Minkyung Bae

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.

Astaxanthin inhibits inflammation and fibrosis in the liver and adipose tissue of mouse models of diet-induced obesity and nonalcoholic steatohepatitis

The objective of this study was to determine if astaxanthin (ASTX), a xanthophyll carotenoid, can prevent obesity-associated metabolic abnormalities, inflammation and fibrosis in diet-induced obesity (DIO) and nonalcoholic steatohepatitis (NASH) mouse models. Male C57BL/6J mice were fed a low-fat (6% fat, w/w), a high-fat/high-sucrose control (HF/HS; 35% fat, 35% sucrose, w/w), or a HF/HS containing ASTX (AHF/HS; 0.03% ASTX, w/w) for 30 weeks. To induce NASH, another set of mice was fed a HF/HS diet containing 2% cholesterol (HF/HS/HC) a HF/HS/HC with 0.015% ASTX (AHF/HS/HC) for 18 weeks. Compared to LF, HF/HS significantly increased plasma total cholesterol, triglyceride and glucose, which were lowered by ASTX. ASTX decreased hepatic mRNA levels of markers of macrophages and fibrosis in both models. The effect of ASTX was more prominent in NASH than DIO mice. In epididymal fat, ASTX also decreased macrophage infiltration and M1 macrophage marker expression, and inhibited hypoxia-inducible factor 1-α and its downstream fibrogenic genes in both mouse models. ASTX significantly decreased tumor necrosis factor α mRNA in the splenocytes from DIO mice upon lipopolysaccharides stimulation compared with those from control mice fed an HF/HS diet. Additionally, ASTX significantly elevated the levels of genes that regulate fatty acid β-oxidation and mitochondrial biogenesis in the skeletal muscle compared with control obese mice, whereas no differences were noted in adipose lipogenic genes. Our results indicate that ASTX inhibits inflammation and fibrosis in the liver and adipose tissue and enhances the skeletal muscles capacity for mitochondrial fatty acid oxidation in obese mice.

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.

Food components with anti-fibrotic activity and implications in prevention of liver disease

Increasing prevalence of nonalcoholic fatty liver disease (NAFLD) in parallel with the obesity epidemic has been a major public health concern. NAFLD is the most common chronic liver disease in the United States, ranging from fatty liver to steatohepatitis, fibrosis and cirrhosis in the liver. In response to chronic liver injury, fibrogenesis in the liver occurs as a protective response; however, prolonged and dysregulated fibrogenesis can lead to liver fibrosis, which can further progress to cirrhosis and eventually hepatocellular carcinoma. Interplay of hepatocytes, macrophages and hepatic stellate cells (HSCs) in the hepatic inflammatory and oxidative milieu is critical for the development of NAFLD. In particular, HSCs play a major role in the production of extracellular matrix proteins. Studies have demonstrated that bioactive food components and natural products, including astaxanthin, curcumin, blueberry, silymarin, coffee, vitamin C, vitamin E, vitamin D, resveratrol, quercetin and epigallocatechin-3-gallate, have antifibrotic effects in the liver. This review summarizes current knowledge of the mechanistic insight into the antifibrotic actions of the aforementioned bioactive food components.

Blackcurrant anthocyanins stimulated cholesterol transport via post-transcriptional induction of LDL receptor in Caco-2 cells

PurposesWe previously showed that polyphenol-rich blackcurrant extract (BCE) showed a hypocholesterolemic effect in mice fed a high fat diet. As direct cholesterol removal from the body via the intestine has been recently appreciated, we investigated the effect of BCE on the modulation of genes involved in intestinal cholesterol transport using Caco-2 cells as an in vitro model.MethodsCaco-2 cells were treated with BCE to determine its effects on mRNA and protein expression of genes important for intestinal cholesterol transport, low-density lipoprotein (LDL) uptake, cellular cholesterol content, and cholesterol transport from basolateral to apical membrane of Caco-2 cell monolayers. Cells were also treated with anthocyanin-rich or -poor fraction of BCE to determine the role of anthocyanin on BCE effects.ResultsBCE significantly increased protein levels of LDL receptor (LDLR) without altering its mRNA, which consequently increased LDL uptake into Caco-2 cells. This post-transcriptional induction of LDLR by BCE was markedly attenuated in the presence of rapamycin, an inhibitor of mechanistic target of rapamycin complex 1 (mTORC1). In addition, BCE altered genes involved in cholesterol transport in the enterocytes, including apical and basolateral cholesterol transporters, in such a way that could enhance cholesterol flux from the basolateral to apical side of the enterocytes. Indeed, BCE significantly increased the flux of LDL-derived cholesterol from the basolateral to the apical chamber of Caco-2 monolayer. LDLR protein levels were markedly increased by anthocyanin-rich fraction, but not by anthocyanin-free fraction.ConclusionmTORC1-dependent post-transcriptional induction of LDLR by BCE anthocyanins drove the transport of LDL-derived cholesterol to the apical side of the enterocytes. This may represent a potential mechanism for the hypocholesterolemic effect of BCE.

Transcriptional and post-transcriptional repression of histone deacetylases by docosahexaenoic acid in macrophages

Histone deacetylation is one of the posttranslational modifications of histones by which eukaryotic cells alter gene transcription. Although fatty acids are the best known macronutrients that modulate gene expression in inflammatory pathways, it is unclear whether common fatty acids in diets can regulate the expression of histone deacetylases (HDACs) in macrophages. We determined the effects of fatty acids, including palmitic acid (PA), oleic acid (OA), linoleic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on the expression of HDAC isoforms in RAW 264.7 macrophages, mouse bone marrow-derived macrophages (BMDM) and human THP-1 cells. In RAW 264.7 macrophages, OA significantly increased mRNA levels of Hdac1, 2 and 3, and EPA induced Hdac2 expression compared with control. Marked repression of Hdac9 mRNA levels by EPA and DHA, with DHA being more potent, was observed in RAW 264.7 macrophages and BMDM. DHA also decreased HDAC3, 4 and 9 protein levels. EPA and DHA facilitated the proteasomal degradation of HDAC3 and 4 protein, while the transcriptional repression of HDAC9 by DHA may be mediated by the repression of myocyte enhancer factor 2 or by the activation of retinoid X receptor. Functionally, inhibition of HDAC activity or knockdown of Hdac9 in macrophages reduced lipopolysaccharide-induced inflammatory gene expression. Our results demonstrate that DHA represses the expression of HDAC3, 4 and 9 at the transcriptional or posttranscriptional levels in murine macrophages. This suggests that the anti-inflammatory effect of DHA may be mediated by the reduction of HDACs.

Astaxanthin exerts anti-inflammatory and antioxidant effects in macrophages in NRF2-dependent and independent manners

Although anti-inflammatory effects of astaxanthin (ASTX) have been suggested, the underlying mechanisms have not been fully understood. Particularly, the modulatory action of ASTX in the interplay between nuclear factor E2-related factor 2 (NRF2) and nuclear factor κB (NFκB) to exert its anti-inflammatory effect in macrophages is unknown. The effect of ASTX on mRNA and protein expression of pro-inflammatory and antioxidant genes and/or cellular reactive oxygen species (ROS) accumulation were determined in RAW 264.7 macrophages, bone marrow-derived macrophages (BMDM) from wild-type (WT) and Nrf2-deficient mice, and/or splenocytes and peritoneal macrophages of obese mice fed ASTX. The effect of ASTX on M1 and M2 macrophage polarization was evaluated in BMDM. ASTX significantly decreased LPS-induced mRNA expression of interleukin 6 (Il-6) and Il-1β by inhibiting nuclear translocation of NFκB p65; and attenuated LPS-induced ROS with an increase in NRF2 nuclear translocation, concomitantly decreasing NADPH oxidase 2 expression in RAW 264.7 macrophages. In BMDM of WT and Nrf2-deficient mice, ASTX decreased basal and LPS-induced ROS accumulation. The induction of Il-6 mRNA by LPS was repressed by ASTX in both types of BMDM while Il-1β mRNA was decreased only in WT BMDM. Furthermore, ASTX consumption lowered LPS sensitivity of splenocytes in obese mice. ASTX decreased M1 polarization of BMDM while increasing M2 polarization. ASTX exerts its anti-inflammatory effect by inhibiting nuclear translocation of NFκB p65 and by preventing ROS accumulation in NRF2-dependent and -independent mechanisms. Thus, ASTX is an agent with anti-inflammatory and antioxidant properties that may be used for the prevention of inflammatory conditions.