Metabolic Profiling of Praziquantel-mediated Prevention of Opisthorchis viverrini-induced Cholangiocyte Transformation in the Hamster Model of Cholangiocarcinoma

Abstract

Background: Opisthorchis viverrini (Ov) infection-induced cholangiocarcinoma (CCA) is a major public health problem in northeastern Thailand. Praziquantel was shown to prevent CCA development in an Ov-infected hamster model; however, the molecular mechanism remains unknown.

Materials and methods: In this study, we used a hamster model with Ov and N-nitrosodimethylamine-induced CCA to study the mechanisms of praziquantel action. The liver tissues from the hamsters with and without praziquantel treatment were analyzed using ¹H nuclear magnetic resonance spectroscopy.

Results: A total of 14 metabolites were found to be significantly different between the two groups. Furthermore, the combination of acetate, inosine and sarcosine was shown to exert an anti-inflammatory effect through interleukin-6 inhibition in a macrophage cell line, suggesting a mechanism by which praziquantel may prevent inflammation caused by Ov, cholangiocyte transformation and further CCA develpoment.

Conclusion: These findings might avail the development of a preventive strategy for CCA in high-risk populations.

Keywords: Praziquantel; anti-inflammation; cholangiocarcinoma; metabolic profiling; nuclear magnetic resonance spectroscopy..

Figure 1. Experimental design of the Opisthorchis viverrini (Ov) infection in the hamster model of N-nitrosodimethylamine (NDMA)- induced cholangiocarcinoma. ON: Ov+NDMA, ONP: Ov+NDMA+praziquantel, PZ: praziquantel, M: months.

Figure 2. Histopathological changes in hamster liver tissues. A: Representative images of histological changes in hamsters with Opisthorchis viverrini and N-nitrosodimethylamine-induced cholangiocarcinoma treated with (ONP) and without praziquantel (ON). B: Bile duct alteration, inflammation and fibrosis scores of the ON and ONP groups. Significantly different at *p<0.05, **p<0.01 and ***p<0.001, respectively, using Student’s t-test.

Figure 3. Principal component analysis (PCA) score plots for liver tissues from untreated (ON; red) and praziquantel-treated (ONP; blue) hamsters with N-nitrosodimethylamine-induced cholangiocarcinoma for all months (A), and at (B) 2 months, (C) 3 months, (D) 4 months and (E) 6 months. Q² represents the predictive ability of the model. The percentage on each PC represents the variance explained by each.

Figure 4. Orthogonal partial least squares discriminant analysis (O-PLS-DA) cross-validated score plot (left) and the loading plot (right) for hamster liver tissues. The O-PLS-DA cross-validated score plot and the O-PLS-DA loading plot demonstrate the significant metabolites contributing to the separation between hamsters with Opisthorchis viverrini and N-nitrosodimethylamine-induced cholangiocarcinoma treated with (ONP) and without praziquantel (ON) considering all months (A) and at 6 months (B), respectively. Peaks pointing upwards represent relatively high levels of metabolites in the ON group, whereas peaks pointing downwards represent relative low levels of metabolites in the ON group. Tosc: T orthogonal signal correction; Tcv: T cross validation.

Figure 5. The relative concentrations of significantly changed metabolites in liver tissues from untreated (ON) and praziquantel-treated (ONP) hamsters with N-nitrosodimethylamine-induced cholangiocarcinoma. The maximum intensities of the significant metabolites from multivariate statistics were analyzed by univariate statistics from entire model (A) and that for 6 months (B). Significantly different at *p<0.05, **p<0.01 and ***p<0.001, respectively.

Figure 6. Orthogonal partial least squares (O-PLS)-regression analysis. O-PLS-regression loading plot of the histopathological results, including severity of bile duct alteration (A), inflammation (B) and fibrosis (C).

Figure 7. A biological network analysis of metabolic profiling of hamster liver tissues. All significant metabolites were mapped onto the Kyoto Encyclopedia of Genes and Genomes (KEGG) database for the biological network analysis using MetaboNetworks software.

Figure 8. The anti-inflammatory effect of a combination of inosine, sarcosine and acetate on the prevention of cholangiocarcinoma development. A: Western blot analysis demonstrated the reduction of expression of interleukin-6 (IL6) after treatment with the combined metabolites in the U937 macrophage cell line. B: Relative expression level of IL6 after treatment with the combined metabolites in macrophage cells. C: IL6 level in culture media after treatment with combined metabolites. Significantly different at *p<0.05, **p<0.01 and ***p<0.001, respectively, using Student’s t-test.