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Background and Purpose: Obesity is often accompanied by hypercholesterolemia characterized by elevated levels of plasma low-density lipoprotein (LDL) and associated with non-alcoholic steatohepatitis, which could progress to hepatic fibrosis. Hepatic stellate cells (HSCs) are the major effectors of hepatic fibrogenesis. This study aims to clarify effects of LDL on activation of HSC, to evaluate roles of curcumin in suppressing these effects and to further elucidate the underlying molecular mechanisms.Experimental Approaches: HSCs were prepared from rats and cell proliferation was measured by cell proliferation assays (MTS assays). Transient transfection assays were performed to evaluate gene promoter activities. Real-time polymerase chain reaction and Western blotting were used to analyse the expression of genes.Key Results: LDL stimulated HSC activation in vitro, which was attenuated by curcumin. Curcumin reduced the abundance of LDL receptor (LDLR) in activated HSCs, decreasing cellular cholesterol. Curcumin-dependent activation of peroxisome proliferator-activated receptor-gamma (PPARgamma) differentially regulated the expression of the transcription factors, sterol regulatory element-binding proteins (SREBPs), in activated HSCs, resulting in the suppression of LDLR gene expression.Conclusions and Implications: Curcumin suppressed LDLR gene expression in activated HSCs in vitro by activating PPARgamma and differentially regulating gene expression of SREBPs, reducing cellular cholesterol and attenuating the stimulatory effects of LDL on HSC activation. These results provide novel insights into the roles and mechanisms of curcumin in the inhibition of LDL-induced HSC activation. This curcumin, a constituent of turmeric, may be useful in preventing hypercholesterolemia-associated hepatic fibrogenesis. [ABSTRACT FROM AUTHOR]

Activation of hepatic stellate cells (HSC), the major effectors of hepatic fibrogenesis, is coupled with sequential alterations in gene expression, including an increase in receptors for transforming growth factor-β (TGF-β) and a dramatic reduction in the peroxisome proliferator-activated receptor-γ (PPAR-γ). The relationship between them remains obscure. We previously demonstrated that curcumin induced gene expression of PPAR-γ in activated HSC, leading to reducing cell proliferation, inducing apoptosis and suppressing expression of extracellular matrix genes. The underlying molecular mechanisms are largely unknown. We recently observed that stimulation of PPAR-γ activation suppressed gene expression of TGF-β receptors in activated HSC, leading to the interruption of TGF-β signaling. This observation supported our assumption of an antagonistic relationship between PPAR-γ activation and TGF-β signaling in HSC. In this study, we further hypothesize that TGF-β signaling might negatively regulate gene expression of PPAR-γ in activated HSC. The present report demonstrates that exogenous TGF-β1 inhibits gene expression of PPAR-γ in activated HSC, which is eliminated by the pretreatment with curcumin likely by interrupting TGF-β signaling. Transfection assays further indicate that blocking TGF-β signaling by dominant negative type II TGF-β receptor increases the promoter activity of PPAR-γ gene. Promoter deletion assays, site-directed mutageneses, and gel shift assays localize two Smad binding elements (SBEs) in the PPAR-γ gene promoter, acting as curcumin response elements and negatively regulating the promoter activity in passaged HSC. The Smad3/4 protein complex specifically binds to the SBEs. Overexpression of Smad4 dose dependently eliminates the inhibitory effects of curcumin on the PPAR-γ gene promoter and TGF-β signaling. Taken together, these results demonstrate that the interruption of TGF-β signaling by curcumin induces gene expression of PPAR-γ in activated HSC in vitro. Our studies provide novel insights into the molecular mechanisms of curcumin in the induction of PPAR-γ gene expression and in the inhibition of HSC activation. [ABSTRACT FROM AUTHOR]

Background and Purpose: Senescence in hepatic stellate cells (HSCs) limits liver fibrosis. Glutaminolysis promotes HSC activation. Here, we investigated how emodin affected HSC senescence involving glutaminolysis. Experimental Approach: Senescence, glutaminolysis metabolites, Nur77 nuclear translocation, glutaminase 1 (GLS1) promoter methylation and related signalling pathways were examined in human HSC‐LX2 cells using multiple cellular and molecular approaches. Fibrotic mice with shRNA‐mediated knockdown of Nur77 were treated with emodin‐vitamin A liposome for investigating the mechanisms in vivo. Human fibrotic liver samples were examined to verify the clinical relevance. Key Results: Emodin upregulated several key markers of senescence and inhibited glutaminolysis cascade in HSCs. Emodin promoted Nur77 nuclear translocation, and knockdown of Nur77 abolished emodin blockade of glutaminolysis and induction of HSC senescence. Mechanistically, emodin facilitated Nur77/DNMT3b interaction and increased GLS1 promoter methylation, leading to inhibited GLS1 expression and blockade of glutaminolysis. Moreover, the glutaminolysis intermediate α‐ketoglutarate promoted extracellular signal‐regulated kinase (ERK) phosphorylation, which in turn phosphorylated Nur77 and reduced its interaction with DNMT3b. This led to decreased GLS1 promoter methylation and increased GLS1 expression, forming an ERK/Nur77/glutaminolysis positive feedback loop. However, emodin repressed ERK phosphorylation and interrupted the feedback cascade, stimulating senescence in HSCs. Studies in mice showed that emodin‐vitamin A liposome inhibited glutaminolysis and induced senescence in HSCs, and consequently alleviated liver fibrosis; but knockdown of Nur77 abrogated these beneficial effects. Similar alterations were validated in human fibrotic liver tissues. Conclusions and Implications: Emodin stimulated HSC senescence through interruption of glutaminolysis. HSC‐targeted delivery of emodin represented a therapeutic option for liver fibrosis. [ABSTRACT FROM AUTHOR]

Activation of hepatic stellate cells (HSCs) is a central event in the development of liver fibrosis, and the elimination of activated HSCs is considered to be an effective anti-fibrotic strategy. Here, we report that dihydroartemisinin (DHA) prevented the activation of HSCs via ferroptosis pathway. Importantly, DHA treatment increased the level of autophagy in HSCs. The inhibition of autophagy by 3-MA dramatically abolished the DHA-induced ferroptosis in HSCs. Mechanistically, the up-regulated m6A modification is essential for the activation of autophagy by DHA through the reduction of fat mass and obesity-associated gene (FTO). Down-regulation of m6A modification by FTO overexpression could impair autophagy and the classical ferroptotic events. Interestingly, the m6A modification of BECN1 mRNA was evidently up-regulated compared with other autophagy-related genes. More importantly, YTHDF1 was identified as a key m6A reader protein for BECN1 mRNA stability, and knockdown of YTHDF1 could prevent DHA-induced HSC ferroptosis. Noteworthy, YTH domain was essential for YTHDF1 to prolong the half-life of BECN1 mRNA in DHA-induced HSC ferroptosis. In mice, DHA treatment alleviated liver fibrosis by triggering HSC ferroptosis. HSC-specific inhibition of m6A modification and autophagy could impair DHA-induced HSC ferroptosis in murine liver fibrosis. Overall, these results provided novel implications to reveal the molecular mechanism of DHA-induced ferroptosis, by which pointed to m6A modification-dependent ferroptosis as a potential target for the treatment of liver fibrosis. [Display omitted] • DHA inhibits HSC activation via ferroptosis pathway. • The activation of autophagy is required for DHA-induced ferroptosis in HSCs. • The m6A modification is associated with DHA-induced autophagy and ferroptosis in HSCs. [ABSTRACT FROM AUTHOR]

Targeting the elimination of activated hepatic stellate cells (HSCs) and blocking excessive deposition of extracellular matrix are recognized as an effective strategy for the treatment of hepatic fibrosis. As a newly discovered programmed cell death mode, the regulatory mechanism of ferroptosis in the clearance of activated HSCs has not been fully elucidated. In the present study, we reported that the induction of ferroptosis in activated HSCs was required for dihydroartemisinin (DHA) to alleviate hepatic fibrosis. Treatment with DHA could improve the damage of hepatic fibrosis in vivo and inhibit the activation of HSCs in vitro. Interestingly, DHA treatment could trigger ferroptosis to eliminate activated HSCs characterized by iron overload, lipid ROS accumulation, glutathione depletion, and lipid peroxidation. Specific ferroptosis inhibitors ferrostatin‐1 and liproxstatin‐1 could impair DHA‐induced ferroptosis and also damage DHA‐mediated the inhibition of activated HSCs. Importantly, autophagy activation may be closely related to DHA‐induced ferroptosis. ATG5 siRNA could prevent DHA‐mediated autophagy activation and ferroptosis induction, whereas ATG5 plasmid could promote the effect of DHA on autophagy and ferroptosis. Of note, the upregulation of nuclear receptor coactivator 4 (NCOA4) may play a critical role in the molecular mechanism. NCOA4 siRNA could impair DHA‐induced ferroptosis, whereas NCOA4 plasmid could enhance the promoting effect of DHA on ferroptosis. Overall, our study revealed the potential mechanism of DHA against hepatic fibrosis and showed that ferroptosis could be a new way to eliminate activated HSCs. [ABSTRACT FROM AUTHOR]

Aims: This study was designated to illustrate the underlying mechanisms of emodin anti‐liver fibrosis via network pharmacology and experiment. Methods: The TSMCP and Genecards database were applied to screen the relevant targets of emodin or liver fibrosis. The essential target was selected by using Cytoscape to analyze the topological network of potential targets. Furthermore, we constructed a preliminary molecule docking study to explore the binding site by Surflex‐Dock suite SYBYL X 2.0. The DAVID database was selected for gene functional annotations and KEGG enrichment analysis. Moreover, we demonstrated the ameliorating effect of emodin on carbon tetrachloride (CCl4)‐induced liver injury in mice. We also verified the network predictions in vitro via various techniques. Results: The collected results showed that 35 targets were related to emodin, and 6,198 targets were associated with liver fibrosis. The Venn analysis revealed that 17 intersection targets were correlated with emodin anti‐liver fibrosis. The topological network analysis suggested that the p53 was the remarkable crucial target. Besides, the molecule docking results showed that emodin could directly interact with p53 by binding the active site residues ASN345, GLN331, and TYR347. Finally, KEGG pathway enrichment results indicated that essential genes were mainly enriched in mitogen‐activated protein kinase (MAPK) signaling pathways. Moreover, our study confirmed that emodin alleviated CCl4‐induced liver injury in mice, inducing hepatic stellate cells (HSCs) apoptosis via regulating the p53/ERK/p38 axis. Conclusions: This study partially verified the network pharmacological prediction of emodin inducing HSCs cell apoptosis through the p53/ERK/p38 axis. [ABSTRACT FROM AUTHOR]

Background and Purpose: Portal hypertension is a lethal complication of cirrhosis. Its mechanism and therapeutic targets remain largely unknown. Hepatic stellate cell (HSC) contraction increases intrahepatic vascular resistance contributing to portal hypertension. We investigated how HSC contraction was regulated by Wnt signalling and the therapeutic implications.Experimental Approach: Liver tissues from cirrhotic patients were examined. Cirrhotic mice with genetic or pharmacological treatments were used for in vivo assessments, and their primary cells were isolated. Cellular functions and signalling pathways were analysed in human HSC-LX2 cells using real-time PCR, Western blotting, siRNA, luciferase reporter assay, chromatin immunoprecipitation, co-immunoprecipitation and site-directed mutagenesis.Key Results: Wnt/β-catenin correlated with HSC contraction in human cirrhotic liver. Wnt3a stimulated Smo-independent Gli1 nuclear translocation followed by LARG-mediated RhoA activation leading to HSC contraction. Suppressor of fused (Sufu) negatively mediated Wnt3a-induced Gli1 nuclear translocation. Wnt/β-catenin repressed transcription of Sufu dependent on β-catenin/TCF4 interaction and TCF4 binding to Sufu promoter. Molecular simulation and site-directed mutagenesis identified the β-catenin residues Lys312 and Lys435 critically involved in this interaction. TCF4 binding to the sequence CACACCTTCC at Sufu promoter was required for transrepression of Sufu. In cirrhotic mice, short-term liver-targeting β-catenin deficiency or acute treatment with β-catenin inhibitors reduced portal pressure via restriction of HSC contraction rather than inhibiting HSC activation. Long-term deficiency or treatments also ameliorated liver injury, fibrosis and inflammation.Conclusion and Implications: Interaction between Wnt/β-catenin and Smo-independent Gli1 pathways promoted HSC contraction via TCF4-dependent transrepression of Sufu. HSC-specific inhibition of β-catenin may have therapeutic benefits for cirrhotic portal hypertension. [ABSTRACT FROM AUTHOR]

Currently, the existing treatments have not cured the liver fibrosis thoroughly. Ferroptosis is a newly discovered way of cell death, which is closely related to many diseases. Previous studies have shown that ferroptosis plays an important role in the occurrence and development of liver fibrosis, but the further mechanism remains to be discovered. LX-2 cells were used as the research object, fibrosis activation index was detected by Western blot, PCR and Immunofluorescence, ferroptosis was detected by kits, the binding and interaction between IRP2 (iron regulatory protein 2) and STUB1 (STIP1 homology and U-box containing protein 1) were detected by Immunoprecipitation and ubiquitin test, and IRP2 knockdown mice were constructed by interfering plasmid to verify the results of in vitro experiment. Our research showed that ART (artemether) had a good anti-fibrosis effect in vivo and in vitro, and ferroptosis played an important role in this process. Further studies have found that ART could lead to the accumulation of IRP 2 a in hepatic stellate cell by inhibiting the ubiquitination of it, thus inducing the increase of iron in HSC (hepatic stellate cell), which could product a large number of ROS (reactive oxide species), resulting the occurrence of ferroptosis in cells. Our findings provided an experimental basis for ART to become a drug for the treatment of liver fibrosis. Our results show that IRP2-Iron-ROS axis is necessary for ART to induce ferroptosis in HSC and play an anti-fibrotic effect. Image 1 • ART exerts its anti-fibrosis effect through ferroptosis pathway. • IRP2 is essential for ART to exert its anti-fibrotic effect. • STUB1 is a new ubiquitin ligase of IRP2 in hepatic stellate cells. • ROS accumulation caused by iron overload is the main cause of ferroptosis in hepatic stellate cells induced by ART. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: Oroxylin A, a natural flavonoid isolated from Scutellaria baicalensis, has been reported to have anti‐hepatic injury effects. However, the effects of oroxylin A on alcoholic liver disease (ALD) remains unclear. The aim of this study was to elucidate the effects of oroxylin A on ALD and the potential mechanisms. Materials and methods: Male ICR mice and human hepatocyte cell line LO2 were used. Yes‐associated protein (YAP) overexpression and knockdown were achieved using plasmid and siRNA technique. Cellular senescence was assessed by analyses of the senescence‐associated β‐galactosidase (SA‐β‐gal), senescence marker p16, p21, Hmga1, cell cycle and telomerase activity. Results: Oroxylin A alleviated ethanol‐induced hepatocyte damage by suppressing activities of supernatant marker enzymes. We found that oroxylin A inhibited ethanol‐induced hepatocyte senescence by decreasing the number of SA‐β‐gal‐positive LO2 cells and reducing the expression of senescence markers p16, p21 and Hmga1 in vitro. Moreover, oroxylin A affected the cell cycle and telomerase activity. Of importance, we revealed that YAP pharmacological inhibitor verteporfin or YAP siRNA eliminated the effect of oroxylin A on ethanol‐induced hepatocyte senescence in vitro, and this was further supported by the evidence in vivo experiments. Conclusion: Therefore, these aggregated data suggested that oroxylin A relieved alcoholic liver injury possibly by inhibiting the senescence of hepatocyte, which was dependent on its activation of YAP in hepatocytes. [ABSTRACT FROM AUTHOR]

Alcoholic liver disease (ALD), characterized by accumulation of fatty acids in liver cells, is usually caused by Chronic alcohol consumption. Our previous study has identified that DA protects against alcoholic liver injury in alcohol-fed rats through alleviating hepatocyte steatosis. It has emerged that saturated fatty acids could provoke endoplasmic reticulum (ER) stress and apoptosis in hepatocytes. This study was aimed to explore the impact of DA on ALD and further elaborate the underlying mechanisms. Results demonstrated that DA attenuates alcoholic liver injury in mice. Our results also indicated that DA attenuated lipid accumulation in hepatocytes exposed to ethanol. DA attenuates ethanol-induced hepatocyte apoptosis. Results demonstrated that DA dose-dependently ameliorated activation of mitochondrial pathway activation, which plays a critical role in apoptosis attributed to lipotoxicity. Further, DA suppressed the activation of JNK and the expression of CHOP, attributed to the inhibition of ER stress. It has emerged that activation of ER stress-JNK/CHOP-mitochondria cascade is considered as the key mechanisms underlying hepatocyte lipoapoptosis. In addition, DA attenuates PI3K/Akt Pathway in hepatocytes, consistent with our previous finding in HSCs. DA effects were reinforced by PI3K specific inhibitor LY294002. In summary, DA significantly protected hepatocytes against lipoapoptosis via a PI3K/Akt Pathway inhibition-dependent mechanism. [ABSTRACT FROM AUTHOR]

Alcoholic liver disease (ALD), characterized by excessive deposition of lipids in hepatocytes, causes heavy health burden personally and socially. Mechanistically, hedgehog signaling was activated during the development of ALD, and exerted compelling role in regulating lipometabolism. The current promising intervention strategy is inhibition of lipid accumulation and apoptosis in hepatocytes. Magnesium isoglycyrrhizinate (MgIG) has been widely used in various liver diseases for its good hepatoprotective activities. However, the role of MgIG in ALD has not been elucidated. Therefore, this study was aimed to explore the role of MgIG and further identify the potential mechanisms. We found for the first time that MgIG reduced lipid accumulation, including triglyceride, and total cholesterol, probably via inducing peroxisome proliferator-activated receptor-alpha and inhibiting sterol regulatory element-binding protein-1c. Further, MgIG alleviated ethanol-induced oxidative stress, evidenced by reduced abundance of reactive oxygen species and increased levels of glutathione, superoxide dismutase, and mitochondrial transmembrane potential. Besides, MgIG protected hepatocytes from ethanol-induced apoptosis. In addition, MgIG dose-dependently suppressed hedgehog signaling. Of note was that disruption of hedgehog signaling could mimic the effects of MgIG, whereas activation of hedgehog signaling abrogated the effects of MgIG. These findings suggested that MgIG prevented ethanol-induced hepatocyte steatosis and apoptosis via a hedgehog signaling inhibition-dependent mechanism. © 2017 IUBMB Life, 69(7):540-552, 2017 [ABSTRACT FROM AUTHOR]

Accumulating evidence indicates that hedgehog signaling plays a pivotal role in pathological angiogenesis and is involved in wound-healing responses in a number of adult tissues, including the liver. We previously demonstrated that hedgehog signaling promoted proliferation and inhibited apoptosis in hepatic stellate cells. This study was aimed to evaluate the effect of tetramethylpyrazine (TMP) on hedgehog signaling and to further examine the molecular mechanisms of TMP-induced antiangiogenesic effects in liver fibrosis. We found that TMP ameliorated the expression of proangiogenic markers vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor receptor 2 (VEGF-R2), platelet-derived growth factor BB (PDGF-BB), platelet-derived growth factor-β receptor (PDGF-βR) and hypoxia inducible factor 1α (HIF-1α), concomitant with reduced abundance of endothelial markers platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), CD34 and von willebrand factor in vivo and in vitro. Interestingly, TMP attenuated the abundance of sonic hedgehog, smoothened (Smo) and glioblastoma but increased the expression of hedgehog-interacting protein in liver sinusoidal endothelial cells, which was underlying mechanism for the antiangiogenesic activity of TMP. Downregulation of Smo activity, using selective Smo inhibitor cyclopamine, lead to a synergistic effect with TMP, whereas Smo overexpression plasmid impaired the induction of antiangiogenesic effects of TMP. Overall, these results provide novel implications to reveal the molecular mechanism of TMP-inhibited liver sinusoidal angiogenesis, by which points to the possibility of using TMP-based antiangiogenic drugs for the treatment of liver fibrosis. © 2017 IUBMB Life, 69(2):115-127, 2017 [ABSTRACT FROM AUTHOR]

BACKGROUND AND PURPOSE Diabetes is characterized by hyperglycaemia, which facilitates the formation of advanced glycation end-products (AGEs). Type 2 diabetes mellitus is commonly accompanied by non-alcoholic steatohepatitis, which could lead to hepatic fibrosis. Receptor for AGEs (RAGE) mediates effects of AGEs and is associated with increased oxidative stress, cell growth and inflammation. The phytochemical curcumin inhibits the activation of hepatic stellate cells (HSCs), the major effectors during hepatic fibrogenesis. The aim of this study was to explore the underlying mechanisms of curcumin in the elimination of the stimulating effects of AGEs on the activation of HSCs. We hypothesize that curcumin eliminates the effects of AGEs by suppressing gene expression of RAGE. EXPERIMENTAL APPROACH Gene promoter activities were evaluated by transient transfection assays. The expression of rage was silenced by short hairpin RNA. Gene expression was analysed by real-time PCR and Western blots. Oxidative stress was evaluated. KEY RESULTS AGEs induced rage expression in cultured HSCs, which played a critical role in the AGEs-induced activation of HSCs. Curcumin at 20 µM eliminated the AGE effects, which required the activation of PPARγ. In addition, curcumin attenuated AGEs-induced oxidative stress in HSCs by elevating the activity of glutamate-cysteine ligase and by stimulating de novo synthesis of glutathione, leading to the suppression of gene expression of RAGE. CONCLUSION AND IMPLICATIONS Curcumin suppressed gene expression of RAGE by elevating the activity of PPARγ and attenuating oxidative stress, leading to the elimination of the AGE effects on the activation of HSCs. LINKED ARTICLE This article is commented on by Stefanska, pp. 2209-2211 of this issue. To view this commentary visit [ABSTRACT FROM AUTHOR]

[Display omitted] Activation of hepatic stellate cells (HSCs) is a central event in the pathogenesis of liver fibrosis and is often accompanied by the disappearance of lipid droplets (LDs). Although interference with LD metabolism can effectively reverse the activation of HSCs, there is currently no effective therapy for liver fibrosis. Our previous evidence indicates that long non-coding RNA (lncRNA)-H19 plays an essential role in LD metabolism of HSC. In this study, we investigated the potential molecular mechanism of dihydroartemisinin (DHA) inhibits LD metabolism and liver fibrosis by regulating H19-AMPK pathway. We found that DHA restores LDs content in activated HSCs via reducing the transcription of H19 driven by hypoxia inducible factor 1 subunit alpha (HIF1α) and inhibiting the lipid oxidation signal mediated by AMP-activated protein kinase (AMPK) phosphorylation. In vivo experiments, we have proved that DHA reduced the deposition of extracellular matrix (ECM) and reduce the level of liver fibrosis in CCl 4 -induced liver fibrosis of mice. In summary, our results emphasize the importance of H19 in liver fibrosis and the potential of DHA to regulate H19 to treat liver fibrosis, providing a new direction for the prevention and treatment of liver fibrosis. [ABSTRACT FROM AUTHOR]

Activation of hepatic stellate cells (HSCs) is a central event in the pathogenesis of liver fibrosis and is characterized by the disappearance of lipid droplets. Although the exogenous supplementation of lipid droplet content can effectively reverse the activation of HSCs, the underlying molecular mechanisms are largely unknown. In our current study, we sought to investigate the role of lncRNA-H19 in the process of lipid droplets disappearance and to further examine the underlying molecular mechanisms. We found that the lncRNA-H19 level was increased in CCl 4 -induced fibrotic liver, which activated HSCs. Further research showed that hypoxia inducible factor-1α (HIF-1α) significantly increased lncRNA-H19 expression by binding to the lncRNA-H19 promoter at two hypoxia response element (HRE) sites located at 492–499 and 515–522 bp. Importantly, lncRNA-H19 knockdown markedly inhibited HSC activation and alleviated liver fibrosis, indicating that lncRNA-H19 may be a potential target for anti-fibrosis therapeutic approaches. Moreover, lncRNA-H19 knockdown could reverse the lipid droplet phenotype of activated HSCs, inhibiting the phosphorylated AMPKα-mediated lipid oxidation signaling pathway. The AMPK agonist AICAR promoted AMPKα phosphorylation and abrogated lipid droplets restoration in HSCs transfected with the lncRNA-H19 knockdown plasmid. Experimental molecular analysis showed that lncRNA-H19 triggered AMPKα to interact with LKB1 and resulted in AMPKα phosphorylation, which accelerating lipid droplets degradation and lipid oxidation. Taken together, our results highlighted the role of lncRNA-H19 in the metabolism of lipid droplets in HSCs, and revealed a new molecular target for alleviating liver fibrosis. • HIF-1α upregulated lncRNA-H19 expression at transcriptional level in HSCs. • Knockdown of LncRNA-H19 inhibited HSC activation and exerts anti-liver fibrosis. • Knockdown of LncRNA-H19 inhibited lipid droplet metabolism through AMPKα pathway in HSCs. [ABSTRACT FROM AUTHOR]

• Oroxylin A decreased the release of inflammatory factors in HSCs. • Oroxylin A exerted anti-inflammatory effect through PI3K/Akt/mTOR signaling. • Oroxylin A exerted anti-inflammatory activity by scavenging ROS in HSCs. More and more evidence showed that autophagy is an inflammation-related defense mechanism against a variety of diseases including liver fibrosis. However, the essential mechanisms remain poorly understood. In this study, we sought to elucidate the impact of Oroxylin A on autophagy and further to identify the potential mechanism of its anti-inflammatory activity. We found that Oroxylin A played a critical role in controlling inflammation in murine liver fibrosis. Moreover, Oroxylin A could inhibit the secretion of pro-inflammatory cytokines in activated hepatic stellate cell (HSCs). We previously reported that Oroxylin A can induce autophagy to alleviate the pathological changes of liver fibrosis and the activation of HSC. Here we further revealed that the inhibition of the PI3K/Akt/mTOR signaling was required for Oroxylin A to induce autophagy activation, which may be the underlying mechanism of the anti-inflammatory activity of Oroxylin A. Interestingly, mTOR overexpression completely impaired the Oroxylin A-mediated autophagy activation, and in turn, damaged the anti-inflammatory activity. Importantly, Oroxylin A inhibited PI3K/Akt/mTOR signaling by scavenging reactive oxygen species (ROS). ROS accumulation by buthionine sulfoximine (BSO) could abrogate the Oroxylin A-mediated ROS elimination, the inhibition of PI3K/Akt/mTOR signaling, and anti-inflammatory activities. Overall, our results provided reliable evidence for the molecular mechanism of Oroxylin A-mediated anti-fibrosis activity, and also identified a new target for drug therapy of liver fibrosis. [ABSTRACT FROM AUTHOR]

Curcumol, a guaiane-type sesquiterpenoid hemiketal extracted from the herb Rhizoma Curcumae, exhibits multiple-pharmacological activities. We previously reported that curcumol ameliorated hepatic fibrosis by inhibiting hepatic stellate cell (HSC) activation. In this study, we aimed to investigate the effect of curcumol on HSC migration and adhesion, and reveal its regulation mechanisms. Cellular viability was determined by Cell Counting Kit-8. Cell migration was detected by boyden chamber and cell scratch experiment. Recombinant human periostin (rh POSTN) and adeno-associated viral (AAV)-GFP-periostin were used to achieve POSTN overexpression in vitro and in vivo , respectively. Nuclear factor kappa B (NF-κB)-p65 overexpression was achieved by using plasmid. ELISA was conducted to detect POSTN level. Immunohistochemistry, qRT-PCR, Western blotting, and immunofluorescence were performed to assess associated factor expression. Curcumol suppressed HSC migration and adhesion, and reduced the secretion and expression of POSTN. By gain of function POSTN in HSCs, using rh POSTN, we found that the inhibition of HSC migration and adhesion by curcumol depended on the decrease of POSTN. Besides, curcumol protection against chronic CCl 4 -caused hepatic fibrosis could be impaired by POSTN overexpression. Moreover, we showed that curcumol repressed NF-κB signaling and the production of pro-inflammatory factor. Importantly, curcumol down-regulation of POSTN was rescued by knock-in of NF-κB, as well as the inhibition of HSC migration and adhesion. These findings reveal the molecular mechanism of curcumol-reduced HSC migration and adhesion, by which points to the possibility of using curcumol based on NF-κB dependent POSTN for the treatment of fibrogenesis. [ABSTRACT FROM AUTHOR]

Proliferation and differentiation of hepatic stellate cells (HSCs) are the most noticeable events in hepatic fibrosis, in which the loss of lipid droplets (LDs) is the most important feature. However, the complex mechanisms of LD disappearance have not been fully elucidated. In the current study, we investigated whether oroxylin A has the pharmacological activity of reversing LDs in activated HSCs, and further examined its potential molecular mechanisms. Using genetic, pharmacological, and molecular biological measure, we found that LD content significantly decreased during HSC activation, whereas oroxylin A markedly reversed LD content in activated HSCs. Interestingly, oroxylin A treatment observably decreased the expression of adipose triglyceride lipase (ATGL) without large differences in classical LD synthesis pathway, LD-related transcription factors, and autophagy pathway. ATGL overexpression could completely impair the effect of oroxylin A on reversing LD content. Importantly, reactive oxygen species (ROS) signaling pathway mediated oroxylin A-induced ATGL downregulation and LD revision in activated HSCs. ROS specific stimulant buthionine sulfoximine (BSO) could dramatically diminish the antioxidant effect of oroxylin A, and in turn, abolish reversal effect of oroxylin A on LD content. Conversely, ROS specific scavenger N-acetyl cystenine (NAC) can significantly enhance the pharmacological effect of oroxylin A on LD revision. Taken together, our study reveals the important molecular mechanism of anti-fibrosis effect of oroxylin A, and also suggests that ROS-ATGL pathway is a potential target for reversing LDs. [ABSTRACT FROM AUTHOR]