Activated Rat Hepatic Stellate Cells Research Articles

Molecular mechanisms of Agardhiella subulata attenuates hepatic fibrosis by modulating hepatic stellate cell activation via the reduction of autophagy

In this study, we investigated the effects of Agardhiella subulata extract (ASE) and its underlying mechanisms in vivo and in vitro. Rats induced hepatic fibrosis with CCl4 and transforming growth factor-β1 (TGF-β1) activated rat hepatic stellate cell line (HSC-T6) was used. The results showed that ASE (oral, 10 or 50 mg/kg) effectively attenuated liver fibrogenesis in the rats. This was evidenced by decreases in serum glutamic oxaloacetic transaminase and glutamic pyruvic transaminase levels, histopathological changes, decreased collagen deposition, and extracellular matrix formation. ASE significantly reduced hepatocyte apoptosis, Kupffer cell activation, and the levels of fibrogenic molecules such as alpha-smooth muscle actin and collagen. Furthermore, ASE downregulated the TGF-β1/Smad signaling pathway and concomitantly inhibited the activation of hepatic stellate cells by reducing autophagy and inducing the regeneration of lipid droplets both in vivo and in vitro. Overall, ASE inhibited various mechanisms that are important targets in the prevention of hepatic fibrosis.

Ginsenoside Rg3 promotes regression from hepatic fibrosis through reducing inflammation-mediated autophagy signaling pathway

Inflammation and autophagy occur during hepatic fibrosis development caused by various pathogens, and effectively curbing of autophage may delay the occurrence of hepatic fibrosis. The current study aimed to unravel the inhibitory effects of Ginsenoside Rg3 (G-Rg3) on inflammation-mediated hepatic autophagy to curb hepatic fibrosis caused by thioacetamide (TAA)-induced subacute and chronic hepatic injury. TAA is mainly metabolized in the liver to cause liver dysfunction. After intraperitoneal injection of TAA for 4 or 10 weeks (TAA-chronic mouse models), severe inflammatory infiltration and fibrosis occurred in the liver. Treatment with G-Rg3 alleviated hepatic pathological changes and reversed hepatic fibrosis in the TAA-chronic models with decreased deposition of collagen fibers, reduced expression of HSCs activation marker (α-SMA), and reduced secretion of profibrogenic factors (TGF-β1). G-Rg3 decreased expressions of autophagy-related proteins in mice of TAA-chronic models. Notably, G-Rg3 inhibited the survival of activated rat hepatic stellate cells (HSC-T6), but had no cytotoxicity on human hepatocytes (L02 cell lines). G-Rg3 dose-dependently inhibited autophagy in vitro with less expression of p62 and fewer LC3a transformation into LC3b in inflammatory inducer lipopolysaccharide (LPS)-induced rat HSC-T6 cells. Furthermore, G-Rg3 enhanced the phosphorylation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) in vivo and in vitro. Besides, mTOR inhibitor Rapamycin and PI3K inhibitors LY294002 were employed in LPS-treated HSC-T6 cell cultures to verify that Rg3 partially reversed the increase in autophagy in hepatic fibrosis in vitro. Taken together, G-Rg3 exerted anti-fibrosis effect through the inhibition of autophagy in TAA-treated mice and LPS-stimulated HSC-T6 cells. These data collectively unravel that G-Rg3 may serve a promising anti-hepatic fibrosis drug.

Phenolic composition, antioxidant and anti-fibrotic effects of Sesbania grandiflora L. (Agastya) - An edible medicinal plant.

Background:Sesbania grandiflora (S. grandiflora/Agastya) is an edible Indian traditional medicinal plant widely used as dietary supplements and possesses various pharmacological activities.Aims:The aim is to evaluate aqueous ethanol extract of S. grandiflora leaves and flowers for its anti-oxidant and anti-proliferative and anti-fibrotic effects using activated rat hepatic stellate cell (HSC)-T6.Material and methods:The antioxidant activities of these plant extracts were assessed as per the standard methods and the total phenolic and flavonoid contents were examined by folin ciocalteu reagent and colorimetric methods respectively. The anti-proliferation assay was conducted by using a cyto X cell viability assay kit. The anti-fibrotic effect was investigated by measuring the hydroxyproline content and gene expression analysis of the two main fibrogenic cytokines in activated HSC-T6 cells: Transforming growth factor-beta (TGF-β) and alpha-smooth muscle actin (α-SMA).Results:The aqueous ethanol extract of S. grandiflora leaves showed the highest antioxidant activity in a concentration-dependent manner. These findings were well correlated with the total phenols and flavonoids contents. The aqueous ethanol extract of S. grandiflora leaf and flower significantly reduced the proliferation of activated HSC-T6 cells. Regarding the anti-fibrotic effect, the hydroxyproline content was significantly reduced in a concentration-dependent manner during the extract treatment. In gene expression analysis, the treatment without extracts drastically up-regulated the fibrogenic cytokines (TGF-β and α-SMA), whereas the treatment with extracts significantly reduced these alterations.Conclusion:Results of present study revealed the significant antioxidant potential of the aqueous ethanol extract of S. grandiflora leaves and flowers. Among two extracts, S. grandiflora leaves demonstrated greater antioxidant, anti-fibrotic capacity with lower inhibiting concentrations corresponding to 50% values than S. grandiflora flowers.

Chrysophanol Prevents Lipopolysaccharide-Induced Hepatic Stellate Cell Activation by Upregulating Apoptosis, Oxidative Stress, and the Unfolded Protein Response.

Hepatic stellate cell (HSC) activation is a vital driver of liver fibrosis. Recent research efforts have emphasized the clearance of activated HSCs by apoptosis, senescence, or reversion to the quiescent state. LPS induces human HSC activation directly and contributes to liver disease progression. Chrysophanol is an anthraquinone with hepatoprotective and anti-inflammatory effects. This study aimed to investigate the pharmacological effects and mechanisms of chrysophanol in an LPS-induced activated rat HSC cell line (HSC-T6). The fibrosis phenotype was identified from the expression of α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), and integrin β1 by western blot analysis. We examined DNA fragmentation by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. We detected the apoptotic markers p53 and cleaved caspase-3 by western blot analysis. Intracellular ROS were labeled with 2′,7′-dichlorofluorescein diacetate (DCF-DA) and the levels were measured by flow cytometry. Finally, we evaluated the ER stress markers binding immunoglobulin protein (BiP) and C/EBP homologous protein (CHOP) by Western blot analysis. Our results showed that chrysophanol decreased HSC-T6 cell viability in LPS-induced activated HSCs. Chrysophanol increased the expression of α-SMA, CTGF, integrin βI, p53, cleaved caspase-3, and DNA fragmentation. Chrysophanol also elevated ROS levels and increased the expression of BiP and CHOP. Pretreatment with chrysophanol prevented LPS-induced HSC-T6 cell activation by upregulating apoptosis, ROS accumulation, unfolded protein response (UPR) activation, and the UPR proapoptotic effect.

Hydrogen sulfide stimulates activation of hepatic stellate cells through increased cellular bio-energetics

Hepatic fibrosis is caused by chronic inflammation and characterized as the excessive accumulation of extracellular matrix (ECM) by activated hepatic stellate cells (HSCs). Gasotransmitters like NO and CO are known to modulate inflammation and fibrosis, however, little is known about the role of the gasotransmitter hydrogen sulfide (H2S) in liver fibrogenesis and stellate cell activation. Endogenous H2S is produced by the enzymes cystathionine β-synthase (CBS), cystathionine γ-lyase (CTH) and 3-mercaptopyruvate sulfur transferase (MPST) [1]. The aim of this study was to elucidate the role of endogenously produced and/or exogenously administered H2S on rat hepatic stellate cell activation and fibrogenesis. Primary rat HSCs were culture-activated for 7 days and treated with different H2S releasing donors (slow releasing donor GYY4137, fast releasing donor NaHS) or inhibitors of the H2S producing enzymes CTH and CBS (DL-PAG, AOAA). The main message of our study is that mRNA and protein expression level of H2S synthesizing enzymes are low in HSCs compared to hepatocytes and Kupffer cells. However, H2S promotes hepatic stellate cell activation. This conclusion is based on the fact that production of H2S and mRNA and protein expression of its producing enzyme CTH are increased during hepatic stellate cell activation. Furthermore, exogenous H2S increased HSC proliferation while inhibitors of endogenous H2S production reduce proliferation and fibrotic makers of HSCs. The effect of H2S on stellate cell activation correlated with increased cellular bioenergetics. Our results indicate that the H2S generation in hepatic stellate cells is a target for anti-fibrotic intervention and that systemic interventions with H2S should take into account cell-specific effects of H2S.

Stable expression of constitutively activated ALK3 suppresses rat hepatic stellate cell activation

Stable expression of constitutively activated ALK3 suppresses rat hepatic stellate cell activation

Lipopolysaccharide Reverses Hepatic Stellate Cell Activation In Vitro and In Vivo via Modulation of c‐Myb and SMAD7

BackgroundDuring liver injury, the perisinusoidal quiescent hepatic stellate cells (HSCs) become activated and transdifferentiate into highly proliferative myofibroblastic phenotype (aHSCs) that expresses α‐smooth muscle actin (αSMA) and platelet‐derived growth factor β receptor (PDGFβR). aHSCs are the major fibrogenic cell type regardless of etiology of chronic liver disease. Their interactions with gut‐derived bacterial lipopolysaccharide (LPS), a potent inflammatory mediator, are implicated in hepatic fibrogenesis. However, evidence also indicates that LPS can attenuate fibrogenic characteristics of aHSCs. We examined molecular mechanisms of anti‐fibrogenic effects of LPS on aHSCs in vitro and in vivo.MethodsCulture‐activated rat HSCs were exposed to 1–100 ng/ml LPS for 24h and parameters of fibrosis and inflammatory cytokines/chemokines were determined by q‐RT‐PCR, Western and immunohistochemical analyses. In vivo, HSC activation and liver fibrosis were induced in rats by CCl4 administration for 3 weeks, after which LPS (5 mg/kg; ip) or vehicle‐PBS was administered. HSCs were isolated 24h later and fibrogenic/inflammatory parameters were analyzed.ResultsLPS, concentration‐dependently, induced phenotypic changes in aHSCs as illustrated by reduction in cell size and rounded shape. LPS strongly down‐regulated mRNA and protein expressions of αSMA, as well as PDGFβR, TGFβRI, Col1α1 and fibronectin. These effects of LPS were prevented by its inhibitor polymyxin B. The LPS‐induced size/shape change of aHSCs was associated with loss of proliferation (Ki67 staining) and down‐regulation of PDGFβR, without any effect on viability, or lipid accumulation (typical of quiescent HSCs). Mechanistically, LPS significantly down‐regulated mRNA and protein expression of c‐Myb, a transcription factor for αSMA, and up‐regulated Smad7 that inhibits Smad2/3–4‐induced transcription of Col1α1. Furthermore, HSCs isolated from in vivo LPS‐treated CCl4‐fibrotic rats demonstrated reduced proliferation, and down‐regulation of αSMA, PDGFβR, TGFβRI and c‐Myb expression, and increased expression of SMAD7. Additionally, in vitro exposure of HSCs isolated from CCl4 and CCl4+LPS‐treated rats to LPS induced further phenotypic change, inhibition of proliferation, and down‐regulation of αSMA, c‐Myb and PDGFβR expression suggesting that the in vivo LPS‐treated HSCs are not desensitized to the subsequent LPS challenge. Interestingly, LPS elicited powerful pro‐inflammatory response (increased TNFα, IL6 and CXCL1 expression) but also strongly up‐regulated expression of anti‐inflammatory IL10 in HSCs.ConclusionLPS limits fibrosis by modulation of c‐Myb and SMAD7 thus down‐regulating the key fibrogenic mechanisms in aHSCs that may have important implication in chronic liver disease.Support or Funding InformationSupported by VA Merit Review Award 1IO1BX001174‐01 and Department of Defense W81XWH‐14.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Anti-fibrosis activity of combination therapy with epigallocatechin gallate, taurine and genistein by regulating glycolysis, gluconeogenesis, and ribosomal and lysosomal signaling pathways in HSC-T6 cells.

A previous study by our group indicated that combined treatment with taurine, epigallocatechin gallate (EGCG) and genistein protects against liver fibrosis. The aim of the present study was to elucidate the antifibrotic mechanism of this combination treatment using isobaric tag for relative and absolute quantification (iTRAQ)-based proteomics in an activated rat hepatic stellate cell (HSC) line. In the present study, HSC-T6 cells were incubated with taurine, EGCG and genistein, and cellular proteins were extracted and processed for iTRAQ labeling. Quantification and identification of proteins was performed using two-dimensional liquid chromatography coupled with tandem mass spectrometry. Proteomic analysis indicated that the expression of 166 proteins were significantly altered in response to combination treatment with taurine, EGCG and genistein. A total 76 of these proteins were upregulated and 90 were downregulated. Differentially expressed proteins were grouped according to their association with specific Kyoto Encyclopedia of Genes and Genomes pathways. The results indicated that the differentially expressed proteins hexokinase-2 and lysosome-associated membrane glycoprotein 1 were associated with glycolysis, gluconeogenesis and lysosome signaling pathways. The expression of these proteins was validated using western blot analysis; the expression of hexokinase-2 was significantly decreased and the expression of lysosome-associated membrane glycoprotein 1 was significantly increased in HSC-T6 cells treated with taurine, EGCG and genistein compared with the control, respectively (P<0.05). These results were in accordance with the changes in protein expression identified using the iTRAQ approach. Therefore, the antifibrotic effect of combined therapy with taurine, EGCG and genistein may be associated with the activation of several pathways in HSCs, including glycolysis, gluconeogenesis, and the ribosome and lysosome signaling pathways. The differentially expressed proteins identified in the current study may be useful for treatment of liver fibrosis in the future.

Hepatoprotective Principles and Other Chemical Constituents from the Mycelium of Phellinus linteus.

In the dimethylnitrosamine (DMN)-induced hepatic fibrosis Wistar rat model, the mycelium extract of Phellinus linteus (PLE) (20 mg/Kg) displayed significant protection against hepatic fibrosis. The present investigation characterized eleven new ionone derivatives, phellinulins D–N (4–14), from the P. linteus mycelium extract and the relative stereochemical structures were constructed according to the spectroscopic and spectrometric analytical results. Some purified compounds were examined for their inhibitory effects on activated rat hepatic stellate cells (HSCs) and several isolates did exhibit significant protection. The results indicated that the mycelium of P. linteus could be explored as a hepatoprotective drug or healthy food candidate in the near future.

Estrogen receptor‑β‑dependent effects of saikosaponin‑d on the suppression of oxidative stress‑induced rat hepatic stellate cell activation.

Saikosaponin-d (SSd) is one of the major triterpenoid saponins derived from Bupleurum falcatum L., which has been reported to possess antifibrotic activity. At present, there is little information regarding the potential target of SSd in hepatic stellate cells (HSCs), which serve an important role in excessive extracellular matrix (ECM) deposition during the pathogenesis of hepatic fibrosis. Our recent study indicated that SSd may be considered a novel type of phytoestrogen with estrogen-like actions. Therefore, the present study aimed to investigate the effects of SSd on the proliferation and activation of HSCs, and the underlying mechanisms associated with estrogen receptors. In the present study, a rat HSC line (HSC-T6) was used and cultured with dimethyl sulfoxide, SSd, or estradiol (E2; positive control), in the presence or absence of three estrogen receptor (ER) antagonists [ICI-182780, methylpiperidinopyrazole (MPP) or (R,R)-tetrahydrochrysene (THC)], for 24 h as pretreatment. Oxidative stress was induced by exposure to hydrogen peroxide for 4 h. Cell proliferation was assessed by MTT growth assay. Malondialdehyde (MDA), CuZn-superoxide dismutase (CuZn-SOD), tissue inhibitor of metalloproteinases-1 (TIMP- 1), matrix metalloproteinase-1 (MMP-1), transforming growth factor-β1 (TGF-β1), hydroxyproline (Hyp) and collagen-1 (COL1) levels in cell culture supernatants were determined by ELISA. Reactive oxygen species (ROS) was detected by flow cytometry. Total and phosphorylated mitogen-activated protein kinases (MAPKs) and α-smooth muscle actin (α-SMA) were examined by western blot analysis. TGF-β1 mRNA expression was determined by RT-quantitative (q)PCR. SSd and E2 were able to significantly suppress oxidative stress-induced proliferation and activation of HSC-T6 cells. Furthermore, SSd and E2 were able to reduce ECM deposition, as demonstrated by the decrease in transforming growth factor-β1, hydroxyproline, collagen-1 and tissue inhibitor of metalloproteinases-1, and by the increase in matrix metalloproteinase-1. These results suggested that the possible molecular mechanism could involve downregulation of the reactive oxygen species/mitogen-activated protein kinases signaling pathway. Finally, the effects of SSd and E2 could be blocked by co-incubation with ICI-182780 or THC, but not MPP, thus indicating that ERβ may be the potential target of SSd in HSC-T6 cells. In conclusion, these findings suggested that SSd may suppress oxidative stress-induced activation of HSCs, which relied on modulation of ERβ.

Combined Methylome and Transcriptome Analysis During Rat Hepatic Stellate Cell Activation.

Hepatic stellate cells (HSCs) are mesenchymal stem cells (MSCs) of the liver. They are unique among MSCs, since HSCs remain in a quiescent, retinoid-storing state in the normal liver but become activated after liver injury and contribute to tissue repair. The epigenetic mechanisms accompanying the transition of HSCs from a quiescent to an activated state are in the focus of the present study. We investigated the methylome and transcriptome during this process and observed profound changes. While the promoter methylation correlated negatively with gene expression, the gene-body methylation revealed no clear correlation. Most genes with altered expression were associated with cell differentiation. Among them, Wilms tumor 1 (Wt1) and Deltex4 (Dtx4) genes were identified as epigenetically regulated. Since HSCs were reported to derive from multipotent Wt1-positive cells and many differentially expressed genes were associated with cell differentiation during their activation, epigenetic alterations are presumably required to enable HSC development.

MiR‑21 promotes α‑SMA and collagen I expression in hepatic stellate cells via the Smad7 signaling pathway.

The aim of the present study was to investigate the role of microRNA (miR)‑21 in regulating collagenI and Smad7 expression in activated rat hepatic stellate cells (HSCs). Rat HSCs were isolated by single‑step density gradient centrifugation with Nycodenz. Cellular content of miR‑21, SMAD7, α‑smooth muscle actin (α‑SMA), collagen type I alpha 1 (COLLA1) and COLL alpha 2 (A2) mRNA was examined by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR), and cellular content of Smad7 and α‑SMA protein was detected by western blotting. Binding of miR‑21 to the 3'‑untranslated region (UTR) of Smad7 was verified by dual‑luciferase assay. The authors observed that, in activated HSCs, expression of miR‑21 was significantly increased in a time‑dependent manner, while expression of Smad7 mRNA and protein was significantly reduced. In addition, miR‑21 mimics significantly enhanced cellular α‑SMA mRNA and protein content, while miR‑21 inhibitor significantly reduced α‑SMA mRNA and protein levels. Similarly, cellular content of COLLA1 and COLLA2 mRNA was significantly elevated by miR‑21 mimics, but reduced by miR‑21 inhibitor, in activated HSCs. Moreover, cellular content of Smad7 mRNA and protein was significantly reduced by miR‑21 mimics, but significantly increased by miR‑21 inhibitor. Furthermore, miR‑21 mimics activated firefly luciferase in HEK293 cells transfected with the wild type 3'‑UTR of Smad7. miR‑21 regulates expression of α‑SMA and collagen I in activated rat HSCs by directly targeting Smad7.

Low concentrations of bilirubin inhibit activation of hepatic stellate cells in vitro

Hepatic stellate cell (HSC) activation serves a key role in liver fibrosis, and is associated with chronic liver diseases. Bilirubin, a product of heme degradation, has been demonstrated to have antioxidant properties. The present study investigated the effects of physiological concentrations of bilirubin on rat HSC activation. Rat HSCs were isolated and cultured for several generations to induce activation. The activated HSCs were subsequently treated with 0, 1, 10 or 20 mg/l bilirubin and assayed for parameters of cell activation. As the bilirubin concentration increased, HSCs demonstrated reduced production of reactive oxygen species, reduced protein expression levels of α-smooth muscle actin, a decreased mRNA expression ratio of tissue inhibitor of matrix metalloproteinase-1/matrix metalloproteinase-2, decreased proliferation and increased apoptosis. In conclusion, elevated bilirubin levels, within its physiological concentration range, appeared to inhibit HSC activation. These findings suggested a potential role for bilirubin in the treatment of fibrosis that requires further investigation.

Galanin receptor 2 mediates antifibrogenic effects of galanin on hepatic stellate cells.

Galanin is an endogenous factor involved in the negative regulation of the biological effects of leptin in bioenergetic metabolism. Leptin promotes fibrogenic effects in hepatic stellate cells (HSCs), however, little is known about the effects of galanin on HSCs. In the present study, the biological functions of galanin and its receptors (GalRs) in HSCs were investigated using cell culture in vitro. It was found that galanin and GalR3 mRNA are expressed in quiescent and activated HSCs. GalR2 expression was undetectable in quiescent HSCs but was markedly induced in activated HSCs. In the HSC-T6 cell line, which is an activated rat HSC cell line, treatment with 100 nmol/l galanin significantly inhibited cell proliferation. It also inhibited transforming growth factor (TGF)-β1 and α-smooth muscle actin (SMA) expression and upregulated peroxisome proliferator-activated receptor (PPAR)-γ expression. Following the knockdown of GalR2 by specific small interfering RNA, the activation of GalR3 by galanin does not influence these effects of galanin on HSCs. However, activation of GalR2 alone by galanin following the knockdown of GalR3 inhibits HSC proliferation and TGF-β1 and α-SMA expression, in addition to inducing PPAR-γ expression. These data suggest that galanin inhibits HSC activation and suppresses the profibrogenic features of these cells, and these effects might be mediated by GalR2. Thus, galanin is a potential endogenous factor in the inhibition of liver fibrosis.

ATGL and DGAT1 are involved in the turnover of newly synthesized triacylglycerols in hepatic stellate cells

Hepatic stellate cell (HSC) activation is a critical step in the development of chronic liver disease. During activation, HSCs lose their lipid droplets (LDs) containing triacylglycerol (TAG), cholesteryl esters (CEs), and retinyl esters (REs). Here we aimed to investigate which enzymes are involved in LD turnover in HSCs during activation in vitro. Targeted deletion of the Atgl gene in mice HSCs had little effect on the decrease of the overall TAG, CE, and RE levels during activation. However, ATGL-deficient HSCs specifically accumulated TAG species enriched in PUFAs and degraded new TAG species more slowly. TAG synthesis and levels of PUFA-TAGs were lowered by the diacylglycerol acyltransferase (DGAT)1 inhibitor, T863. The lipase inhibitor, Atglistatin, increased the levels of TAG in both WT and ATGL-deficient mouse HSCs. Both Atglistatin and T863 inhibited the induction of activation marker, α-smooth muscle actin, in rat HSCs, but not in mouse HSCs. Compared with mouse HSCs, rat HSCs have a higher turnover of new TAGs, and Atglistatin and the DGAT1 inhibitor, T863, were more effective. Our data suggest that ATGL preferentially degrades newly synthesized TAGs, synthesized by DGAT1, and is less involved in the breakdown of preexisting TAGs and REs in HSCs. Furthermore a large change in TAG levels has modest effect on rat HSC activation.

Hepatic stellate cell transdifferentiation involves genome-wide remodeling of the DNA methylation landscape

DNA methylation (5-mC) is an epigenetic mark that is an established regulator of transcriptional repression with an important role in liver fibrosis. Currently, there is very little knowledge available as to how DNA methylation controls the phenotype of hepatic stellate cell (HSC), the key cell type responsible for onset and progression of liver fibrosis. Moreover, recently discovered DNA hydroxymethylation (5-hmC) is involved in transcriptional activation and its patterns are often altered in human diseases. The aim of this study is to investigate the role of DNA methylation/hydroxymethylation in liver fibrosis. Levels of 5-mC and 5-hmC were assessed by slot blot in a range of animal liver fibrosis models and human liver diseases. Expression levels of TET and DNMT enzymes were measured by qRT-PCR and Western blotting. Reduced representation bisulfite sequencing (RRBS) method was used to examine 5-mC and 5-hmC patterns in quiescent and in vivo activated rat HSC. We demonstrate global alteration in 5-mC and 5-hmC and their regulatory enzymes that accompany liver fibrosis and HSC transdifferentiation. Using RRBS, we show exact genomic positions of changed methylation patterns in quiescent and in vivo activated rat HSC. In addition, we demonstrate that reduction in DNMT3a expression leads to attenuation of pro-fibrogenic phenotype in activated HSC. Our data suggest that DNA 5-mC/5-hmC is a crucial step in HSC activation and therefore fibrogenesis. Changes in DNA methylation during HSC activation may bring new insights into the molecular events underpinning fibrogenesis and may provide biomarkers for disease progression as well as potential new drug targets.

Effects of the total flavone of litchi chinensis sonn on expressions of NF-κB and α-SMA in TGF-β1 activated rat hepatic stellate cells#br#

Effects of the total flavone of litchi chinensis sonn on expressions of NF-κB and α-SMA in TGF-β1 activated rat hepatic stellate cells#br#

Non-Canonical Wnt Predominates in Activated Rat Hepatic Stellate Cells, Influencing HSC Survival and Paracrine Stimulation of Kupffer Cells.

The Wnt system is highly complex and is comprised of canonical and non-canonical pathways leading to the activation of gene expression. Our aim was to examine changes in the expression of Wnt ligands and regulators during hepatic stellate cell (HSC) transdifferentiation and assess the relative contributions of the canonical and non-canonical Wnt pathways in fibrogenic activated HSC. The expression profile of Wnt ligands and regulators in HSC was not supportive for a major role for β-catenin-dependent canonical Wnt signalling, this verified by inability to induce Topflash reporter activity in HSC even when expressing a constitutive active β-catenin. We detected expression of Wnt5a in activated HSC which can signal via non-canonical mechanisms and showed evidence for non-canonical signalling in these cells involving phosphorylation of Dvl2 and pJNK. Stimulation of HSC or Kupffer cells with Wnt5a regulated HSC apoptosis and expression of TGF-β1 and MCP1 respectively. We were unable to confirm a role for β-catenin-dependent canonical Wnt in HSC and instead propose autocrine and paracrine functions for Wnts expressed by activated HSC via non-canonical pathways. The data warrant detailed investigation of Wnt5a in liver fibrosis.

Activated rat hepatic stellate cells influence Th1/Th2 profile in vitro

To investigate the effects of activated rat hepatic stellate cells (HSCs) on rat Th1/Th2 profile in vitro. Growth and survival of activated HSCs and CD4(+) T lymphocytes cultured alone or together was assessed after 24 or 48 h. CD4(+) T lymphocytes were then cultured with or without activated HSCs for 24 or 48 h and the proportion of Th1 [interferon (IFN)-γ(+)] and Th2 [interleukin (IL)-4(+)] cells was assessed by flow cytometry. Th1 and Th2 cell apoptosis was assessed after 24 h of co-culture using a caspase-3 staining procedure. Differentiation rates of Th1 and Th2 cells from CD4(+) T lymphocytes that were positive for CD25 but did not express IFN-γ or IL-4 were also assessed after 48 h of co-culture with activated HSCs. Galectin-9 expression in HSCs was determined by immunofluorescence and Western blotting. ELISA was performed to assess galectin-9 secretion from activated HSCs. Co-culture of CD4(+) T lymphocytes with activated rat HSCs for 48 h significantly reduced the proportion of Th1 cells compared to culture-alone conditions (-1.73% ± 0.71%; P < 0.05), whereas the proportion of Th2 cells was not altered; the Th1/Th2 ratio was significantly decreased (-0.44 ± 0.13; P < 0.05). In addition, the level of IFN-γ in Th1 cells was decreased (-65.71 ± 9.67; P < 0.01), whereas the level of IL-4 in Th2 cells was increased (82.79 ± 25.12; P < 0.05) by co-culturing, as measured by mean fluorescence intensity by flow cytometry. Apoptosis rates in Th1 (12.27% ± 0.99%; P < 0.01) and Th2 (1.71% ± 0.185%; P < 0.01) cells were increased 24 h after co-culturing with activated HSCs; the Th1 cell apoptosis rate was significantly higher than in Th2 cells (P < 0.01). Galectin-9 protein expression was significantly decreased in HSCs only 24 h after co-culturing (P < 0.05) but not after 48 h. Co-culture for 48 h significantly increased the differentiation of Th1 and Th2 cells; however, the increase in the proportion of Th2 cells was significantly higher than that of Th1 cells (1.85% ± 0.48%; P < 0.05). Activated rat HSCs lower the Th1/Th2 profile, inhibiting the Th1 response and enhancing the Th2 response, and this may be a novel pathway for liver fibrogenesis.

P0452 : Changes in DNA methylation and hydroxymethylation during HSC activation

Background and Aims: DNA methylation (5mC) is an epigenetic mark that is an established regulator of gene transcription with an important role in liver fibrosis. Currently, there is only very basic knowledge available as to how DNA methylation controls the phenotype of hepatic stellate cells (HSC), the key cell type responsible for onset and progression of liver fibrosis. Evidence has shown that presence of higher levels of DNA methylation around gene promoters correlate with low or no transcriptional activity. However, recently discovered 5-hydroxymethylcytosine (5hmC), generated by oxidation of 5mC through action of TET enzymes, is actually involved in gene activation and its patterns are often altered in human diseases. In this study we investigated the role of 5mC and 5hmC in liver fibrosis. Methods: Levels of 5mC and 5hmC were assessed by slot blot in a range of animal liver fibrosis models and diseased human livers. Expression levels of TET enzymes and DNA methyltransferases (DNMTs) were measured by qRT-PCR and WB. Reduced representation bisulfide sequencing (RRBS) method was used to examine 5mC and 5hmC patterns in quiescent and in vivo activated rat HSC. Results: Expression of TET enzymes and therefore global levels of 5hmC, are both downregulated in activated rat HSC and in diseased human livers. Moreover, DNMTs expression is changed in human and rat samples suggesting changed levels of 5mC. Using RRBS, we examined the exact genomic positions of changed methylation patterns in quiescent and in vivo activated rat HSC. Conclusions: We found significant differences in TET and DNMT protein expression between quiescent and activated rat HSC as well as in human samples, which in turn cause alteration in the HSC DNA methylome and hydroxymethylome. Changes in DNA methylation during HSC activation may bring new insights into the molecular events underpinning fibrogenesis and may provide biomarkers for disease progression as well as potential new drug targets.