Lactobacillus acidophilus suppresses non-alcoholic fatty liver disease-associated hepatocellular carcinoma through producing valeric acid.

BACKGROUND: Gut probiotic depletion is associated with non-alcoholic fatty liver disease-associated hepatocellular carcinoma (NAFLD-HCC). Here, we investigated the prophylactic potential of Lactobacillus acidophilus against NAFLD-HCC. METHODS: NAFLD-HCC conventional and germ-free mice were established by diethylnitrosamine (DEN) injection with feeding of high-fat high-cholesterol (HFHC) or choline-deficient high-fat (CDHF) diet. Orthotopic NAFLD-HCC allografts were established by intrahepatic injection of murine HCC cells with HFHC feeding. Metabolomic profiling was performed using liquid chromatography-mass spectrometry. Biological functions of L. acidophilus conditional medium (L.a CM) and metabolites were determined in NAFLD-HCC human cells and mouse organoids. FINDINGS: L. acidophilus supplementation suppressed NAFLD-HCC formation in HFHC-fed DEN-treated mice. This was confirmed in orthotopic allografts and germ-free tumourigenesis mice. L.a CM inhibited the growth of NAFLD-HCC human cells and mouse organoids. The protective function of L. acidophilus was attributed to its non-protein small molecules. By metabolomic profiling, valeric acid was the top enriched metabolite in L.a CM and its upregulation was verified in liver and portal vein of L. acidophilus-treated mice. The protective function of valeric acid was demonstrated in NAFLD-HCC human cells and mouse organoids. Valeric acid significantly suppressed NAFLD-HCC formation in HFHC-fed DEN-treated mice, accompanied by improved intestinal barrier integrity. This was confirmed in another NAFLD-HCC mouse model induced by CDHF diet and DEN. Mechanistically, valeric acid bound to hepatocytic surface receptor GPR41/43 to inhibit Rho-GTPase pathway, thereby ablating NAFLD-HCC. INTERPRETATION: L. acidophilus exhibits anti-tumourigenic effect in mice by secreting valeric acid. Probiotic supplementation is a potential prophylactic of NAFLD-HCC. FUNDING: Shown in Acknowledgments.

Lactobacillus gallinarum modulates the gut microbiota and produces anti-cancer metabolites to protect against colorectal tumourigenesis.

OBJECTIVE: Using faecal shotgun metagenomic sequencing, we identified the depletion of Lactobacillus gallinarum in patients with colorectal cancer (CRC). We aimed to determine the potential antitumourigenic role of L. gallinarum in colorectal tumourigenesis. DESIGN: The tumor-suppressive effect of L. gallinarum was assessed in murine models of CRC. CRC cell lines and organoids derived from patients with CRC were cultured with L. gallinarum or Escherichia coli MG1655 culture-supernatant to evaluate cell proliferation, apoptosis and cell cycle distribution. Gut microbiota was assessed by 16S ribosomal DNA sequencing. Antitumour molecule produced from L. gallinarum was identified by liquid chromatography mass spectrometry (LC-MS/MS) and targeted mass spectrometry. RESULTS: L. gallinarum significantly reduced intestinal tumour number and size compared with E. coli MG1655 and phosphate-buffered saline in both male and female murine intestinal tumourigenesis models. Faecal microbial profiling revealed enrichment of probiotics and depletion of pathogenic bacteria in L. gallinarum-treated mice. Culturing CRC cells with L. gallinarum culture-supernatant (5%, 10% and 20%) concentration-dependently suppressed cell proliferation and colony formation. L. gallinarum culture-supernatant significantly promoted apoptosis in CRC cells and patient-derived CRC organoids, but not in normal colon epithelial cells. Only L. gallinarum culture-supernatant with fraction size <3 kDa suppressed proliferation in CRC cells. Using LC-MS/MS, enrichments of indole-3-lactic acid (ILA) was identified in both L. gallinarum culture-supernatant and the gut of L. gallinarum-treated mice. ILA displayed anti-CRC growth in vitro and inhibited intestinal tumourigenesis in vivo. CONCLUSION: L. gallinarum protects against intestinal tumourigenesis by producing protective metabolites that can promote apoptosis of CRC cells.

Black blood T1rho MR imaging may diagnose early stage liver fibrosis: a proof-of-principle study with rat biliary duct ligation model.

BACKGROUND: To explore black blood T1rho (T1ρ) liver imaging and investigate the earliest stage when biliary duct ligation (BDL) induced liver fibrosis can be diagnosed. METHODS: MR was performed at 3 Tesla. A T1ρ prepared 2D fast spin echo (FSE) sequence with acquisition of four spin lock times (TSLs: 1, 10, 30, and 50 msec) and spin-lock frequency of 500 Hz was applied. Inherent black blood effect of FSE and double inversion recovery (DIR) achieved blood signal suppression, and 3 axial sections per liver were obtained. Male Sprague-Dawley rats were scanned at baseline (n=32), and on day-3 (n=13), day-5 (n=11), day-7 (n=10), day-10 (n=4) respectively after BDL. Hematoxylin-eosin (HE) and picrosirius red staining liver histology was obtained at these time points. RESULTS: The physiological liver parenchyma T1ρ was 38.38±1.53 msec (range, 36.05-41.53 msec). Liver T1ρ value elevated progressively after BDL. On day-10 after BDL all experimental animals can be separated from normal liver based on T1ρ measurement with lowest value being 42.82 msec. Day-7 and day-10 liver resembled METAVIR stage-F1/F2 fibrosis, and fibrous area counted for 0.22%±0.13% and 0.38%±0.44% of liver parenchyma area, respectively. CONCLUSIONS: This study provides the first proof-of-principle that T1ρ might diagnose early stage liver fibrosis.

Interaction of adipokines and hepatitis B virus on histological liver injury in the Chinese.

OBJECTIVES: Chronic hepatitis B patients with diabetes and metabolic syndrome are at increased risk of cirrhosis and hepatocellular carcinoma, but the underlying mechanism is unclear. Our objective was to test whether dysregulation of adipokines contributes to liver injury. We also studied whether viral factors affected adipokines, insulin resistance, and hepatic steatosis. METHODS: A prospective cohort of 266 chronic hepatitis B patients undergoing liver biopsy was studied. Fasting blood was taken for the analysis of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), adiponectin, leptin, and resistin. Insulin resistance was assessed by the homeostasis model assessment of insulin resistance (HOMA-IR). Factors associated with significant necroinflammation and cirrhosis were identified. RESULTS: Histological activity index was correlated with serum TNF-alpha (R=0.40, P<0.0001) and IL-6 (R=0.32, P<0.0001) but not with adiponectin, leptin, or resistin. By multivariate analysis, TNF-alpha was associated with significant necroinflammation after adjusting for age and viral factors (odds ratio (OR) 1.041, 95% confidence interval (CI) 1.002-1.082, P=0.04). Serum adiponectin had positive correlation with hepatitis B virus DNA (R=0.17, P=0.007) and was decreased in patients with insulin resistance and hepatic steatosis. On the other hand, viral load, hepatitis B e-antigen status, and genotypes had no association with insulin resistance, hepatic steatosis, and the levels of TNF-alpha and IL-6. A total of 68 (25.6%) patients had cirrhosis. HOMA-IR, but not adipokine dysregulation, was independently associated with cirrhosis (OR 1.09, 95% CI 1.02-1.15, P=0.006). CONCLUSIONS: TNF-alpha and/or IL-6 contribute to hepatic necroinflammation in chronic hepatitis B patients. Adiponectin protects against insulin resistance and hepatic steatosis but does not affect liver injury. Adipokines and viral factors contribute to liver injury independently.

High serum interleukin-6 level predicts future hepatocellular carcinoma development in patients with chronic hepatitis B.

Increased interleukin-6 (IL-6) production is implicated in the pathogenesis of hepatocellular carcinoma (HCC) in animal models. Although previous studies showed that HCC patients had higher serum IL-6 level at the time of diagnosis, it is unclear if the cytokine contributes to the development of HCC or is just a reaction to cancer. To address this question, we performed a nested case-control study. Consecutive chronic hepatitis B patients were recruited from 1997 to 2000 and followed till 2008. Profiling of 27 cytokines, chemokines and growth factors was performed at baseline, date of peak alanine aminotransferase (ALT) level and the last visit. Thirty-seven patients developed HCC at a median follow-up of 62 months (interquartile range: 41-110). Serum IL-6 was higher in patients with HCC than controls both during peak ALT and at the last visit (both p = 0.02). Patients with IL-6 above 7 pg/ml during peak ALT had increased risk of HCC or death (adjusted hazard ratio 3.0; 95% confidence interval 1.2, 7.8; p = 0.02). The sensitivity, specificity, positive and negative predictive values of this cutoff to predict future HCC development were 70%, 73%, 72% and 71%, respectively. Combination of IL-6 and AFP improved the sensitivity in diagnosing HCC or predicting future HCC development. In conclusion, high serum IL-6 level predates the development of HCC in chronic hepatitis B patients, and has moderate accuracy in predicting future cancer. This may assist clinicians in selecting high-risk patients for HCC surveillance program.

Effect of celecoxib on experimental liver fibrosis in rat.

BACKGROUND/AIM: Cyclooxygenase-2 (COX-2), an inducible enzyme that catalyzes prostaglandin synthesis, has been implicated in a number of hepatic stellate cell (HSC) functions. In the current study, we assessed the in vivo effect of celecoxib, a COX-2-selective inhibitor, in experimental liver fibrosis in rats. METHODS: Male Sprague-Dawley rats received experimental treatments for 5 weeks. Serum alanine transminase at the time of sacrifice was measured. Quantitative assessment of liver fibrosis was performed by computerized morphometry. Expression of COX-2, alpha smooth muscle actin and connective tissue growth factor (CTGF) was evaluated by immunohistochemistry. Real-time quantitative PCR was used to determine the expression of genes associated with fibrogenesis and extracellular matrix degradation. RESULTS: Liver fibrosis was significantly worse in rats that received both carbon tetrachloride (CCl4) and celecoxib, compared with rats that received CCl4 and gavage of water (P = 0.037). There was also more HSC activation, and upregulation of collagen alpha1(I), heat-shock protein 47, alphaB crystallin, matrix metalloproteinase (MMP)-2, MMP-9 and tissue inhibitor of MMP (TIMP)-2. The expression of TIMP-1 and CTGF was not significantly different between the two groups. The pro-fibrogenic effect of celecoxib in toxin-induced liver fibrosis in rats was further confirmed in thioacetamide model of liver injury. CONCLUSIONS: Celecoxib potentiates experimental liver fibrosis; further studies are warranted to investigate the potential pro-fibrogenic effect of celecoxib in other animal models of liver fibrosis and in patients with chronic hepatitis.