Initial assessment of potential carcinogenic constituents in the excretory and secretory products of Clonorchis sinensis

Background Clonorchis sinensis (Cs) is a carcinogenic liver fluke in humans. The excretory-secretory products(ESPs) play a pivotal role in the carcinogenic process. However, its precise composition and the specific targets through which it exerts its effects remain unclear. Therefore, the identification of carcinogenic components and their molecular targets in ESPs is instrumental in elucidating the specific molecular pathways underlying Cs-induced cholangiocarcinoma, thereby providing novel insights for targeted therapy. Methods Analysis the potential candidate proteins with carcinogenic effects in the excretory and secretory products of Clonorchis sinensis (CsESPs) based on bioinformatics methods. The candidate gene was obtained from the adult of Cs by the reverse transcriptase-polymerase chain reaction (RT-PCR) method, and molecular cloning to prokaryotic expression plasmid pET-28a by recombination strategy. The prokaryotic expression plasmid pET-28a-CsCTSB was identified by restriction enzyme digestion and sequenced. The pET-28a-CsCTSB was transformed into E. coli BL21 (DE3) by the CaCl ₂ transformation method. After purification and renaturation, the recombinant protein was used for further experimental research. An in vivo animal model of Cs infection was established, and the CsESPs were obtained by differential centrifugation. H&E staining was performed to test the pathological changes in hepatobiliary tissues after Cs infection. The expression of cathepsin B (CTSB) in hepatobiliary tissues and the expression of sphingosine 1-phosphate receptor 2 (S1PR2) in cholangiocarcinoma cells were detected by Western blotting assay. Immunohistochemical staining was performed to test the expression of S1PR2 in hepatobiliary tissues. The interaction between CsCTSB and S1PR2 were predicted and analyzed by bioinformatics methods. Results CTSB was predicte as a candidate protein with carcinogenic effects in the CsESPs by bioinformatics analysis. The recombinant CsCTSB was expressed in E. coli BL21, induced with IPTG, purified via Ni-NTA affinity chromatography, and confirmed by SDS-PAGE and Western blot. A single band of approximately 37 kDa was observed at the expected position by SDS-PAGE, and its activity was evaluated by enzymology ( P  < 0.001). After Cs infection, significant pathological changes showed in the hepatobiliary tissues, and the expression of CTSB was significantly increased ( P  < 0.05). Both immunohistochemical and Western blotting confirmed that the expression of S1PR2 was significantly increased in the hepatobiliary tissues infected by Cs and in cholangiocarcinoma cells stimulated by CsESPs. Bioinformatics analysis showed that there might be an interaction region between CsCTSB and S1PR2. Conclusions This study suggests that CTSB in CsESPs may be one of the effective components with carcinogenic effects. Based on the expression patterns of CTSB and S1PR2 in the hepatobiliary tissues of mice and cells infected with Cs, as well as the bioinformatics prediction analysis, CTSB and S1PR2 may jointly promote the hepatobiliary injury and even carcinogenesis induced by Cs.