Treatment of the rat hepatic stellate cell line,PAV-1, by retinol and palmitic acid leads to a convenient model to study retinoids metabolism

The main site of vitamin A storage in the liver is the hepatic stellate cells (HSC). Involvement of HSC in vitamin A metabolism has mainly been studied using primary culture, which represents the most physiological model but technically suffers several drawbacks (yield, low reproducibility, etc.). To circumvent these problems, we have previously established and characterised an immortalised rat HSC line named PAV-1. This study aimed to investigate in PAV-1 and in primary HSC (i) the incorporation of retinol and its esterification, (ii) the cellular retinol-binding protein (CRBP) content, (iii) the acid retinyl ester hydrolase activity (aREH), (iv) the thermal susceptibility and (v) the lipid composition of the membranes, which may play a crucial role in retinol transport across cellular membrane. In routine conditions of culture, the rate of retinol esterification in PAV-1 was low (5.2%) compared to that obtained with primary HSC (69.9%). Retinol pre-treatment doubled this esterification rate (10.7%) and the CRBP content in PAV-1. The co-incubation with retinol and palmitic acid enabled PAV-1 to esterify retinol with a rate close to that of primary HSC (66.2% vs. 69.9%) and with similar retinyl ester profiles. aREH activity was higher in primary HSC than in PAV-1. Thermal susceptibility and phospholipid composition of membranes in PAV-1 treated cells were similar to those of primary HSC. In conclusion, our study shows that PAV-1 cells treated with retinol and palmitic acid is a sound and convenient model for studying vitamin A mobilisation, a fundamental physiological event occurring in HSC.     

All‐trans retinoic acid ameliorates hepatic stellate cell activation via suppression of thioredoxin interacting protein expression

Activation of hepatic stellate cells (HSCs) is the effector factor of hepatic fibrosis and hepatocellular carcinoma (HCC) development. Accumulating evidence suggests that retinoic acids (RAs), derivatives of vitamin A, contribute to prevention of liver fibrosis and carcinogenesis, however, regulatory mechanisms of RAs still remain exclusive. To elucidate RA signaling pathway, we previously performed a genome‐wide screening of RA‐responsive genes by in silico analysis of RA‐response elements, and identified 26 RA‐responsive genes. We found that thioredoxin interacting protein (TXNIP), which inhibits antioxidant activity of thioredoxin (TRX), was downregulated by all‐trans retinoic acid (ATRA). In the present study, we demonstrate that ATRA ameliorates activation of HSCs through TXNIP suppression. HSC activation was attenuated by TXNIP downregulation, whereas potentiated by TXNIP upregulation, indicating that TXNIP plays a crucial role in activation of HSCs. Notably, we showed that TXNIP‐mediated HSC activation was suppressed by antioxidant N‐acetylcysteine. In addition, ATRA treatment or downregulation of TXNIP clearly declined oxidative stress levels in activated HSCs. These data suggest that ATRA plays a key role in inhibition of HSC activation via suppressing TXNIP expression, which reduces oxidative stress levels.     

Decursin attenuates hepatic fibrogenesis through interrupting TGF-beta-mediated NAD(P)H oxidase activation and Smad signaling in vivo and in vitro

Aims

We studied that a potent antifibrotic effect of decursin on in vivo liver damage model and the mechanism in inhibiting which transforming growth factor (TGF)-β1-induced human hepatic stellate cells (HSCs) activation.

Main methods

Liver injury was induced in vivo by intraperitoneal injection of carbon tetrachloride (CCl₄) with or without decursin for 4 weeks in mice. Human hepatic stellate cell line, an immortalized human HSC line, was used in in vitro assay system. The effects of decursin on HSC activation were measured by analyzing the expression of α-smooth muscle actin (α-SMA) and collagen I in liver tissue and human HSCs.

Key findings

Decursin treatment significantly reduced the ratio of liver/body weight, α-SMA activation, and type I collagen overexpression in CCl₄ treated mice liver. The elevated serum levels, including ALT, AST, and ALP, were also decreased by decursin treatment. Treatment of decursin markedly proved the generation of reactive oxygen species, NAD(P)H oxidase (NOX) protein (1, 2, and 4) upregulation, NOX activity, and superoxide anion production in HSCs by TGF-β1. It also significantly reduced TGF-β1-induced Smad 2/3 phosphorylation, nuclear translocation of Smad 4, and association of Smad 2/3–Smad 4 complex. Consistent with in vitro results, decursin treatment effectively blocked the levels of NOX protein, and Smad 2/3 phosphorylation in injured mice liver.

Significance

Decursin blocked CCl₄-induced liver fibrosis and inhibited TGF-β1-mediated HSC activation in vitro. These data demonstrated that decursin exhibited hepatoprotective effects on experimental fibrosis, potentially by inhibiting the TGF-β1 induced NOX activation and Smad signaling.     

The plasticity of p19 ARF null hepatic stellate cells and the dynamics of activation

In the healthy adult liver, quiescent hepatic stellate cells (HSCs) present the major site for vitamin A storage in cytoplasmic lipid droplets. During liver injury due to viral infection or alcohol intoxication, HSCs get activated and produce high amounts of extracellular matrix components for tissue repair and fibrogenesis. Employing p19 ARF deficiency, we established a non-transformed murine HSC model to investigate their plasticity and the dynamics of HSC activation. Primary HSCs isolated from livers of adult p19 ARF null mice underwent spontaneous activation through long-term passaging without an obvious replicative limit. The immortalized cell line, referred to as M1-4HSC, showed stellate cell characteristics including the expression of desmin, glial fibrillary acidic protein, alpha-smooth muscle actin and pro-collagen I. Treatment of these non-tumorigenic M1-4HSC with pro-fibrogenic TGF-beta1 provoked a morphological transition to a myofibroblastoid cell type which was accompanied by enhanced cellular turnover and impaired migration. In addition, M1-4HSCs expressed constituents of cell adhesion complexes such as p120(ctn) and beta-catenin at cell borders, which dislocalized in the cytoplasm during stimulation to myofibroblasts, pointing to the epitheloid characteristics of HSCs. By virtue of its non-transformed phenotype and unlimited availability of cells, the p19(ARF) deficient model of activated HSCs and corresponding myofibroblasts render this system a highly valuable tool for studying the cellular and molecular basis of hepatic fibrogenesis.     

Nicotinamide riboside,an NAD+ precursor,attenuates the development of liver fibrosis in a diet-induced mouse model of liver fibrosis

ObjectiveLiver fibrosis is part of the non-alcoholic fatty liver disease (NAFLD) spectrum, which currently has no approved pharmacological treatment. In this study, we investigated whether supplementation of nicotinamide riboside (NR), a nicotinamide adenine dinucleotide (NAD+) precursor, can reduce the development of liver fibrosis in a diet-induced mouse model of liver fibrosis.MethodsMale C57BL/6 J mice were fed a low-fat control (LF), a high-fat/high-sucrose/high-cholesterol control (HF) or a HF diet supplemented with NR at 400 mg/kg/day (HF-NR) for 20 weeks. Features of liver fibrosis were assessed by histological and biochemical analyses. Whole-body energy metabolism was also assessed using indirect calorimetry. Primary mouse and human hepatic stellate cells were used to determine the anti-fibrogenic effects of NR in vitro.ResultsNR supplementation significantly reduced body weight of mice only 7 weeks after mice were on the supplementation, but did not attenuate serum alanine aminotransferase levels, liver steatosis, or liver inflammation. However, NR markedly reduced collagen accumulation in the liver. RNA-Seq analysis suggested that the expression of genes involved in NAD+ metabolism is altered in activated hepatic stellate cells (HSCs) compared to quiescent HSCs. NR inhibited the activation of HSCs in primary mouse and human HSCs. Indirect calorimetry showed that NR increased energy expenditure, likely by upregulation of β-oxidation in skeletal muscle and brown adipose tissue.ConclusionNR attenuated HSC activation, leading to reduced liver fibrosis in a diet-induced mouse model of liver fibrosis. The data suggest that NR may be developed as a potential preventative for human liver fibrosis.     

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

Background

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.

Methods

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.

Results

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.

Conclusion

Taken together, ASTX exerted anti-fibrogenic effects by blocking TGFβ1-signaling, consequently inhibiting the activation of Smad3 pathway in HSCs.

General significance

This study suggests that ASTX may be used as a preventive/therapeutic agent to prevent hepatic fibrosis.     

In vitro modeling of liver fibrosis with 3D co-culture system using a novel human hepatic stellate cell line

Hepatic stellate cells (HSCs) play an important role in liver fibrosis; however, owing to the heterogeneity and limited supply of primary HSCs, the development of in vitro liver fibrosis models has been impeded. In this study, we established and characterized a novel human HSC line (LSC-1), and applied it to various types of three-dimensional (3D) co-culture systems with differentiated HepaRG cells. Furthermore, we compared LSC-1 with a commercially available HSC line on conventional monolayer culture. LSC-1 exhibited an overall upregulation of the expression of fibrogenic genes along with increased levels of matrix and adhesion proteins, suggesting a myofibroblast-like or transdifferentiated state. However, activated states reverted to a quiescent-like phenotype when cultured in different 3D culture formats with a relatively soft microenvironment. Additionally, LSC-1 exerted an overall positive effect on co-cultured differentiated HepaRG, which significantly increased hepatic functionality upon long-term cultivation compared with that achieved with other HSC line. In 3D spheroid culture, LSC-1 exhibited enhanced responsiveness to transforming growth factor beta 1 exposure that is caused by a different matrix-related protein expression mechanism. Therefore, the LSC-1 line developed in this study provides a reliable candidate model that can be used to address unmet needs, such as development of antifibrotic therapies.     

Oxidative stress-mediated mitochondrial fission promotes hepatic stellate cell activation via stimulating oxidative phosphorylation

Previous studies have demonstrated dysregulated mitochondrial dynamics in fibrotic livers and hepatocytes. Little is currently known about how mitochondrial dynamics are involved, nor is it clear how mitochondrial dynamics participate in hepatic stellate cell (HSC) activation. In the present study, we investigated the role of mitochondrial dynamics in HSC activation and the underlying mechanisms. We verified that mitochondrial fission was enhanced in human and mouse fibrotic livers and active HSCs. Moreover, increased mitochondrial fission driven by fis1 overexpression could promote HSC activation. Inhibiting mitochondrial fission using mitochondrial fission inhibitor-1 (Mdivi-1) could inhibit activation and induce apoptosis of active HSCs, indicating that increased mitochondrial fission is essential for HSC activation. Mdivi-1 treatment also induced apoptosis in active HSCs in vivo and thus ameliorated CCl₄-induced liver fibrosis. We also found that oxidative phosphorylation (OxPhos) was increased in active HSCs, and OxPhos inhibitors inhibited activation and induced apoptosis in active HSCs. Moreover, increasing mitochondrial fission upregulated OxPhos, while inhibiting mitochondrial fission downregulated OxPhos, suggesting that mitochondrial fission stimulates OxPhos during HSC activation. Next, we found that inhibition of oxidative stress using mitoquinone mesylate (mitoQ) and Tempol inhibited mitochondrial fission and OxPhos and induced apoptosis in active HSCs, suggesting that oxidative stress contributes to excessive mitochondrial fission during HSC activation. In conclusion, our study revealed that oxidative stress contributes to enhanced mitochondrial fission, which triggers OxPhos during HSC activation. Importantly, inhibiting mitochondrial fission has huge prospects for alleviating liver fibrosis by eliminating active HSCs.Subject terms: Endocrine system and metabolic diseases, Cell biology     

Wnt signaling enhances the activation and survival of human hepatic stellate cells

Wnt signaling was implicated in pulmonary and renal fibrosis. Since Wnt activity is enhanced in liver cirrhosis, Wnt signaling may also participate in hepatic fibrogenesis. Thus, we determined if Wnt signaling modulates hepatic stellate cell (HSC) activation and survival. Wnt3A treatment significantly activated human HSCs, while this was inhibited in secreted frizzled-related protein 1 (sFRP1) overexpressing cells. Wnt3A treatment significantly suppressed TRAIL-induced apoptosis in control HSCs versus sFRP1 over-expressing cells. Particularly, caspase 3 was more activated in sFRP1 over-expressing cells following TRAIL and Wnt3A treatment. These observations imply that Wnt signaling promotes hepatic fibrosis by enhancing HSC activation and survival.     

Beta-carotene storage, conversion to retinoic acid, and induction of the lipocyte phenotype in hepatic stellate cells

Hepatic stellate cells (HSCs) are the major site of retinol (ROH) metabolism and storage. GRX is a permanent murine myofibroblastic cell line, derived from HSCs, which can be induced to display the fat-storing phenotype by treatment with retinoids. Little is known about hepatic or serum homeostasis of beta-carotene and retinoic acid (RA), although the direct biogenesis of RA from beta-carotene has been described in enterocytes. The aim of this study was to identify the uptake, metabolism, storage, and release of beta-carotene in HSCs. GRX cells were plated in 25 cm(2) tissue culture flasks, treated during 10 days with 3 micromol/L beta-carotene and subsequently transferred into the standard culture medium. beta-Carotene induced a full cell conversion into the fat-storing phenotype after 10 days. The total cell extracts, cell fractions, and culture medium were analyzed by reverse phase high-performance liquid chromatography for beta-carotene and retinoids. Cells accumulated 27.48 +/- 6.5 pmol/L beta-carotene/10(6) cells, but could not convert it to ROH nor produced retinyl esters (RE). beta-Carotene was directly converted to RA, which was found in total cell extracts and in the nuclear fraction (10.15 +/- 1.23 pmol/L/10(6) cells), promoting the phenotype conversion. After 24-h chase, cells contained 20.15 +/- 1.12 pmol/L beta-carotene/10(6) cells and steadily released beta-carotene into the medium (6.69 +/- 1.75 pmol/ml). We conclude that HSC are the site of the liver beta-carotene storage and release, which can be used for RA production as well as for maintenance of the homeostasis of circulating carotenoids in periods of low dietary uptake.     

Astragaloside IV suppresses collagen production of activated hepatic stellate cells via oxidative stress-mediated p38 MAPK pathway

Oxidative stress is involved in hepatic fibrogenesis. Activation of hepatic stellate cells (HSCs), the key effectors in hepatic fibrogenesis, is characterized by overproduction of extracellular matrix. Astragaloside IV, the active component of Radix Astragali, has antioxidant properties and antifibrotic potential in renal fibrosis. Little is known about the role of astragaloside IV in liver and its involvement in hepatic fibrosis. This study aims at evaluating the antifibrotic potential of astragaloside IV and characterizing involved signal transduction pathways in culture-activated HSCs. Our results show that astragaloside IV attenuates oxidative stress in culture-activated HSCs, as demonstrated by scavenging reactive oxygen species and reducing lipid peroxidation, and elevates the level of cellular glutathione by stimulating Nrf2gene expression. Depletion of cellular glutathione by buthionine sulfoximine or abrogation of p38 MAPK by SB-203580 evidently eliminates the inhibitory effects of astragaloside IV on genes relevant to HSC activation. These results demonstrate that astragaloside IV inhibits HSC activation by inhibiting generation of oxidative stress and associated p38 MAPK activation and provide novel insights into the mechanisms of astragaloside IV as an antifibrogenic candidate in the prevention and treatment of liver fibrosis.     

Tenascin-C promotes migration of hepatic stellate cells and production of type I collagen

Tenascin-C (TN-C) is an extracellular matrix glycoprotein markedly upregulated during liver fibrosis. The study is performed to explore the role of TN-C during the growth and activation of hepatic stellate cells (HSCs). We found that TN-C was accumulated accompanying with the HSC activation. Our data on cell migration assay revealed that the rTN-C treatment enhanced HSC migration in a dose- and time-dependent manner, but did not influence their proliferation. HSCs transfected with pTARGET-TN-C overexpression vector displayed increased the type I collagen (Col I) production. TN-C overexpression enhanced the process of HSC activation through TGF-β1 signaling. Moreover, the anti-α9β1 integrin antibody treatment blocked the TN-C-driven Col I increase in rat HSCs. Collectively, TN-C had a positive role in activation of HSCs mediated by TGF-β1 and α9β1 integrin, manifesting elevation of Col I production and promotion of cell migration. Our results provide a potential insight for the therapy of hepatic fibrosis.     

Delta-like 1 serves as a new target and contributor to liver fibrosis down-regulated by mesenchymal stem cell transplantation

Chronic liver injury always progresses to fibrosis and eventually to cirrhosis, a massive health care burden worldwide. Delta-like 1 (Dlk1) is well known as an inhibitor of adipocyte differentiation. However, whether it is involved in liver fibrosis remains unclear. Here, we provide the first evidence that Dlk1 is a critical contributor to liver fibrosis through promoting activation of hepatic stellate cells (HSCs) during chronic liver injury. We found that upon liver injury, Dlk1 was dramatically induced and initially expressed in hepatocytes and then into the HSCs by a paracrine manner. It leads to the activation of HSCs, which is considered to be a pivotal event in liver fibrogenesis. Two forms (～50 and ～25 kDa) of the Dlk1 protein were detected by Western blot analysis. In vitro administration of Dlk1 significantly promoted HSC activation, whereas in vivo knockdown of Dlk1 dramatically inhibited HSC activation and the subsequent fibrosis. The large soluble form (～50 kDa) of Dlk1 was shown to contribute to HSC activation. We were encouraged to find the Dlk1-promoted HSC activation and liver fibrosis can be depressed by transplantation of bone marrow-mesenchymal stem cells (BM-MSCs). Furthermore, we demonstrated that FGF2 was up-regulated in BM-MSCs under injury stimulation, and it probably participated in the inhibition of Dlk1 by BM-MSCs. Our findings provide a novel role of Dlk1 in liver fibrosis leading to a better understanding of the molecular basis in fibrosis and cirrhosis and also give insights into the cellular and molecular mechanisms of MSC biology in liver repair.     

Loss of expression of miR-335 is implicated in hepatic stellate cell migration and activation

Activation and migration of resident stellate cells (HSCs) within the hepatic space of Disse play an important role in hepatic fibrosis, which accounts for the increased numbers of activated HSCs in areas of inflammation during hepatic fibrosis. Currently, microRNAs have been found to play essential roles in HSC differentiation, proliferation, apoptosis, fat accumulation and collagen production. However, little is known about microRNA mediated HSC activation and migration. In this study, the miRNA expression profiles of quiescent HSCs, partially activated HSCs and fully activated HSCs were compared in pairs. Gene ontology (GO) and GO-Map network analysis indicated that the activation of HSCs was regulated by microRNAs. Among them miR-335 was confirmed to be significantly reduced during HSC activation by qRT-PCR, and restoring expression of miR-335 inhibited HSC migration and reduced α-SMA and collagen type I. Previous study revealed that tenascin-C (TNC), an extracellular matrix glycoprotein involved in cell migration, might be a target of miR-335. Therefore, we further studied the TNC expression in miR-335 over-expressed HSCs. Our data showed that exogenous TNC could enhance HSC migration in vitro and miR-335 restoration resulted in a significant inhibition of TNC expression. These results demonstrated that miR-335 restoration inhibited HSC migration, at least in part, via downregulating the TNC expression.     

Profibrotic effect of miR-33a with Akt activation in hepatic stellate cells

MicroRNAs (miRNAs) attract more attention in the pathophysiology of liver fibrosis and miR-33a has been previously demonstrated as involved in the regulation of cholesterol and lipid metabolism. Transforming growth factor-beta1 (TGF-β1) is generally accepted to be the main stimulating factor in the hepatic stellate cells (HSCs) activation, which plays an important role in hepatic fibrosis. However, the involvement and underlying mechanism of miR-33a and its role in TGF-β1-induced hepatic fibrogenesis remains unknown. Here, we investigate the role of miR-33a in the activation of immortalized human HSCs, Lx-2 cells. Our findings have shown that the expression of miR-33a with its host gene sterol regulatory element-binding protein 2 (SREBP2) was more highly expressed in activation of Lx-2 cells than in quiescent cells. The expression of miR-33a on TGF-β1-induced HSCs activation may be modulated via the activation of PI3K/Akt pathway. In addition, miR-33a significantly correlated with TGF-β1-induced expression of α1 (I) collagen (Col1A1) and α-SMA in HSCs. Bioinformatics analyses predict that peroxisome proliferator activated receptor-alpha (PPAR-α) is the potential target of miR-33a. We further found that anti-miR-33a significantly increases target gene PPAR-α mRNA and protein level, suggesting that miR-33a involved in HSCs function might be modulated by targeting PPAR-α. Finally, our results indicate that the expression of miR-33a increased with the progression of liver fibrosis. These results suggested that anti-miR-33a inhibit activation and extracellular matrix production, at least in part, via the activation of PI3K/Akt pathway and PPAR-α and anti sense of miR-33a may be a novel potential therapeutic approach for treating hepatic fibrosis in the future.     

Activation of Mir-29a in Activated Hepatic Stellate Cells Modulates Its Profibrogenic Phenotype through Inhibition of Histone Deacetylases 4

Background

Recent studies have shown that microRNA-29 (miR-29) is significantly decreased in liver fibrosis and that its downregulation influences the activation of hepatic stellate cells (HSCs). In addition, inhibition of the activity of histone deacetylases 4 (HDAC4) has been shown to strongly reduce HSC activation in the context of liver fibrosis.

Objectives

In this study, we examined whether miR-29a was involved in the regulation of HDAC4 and modulation of the profibrogenic phenotype in HSCs.

Methods

We employed miR-29a transgenic mice (miR-29aTg mice) and wild-type littermates to clarify the role of miR-29a in cholestatic liver fibrosis, using the bile duct-ligation (BDL) mouse model. Primary HSCs from both mice were treated with a miR-29a mimic and antisense inhibitor in order to analyze changes in profibrogenic gene expression and HSC activation using real-time quantitative RT-PCR, immunofluorescence staining, western blotting, and cell proliferation and migration assays.

Results

After BDL, overexpression of miR-29a decreased collagen-1α1, HDAC4 and activated HSC markers of glial fibrillary acidic protein expression in miR-29aTg mice compared to wild-type littermates. Overexpression of miR-29a and HDAC4 RNA-interference decreased the expression of fibrotic genes, HDAC4 signaling, and HSC migration and proliferation. In contrast, knockdown of miR-29a with an antisense inhibitor increased HDAC4 function, restored HSC migration, and accelerated HSC proliferation.

Conclusions

Our results indicate that miR-29a ameliorates cholestatic liver fibrosis after BDL, at least partially, by modulating the profibrogenic phenotype of HSCs through inhibition of HDAC4 function.     

Tricin inhibits proliferation of human hepatic stellate cells in vitro by blocking tyrosine phosphorylation of PDGF receptor and its signaling pathways

4',5,7-Trihydroxy-3',5'-dimethoxyflavone (Tricin), a naturally occurring flavone, has anti-inflammatory potential and exhibits diverse biological activities including antigrowth activity in several human cancer cell lines and cancer chemopreventive effects in the gastrointestinal tract of mice. The present study aimed to investigate the biological actions of tricin on hepatic stellate cells (HSCs) in vitro, exploring its potential as a treatment of liver fibrosis, since HSC proliferation is closely related to the progression of hepatic fibrogenesis in chronic liver diseases leading to irreversible liver cirrhosis and hepatocellular carcinoma. Tricin inhibited platelet-derived growth factor (PDGF)-BB-induced cell proliferation by blocking cell cycle progression and cell migration in the human HSC line LI90 and culture-activated HSCs. It also reduced the phosphorylation of PDGF receptor β and the downstream signaling molecules ERK1/2 and Akt, which might be due to its tyrosine kinase inhibitor properties rather than inhibition of the direct binding between PDGF-BB and its receptor. Our findings suggest that tricin might be beneficial in HSC-targeting therapeutic or chemopreventive applications for hepatic fibrosis.