Immortalized Rat Hepatic Stellate Cell Research Articles

Synthesis of Novel 2-(Pyridin-2-yl) Pyrimidine Derivatives and Study of Their Anti-Fibrosis Activity.

A pyrimidine moiety exhibiting a wide range of pharmacological activities has been employed in the design of privileged structures in medicinal chemistry. To prepare libraries of novel heterocyclic compounds with potential biological activities, a series of novel 2-(pyridin-2-yl) pyrimidine derivatives were designed, synthesized and their biological activities were evaluated against immortalized rat hepatic stellate cells (HSC-T6). Fourteen compounds were found to present better anti-fibrotic activities than Pirfenidone and Bipy55′DC. Among them, compounds ethyl 6-(5-(p-tolylcarbamoyl)pyrimidin-2-yl)nicotinate (12m) and ethyl 6-(5-((3,4-difluorophenyl)carbamoyl)pyrimidin-2-yl)nicotinate (12q) show the best activities with IC50 values of 45.69 μM and 45.81 μM, respectively. Furthermore, the study of anti-fibrosis activity was evaluated by Picro-Sirius red staining, hydroxyproline assay and ELISA detection of Collagen type I alpha 1 (COL1A1) protein expression. Our study showed that compounds 12m and 12q effectively inhibited the expression of collagen, and the content of hydroxyproline in cell culture medium in vitro, indicating that compounds 12m and 12q might be developed the novel anti-fibrotic drugs.

Resveratrol Regulates Activated Hepatic Stellate Cells by Modulating NF-κB and the PI3K/Akt Signaling Pathway.

In the present study, we investigated whether resveratrol could suppress the hepatic fibrogenesis in activated hepatic stellate cells. The immortalized rat hepatic stellate cells, t-HSC/Cl-6, were treated with resveratrol 1 h prior to lipopolysaccharide (LPS, 1 μg/mL). Resveratrol decreased t-HSC/Cl-6 cell viability at much lower concentrations within 24 h. Resveratrol pretreatment also decreased the LPS-induced protein expression of α-SMA and collagen I. In addition, resveratrol significantly reduced the protein expression of Toll-like receptor 4 (TLR4) and myeloid differentiation primary response gene 88 (MyD88), and the expression of phosphorylated phosphatidylinositol 3-kinase (PI3K) and phosphorylated serine/threonine kinase B (Akt). Moreover, resveratrol markedly blocked the translocation of nuclear factor (NF)-κB in LPS-activated HSCs. Furthermore, resveratrol inhibited HSCs activation through stimulating LXRβ, but did not influence LXRα. Overall, we conclude that the antifibrotic effect of resveratrol is the result of blocking NF-κB activation and PI3K/Akt phosphorylation, which inhibits HSC activation to obstruct liver fibrosis. Thus, resveratrol may be a natural agent for preventing hepatic fibrosis.

Tissue-type plasminogen activator suppresses activated stellate cells through low-density lipoprotein receptor-related protein 1

Hepatic stellate cell (HSC) activation and trans-differentiation into myofibroblast (MFB)-like cells is key for fibrogenesis after liver injury and a potential therapeutic target. Recent studies demonstrated that low-density lipoprotein receptor-related protein 1 (LRP1)-dependent signaling by tissue-type plasminogen activator (t-PA) is a pro-fibrotic regulator of the MFB phenotype in kidney. This study investigated whether LRP1 signaling by t-PA is also relevant to HSC activation following injury. Primary and immortalized rat HSCs were treated with t-PA and assayed by western blot, MTT, and TUNEL. In vitro results were then verified using an in vivo, acute carbon tetrachloride (CCl4) injury model that examined the phenotype and recovery kinetics of MFBs from wild-type animals vs mice with a global (t-PA) or HSC-targeted (LRP1) deletion. In vitro, in contrast to kidney MFBs, exogenous, proteolytically inactive t-PA suppressed, rather than induced, activation markers in HSCs following phosphorylation of LRP1. This process was mediated by LRP1 as inhibition of t-PA binding to LRP1 blocked the effects of t-PA. In vivo, following acute injury, phosphorylation of LRP1 on activated HSCs occurred immediately prior to their disappearance. Mice lacking t-PA or LRP1 retained higher densities of activated HSCs for a longer time period compared with control mice after injury cessation. Hence, t-PA, an FDA-approved drug, contributes to the suppression of activated HSCs following injury repair via signaling through LRP1. This renders t-PA a potential target for exploitation in treating patients with fibrosis.

Aldosterone antagonist inhibits fibrosis-induced NOX4 protein expression in hepatic cells and tissues of rats

To investigate the inhibitory potential of aldosterone antagonist on NOX4 protein expression in hepatic fibrosis by using a rat model of carbon tetrachloride (CCl4)-induced hepatotoxicity. Twenty-four male Wistar rats were randomly divided into three equal groups: fibrosis model group (receiving three subcutaneous injections per week of 2.5 ml/kg 40% CCl4); spironolactone (Sp)-treated fibrosis model group (receiving CCl4 regimen plus three injections per day of 20 mg/kg Sp in olive oil); negative-treatment fibrosis model group (receiving CCl4 regimen plus three injections per day of olive oil alone). Unmanipulated rats (receiving no CCl4 and no supplemental treatments) served as normal controls. After 4 weeks, liver histology was carried out to assess cytotoxicity (by hematoxylin-eosin staining), fibrosis (by Masson staining and METAVIR scoring), and NOX4 protein expression (by immunohistochemistry). In addition, in vitro analyses of immortalized rat hepatic stellate cells, HSC-T6, were performed to evaluate dose-response (10-9, 10-7 and 10-5 mol/L) and time-response (6, 12 and 24 h) of aldosterone agonist (Ald) and an aldosterone antagonist, eplerenone (EPLE). Effects on NOX4 protein expression were evaluated by western blotting. The fibrosis model group showed significantly more fibrosis than the normal control group (16.060 +/- 0.300 vs. 2.471 +/- 0.160, P = 0.000]; however, the Sp-treated fibrosis model group showed significantly less CCl4-induced fibrosis (5.761 +/- 0.152 vs. model: 16.060 +/- 0.300, P = 0.000). The fibrosis model group also showed significantly higher NOX4 protein expression in liver tissues than the normal control group (7.231 +/- 0.211 vs. 1.350 +/- 0.252, P = 0.000), and the Sp-treated fibrosis model tissues showed significantly less CCl4-induced up-regulated NOX4 protein expression (4.270 +/- 0.242 vs. model: 7.231 +/- 0.211, P = 0.000]. Ald induced up-regulated NOX4 protein expression in HSC-T6 cells in dose- and concentration-dependent manners, with the peak expression being induced by the 10-5 mol/L concentration and 24 h exposure. The Ald-treated cells expressed significantly more NOX4 protein than the untreated control cells (0.710 +/- 0.011 vs. 0.316 +/- 0.015, P = 0.000]. and the EPLE-treated cells showed significantly less Ald-induced up-regulated NOX4 expression (0.615 +/- 0.014 vs. 0.710 +/- 0.011, P = 0.000]. Aldosterone antagonists inhibit the fibrosis-induced NOX4 protein expression in rat hepatic cells.

RNA Interference against Discoidin Domain Receptor 2 Ameliorates Alcoholic Liver Disease in Rats

Discoidin domain receptor 2 (DDR2) is involved in fibrotic disease. However, the exact pathogenic implications of the receptor in early alcoholic liver disease are still controversial. We constructed plasmid vectors encoding short-hairpin RNA against DDR2 to investigate its role in alcoholic liver disease in an immortalized rat hepatic stellate cell line, HSC-T6, and in rats by MTT, RT-PCR and western blot analyses; immunohistochemistry and electron microscopy. Alcohol-induced upregulation of DDR2 was associated with the expression of matrix metalloproteinase 2, the transforming growth factor β1 signaling pathway and tissue inhibitor of metalloproteinase 1; collagen deposition; and extracellular matrix remodeling. Inhibition of DDR2 decreased HSC-T6 cell proliferation and liver injury in rats with 10-week-induced alcoholic liver disease. DDR2 may have an important role in the pathogenesis of early-stage alcoholic liver disease. Silencing DDR2 may be effective in preventing early-stage alcoholic liver disease.

Helicobacter pylori accelerates hepatic fibrosis by sensitizing transforming growth factor-β1-induced inflammatory signaling

Our earlier report has shown that Helicobacter pylori promoted hepatic fibrosis in a murine model. Herein, in order to elucidate the mechanism by which H. pylori accelerate liver fibrosis, the authors investigated the changes in expression levels of mitogen-activated protein kinases (MAPKs), p53-related proteins, antioxidants, and proinflammatory cytokines in liver samples. H. pylori infection enhanced CCl4-induced MAP kinase activation and p53 signaling pathway as well as Bax- and proliferating-cell nuclear antigen expressions, whereas H. pylori alone induced neither of these expressions nor hepatic fibrosis. Moreover, mRNA expressions of inflammatory cytokines, glutathione peroxidase expression, and the proliferative index were strongly augmented in livers of the H. pylori with CCl4 treatment group compared with those of the CCl4-alone treatment group, whereas there was no difference in apoptotic index between the two groups. Interestingly, H. pylori treatment increased the number of α-fetoprotein-expressing hepatocytes independently of CCl4 intoxication. In vitro analyses, using an immortalized rat hepatic stellate cell (HSC) line, revealed that H. pylori lysates increased the proliferation of HSCs, which was boosted by the addition of transforming growth factor-beta1 (TGF-β1). Furthermore, the treatment of H. pylori lysates promoted the translocation of nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) into the nucleus based on an increase in the degradation of NF-κB inhibitor alpha, in the presence of TGF-β1, as did H2O2 treatment. In conclusion, H. pylori infection along with an elevated TGF-β1 may accelerate hepatic fibrosis through increased TGF-β1-induced pro-inflammatory signaling pathways in HSCs. Moreover, H. pylori infection might increase the risk of TGF-β1-mediated tumorigenesis by disturbing the balance between apoptosis and proliferation of hepatocytes.

Antifibrotic Phenanthrenes of Dendrobium nobile Stems

Two new phenanthrenes ( 1 and 6) and four new dihydrophenanthrenes ( 2- 5) were isolated from a methanolic extract of Dendrobium nobile stems, along with 13 known phenanthrenes and dihydrophenanthrenes ( 7- 19). By spectroscopic analysis, the structures of compounds 1- 6 were determined as 2,8-dihydroxy-3,4,7-trimethoxyphenanthrene ( 1), 3-hydroxy-2,4,7-trimethoxy-9,10-dihydrophenanthrene ( 2), 2,8-dihydroxy-3,4,7-trimethoxy-9,10-dihydrophenanthrene ( 3), 2-hydroxy-4,7-dimethoxy-9,10-dihydrophenanthrene ( 4), 2,2'-dihydroxy-3,3',4,4',7,7'-hexamethoxy-9,9',10,10'-tetrahydro-1,1'-biphenanthrene ( 5), and 2,3,5-trihydroxy-4,9-dimethoxyphenanthrene ( 6), respectively. Antifibrotic activity of compounds 1- 19 was evaluated employing HSC-T6, an immortalized rat hepatic stellate cell line, as an in vitro assay system by assessing cell proliferation.

Inhibitory Effect of Emodin on Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) Expression in Rat Hepatic Stellate Cells

Emodin inhibited expression of both transforming growth factor beta1 (TGFbeta1)- and phorbol ester (PMA)-induced tissue inhibitors of metalloproteinase-1 (TIMP-1) in an immortalized rat hepatic stellate cell line, HSC-T6, by Western blot and reverse transcription polymerase chain reaction. Reporter gene assays showed that emodin reduced both basal and PMA-induced activated protein-1 (AP-1) promoter activities. Electrophoretic mobility shift assay revealed that emodin reduced AP-1 DNA binding activities in HSC-T6 cells. AP-1 components analysis showed that emodin also attenuated JunD mRNA expression. Furthermore, emodin markedly inhibited TGFbeta1-induced p42/p44 mitogen-activated protein kinase phosphorylation but did not alter PMA induction. We conclude that emodin effectively inhibits PMA- and TGFbeta1-stimulated TIMP-1 expression in hepatic stellate cells by suppressing the AP-1 signaling pathway and extracellular signal-regulated kinase activation, respectively. These data provide new insight into the cellular and molecular mechanisms of emodin against liver fibrosis.

Novel Inhibitors of Prolyl 4-Hydroxylase; Solid-phase Synthesis of 2,2-Dimethyl-3,4-Dialkoxy-Substituted 6-Aminobenzopyran Derivatives

2,2-Dimethyl-3,4-dialkoxy-substituted 6-aminobenzopyran analogues (eg., 7 and 8) were identified as prolyl 4-hydroxylase inhibitors via a screening process using HSC-T6 and LI 90 cells that express an immortalized rat hepatic stellate cell line and as part of a test of the type I collagen contents employing the ELISA method. A subsequent lead optimization effort based on solid-phase parallel synthesis led to the identification of 2,2-dimethyl-3,4-dialkoxy-substituted 6-aminobenzopyrans as potent inhibitors of prolyl 4-hydroxylase.

Establishment and identification of a novel immortalized rat hepatic stellate cell line HSC-PQ

Establishment and identification of a novel immortalized rat hepatic stellate cell line HSC-PQ

Triiodothyronine and interleukin-6 (IL-6) induce expression of HGF in an immortalized rat hepatic stellate cell line.

Despite its being considered a primary mitogen for hepatocytes, triiodothyronine (T3) has no effect on the proliferation of hepatocytes in vitro, and in our studies, induces significant in vivo hepatocyte proliferation only during liver injury. We hypothesized that T3 may affect hepatocytes proliferation indirectly, by inducing other cells in the liver to secrete hepatic mitogens. In vivo studies: Lipopolysaccharide, T3 and a combination of the two were injected into rats, and hepatocyte proliferation was determined by PCNA staining and mitotic index. a rat hepatic stellate cell line (HSC-6T) was cultured with T3, IL-6 and a combination of the two, and we assessed the effect of these cytokine/hormone combinations on the cell proliferation and on secretion of IL-6 and HGF, measured by ELISA. Expression of thyroid hormone receptors was assessed by RT-PCR. In vivo: T3, together with lipopolysaccharide, enhances PCNA staining and the mitotic index of hepatocytes in the treated rats. In vitro: the hepatic stellate cell line expresses thyroid hormone receptor alpha 1, but not beta 1. Proliferation of stellate cells is not affected by T3, with or without IL-6. T3 has no effect on secreted levels of IL-6 in the stellate cell line. Hepatic stellate cells cultured with T3 and IL-6 show significantly increased amounts of secreted HGF after 48 h in culture. T3 may induce hepatocyte proliferation in vivo during injury by turning on expression of HGF in stellate cells and acting together with IL-6.

Hypoxic stimulation of vascular endothelial growth factor expression in activated rat hepatic stellate cells.

The tissue repair response to hypoxic stimuli during wound healing includes enhanced production of angiogenic factors, such as vascular endothelial growth factor (VEGF). Hepatic stellate cells are oxygen-sensing cells, capable of producing VEGF. We hypothesized that hypoxia-stimulated signaling in activated stellate cells mediate VEGF secretion during liver injury. The specific aim was to evaluate the effect of hypoxia on the gene expression of VEGF in HSC-T6 cells, an immortalized rat hepatic stellate cell line, and in rat primary cultures of stellate cells. Hypoxic induction of VEGF mRNA was dose- and time-dependent. The hypoxic stimulation of VEGF messenger RNA (mRNA) correlated with the secretion of VEGF protein in conditioned media by hypoxic T6 cells. S-Nitroso-N-acetyl-D, L-penicillamine (SNAP), a nitric oxide (NO) donor, and desferrioxamine (DFx) and cobalt chloride, mimics of cellular hypoxia, similarly stimulated VEGF mRNA expression and secretion. Four previously described splice variants of the VEGF mRNA (VEGF-120, 144, 164, 188) were detected in both normoxic- or hypoxic-activated stellate cells. There was differential expression of the VEGF receptors, Flt-1 and Flk-1, in hypoxic T6 cells. Hypoxic conditions selectively stimulated Flt-1 mRNA expression, whereas Flk-1 mRNA remained unchanged. Hypoxic induction of VEGF was also demonstrated in primary stellate cell cultures and after in vivo injury. Hypoxia stimulates cell signaling in stellate cells, culminating in the rapid induction of VEGF and Flt-1 mRNA expression and VEGF secretion. The hypoxic induction of VEGF is mimicked by NO and may be of mechanistic importance in the pathogenesis of hepatic wound healing and hepatocarcinogenesis.

Characterization of immortalized rat hepatic stellate cell lines transfected with SV40 T antigen

Hepatic stellate cells (HSCs) play an important role in fibrogenesis of the liver through their activity of biosynthesizing a widely variety of extracellular matrix proteins. However, in vitro experiments using primary cultured HSCs are limited, because HSCs in primary culture tend to dedifferentiate quickly. We recently established several HSC lines by transfection with SV 40 large T antigen (SV40 TAg) without any culture crisis. The cell lines have both morphological and functional characteristics of HSCs. Immunohistochemically, these cell lines showed positive staining of SV 40 TAg in nuclei and desmin and type IV collagen in cytoplasms. Cell lines also had messenger RNA expressions of type I collagen α1 chain, α-smooth muscle actin, hepatocyte growth factor, transforming growth factor-β 1. These cell lines would be useful for in vitro studies in regard to the mechanism of differentiation or transformation of rat HSCs.