Bile acid metabolism regulated by the gut microbiota promotes non-alcoholic steatohepatitis-associated hepatocellular carcinoma in mice

Abstract

Gut microbiota plays a significant role in the development of hepatocellular carcinoma (HCC) in non-alcoholic steatohepatitis (NASH). However, understanding of the precise mechanism of this process remains incomplete. A new class steatohepatitis-inducing high-fat diet (HFD), namely STHD-01, can promote the development of HCC without the administration of chemical carcinogens. Using this diet, we comprehensively analyzed changes in the gut microbiota and its metabolic functions during the development of HCC in NASH. Mice fed the STHD-01 developed NASH within 9 weeks. NASH further progressed into HCC by 41 weeks. Treatment with antibiotics significantly attenuated liver pathology and suppressed tumor development, indicating the critical role of the gut microbiota in tumor development in this model. Accumulation of cholesterol and bile acids in the liver and feces increased after feeding the mice with STHD-01. Treatment with antibiotics did not reverse these phenotypes. In contrast, accumulation of secondary bile acids was dramatically reduced after the treatment with antibiotics, suggesting the critical role of the gut microbiota in the conversion of primary bile acids to secondary bile acids. Secondary bile acids such as deoxycholic acid activated the mTOR, pathway in hepatocytes. Activation of mTOR was observed in the liver of mice fed STHD-01, and the activation was reduced when mice were treated with antibiotics. Collectively, bile acid metabolism by the gut microbiota promotes HCC development in STHD-01-induced NASH.

Keywords: bile acid metabolites; deoxycholic acid; hepatocellular carcinoma; mTOR.

Figure 1. Body characteristics and enzyme-related metabolism changed after feeding of STHD-01

(A) Experimental protocol. (B) Body weight, total calorie intake, liver/body weight, and plasma triiodothyronine (T3) and thyroxin (T4) levels were measured at 41 weeks post STHD-01 feeding. Data are shown as mean ± SD (CONT, n = 5; STHD-01, n = 9; STHD-01 + Abx, n = 7). ^**p < 0.01, ^*** p < 0.001 by Tukey’s test.

Figure 2. STHD-01 promotes the development of NASH-associated HCC through induction of gut dysbiosis

(A) The total number of hepatocellular carcinomas (Top), whole liver tissue images (Middle), and representative histology of the liver (Bottom) in each group are shown. (B) The plasma level of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and the mRNA transcription of Tnf-α, Il-1β, α-SMA, Col1α1, Tgf-β in the liver tissue were measured. (A, B) Data are presented as mean ± SD (CONT, n = 5; STHD-01, n = 9; STHD-01 + Abx, n = 7). ^*p < 0.05, ^**p < 0.01, ^*** p < 0.001 by Tukey’s test. (C) Fecal samples were collected from each individual mouse (CONT, n = 5; STHD-01, n = 9; STHD-01 + Abx, n = 7) at 41 weeks post STHD-01 feeding. Fecal samples within the same group of mice were then pooled and the gut microbiome was analyzed by T-RFLP. The abundance of bacterial genus is shown. The total number of bacteria is given at the top of each column.

Figure 3. Bile acid synthesis from cholesterol was up-regulated upon the feeding of STHD-01

(A) Pathway of bile acid synthesis from cholesterol and related enzymes are shown. (B) Total bile acid concentration in plasma, feces, and liver. Liver samples were collected from all mice in each group and pooled. Data are expressed as mean ± SD (CONT, n = 5; STHD-01, n = 9; STHD-01 + Abx, n = 7). ^**p < 0.01, ^***p < 0.001 by Tukey’s test.

Figure 4. The alternation of bile acids reabsorption transporters by feeding of STHD-01

(A) Transporters and transport-proteins related to bile acid secretion/reabsorption into/from the plasma and bile duct are shown. (B) The mRNA transcription level of bile acid transporters in the liver and ileum were analyzed. The fold expression relative to 9 week control mice are shown. Data are expressed as mean ± SD (CONT, n = 5; STHD-01, n = 9; STHD-01 + Abx, n = 7). ^*p < 0.05, ^**p < 0.01 by Tukey’s test.

Figure 5. Changes in the composition of bile acids in the liver and feces after feeding of STHD-01

The composition of bile acids in the liver (left) and feces (right) at 9 weeks (A) and 41 weeks (B) post STHD-01 feeding. Top panel shows the concentration of the total bile acid in each group. Middle panel shows the percent abundance of different primary and secondary bile acids within the total bile acid. Bottom panel shows the percent abundance of different secondary bile acids within the whole pool of secondary bile acids. Sample from all the mice in the same group were pooled and analyzed.

Figure 6. Abundance of secondary bile acids in the liver was regulated by the gut microbiota

(A) Bile acid metabolic pathways. (B) A heat-map of the abundance of detected secondary bile acids in the liver. Sample from all the mice in the same group were pooled and analyzed.

Figure 7. DCA activated the mTOR signaling in the HepG2 cells

(A) Viability of HepG2 cells after stimulation with primary and secondary bile acids (n = 3). (B) Phosphorylation of mTOR in HepG2 cells after the stimulation by primary and secondary bile acids (CA, CDCA n = 3; DCA n = 5). (C) Phosphorylation of mTOR in HepG2 cells after the stimulation by the different concentrations of DCA (n = 5). Data are presented as mean ± SD. ^*p < 0.05 ^**p < 0.01 by the Tukey’s test.

Figure 8. Elevated mTOR activation in the liver in mice fed the STHD-01

(Top) The liver was harvested from mice at 41 weeks post STHD-01 feeding. The activation of mTOR-related pathways was analyzed by western blotting (CONT, n = 16; STHD-01, n = 16; STHD-01 + Abx, n = 7). The representative blots are shown. (Bottom) The quantification of western blot analysis. Data are presented as mean ± SD (CONT, n = 16; STHD-01, n = 16; STHD-01 + Abx, n = 7). ^*p < 0.05, ^** p < 0.01 , ^***p < 0.001 by Tukey’s test.