Comparison of Carotenoids for Their Antifibrogenic Effects in Hepatic Stellate Cells.

Hepatic stellate cells (HSC) have an important role in the development of liver fibrosis by producing extracellular matrix proteins when they are activated upon liver injury. We previously demonstrated that astaxanthin (ASTX), a xanthophyll carotenoid, attenuates HSC activation. The objective of this study was to compare the anti-fibrogenic effects of ASTX with those of other common carotenoids. LX-2 cells, a human HSC cell line, were treated with ASTX, lycopene, lutein (LT), zeaxanthin, or canthaxanthin, to measure messenger RNA (mRNA) and protein expression of pro-fibrogenic genes. Pro-fibrogenic gene expressions were also measured in ASTX- or LT-treated primary mouse HSC. To determine the underlying mechanisms of the anti-fibrogenic effect of ASTX and LT, SMA-related and MAD-related protein 3 (SMAD3) pathways and the accumulation of reactive oxygen species (ROS) were measured in LX-2 cells. Among five carotenoids tested, ASTX and LT attenuated HSC activation in LX-2 cells by reducing the mRNA and protein levels of pro-fibrogenic genes, such as smooth muscle α actin and procollagen type I α1 (COL1A1). In addition, both ASTX and LT significantly decreased the expression of pro-fibrogenic genes, including COL1A1, COL3A1, and COL6A1, in activated primary mouse HSC, with ASTX being more potent than LT. The anti-fibrogenic effect of ASTX was mediated by inhibiting the phosphorylation of SMAD3 and cellular ROS accumulation, while LT only prevented the accumulation of ROS in LX-2 cells. In conclusion, ASTX showed the most potent anti-fibrogenic effect among the five carotenoids via inhibition of SMAD3 phosphorylation and cellular ROS accumulation while LT only prevented ROS levels in HSC.

Bae M ，Kim MB ，Kang H ，Park YK ，Lee JY ... -  《-》

Astaxanthin prevents TGFβ1-induced pro-fibrogenic gene expression by inhibiting Smad3 activation in hepatic stellate cells.

Non-alcoholic steatohepatitis (NASH) is a subset of non-alcoholic fatty liver disease, the most common chronic liver disease in the U.S. Fibrosis, a common feature of NASH, results from the dysregulation of fibrogenesis in hepatic stellate cells (HSCs). In this study, we investigated whether astaxanthin (ASTX), a xanthophyll carotenoid, can inhibit fibrogenic effects of transforming growth factor β1 (TGFβ1), a key fibrogenic cytokine, in HSCs. Reactive oxygen species (ROS) accumulation was measured in LX-2, an immortalized human HSC cell line. Quantitative realtime PCR, Western blot, immunocytochemical analysis, and in-cell Western blot were performed to determine mRNA and protein of fibrogenic genes, and the activation of Smad3 in TGFβ1-activated LX-2 cells and primary mouse HSCs. In LX-2 cells, ROS accumulation induced by tert-butyl hydrogen peroxide and TGFβ1 was abolished by ASTX. ASTX significantly decreased TGFβ1-induced α-smooth muscle actin (α-SMA) and procollagen type 1, alpha 1 (Col1A1) mRNA as well as α-SMA protein levels. Knockdown of Smad3 showed the significant role of Smad3 in the expression of α-SMA and Col1A1, but not TGFβ1, in LX-2 cells. ASTX attenuated TGFβ1-induced Smad3 phosphorylation and nuclear translocation with a concomitant inhibition of Smad3, Smad7, TGFβ receptor I (TβRI), and TβRII expression. The inhibitory effect of ASTX on HSC activation was confirmed in primary mouse HSCs as evidenced by decreased mRNA and protein levels of α-SMA during activation. Taken together, ASTX exerted anti-fibrogenic effects by blocking TGFβ1-signaling, consequently inhibiting the activation of Smad3 pathway in HSCs. This study suggests that ASTX may be used as a preventive/therapeutic agent to prevent hepatic fibrosis.

Yang Y ，Kim B ，Park YK ，Koo SI ，Lee JY ... -  《-》

Histone deacetylase 9 plays a role in the antifibrogenic effect of astaxanthin in hepatic stellate cells.

Activation of hepatic stellate cells (HSCs) is critical for liver fibrosis development. Previously, we showed that astaxanthin (ASTX), a xanthophyll carotenoid, has antifibrogenic effects in LX-2 cells, a human HSC cell line. We sought to determine the effect of ASTX on HSC activation, and to identify molecular mediators that are critically involved in the processes. ASTX prevented the activation of mouse primary HSCs, as evidenced by attenuated induction of procollagen type I α1. In human primary HSCs, ASTX also inhibited transforming growth factor β1 (TGFβ1)-induced fibrogenic gene expression. Among 11 classical histone deacetylases (HDACs), difference in HDAC9 mRNA levels between quiescent and activated HSCs was most evident while ASTX significantly decreased the expression of HDAC9 and its transcriptional regulator myocyte enhancer factor 2 (MEF2). ASTX decreased HDAC9 protein as well. In the activated HSCs, ASTX significantly reduced mRNA of HDAC9 and MEF2. Human primary biliary cirrhosis livers showed significantly higher HDAC9 mRNA and protein levels than normal livers, and other liver pathologies also exhibited induced HDAC9 expression. HDAC9 knockdown in LX-2 cells decreased TGFβ1-induced fibrogenic gene expression. In conclusion, ASTX inhibits HSC activation and facilitates HSC inactivation, which is attributable to its inhibitory action on HDAC9 expression.

Yang Y ，Bae M ，Park YK ，Lee Y ，Pham TX ，Rudraiah S ，Manautou J ，Koo SI ，Lee JY ... -  《-》

Astaxanthin prevents and reverses the activation of mouse primary hepatic stellate cells.

Activation of hepatic stellate cells (HSCs) is a critical step that leads to the development of liver fibrosis. We showed that astaxanthin (ASTX), a xanthophyll carotenoid, displays antifibrogenic effects in LX-2 cells, a human HSC cell line. In this study, we further determined the effect of ASTX on HSC activation and inactivation using primary HSCs from C57BL/6J mice. Quiescent and activated HSCs were incubated with ASTX (25μM) at different stages of activation. ASTX prevented the activation of quiescent HSCs, as evidenced by the presence of intracellular lipid droplets and reduction of α-smooth muscle actin, an HSC activation marker. Also, ASTX reverted activated HSCs to a quiescent phenotype with the reappearance of lipid droplets with a concomitant increase in lecithin retinol acyltransferase mRNA. Cellular accumulation of reactive oxygen species was significantly reduced by ASTX, which was attributable to a decrease in NADPH oxidase 2 expression. The antifibrogenic effect of ASTX was independent of nuclear erythroid 2-related factor 2 as it was observed in HSCs from wild-type and Nrf2(-/-) mice. In conclusion, ASTX inhibits HSC activation and reverts activated HSCs to a quiescent state. Further investigation is warranted to determine if ASTX effectively prevents the development of liver fibrosis.

Yang Y ，Bae M ，Kim B ，Park YK ，Koo SI ，Lee JY ... -  《-》

Astaxanthin inhibits the reduction of glycolysis during the activation of hepatic stellate cells.

Hepatic stellate cells (HSCs) play an essential role in the development of liver fibrosis by producing extracellular matrix proteins, growth factors, and pro-inflammatory and pro-fibrogenic cytokines once activated. We previously demonstrated that astaxanthin (ASTX), a xanthophyll carotenoid, attenuates HSC activation. The objective of this study was to investigate whether there is a difference in glycolysis between quiescent and activated HSCs and the effect of ASTX on glycolysis during HSC activation. Mouse primary HSCs were activated for 7 days in the presence or absence of 25 μM of ASTX. Quiescent HSCs (qHSCs), 1 day after isolation, and activated HSCs (aHSCs) treated with/without ASTX were plated in a Seahorse XF24 cell culture microplate for Glycolysis Stress tests. aHSCs had significantly lower glycolysis, but higher glycolytic capacity, maximum capacity of glycolysis, and non-glycolytic acidification than qHSCs. Importantly, ASTX markedly increased glycolysis during HSC activation with a concomitant increase in lactate formation and secretion. Compared with qHSCs, aHSCs had significantly lower expression of glucose transporter 1, the major glucose transporter in HSCs, and its transcription factor hypoxia-inducible factor 1α, which was markedly increased by ASTX in aHSCs. Our data suggest that ASTX may prevent the activation of HSCs by altering glycolysis and the expression of genes involved in the pathways.

Bae M ，Lee Y ，Pham TX ，Hu S ，Park YK ，Lee JY ... -  《-》