in the liver, enriching pathways related to cancer, inflammation, and metabolism, including AMPK, PPAR, mTOR, and FoxO pathways. CONCLUSIONS: C. sinensis can effectively promote the pathogenesis of CCA and significantly increase the expression of CCA-related genes (e.g., CK19 and CK7). The inflammation, disrupting circadian rhythms and altering energy metabolism caused by C. sinensis infection, may promote the progression of CCA. This study provides a foundational experimental basis for diagnosing and intervening in C. sinensis-related CCA.