A Dual Coverage Monitoring of the Bile Acids Profile in the Liver-Gut Axis throughout the Whole Inflammation-Cancer Transformation Progressive: Reveal Hepatocellular Carcinoma Pathogenesis

Abstract

Hepatocellular carcinoma (HCC) is the terminal phase of multiple chronic liver diseases, and evidence supports chronic uncontrollable inflammation being one of the potential mechanisms leading to HCC formation. The dysregulation of bile acid homeostasis in the enterohepatic circulation has become a hot research issue concerning revealing the pathogenesis of the inflammatory-cancerous transformation process. We reproduced the development of HCC through an N-nitrosodiethylamine (DEN)-induced rat model in 20 weeks. We achieved the monitoring of the bile acid profile in the plasma, liver, and intestine during the evolution of "hepatitis-cirrhosis-HCC" by using an ultra-performance liquid chromatography-tandem mass spectrometer for absolute quantification of bile acids. We observed differences in the level of primary and secondary bile acids both in plasma, liver, and intestine when compared to controls, particularly a sustained reduction of intestine taurine-conjugated bile acid level. Moreover, we identified chenodeoxycholic acid, lithocholic acid, ursodeoxycholic acid, and glycolithocholic acid in plasma as biomarkers for early diagnosis of HCC. We also identified bile acid-CoA:amino acid N-acyltransferase (BAAT) by gene set enrichment analysis, which dominates the final step in the synthesis of conjugated bile acids associated with the inflammatory-cancer transformation process. In conclusion, our study provided comprehensive bile acid metabolic fingerprinting in the liver-gut axis during the inflammation-cancer transformation process, laying the foundation for providing a new perspective for the diagnosis, prevention, and treatment of HCC.

Keywords: BAAT; bile acids; enterohepatic circulation; hepatocellular carcinoma; inflammation-cancer transformation process; liver–gut axis.

Figure 1

Histological examination of the liver in DEN-induced HCC rats during the inflammation-cancer transformation process. (A) The healthy control group showed a normal arrangement of liver cells and a regular structure of liver lobules. (B–E) Representative micrographs of liver tissue sections at various carcinogenic stages (H & E stain, ×200).

Figure 2

The variations of TBA, total primary BAs, and total secondary BAs in different specimens of DEN-induced HCC rats at healthy, hepatitis, cirrhosis, HCC, and advanced cancer stages. (A) TBA concentrations were significantly higher than blank controls in plasma, liver, and intestinal contents samples, and positively correlated with disease progression in plasma and liver. (B) The level change of total primary BAs was basically identical between plasma, liver, and intestinal contents, which were significantly elevated at all stages. (C) Total secondary BA levels were elevated in plasma and intestinal contents samples while significantly decreased in liver samples over the full course of the disease. The figure indicates statistical significance compared with healthy controls by t-tests. * p < 0.05, ** p < 0.01.

Figure 3

Significant changes in the composition of the free bile acid pool in DEN-induced HCC model rats. Hydrophobic BAs were elevated, and hydrophilic BAs were downregulated in the liver (B) and intestinal contents (C). In addition, plasma (A) was elevated except for LCA. The figure indicates statistical significance compared with healthy controls by t-tests, * p < 0.05, ** p < 0.01.

Figure 4

The changes of conjugated BAs in the plasma and liver of DEN-induced model rats were approximately the same as those of prototype BAs. However, the trend in intestinal contents was the opposite, where all taurine-conjugated BAs were significantly reduced. (A–E): Distribution of glycio-conjugated BAs in three samples. (F–J): Distribution of taurine-conjugated BAs in three samples. The figure indicates statistical significance compared with healthy controls by t-tests, * p < 0.05, ** p < 0.01.

Figure 5

(A–C): PCA score plots and (D–F): OPLS-DA score plots of plasma, liver, and intestinal contents samples. In the PCA score plot, the distance between points indicates the difference between samples. The fact that the disease groups are clustered together and separated from the healthy groups indicates that the different groups in the experiment can be well distinguished from each other. R²X and R²Y in the OPLS-DA score plot indicate the explanation rate of the proposed model for the X and Y matrices, respectively, and Q² marks the predictive power of the model. Usually, the values of R²X, R²Y, and Q² are higher than 0.5 can indicate that the model fits with acceptable accuracy.

Figure 6

PPI network construction for 125 enriched genes using Cytoscape. The lines in the network diagram represent the connectivity of each gene, and the color represents their scores, with darker nodes representing higher scores and higher rankings. BAAT is the top-ranked most critical node gene based on the MCC algorithm, located in the center of the network diagram.