Autophagy fuels tissue fibrogenesis

Activation of hepatic stellate cells (HSC), a resident pericytic cell in liver, into a proliferative and fibrogenic cell type, is the principal event underlying hepatic fibrosis following injury. Release of lipid droplets (LD) containing retinyl esters and triglyceride is a defining feature of HSC activation, yet the basis for this release has remained mysterious. Here we offer a surprising discovery that autophagy is the missing link underlying LD release, by stimulating metabolism of their contents to provide the energy vital to fuel HSC activation. By specifically inhibiting the autophagic pathway in activated HSC, LD release is impaired and cellular ATP levels are decreased. Moreover, animals with HSC-specific deletion of Atg7 display attenuated activation following liver injury, leading to reduced fibrosis in vivo. We further demonstrate that fibrogenic cells from other organs, including kidney and lung, also rely on autophagy as a core pathway driving the scarring response. Our results provide a novel framework for understanding pathways underlying fibrogenic cell responses to tissue injury.     

CXCL6‐EGFR‐induced Kupffer cells secrete TGF‐β1 promoting hepatic stellate cell activation via the SMAD2/BRD4/C‐MYC/EZH2 pathway in liver fibrosis

Liver fibrosis is the excessive accumulation of extracellular matrix proteins in response to the inflammatory response that accompanies tissue injury, which at an advanced stage can lead to cirrhosis and even liver failure. This study investigated the role of the CXC chemokine CXCL6 (GCP‐2) in liver fibrosis. The expression of CXCL6 was found to be elevated in the serum and liver tissue of high stage liver fibrosis patients. Furthermore, treatment with CXCL6 (100 ng/mL) stimulated the phosphorylation of EGFR and the expression of TGF‐β in cultured Kupffer cells (KCs). Although treatment with CXCL6 directly did not activate the hepatic stellate cell (HSC) line, HSC‐T6, HSCs cultured with media taken from KCs treated with CXCL6 or TGF‐β showed increased expression of α‐SMA, a marker of HSC activation. CXCL6 was shown to function via the SMAD2/BRD4/C‐MYC/EZH2 pathway by enhancing the SMAD3‐BRD4 interaction and promoting direct binding of BRD4 to the C‐MYC promoter and CMY‐C to the EZH2 promoter, thereby inducing profibrogenic gene expression in HSCs, leading to activation and transdifferentiation into fibrogenic myofibroblasts. These findings were confirmed in a mouse model of CCl₄‐induced chronic liver injury and fibrosis in which the levels of CXCL6 and TGF‐β in serum and the expression of α‐SMA, SMAD3, BRD4, C‐MYC, and EZH2 in liver tissue were increased. Taken together, our results reveal that CXCL6 plays an important role in liver fibrosis through stimulating the release of TGF‐β by KCs and thereby activating HSCs.     

Lipopolysaccharide mediates hepatic stellate cell activation by regulating autophagy and retinoic acid signaling

Bacterial translocation and lipopolysaccharide (LPS) leakage occur at a very early stage of liver fibrosis in animal models. We studied the role of LPS in hepatic stellate cell (HSC) activation and the underlying mechanisms in vitro and in vivo. Herein, we demonstrated that LPS treatment led to a dramatic increase in autophagosome formation and autophagic flux in LX-2 cells and HSCs, which was mediated through the AKT-MTOR and AMPK-ULK1 pathway. LPS significantly decreased the lipid content, including the lipid droplet (LD) number and lipid staining area in HSCs; pretreatment with macroautophagy/autophagy inhibitors or silencing ATG5 attenuated this decrease. Furthermore, lipophagy was induced by LPS through the autophagy-lysosomal pathway in LX-2 cells and HSCs. Additionally, LPS-induced autophagy further reduced retinoic acid (RA) signaling, as demonstrated by a decrease in the intracellular RA level and Rar target genes, resulting in the downregulation of Bambi and promoting the sensitization of the HSC's fibrosis response to TGFB. Compared with CCl₄ injection alone, CCl₄ plus LPS injection exaggerated liver fibrosis in mice, as demonstrated by increased Col1a1 (collagen, type I, α 1), Acta2, Tgfb and Timp1 mRNA expression, ACTA2/α-SMA and COL1A1 protein expression, and Sirius Red staining area, which could be attenuated by injection of an autophagy inhibitor. LPS also reduced lipid content in HSCs in vivo, with this change being attenuated by chloroquine (CQ) administration. In conclusion, LPS-induced autophagy resulted in LD loss, RA signaling dysfunction, and downregulation of the TGFB pseudoreceptor Bambi, thus sensitizing HSCs to TGFB signaling.     

Effect of human amniotic epithelial cells on pro‐fibrogenic resident hepatic cells in a rat model of liver fibrosis

Myofibroblasts are key fibrogenic cells responsible for excessive extracellular matrix synthesis characterizing the fibrotic lesion. In liver fibrosis, myofibroblasts derive either from activation of hepatic stellate cells (HSC) and portal fibroblasts (PF), or from the activation of fibroblasts that originate from ductular epithelial cells undergoing epithelial–mesenchymal transition. Ductular cells can also indirectly promote myofibroblast generation by activating TGF‐β, the main fibrogenic growth factor, through αvβ6 integrin. In addition, after liver injury, liver sinusoidal cells can lose their ability to maintain HSC quiescence, thus favouring HSC differentiation towards myofibroblasts. The amniotic membrane and epithelial cells (hAEC) derived thereof have been shown to decrease hepatic myofibroblast levels in rodents with liver fibrosis. In this study, in a rat model of liver fibrosis, we investigated the effects of hAEC on resident hepatic cells contributing to myofibroblast generation. Our data show that hAEC reduce myofibroblast numbers with a consequent reduction in fibronectin and collagen deposition. Interestingly, we show that hAEC strongly act on specific myofibroblast precursors. Specifically, hAEC reduce the activation of PF rather than HSC. In addition, hAEC target reactive ductular cells by inhibiting their proliferation and αvβ6 integrin expression, with a consequent decrease in TGF‐β activation. Moreover, hAEC counteract the transition of ductular cells towards fibroblasts, while it does not affect injury‐induced and fibrosis‐promoting sinusoidal alterations. In conclusion, among the emerging therapeutic applications of hAEC in liver diseases, their specific action on PF and ductular cells strongly suggests their application in liver injuries involving the expansion and activation of the portal compartment.     

The Autoregulatory Feedback Loop of MicroRNA-21/Programmed Cell Death Protein 4/Activation Protein-1 (MiR-21/PDCD4/AP-1) as a Driving Force for Hepatic Fibrosis Development

Sustained activation of hepatic stellate cells (HSCs) leads to hepatic fibrosis, which is characterized by excessive collagen production, and for which there is no available drug clinically. Despite tremendous progress, the cellular activities underlying HSC activation, especially the driving force in the perpetuation stage, are only partially understood. Recently, microRNA-21 (miR-21) has been found to be prevalently up-regulated during fibrogenesis in different tissues, although its detailed role needs to be further elucidated. In the present study, miR-21 expression was examined in human cirrhotic liver samples and in murine fibrotic livers induced by thioacetamide or carbon tetrachloride. A dramatic miR-21 increase was noted in activated HSCs. We further found that miR-21 maintained itself at constant high levels by using a microRNA-21/programmed cell death protein 4/activation protein-1 (miR-21/PDCD4/AP-1) feedback loop. Disrupting this loop with miR-21 antagomir or AP-1 inhibitors significantly suppressed fibrogenic activities in HSCs and ameliorated liver fibrosis. In contrast, reinforcing this loop with small interfering RNA (siRNA) against PDCD4 promoted fibrogenesis in HSCs. Further analysis indicated that the up-regulated miR-21 promoted the central transforming growth factor-β (TGF-β) signaling pathway underlying HSC activation. In summary, we suggest that the miR-21/PDCD4/AP-1 autoregulatory loop is one of the main driving forces for hepatic fibrosis progression. Targeting this aberrantly activated feedback loop may provide a new therapeutic strategy and facilitate drug discovery against hepatic fibrosis.     

Induction of cell death in activated hepatic stellate cells by targeted gene expression of the thymidine kinase/ganciclovir system

Liver fibrosis is the result from a relative imbalance between synthesis and degradation of matrix proteins. Following liver injury of any etiology, hepatic stellate cells undergo a response known as activation, which is the transition of quiescent cells into proliferative, fibrogenic, and contractile myofibroblasts. Upon this cellular transdifferentiation the effector cell becomes the major source of fibrillar and non-fibrillar matrix proteins resulting in excessive scar formation and cirrhosis, the end stage of fibrosis. Concomitant with progressive liver fibrosis, the tissue inhibitor of metalloproteinases-1 (TIMP-1) is strongly activated in hepatic stellate cells. We have developed a recombinant replication-defective adenovirus in which the TIMP-1 promoter is coupled to the herpes simplex virus thymidine kinase gene rendering activated hepatic stellate cells susceptible to ganciclovir. This novel targeted suicide gene approach was validated in a culture model considered to reflect an accelerated time course of the cellular and molecular events that occur during liver fibrosis. We demonstrate that transfer of the suicide gene to culture-activated hepatic stellate cells results in a strong expression of the respective transgene as assessed by Northern blot and Western blot analyses. The enzyme catalyzed the proper conversion of its prodrug subsequently initiating programmed cell death as estimated by caspase-3 assay and Annexin V-Fluos staining. Altogether, these results indicate that induction of programmed cell death is a promising approach to eliminate fibrogenic HSC.     

Activation of peroxisome proliferator-activated receptor-gamma contributes to the inhibitory effects of curcumin on rat hepatic stellate cell growth

Hepatic fibrogenesis occurs as a wound-healing process after many forms of chronic liver injury. Hepatic fibrosis ultimately leads to cirrhosis if not treated effectively. During liver injury, quiescent hepatic stellate cells (HSC), the most relevant cell type, become active and proliferative. Oxidative stress is a major and critical factor for HSC activation. Activation of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) inhibits the proliferation of nonadipocytes. The level of PPAR-gamma is dramatically diminished along with activation of HSC. Curcumin, the yellow pigment in curry, is a potent antioxidant. The aims of this study were to evaluate the effect of curcumin on HSC proliferation and to begin elucidating underlying mechanisms. It was hypothesized that curcumin might inhibit the proliferation of activated HSC by inducing PPAR-gamma gene expression and reviving PPAR-gamma activation. Our results indicated that curcumin significantly inhibited the proliferation of activated HSC and induced apoptosis in vitro. We demonstrated, for the first time, that curcumin dramatically induced the gene expression of PPAR-gamma and activated PPAR-gamma in activated HSC. Blocking its trans-activating activity by a PPAR-gamma antagonist markedly abrogated the effects of curcumin on inhibition of cell proliferation. Our results provide a novel insight into mechanisms underlying the inhibition of activated HSC growth by curcumin. The characteristics of curcumin, including antioxidant potential, reduction of activated HSC growth, and no adverse health effects, make it a potential antifibrotic candidate for prevention and treatment of hepatic fibrosis.     

Elucidation of the role of COX-2 in liver fibrogenesis using transgenic mice

Hepatic COX-2 overexpression is sufficient to induce hepatitis, but its role on liver fibrosis remains unknown. We aim to elucidate possible biological effects of COX-2 in liver fibrosis using both gain-of-function and loss-of-function mouse models. COX-2 transgenic (TG) mice that specifically overexpress the human COX-2 cDNA in the liver, knockout (KO), and wild type (WT) mice were studied in two different murine fibrosis models induced by carbon tetrachloride (CCl₄) injection or methionine and choline-deficient (MCD) diet. Liver injury was assessed by serum ALT and bilirubin levels and histological examination. Hepatic collagen content was determined by picrosirius red stain morphometry assay and quantitation of hydroxyproline. Hepatic stellate cell (HSC) activation was determined by immunohistochemical analysis of α-smooth muscle actin (α-SMA). mRNA expression of fibrogenic genes was assayed by real-time quantitative PCR. COX-2 protein was overexpressed in the liver of TG mice compared with WT littermates. CCl₄ or MCD-induced liver fibrotic injury was equally severe in TG and WT mice, as demonstrated by similar elevated levels of hepatic collagen contents. Enhanced COX-2 expression in TG liver did not affect HSC activation and fibrogenic gene expression upon CCl₄ or MCD treatment. Importantly, CCl₄-treated KO mice did not show significant difference in liver fibrotic damage and fibrogenic gene expression compared with the WT counterparts. This is the first report on the effect of COX-2 in liver fibrosis based on genetic mouse models. The results suggest that COX-2 does not appear to mediate the development of liver fibrosis.     

Localization of liver myofibroblasts and hepatic stellate cells in normal and diseased rat livers: distinct roles of (myo-)fibroblast subpopulations in hepatic tissue repair

Previous in vitro studies indicated that hepatic stellate cells (HSC) and rat liver myofibroblasts (rMF) have to be regarded as different cell populations of the myofibroblastic lineage with fibrogenic potential. Employing the discrimination features defined by these studies the localization of HSC and rMF was analyzed in diseased livers. Normal and acutely as well as chronically carbon tetrachloride-injured livers were analyzed by immunohistochemistry and by in situ hybridization. In normal livers HSC [desmin/glial fibrillary acid protein (GFAP)-positive cells] were distributed in the hepatic parenchyma, while rMF (desmin/smooth muscle alpha actin-positive, GFAP-negative cells colocalized with fibulin-2) were located in the portal field, the walls of central veins, and only occasionally in the parenchyma. Acute liver injury was characterized almost exclusively by an increase in the number of HSC, while the amount of rMF was nearly unchanged. In early stages of fibrosis, HSC and rMF were detected within the developing scars. In advanced stages of fibrosis, HSC were mainly present at the scar–parenchymal interface, while rMF accounted for the majority of the cells located within the scar. At every stage of fibrogenesis, rMF, in contrast to HSC, were only occasionally detected in the hepatic parenchyma. HSC and rMF are present in normal and diseased livers in distinct compartments and respond differentially to tissue injury. Acute liver injury is followed by an almost exclusive increase in the number of HSC, while in chronically injured livers not only HSC but also rMF are involved in scar formation. Accepted: 16 September 1999     

PPARγ/NF-κB and TGF-β1/Smad pathway are involved in the anti-fibrotic effects of levo-tetrahydropalmatine on liver fibrosis

Liver fibrosis is a necessary stage in the development of chronic liver diseases to liver cirrhosis. This study aims to investigate the anti-fibrotic effects of levo-tetrahydropalmatine (L-THP) on hepatic fibrosis in mice and cell models and its underlying mechanisms. Two mouse hepatic fibrosis models were generated in male C57 mice by intraperitoneal injection of carbon tetrachloride (CCl4) for 2 months and bile duct ligation (BDL) for 14 days. Levo-tetrahydropalmatine was administered orally at doses of 20 and 40 mg/kg. An activated LX2 cell model induced by TGF-β1 was also generated. The results showed that levo-tetrahydropalmatine alleviated liver fibrosis by inhibiting the formation of extracellular matrix (ECM) and regulating the balance between TIMP1 and MMP2 in the two mice liver fibrosis models and cell model. Levo-tetrahydropalmatine inhibited activation and autophagy of hepatic stellate cells (HSCs) by modulating PPARγ/NF-κB and TGF-β1/Smad pathway in vivo and in vitro. In conclusion, levo-tetrahydropalmatine attenuated liver fibrosis by inhibiting ECM deposition and HSCs autophagy via modulation of PPARγ/NF-κB and TGF-β1/Smad pathway.     

Macrophage autophagy protects against liver fibrosis in mice

Autophagy is a lysosomal degradation pathway of cellular components that displays antiinflammatory properties in macrophages. Macrophages are critically involved in chronic liver injury by releasing mediators that promote hepatocyte apoptosis, contribute to inflammatory cell recruitment and activation of hepatic fibrogenic cells. Here, we investigated whether macrophage autophagy may protect against chronic liver injury. Experiments were performed in mice with mutations in the autophagy gene Atg5 in the myeloid lineage (Atg5^fl/fl LysM-Cre mice, referred to as atg5^−/−) and their wild-type (Atg5^fl/fl, referred to as WT) littermates. Liver fibrosis was induced by repeated intraperitoneal injection of carbon tetrachloride. In vitro studies were performed in cultures or co-cultures of peritoneal macrophages with hepatic myofibroblasts. As compared to WT littermates, atg5^−/− mice exposed to chronic carbon tetrachloride administration displayed higher hepatic levels of IL1A and IL1B and enhanced inflammatory cell recruitment associated with exacerbated liver injury. In addition, atg5^−/− mice were more susceptible to liver fibrosis, as shown by enhanced matrix and fibrogenic cell accumulation. Macrophages from atg5^−/− mice secreted higher levels of reactive oxygen species (ROS)-induced IL1A and IL1B. Moreover, hepatic myofibroblasts exposed to the conditioned medium of macrophages from atg5^−/− mice showed increased profibrogenic gene expression; this effect was blunted when neutralizing IL1A and IL1B in the conditioned medium of atg5^−/− macrophages. Finally, administration of recombinant IL1RN (interleukin 1 receptor antagonist) to carbon tetrachloride-exposed atg5^−/− mice blunted liver injury and fibrosis, identifying IL1A/B as central mediators in the deleterious effects of macrophage autophagy invalidation. These results uncover macrophage autophagy as a novel antiinflammatory pathway regulating liver fibrosis.     

Antiproliferative effect of catechin in GRX cells

The phenolic compounds present in cocoa seeds have been studied regarding health benefits, such as antioxidant and anti-inflammatory activities. Fibrosis is a wound healing response that occurs in almost all patients with chronic liver injury. A large number of cytokines and soluble intercellular mediators are related to changes in the behavior and phenotype of the hepatic stellate cell (HSC) that develop a fibrogenic and contractile phenotype leading to the development of fibrosis. The objective of this study was to assess the catechin effect in GRX liver cells in activities such as cell growth and inflammation. The GRX cells treatment with catechin induced a significant decrease in cell growth. This mechanism does not occur by apoptosis or even by autophagy because there were no alterations in expression of caspase 3 and PARP (apoptosis), and LC3 (autophagy). The expression of p27 and p53 proteins, regulators of the cell cycle, showed increased expression, while COX-2 and IL-6 mRNA showed a significant decrease in expression. This study shows that catechin decreases cell growth in GRX cells and, probably, this decrease does not occur by apoptosis or autophagy but through an anti-inflammatory effect and cell cycle arrest. Catechin also significantly decreased the production of TGF-β by GRX cells, showing a significant antifibrotic effect.     

Hepatic stellate cells and astrocytes

Scar formation inhibits tissue repair and regeneration in the liver and central nervous system. Activation of hepatic stellate cells (HSCs) after liver injury or of astrocytes after nervous system damage is considered to drive scar formation. HSCs are the fibrotic cells of the liver, as they undergo activation and acquire fibrogenic properties after liver injury. HSC activation has been compared to reactive gliosis of astrocytes, which acquire a reactive phenotype and contribute to scar formation after nervous system injury, much like HSCs after liver injury. It is intriguing that a wide range of neuroglia-related molecules are expressed by HSCs. We identified an unexpected role for the p75 neurotrophin receptor in regulating HSC activation and liver repair. Here we discuss the molecular mechanisms that regulate HSC activation and reactive gliosis and their contributions to scar formation and tissue repair. Juxtaposing key mechanistic and functional similarities in HSC and astrocyte activation might provide novel insight into liver regeneration and nervous system repair.     

肝纤维化的表观遗传学研究进展

肝纤维化是常见的慢性进行性肝病,是慢性肝病发展到肝硬化阶段的必经阶段,却有逆转的可能性。肝纤维化的中心环节是肝星状细胞的激活。关于肝星状细胞的激活,除了经典的肝纤维化通路,不断有新的通路和机制出现,包括自噬、内质网应激、氧化应激、胆固醇代谢和表观遗传,这些都揭示了肝星状细胞的活化机制。表观遗传包括DNA甲基化、组蛋白修饰和调节性非编码RNAs,这些机制也参与调节肝星状细胞活化和肝纤维化发生,对表观遗传和肝纤维化治疗之间的关系研究具有重要意义。     

Acetylcholine promotes the proliferation and collagen gene expression of myofibroblastic hepatic stellate cells

The mechanisms that initiate and perpetuate the fibrogenic response, during liver injury, are unclear. Animal studies, however, strongly support a role for the autonomic nervous system (ANS) in wound healing. Therefore, the ANS may also mediate the development of cirrhosis. Hepatic stellate cells (HSC), the liver's major matrix-producing cells, are activated by injury to become proliferative, fibrogenic myofibroblasts. HSC respond to sympathetic neurotransmitters by changing phenotype, suggesting that HSC may be the cellular effectors of ANS signals that modulate hepatic fibrogenesis during recovery from liver damage. We show here that the parasympathetic neurotransmitter acetylcholine markedly stimulates the proliferation of myofibroblastic HSC and induces HSC collagen gene expression in these cells. By extending evidence that HSC are direct targets of the ANS, these results support the proposed neuroglial role of HSC in the liver and suggest that interrupting ANS signalling may be useful in constraining the fibrogenic response to liver injury.     

H19/miR-148a/USP4 axis facilitates liver fibrosis by enhancing TGF-β signaling in both hepatic stellate cells and hepatocytes

Liver fibrosis is a wound-healing response represented by excessive extracellular matrix deposition. Activation of hepatic stellate cell (HSC) is the critical cellular basis for hepatic fibrogenesis, whereas hepatocyte undergoes epithelial-mesenchymal transition (EMT) which is also involved in chronic liver injury. Long noncoding RNA H19 has been found to be associated with cholestatic liver fibrosis lately. However, the role of H19 in liver fibrosis remains largely to be elucidated. In this study, we found that the expression of H19 was significantly upregulated in the liver tissue of CCl₄-induced mice, a toxicant-induced liver fibrogenesis model. Overexpression of H19 significantly aggravated activation of HSC and EMT of hepatocyte both by stimulating transforming growth factor-β (TGF-β) pathway. In terms of mechanism, H19 functioned as a competing endogenous RNA to sponge miR-148a and subsequently sustained the level of ubiquitin-specific protease 4 (USP4), which was an identified target of miR-148a and was able to stabilize TGF-β receptor I. In conclusion, our findings revealed a novel H19/miR-148a/USP4 axis which promoted liver fibrosis via TGF-β pathway in both HSC and hepatocyte, indicating that H19 could become a promising target for the treatment of liver fibrosis.     

The tyrosine phosphatase SHP-1 inhibits proliferation of activated hepatic stellate cells by impairing PDGF receptor signaling

The dimerization and auto-transphosphorylation of platelet-derived growth factor receptor (PDGFR) upon engagement by platelet-derived growth factor (PDGF) activates signals promoting the mitogenic response of hepatic stellate cells (HSCs) due to liver injury, thus contributing to the development of hepatic fibrosis. We demonstrate that the tyrosine phosphatases Src homology 2 domain-containing phosphatase 1 and 2 (SHP-1 and SHP-2) act as crucial regulators of a complex signaling network orchestrated by PDGFR activation in a spatio-temporal manner with diverse and opposing functions in HSCs. In fact, silencing of either phosphatase shows that SHP-2 is committed to PDGFR-mediated cell proliferation, whereas SHP-1 dephosphorylates PDGFR hence abrogating the downstream signaling pathways that result in HSC activation. In this regard, SHP-1 as an off-switch of PDGFR signaling appears to emerge as a valuable molecular target to trigger as to prevent HSC proliferation and the fibrogenic effects of HSC activation. We show that boswellic acid, a multitarget compound with potent anti-inflammatory action, exerts an anti-proliferative effect on HSCs, as in other cell models, by upregulating SHP-1 with subsequent dephosphorylation of PDGFR-β and downregulation of PDGF-dependent signaling after PDGF stimulation. Moreover, the synergism resulting from the combined use of boswellic acid and imatinib, which directly inhibits PDGFR-β activity, on activated HSCs offers new perspectives for the development of therapeutic strategies that could implement molecules affecting diverse players of this molecular circuit, thus paving the way to multi-drug low-dose regimens for liver fibrosis.     

Hepatic stellate cells and astrocytes: Stars of scar formation and tissue repair

Scar formation inhibits tissue repair and regeneration in the liver and central nervous system. Activation of hepatic stellate cells (HSCs) after liver injury or of astrocytes after nervous system damage is considered to drive scar formation. HSCs are the fibrotic cells of the liver, as they undergo activation and acquire fibrogenic properties after liver injury. HSC activation has been compared to reactive gliosis of astrocytes, which acquire a reactive phenotype and contribute to scar formation after nervous system injury, much like HSCs after liver injury. It is intriguing that a wide range of neuroglia-related molecules are expressed by HSCs. We identified an unexpected role for the p75 neurotrophin receptor in regulating HSC activation and liver repair. Here we discuss the molecular mechanisms that regulate HSC activation and reactive gliosis and their contributions to scar formation and tissue repair. Juxtaposing key mechanistic and functional similarities in HSC and astrocyte activation might provide novel insight into liver regeneration and nervous system repair.Key words: p75 neurotrophin receptor, transforming growth factor-β, neurotrophins, epidermal growth factor, extracellular matrix, collagen, chondroitin sulfate proteoglycans, matrix metalloproteinases, scar, neurons, hepatocytes     

De novo synthesis of glutathione is a prerequisite for curcumin to inhibit hepatic stellate cell (HSC) activation

On liver injury, quiescent hepatic stellate cells (HSC), the most relevant cell type for hepatic fibrogenesis, become active, characterized by enhanced cell growth and overproduction of extracellular matrix (ECM). Oxidative stress facilitates HSC activation and the pathogenesis of hepatic fibrosis. Glutathione (GSH) is the most important intracellular antioxidant. We previously showed that curcumin, the yellow pigment in curry from turmeric, significantly inhibited HSC activation. The aim of this study is to elucidate the underlying mechanisms. It is hypothesized that curcumin might inhibit HSC activation mainly by its antioxidant capacity. Results from this study demonstrate that curcumin dose and time dependently attenuates oxidative stress in passaged HSC demonstrated by scavenging reactive oxygen species and reducing lipid peroxidation. Curcumin elevates the level of cellular GSH and induces de novo synthesis of GSH in HSC by stimulating the activity and gene expression of glutamate-cysteine ligase (GCL), a key rate-limiting enzyme in GSH synthesis. Depletion of cellular GSH by the inhibition of GCL activity using L-buthionine sulfoximine evidently eliminates the inhibitory effects of curcumin on HSC activation. Taken together, our results demonstrate, for the first time, that the antioxidant property of curcumin mainly results from increasing the level of cellular GSH by inducing the activity and gene expression of GCL in activated HSC in vitro. De novo synthesis of GSH is a prerequisite for curcumin to inhibit HSC activation. These results provide novel insights into the mechanisms of curcumin as an antifibrogenic candidate in the prevention and treatment of hepatic fibrosis.     

Elevated mitochondrial activity distinguishes fibrogenic hepatic stellate cells and sensitizes for selective inhibition by mitotropic doxorubicin

Activation of hepatic stellate cells (HSCs) is an integral component of the wound‐healing process in liver injury/inflammation. However, uncontrolled activation of HSCs leads to constant secretion of collagen‐rich extracellular matrix (ECM) proteins, resulting in liver fibrosis. The enhanced ECM synthesis/secretion demands an uninterrupted supply of intracellular energy; however, there is a paucity of data on the bioenergetics, particularly the mitochondrial (mito) metabolism of fibrogenic HSCs. Here, using human and rat HSCs in vitro, we show that the mito‐respiration, mito‐membrane potential (Δψm) and cellular ‘bioenergetic signature’ distinguish fibrogenic HSCs from normal, less‐active HSCs. Ex vivo, HSCs from mouse and rat models of liver fibrosis further confirmed the altered ‘bioenergetic signature’ of fibrogenic HSCs. Importantly, the distinctive elevation in mito‐Δψm sensitized fibrogenic HSCs for selective inhibition by mitotropic doxorubicin while normal, less‐active HSCs and healthy human primary hepatocytes remained minimally affected if not, unaffected. Thus, the increased mito‐Δψm may provide an opportunity to selectively target fibrogenic HSCs in liver fibrosis.