Analysis of the anti-fibrotic effects of hop bitter acids on hepatic stellate cells and their anti-tumorigenic effects on hepatocellular carcinoma cells

Bitter acids (BA) from the hop plant Humulus lupulus L. exhibit multiple beneficial biological properties. However, no studies are available regarding the effects of hop bitter acids on liver cells or chronic liver diseases, respectively. Chronic liver diseases are accompanied by chronic hepatic inflammation which can lead to liver fibrosis. If left untreated, liver fibrosis can progress to cirrhosis, which can cause liver failure. Importantly, cirrhosis is also the main risk factor for the development of hepatocellular carcinoma (HCC). Both cirrhosis and HCC are associated with high morbidity and mortality, and up until now, no effective treatment options are available. The aim of this thesis was to assess the effects of BA on hepatic stellate cells, the central mediators of liver fibrosis, and on hepatocellular carcinoma cells. First, the effects of hop bitter acids on hepatic stellate cells (HSC) were analyzed. HSC were isolated from murine and human liver tissues and were incubated with a standardized hop bitter acid extract. At a concentration of 25 µg/ml hop bitter acids (BA) started to induce lactate dehydrogenase leakage. Already at lower concentrations BA led to a dose dependent inhibition of HSC proliferation and inhibited IkappaB-alpha-phosphorylation and nuclear p65 translocation in a dose dependent way. Accordingly, the same doses of BA inhibited the expression of pro-inflammatory and NFkappaB-regulated genes, but did not affect expression of genes not related to NFkappaB signaling. In addition to these effects on activated HSC, BA inhibited the in vitro activation of non-activated HSC as evidenced by delayed expression of collagen I and alpha-sma mRNA and protein. Together, these findings indicate that BA inhibit NFkappaB activation, and herewith, the activation and development of pro-fibrogenic phenotype of HSC in vitro. Next, we analyzed the effects of BA on hepatocellular carcinoma (HCC) cells. Here, we used two different standardized BA extracts enriched for either alpha-acids or beta-acids to get a first insight whether biological activity vary between these two groups of bitter acids. At a concentration of 25 µg/ml, only the beta-acid rich extract started to induce aspartate aminotransferase (AST) release as marker for cell injury, whereas at a dose of 50 µg/ml both extracts led to a significant increase of AST liberation. Already at lower concentrations both extracts dose-dependently inhibited proliferation and migration of HCC cells. Analysis of different signaling pathways revealed an inhibition of ERK1/2 phosphorylation, down-regulation of AP-1 activity and an alleviation of NFkappaB activity in HCC cells in vitro. Hereby, the beta-acid rich extract showed more pronounced effects. Furthermore, the stability of BA in a rodent chow supplemented with an alpha-acid rich extract was assessed applying high pressure liquid chromatography diode array detection technology. Mimicking feasible storage conditions we observed a very poor recovery, indicative of a strong degradation of BA. This prompted us not to proceed with application of this particular BA supplemented chow in rodent models, since in addition to imprecise BA-concentration also potential ill-defined degradation products would not have allowed a sound interpretation of such studies. In conclusion, hop bitter acids alleviate the pro-fibrogenic phenotype of hepatic stellate cells and inhibit central signaling pathways, which are known to play important roles in tumor development and progression, in hepatocellular carcinoma cells. These data suggest the potential use of hop bitter acids as functional nutrient or therapeutical agent for both prevention and treatment of chronic liver diseases. Further studies are needed to verify the beneficial effects in experimental models of liver injury in vivo. However, analysis of a newly designed BA-supplemented experimental chow for rodents revealed strong degradation processes even under optimal storage conditions. Therefore, special care has to be taken when planning long term BA-application experiments

Discrimination of steatosis and NASH in mice using nuclear magnetic resonance spectroscopy

Nonalcoholic fatty liver disease (NAFLD) is a common cause of hepatic dysfunction. The disease spectrum ranges from hepatic steatosis to nonalcoholic steatohepatitis (NASH). The aim of this study was to identify metabolic differences in murine models of simple hepatic steatosis and NASH for the distinction of these NAFLD stages. For 12 weeks, male BALB/c mice were fed either a control or two different high-fat diets leading to hepatic steatosis and NASH, respectively. Metabolic differences were determined by independent component analysis (ICA) of nuclear magnetic resonance (NMR) spectra of lipophilic and hydrophilic liver extracts, and urine specimens. The results from ICA clearly discriminated the three investigated groups. Discriminatory biomarkers in the lipophilic liver extracts were free cholesterol, cholesterol ester and lipid methylene. Discrimination of the hydrophilic liver extracts was mainly mediated by betaine, glucose, and lactate, whereas in urine taurine, trimethylamine-N-oxide, and trimethylamine were the most discriminatory biomarkers. In conclusion, NMR metabolite fingerprinting of spot urine specimens may allow the noninvasive distinction of steatosis and NASH

Expression and function of methylthioadenosine phosphorylase in chronic liver disease

To study expression and function of methylthioadenosine phosphorylase (MTAP), the rate-limiting enzyme in the methionine and adenine salvage pathway, in chronic liver disease. DESIGN: MTAP expression was analyzed by qRT-PCR, Western blot and immunohistochemical analysis. Levels of MTA were determined by liquid chrmatography-tandem mass spectrometry. RESULTS: MTAP was downregulated in hepatocytes in murine fibrosis models and in patients with chronic liver disease, leading to a concomitant increase in MTA levels. In contrast, activated hepatic stellate cells (HSCs) showed strong MTAP expression in cirrhotic livers. However, also MTA levels in activated HSCs were significantly higher than in hepatocytes, and there was a significant correlation between MTA levels and collagen expression in diseased human liver tissue indicating that activated HSCs significantly contribute to elevated MTA in diseased livers. MTAP suppression by siRNA resulted in increased MTA levels, NFκB activation and apoptosis resistance, while overexpression of MTAP caused the opposite effects in HSCs. The anti-apoptotic effect of low MTAP expression and high MTA levels, respectively, was mediated by induced expression of survivin, while inhibition of survivin abolished the anti-apoptotic effect of MTA on HSCs. Treatment with a DNA demethylating agent induced MTAP and reduced survivin expression, while oxidative stress reduced MTAP levels but enhanced survivin expression in HSCs. CONCLUSION: MTAP mediated regulation of MTA links polyamine metabolism with NFκB activation and apoptosis in HSCs. MTAP and MTAP modulating mechanisms appear as promising prognostic markers and therapeutic targets for hepatic fibrosis