Primary Rat Hepatic Stellate Cells Research Articles

The heavy subunit of ferritin stimulates NLRP3 inflammasomes in hepatic stellate cells through ICAM-1 to drive hepatic inflammation.

Serum ferritin concentrations increase during hepatic inflammation and correlate with the severity of chronic liver disease. Here, we report a molecular mechanism whereby the heavy subunit of ferritin (FTH) contributes to hepatic inflammation. We found that FTH induced activation of the NLRP3 inflammasome and secretion of the proinflammatory cytokine interleukin-1β (IL-1β) in primary rat hepatic stellate cells (HSCs) through intercellular adhesion molecule-1 (ICAM-1). FTH-ICAM-1 stimulated the expression of Il1b, NLRP3 inflammasome activation, and the processing and secretion of IL-1β in a manner that depended on plasma membrane remodeling, clathrin-mediated endocytosis, and lysosomal destabilization. FTH-ICAM-1 signaling at early endosomes stimulated Il1b expression, implying that this endosomal signaling primed inflammasome activation in HSCs. In contrast, lysosomal destabilization was required for FTH-induced IL-1β secretion, suggesting that lysosomal damage activated inflammasomes. FTH induced IL-1β production in liver slices from wild-type mice but not in those from Icam1-/- or Nlrp3-/- mice. Thus, FTH signals through its receptor ICAM-1 on HSCs to activate the NLRP3 inflammasome. We speculate that this pathway contributes to hepatic inflammation, a key process that stimulates hepatic fibrogenesis associated with chronic liver disease.

Extracellular vesicles derived from liver sinusoidal endothelial cells inhibit the activation of hepatic stellate cells and Kupffer cells in vitro

Liver sinusoidal endothelial cells (LSECs) play a crucial role in maintaining liver microcirculation and exchange of nutrients in the liver and are thought to be involved in the pathogenesis of metabolic dysfunction-associated steatotic liver disease (MASLD). The activation of hepatic stellate cells (HSCs) and Kupffer cells (KCs) has been considered to be responsible for the onset of liver fibrosis and the aggravation of liver injury. However, the paracrine regulatory effects of LSECs in the development of MASLD, in particular the role of LSEC-derived extracellular vesicles (EVs) remains unclear. Therefore, the aim of the present study was to investigate the influence of LSEC-derived EVs on HSCs and KCs. Primary rat LSECs, HSCs and KCs were isolated from male Wistar rats. LSEC-derived EVs were isolated from conditioned medium by ultracentrifugation and analyzed by nanoparticle tracking analysis, and expression of specific markers. LSEC-derived EVs reduced the expression of activation markers in activated HSCs but did not affect quiescent HSCs. Also, LSEC-derived EVs suppressed proliferation of activated HSCs activation, as assessed by Xcelligence and BrdU assay. LSEC-derived EVs also increased the expression of inflammatory genes in HSCs that normally are lowly expression during their activation. In contrast, EVs decreased the expression of inflammatory genes in activated KCs. In summary, our results suggest that LSEC-derived EVs may attenuate the fibrogenic phenotype of activated HSCs and the inflammatory phenotype of KCs. Our results show promise for LSEC-derived EVs as therapeutic moieties to treat MASLD. In addition, these EVs might prove of diagnostic value.

Polyenylphosphatidylcholine as bioactive excipient in tablets for the treatment of liver fibrosis

Liver fibrosis is a condition characterized by the accumulation of extracellular matrix (ECM) arising from the myofibroblastic transdifferentiation of hepatic stellate cells (HSCs) occurring as the natural response to liver damage. To date, no pharmacological treatments have been specifically approved for liver fibrosis. We recently reported a beneficial effect of polyenylphosphatidylcholines (PPCs)-rich formulations in reverting fibrogenic features of HSCs. However, unsaturated phospholipids’ properties pose a constant challenge to the development of tablets as preferred patient-centric dosage form. Profiting from the advantageous physical properties of the PPCs-rich Soluthin® S 80 M, we developed a tablet formulation incorporating 70% w/w of this bioactive lipid. Tablets were characterized via X-ray powder diffraction, thermogravimetry, and Raman confocal imaging, and passed the major compendial requirements. To mimic physiological absorption after oral intake, phospholipids extracted from tablets were reconstituted as protein-free chylomicron (PFC)-like emulsions and tested on the fibrogenic human HSC line LX-2 and on primary cirrhotic rat hepatic stellate cells (PRHSC). Lipids extracted from tablets and reconstituted in buffer or as PFC-like emulsions exerted the same antifibrotic effect on both activated LX-2 and PRHSCs as observed with plain S 80 M liposomes, showing that the manufacturing process did not interfere with the bioactivity of PPCs.

Hepatic stellate cells induce an inflammatory phenotype in Kupffer cells via the release of extracellular vesicles.

Liver fibrosis is the response of the liver to chronic liver inflammation. The communication between the resident liver macrophages (Kupffer cells [KCs]) and hepatic stellate cells(HSCs) has been mainly viewed as one-directional: from KCs to HSCs with KCs promoting fibrogenesis. However, recent studies indicated that HSCs may function as a hub of intercellular communications. Therefore, the aim of the present study was to investigate the role of HSCs on the inflammatory phenotype of KCs. Primary rat HSCs and KCs were isolated from male Wistar rats. HSCs-derived conditioned medium (CM) was harvested from different time intervals (Day 0-2: CM-D2 and Day 5-7: CM-D7) during the activation of HSCs. Extracellular vesicles (EVs) were isolated from CM by ultracentrifugation and evaluated by nanoparticle tracking analysis and western blot analysis. M1 and M2 markers of inflammation were measured by quantitative PCR and macrophage function by assessing phagocytic capacity. CM-D2 significantly induced the inflammatory phenotype in KCs, but not CM-D7. Neither CM-D2 nor CM-D7 affected the phagocytosis of KCs. Importantly, the proinflammatory effect of HSCs-derived CM is mediated via EVs released from HSCs since EVs isolated from CMmimicked the effect of CM, whereas EV-depleted CM lost its ability to induce a proinflammatory phenotype in KCs. In addition, when the activation of HSCs was inhibited, HSCs produced less EVs. Furthermore, the proinflammatory effects of CM and EVs are related to activating Toll-like receptor 4 (TLR4) in KCs. In conclusion, HSCs at an early stage of activation induce a proinflammatory phenotype in KCs via the release of EVs. This effect is absent in CM derived from HSCs at a later stage of activation and is dependent on the activation of TLR4 signaling pathway.

Hydroxyurea attenuates hepatic stellate cell proliferation in vitro and liver fibrogenesis in vivo.

Liver fibrosis results from excessive proliferation of, and collagen production by hepatic stellate cells (HSCs) that is caused by chronic liver injury. No drugs are available to cure liver fibrosis. Hydroxyurea is an anti-proliferative drug that is used in benign and malignant disorders. Here, we studied the effect of hydroxyurea on primary HSCs and its anti-fibrotic effect in the CCl4 mouse model of liver fibrosis. Primary rat HSCs were cultured in the absence or presence of hydroxyurea (0.1-1.0 mmol/L). CCl4 or vehicle was administered to C57BL/6/J mice for 4 weeks, with or without hydroxyurea (100 mg/kg/day) co-treatment. We used real-time cell proliferation analysis, Oil Red O (lipid droplet) staining, immunohistochemistry, Acridine Orange staining (apoptosis), Sytox green staining (necrosis), RT-qPCR, ELISA, and Western Blotting for analysis. Hydroxyurea dose-dependently suppressed lipid droplet-loss and mRNA levels of Col1α1 and Acta2 in transdifferentiating HSCs. In fully-activated HSCs, hydroxyurea dose-dependently attenuated PCNA protein levels and BrdU incorporation, but did not reverse Col1α1 and Acta2 mRNA expression. Hydroxyurea did not induce apoptosis or necrosis in HSCs or hepatocytes. Hydroxyurea suppressed accumulation of desmin-positive HSCs and hepatic collagen deposition after CCl4 treatment. CCl4 -induced regenerative hepatocyte proliferation, Col1α1 and Acta2 mRNA expression and α-SMA protein levels were not affected. This study demonstrates that hydroxyurea inhibits HSC proliferation in vitro and attenuates early development of liver fibrosis in vivo, while preserving hepatocyte regeneration after toxic insults by CCl4. Thus, hydroxyurea may have therapeutic value against liver fibrosis.

Integrated analysis and validation of TRIM23/p53 signaling pathway in hepatic stellate cells ferroptosis and liver fibrosis

BackgroundTripartite motif containing proteins 23(TRIM23) is identified as an E3 ubiquitin ligase involved in signal transduction, but its role in liver fibrosis remains unknown. AimsTo investigate the effects and mechanisms of TRIM23 on hepatic stellate cells(HSCs) ferroptosis and liver fibrosis. MethodsWe utilized the Gene Expression Omibus database to identify differentially expressed genes and downstream pathways. TRIM23 expression was examined in fibrotic liver tissues. The effects of TRIM23 on HSCs ferroptosis were validated through assessing cell viability, lipid peroxidation, and ferroptotic markers using HSC-T6 cell lines and primary rat HSCs. Co-immunoprecipitation assays were conducted to analyze the interactions between TRIM23 and p53. A CCl4-induced liver fibrosis rat model was employed to confirm the in vivo effects. ResultsTRIM23 expression was positively correlated with the severity of liver fibrosis. Upregulated TRIM23 expression promoted HSCs viability and activation by attenuating ferroptosis. Furthermore, the upregulation of TRIM23 expression significantly enhanced p53 ubiquitination. In contrast, TRIM23 knockdown induced HSCs ferroptosis by regulating p53, leading to the suppression of cell viability and activation. Silencing TRIM23 led to the regression of liver fibrosis induced by CCl4 treatment in vivo. ConclusionOur study uncovers a novel mechanism in which TRIM23 inhibits HSCs ferroptosis, promotes cell activation and contributes to liver fibrosis by regulating p53 ubiquitination.

Arginase 1 expression is increased during hepatic stellate cell activation and facilitates collagen synthesis.

Activation of hepatic stellate cells (HSC) is a key event in the initiation of liver fibrosis. Activated HSCs proliferate and secrete excessive amounts of extracellular matrix (ECM), disturbing liver architecture and function, leading to fibrosis and eventually cirrhosis. Collagen is the most abundant constituent of ECM and proline is the most abundant amino acid of collagen. Arginine is the precursor in the biosynthetic pathway of proline. Arginine is the exclusive substrate of both nitric oxide synthase (NOS) and arginase. NOS is an M1 (proinflammatory) marker of macrophage polarization whereas arginase-1 (Arg1) is an M2 (profibrogenic) marker of macrophage polarization. Differential expression of NOS and Arg1 has not been studied in HSCs yet. To identify the expression profile of arginine catabolic enzymes during HSC activation and to investigate their role in HSC activation, primary rat HSCs were cultured-activated for 7 days and expression of iNOS and Arg1 were investigated. Nor-NOHA was used as a specific and reversible arginase inhibitor. During HSC activation, iNOS expression decreased whereas Arg1 expression increased. Inhibition of Arg1 in activated HSCs efficiently inhibited collagen production but not cell proliferation. HSC activation is accompanied by a switch of arginine catabolism from iNOS to Arg1. Inhibition of Arg1 decreases collagen synthesis. Therefore, we conclude that Arg1 can be a therapeutic target for the inhibition of liver fibrogenesis.

Lipidomic profiling of rat hepatic stellate cells during activation reveals a two-stage process accompanied by increased levels of lysosomal lipids

Hepatic stellate cells (HSCs) are liver-resident cells best known for their role in vitamin A storage under physiological conditions. Upon liver injury, HSCs activate into myofibroblast-like cells, a key process in the onset of liver fibrosis. Lipids play an important role during HSC activation. Here, we provide a comprehensive characterization of the lipidomes of primary rat HSCs during 17days of activation invitro. For lipidomic data interpretation, we expanded our previously described Lipid Ontology (LION) and associated web application (LION/Web) with the LION-PCA heatmap module, which generates heatmaps of the most typical LION-signatures in lipidomic datasets. Furthermore, we used LION to perform pathway analysis to determine the significant metabolic conversions in lipid pathways. Together, we identify two distinct stages of HSC activation. In the first stage, we observe a decrease of saturated phosphatidylcholine, sphingomyelin, and phosphatidic acid and an increase in phosphatidylserine and polyunsaturated bis(monoacylglycero)phosphate (BMP), a lipid class typically localized at endosomes and lysosomes. In the second activation stage, BMPs, hexosylceramides, and ether-linked phosphatidylcholines are elevated, resembling a lysosomal lipid storage disease profile. The presence of isomeric structures of BMP in HSCs was confirmed exvivo in MS-imaging datasets of steatosed liver sections. Finally, treatment with pharmaceuticals targeting the lysosomal integrity led to cell death in primary HSCs but not in HeLa cells. In summary, our combined data suggest that lysosomes play a critical role during a two-stage activation process of HSCs.

Glycocholic acid aggravates liver fibrosis by promoting the up-regulation of connective tissue growth factor in hepatocytes

AimsThe precise role of bile acid in the progression of liver fibrosis has yet to be elucidated. In this study, common bile duct ligation was used as an in vivo mouse model for the evaluation of bile acids that promote liver connective tissue growth factor expression. Main methodsPrimary rat and mice hepatocytes, as well as primary rat hepatic stellate and HepaRG cells were evaluated as in vitro models for promoting the expression of connective tissue growth factor by bile acids. Key findingsCompared with taurochenodeoxycholic acid, glycochenodeoxycholic acid, and taurocholic acid, glycocholic acid (GCA) most strongly promoted the secretion of connective tissue growth factor in mouse primary hepatocytes, rat primary hepatocytes and HepaRGs. GCA did not directly promote the activation of hepatic stellate cells. The administration of GCA in mice with ligated bile ducts promotes the progression of liver fibrosis, which may promote the yes-associated protein of hepatocytes into the nucleus, resulting in the hepatocytes secreting more connective tissue growth factor for hepatic stellate cell activation. In conclusion, our data showed that GCA can induce the expression of connective tissue growth factor in hepatocytes by promoting the nuclear translocation of yes-associated protein, thereby activating hepatic stellate cells. SignificanceOur findings help to elucidate the contribution of GCA to the progression of hepatic fibrosis in cholestatic disease and aid the clinical monitoring of cholestatic liver fibrosis development.

Characterization of focal adhesion proteins in rodent hepatic stellate cells.

Ongoing liver injury leads to fibrosis and ultimately cirrhosis, a leading cause of death worldwide. The primary mechanism underlying the fibrogenic response is the activation of cells known as hepatic stellate cells (HSCs) which are "quiescent" in the normal liver but become "activated" after injury by transdifferentiating into extracellular matrix-secreting myofibroblasts. Since integrins (extracellular matrix binding receptors) are important mediators of HSC activation and fibrogenesis, we hypothesized that focal adhesion (FA) proteins, which link integrins to the intracellular protein machinery, may be important in the activation process. Therefore, using both an in vitro model of activation in primary rat HSCs and an in vivo model of liver injury, we examined three FA proteins: vinculin, FAK, and talin. All three proteins were significantly upregulated during HSC activation at both the messenger RNA (mRNA) and protein levels. Confocal microscopy demonstrated that the proteins had a widespread expression throughout HSCs with prominent localization at the end of actin filaments. Finally, we stimulated HSCs with the profibrotic ligands endothelin-1 (ET-1) and transforming growth factor beta (TGF-β) and observed an increase in the size of vinculin-containing FAs and the cell area occupied by them. The data indicate that HSCs possess a broad array of FA proteins, and given their upregulation during activation, this raises the possibility that they play a role in the fibrogenic response to injury.

Disulfiram inhibits liver fibrosis in rats by suppressing hepatic stellate cell activation and viability

BackgroundLiver fibrosis is a wound-healing response to chronic injury, featuring with excess accumulation of extracellular matrix secreted by the activated hepatic stellate cells (HSC). Disulfiram (DSF), also known as Antabuse, has been used for the treatment of alcohol addiction and substance abuse. Recently, overwhelming studies had revealed anti-cancer effects of DSF in multiple cancers, including liver cancer. But the actual effects of DSF on liver fibrosis and liver function remain unknown.MethodsIn this study, we evaluated the effects of low-dose DSF in CCl4- and Bile Duct Ligation (BDL)—induced hepatic fibrosis rat models. Cell proliferation was detected by using the Cell-Light™ EdU Apollo®567 Cell Tracking Kit. Cell apoptosis was analyzed using a TdT-mediated dUTP nick end labeling (TUNEL) kit, viability was measured with Cell Counting Kit-8(CCK8). Relative mRNA expression of pro-fibrogenic was assessed using quantitative RT-PCR. The degree of liver fibrosis, activated HSCs, were separately evaluated through Sirius Red-staining, immunohistochemistry and immunofluorescence. Serum alanine aminotransferase (ALT) and asparagine aminotransferase (AST) activities were detected with ALT and AST detecting kits using an automated analyzer.ResultsLiver fibrosis was distinctly attenuated while liver functions were moderately ameliorated in the DSF-treated group. Activation and proliferation of primary rat HSCs isolated from rat livers were significantly suppressed by low-dose DSF. DSF also inhibited the viability of in vitro cultured rat or human HSC cells dose-dependently but had no repressive role on human immortalized hepatocyte THLE-2 cells. Interestingly, upon DSF treatment, the viability of LX-2 cells co-cultured with THLE-2 was significantly inhibited, while that of THLE-2 co-cultured with LX-2 was increased. Further study indicated that HSCs apoptosis was increased in DSF/CCl4-treated liver samples. These data indicated that DSF has potent anti-fibrosis effects and protective effects toward hepatocytes and could possibly be repurposed as an anti-fibrosis drug in the clinic.ConclusionsDSF attenuated ECM remodeling through suppressing the transformation of quiet HSCs into proliferative, fibrogenic myofibroblasts in hepatic fibrosis rat models. DSF provides a novel approach for the treatment of liver fibrosis.

Effects of microRNA-129-5p on hepatic fibrosis and primary hepatic stellate cell proliferation and migration in rats

Purpose: To investigate the effects and mechanism of microRNA (miR)-129-5p in hepatic fibrosis and hepatic stellate cell proliferation and migration in rats.Methods: Forty Sprague-Dawley rats were randomly divided into control and model groups. Carbon tetrachloride (CCl4)-induced hepatic fibrosis was established in the model group. Hematoxylin and eosin staining was used to observe rat liver pathological sections. Sirius red staining was used to assess collagen deposition, while quantitative real-time polymerase chain reaction was used to evaluate miR- 129-5p expression in the rat liver during hepatic fibrosis. Three groups of rat primary hepatic stellate cells (HSCs) (miR-129-5p overexpression group, negative control lentivirus group and blank control group) were prepared. Cell proliferation and migration were determined using Cell Counting Kit-8 and Transwell assay, respectively. Serum- and glucocorticoid-regulated kinase 3 (SGK3), β-catenin, and α-smooth muscle actin (α-SMA) expression were evaluated by Western blotting, while dual luciferasereporter gene assay was used to evaluate whether SGK3 is an miR-129-5p target.Results: miR-129-5p expression was significantly reduced during the progression of CCl4-induced rat hepatic fibrosis (p < 0.05). The proliferation rate and migration ability of the primary HSCs in the miR- 129-5p mimics group were significantly lower than those in the miR-129-5p NC and blank control groups (p < 0.05). Protein expression of SGK3, β-catenin, and α-SMA in the miR-129-5p mimics group was reduced (p < 0.05), and miR-129-5p showed targeted binding to SGK3.Conclusion: MiR-129-5p down-regulates the expression of β-catenin through targeted regulation of SGK3 to inhibit HSC activation, providing novel insight into design of a potential treatment strategy for hepatic fibrosis.

The miR-23b/27b/24-1 Cluster Inhibits Hepatic Fibrosis by Inactivating Hepatic Stellate Cells.

Background & AimsHepatic fibrosis is characterized by hepatic stellate cell (HSC) activation and transdifferentiation-mediated extracellular matrix (ECM) deposition, which both contribute to cirrhosis. However, no antifibrotic regimen is available in the clinic. microRNA-23b/27b/24-1 cluster inhibition of transforming growth factor-β (TGF-β) signaling during hepatic development prompted us to explore whether this cluster inhibits HSC activation and hepatic fibrosis.MethodsExperimental fibrosis was studied in carbon tetrachloride (CCl4)-treated C57BL/6 mice. After administration of miR-23b/27b/24-1 lentivirus or vehicle, animals were euthanized for liver histology. In primary rat HSC and HSC-T6, the anti-fibrotic effect of miR-23b/27b/24-1 cluster was furtherly investigated by RNA-sequencing, luciferase reporter assay, western blotting and bioinformatic means.ResultsIn this study, we showed that increasing the miR-23b/27b/24-1 level through intravenous delivery of miR-23b/27b/24-1 lentivirus ameliorated mouse hepatic fibrosis. Mechanistically, the miR-23b/27b/24-1 cluster directly targeted messenger RNAs, which reduced the protein expression of 5 secretory profibrotic genes (TGF-β2, Gremlin1, LOX, Itgα2, and Itgα5) in HSCs. Suppression of the TGF-β signaling pathway by down-regulation of TGF-β2, Itgα2, and Itgα5, and activation of the bone morphogenetic protein signaling pathway by inhibition of Gremlin1, decreased extracellular matrix secretion of HSCs. Furthermore, down-regulation of LOX expression softened the ECM. Moreover, a reduction in tissue inhibitors of metalloproteinase 1 expression owing to weakened TGF-β signaling increased ECM degradation.ConclusionsHepatic overexpression of the miR-23b/27b/24-1 cluster blocked hepatic fibrosis and may be a novel therapeutic regimen for patients with hepatic fibrosis.

Isolation and Culture of Primary Rat Hepatic Stellate Cells

Isolation and Culture of Primary Rat Hepatic Stellate Cells

Alpha-single chains of collagen type VI inhibit the fibrogenic effects of triple helical collagen VI in hepatic stellate cells

The interaction of extracellular matrix (ECM) components with hepatic stellate cells (HSCs) is thought to perpetuate fibrosis by stimulating signaling pathways that drive HSC activation, survival and proliferation. Consequently, disrupting the interaction between ECM and HSCs is considered a therapeutical avenue although respective targets and underlying mechanisms remain to be established. Here we have interrogated the interaction between type VI collagen (CVI) and HSCs based on the observation that CVI is 10-fold upregulated during fibrosis, closely associates with HSCs in vivo and promotes cell proliferation and cell survival in cancer cell lines. We exposed primary rat HSCs and a rat hepatic stellate cell line (CFSC) to soluble CVI and determined the rate of proliferation, apoptosis and fibrogenesis in the absence of any additional growth factors. We find that CVI in nanomolar concentrations prevents serum starvation-induced apoptosis. This potent anti-apoptotic effect is accompanied by induction of proliferation and acquisition of a pronounced pro-fibrogenic phenotype characterized by increased α-smooth muscle actin, TGF-β, collagen type I and TIMP-1 expression and diminished proteolytic MMP-13 expression. The CVI-HSC interaction can be disrupted with the monomeric α2(VI) and α3(VI) chains and abrogates the activating CVI effects. Further, functional relevant α3(VI)—derived 30 amino acid peptides lead to near-complete inhibition of the CVI effect. In conclusion, CVI serves as a potent mitogen and activating factor for HSCs. The antagonistic effects of the CVI monomeric chains and peptides point to linear peptide sequences that prevent activation of CVI receptors which may allow a targeted antifibrotic therapy.

Histone H3K27 methyltransferase EZH2 and demethylase JMJD3 regulate hepatic stellate cells activation and liver fibrosis.

Rationale: As the central hallmark of liver fibrosis, transdifferentiation of hepatic stellate cells (HSCs), the predominant contributor to fibrogenic hepatic myofibroblast responsible for extracellular matrix (ECM) deposition, is characterized with transcriptional and epigenetic remodeling. We aimed to characterize the roles of H3K27 methyltransferase EZH2 and demethylase JMJD3 and identify their effective pathways and novel target genes in HSCs activation and liver fibrosis.Methods: In primary HSCs, we analyzed effects of pharmacological inhibitions and genetic manipulations of EZH2 and JMJD3 on HSCs activation. In HSCs cell lines, we evaluated effects of EZH2 inhibition by DZNep on proliferation, cell cycling, senescence and apoptosis. In CCl4 and BDL murine models of liver fibrosis, we assessed in vivo effects of DZNep administration and Ezh2 silencing. We profiled rat primary HSCs transcriptomes with RNA-seq, screened the pathways and genes associated with DZNep treatment, analyzed EZH2 and JMJD3 regulation towards target genes by ChIP-qPCR.Results: EZH2 inhibition by DZNep resulted in retarded growth, lowered cell viability, cell cycle arrest in S and G2 phases, strengthened senescence, and enhanced apoptosis of HSCs, decreased hepatic collagen deposition and rescued the elevated serum ALT and AST activities of diseased mice, and downregulated cellular and hepatic expressions of H3K27me3, EZH2, α-SMA and COL1A. Ezh2 silencing by RNA interference in vitro and in vivo showed similar effects. JMJD3 inhibition by GSK-J4 and overexpression of wild-type but not mutant Jmjd3 enhanced or repressed HSCs activation respectively. EZH2 inhibition by DZNep transcriptionally inactivated TGF-β1 pathway, cell cycle pathways and vast ECM components in primary HSCs. EZH2 inhibition decreased H3K27me3 recruitment at target genes encoding TGF-β1 pseudoreceptor BAMBI, anti-inflammatory cytokine IL10 and cell cycle regulators CDKN1A, GADD45A and GADD45B, and increased their expressions, while Jmjd3 overexpression manifested alike effects.Conclusions: EZH2 and JMJD3 antagonistically modulate HSCs activation. The therapeutic effects of DZNep as epigenetic drug in liver fibrosis are associated with the regulation of EZH2 towards direct target genes encoding TGF-β1 pseudoreceptor BAMBI, anti-inflammatory cytokine IL10 and cell cycle regulators CDKN1A, GADD45A and GADD45B, which are also regulated by JMJD3. Our present study provides new mechanistic insight into the epigenetic modulation of EZH2 and JMJD3 in HSCs biology and hepatic fibrogenesis.

Phosphate Groups in the Lipid A Moiety Determine the Effects of LPS on Hepatic Stellate Cells: A Role for LPS-Dephosphorylating Activity in Liver Fibrosis.

Alkaline phosphatase (AP) activity is highly upregulated in plasma during liver diseases. Previously, we demonstrated that AP is able to detoxify lipopolysaccharide (LPS) by dephosphorylating its lipid A moiety. Because a role of gut-derived LPS in liver fibrogenesis has become evident, we now examined the relevance of phosphate groups in the lipid A moiety in this process. The effects of mono-phosphoryl and di-phosphoryl lipid A (MPLA and DPLA, respectively) were studied in vitro and LPS-dephosphorylating activity was studied in normal and fibrotic mouse and human livers. The effects of intestinal AP were studied in mice with CCL4-induced liver fibrosis. DPLA strongly stimulated fibrogenic and inflammatory activities in primary rat hepatic stellate cells (rHSCs) and RAW264.7 macrophages with similar potency as full length LPS. However, MPLA did not affect any of the parameters. LPS-dephosphorylating activity was found in mouse and human livers and was strongly increased during fibrogenesis. Treatment of fibrotic mice with intravenous intestinal-AP significantly attenuated intrahepatic desmin+− and αSMA+ −HSC and CD68+− macrophage accumulation. In conclusion, the lack of biological activity of MPLA, contrasting with the profound activities of DPLA, shows the relevance of LPS-dephosphorylating activity. The upregulation of LPS-dephosphorylating activity in fibrotic livers and the protective effects of exogenous AP during fibrogenesis indicate an important physiological role of intestinal-derived AP during liver fibrosis.

SPOCK1 overexpression induced by platelet-derived growth factor-BB promotes hepatic stellate cell activation and liver fibrosis through the integrin α5β1/PI3K/Akt signaling pathway

AbstractSparc/osteonectin, cwcv, and kazal-like domain proteoglycan 1 (SPOCK1) is a matricellular protein which regulates cell proliferation, invasion, and survival but the function of SPOCK1 in liver fibrosis is obscure. In this study, we found that SPOCK1 expression increased significantly in fibrotic liver tissues and activated primary rat hepatic stellate cells (R-HSCs). SPOCK1 co-localized with α-smooth muscle actin (α-SMA) in the cytoplasm. Mechanistically, we found platelet-derived growth factor-BB (PDGF-BB) induced SPOCK1 expression by activating the PI3K/Akt/forkhead box M1 (FoxM1) signaling pathway. Intracellular SPOCK1 downregulation decreased the HSC activation, proliferation, and migration induced by PDGF-BB. Furthermore, intracellular SPOCK1 overexpression or recombinant SPOCK1 treatment promoted HSC activation, proliferation, and migration by activating the PI3K/Akt signaling pathway. Co-immunoprecipitation, double immunofluorescence staining indicated that SPOCK1 interacted with integrin α5β1, and neutralization of integrin α5β1 significantly reduced the role of recombinant SPOCK1 in HSCs. In vivo HSC-specific SPOCK1 knockdown following lentivirus administration dramatically ameliorated thioacetamide (TAA)-induced collagen deposition in rat livers. Collectively, our study indicates that SPOCK1 is crucial for hepatic fibrosis and it might be a promising therapeutic target.Matricellular protein SPOCK1 expression positively correlates with liver fibrosis and hepatic stellate cell activation. SPOCK1 is upregulated by PDGF-BB via the PI3K/Akt/FoxM1 signaling pathway and induces pro-fibrogenic responses. Additionally, it promotes stellate cell pro-fibrogenic responses through the integrin α5β1/PI3K/Akt signaling pathway. SPOCK1 is therefore a promising therapeutic target for liver fibrosis.

Aster tataricus Linn., Suppresses Hepatic Stellate Cell Activation and Protects Against Thioacetamide-induced Liver Fibrosis in Rats

Aster tataricus Linn., (Asteraceae), an oriental and nutrient potential herb used in Asian countries for various health benefits. The present study focused on the protective effects of Aster tataricus against liver fibrosis in cellular and experimental rat model. Cell cytotoxicity, cell cycle and apoptosis functions were analyzed using hepatic stellate cell line following MTT assay, flow cytometry, and Annexin V-FITC/PI staining methods. For in vivo evaluation, thioacetamide-induced hepatofibrosis rat model was established. Sprague-Dawley rats were divided into 5 groups of 10 each (control, thioacetamide, thioacetamide with Aster tataricus extract 100, 500 mg/kg and silymarin 50 mg/kg groups, respectively). Fibrosis was induced by thioacetamide treatment (200 mg/kg, ip) 3 times per week for 13 weeks except for control group. Aster tataricus extract (100 and 500 mg/kg), and silymarin was administrated orally to each group 6 times per week from 7th week to 13th week and various fibrosis related parameters were estimated by real-time polymerase chain reaction using TRIzol Plus RNA purification kit. Results indicated that hepatic stellate cells treated with Aster tataricus extract (0.5 mg/ml) and silymarin (0.05 mg/ml) significantly (p<0.05) induced apoptosis (19.04 and 24.82 %, respectively) compared to the control group (9.78 %). Moreover, rat primary hepatic stellate cells showed morphological changes and degradation of collagen and fibronectin with treatment of Aster tataricus extract at 0.5 mg/kg. In in vivo evaluation Aster tataricus extract at concentrations of 100 and 500 mg/ml attenuated the increased serum levels of alanine transaminase, aspartate transaminase and hydroxyproline and restored the decreased glutathione levels significantly in thioacetamide-induced fibrotic rats (p<0.05). The altered histopathology in thioacetamide-induced liver tissues and changes in fibrosis-related gene expression (TGF-β, α-SMA and Col1α1) were also restored by Aster tataricus extract treatment. In conclusion, A. tataricus can be developed as a potential therapeutic agent to treat liver fibrosis.

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.