RESUMO
BACKGROUND Hepatic stellate cells (HSCs) play a vital role in hepatic fibrogenesis. Our recent clinical study indicated that the Zi Qi decoction, a Traditional Chinese Medicine formula, exhibited good efficacy in alleviating liver fibrosis, but the underlying mechanism remains elusive. MATERIAL AND METHODS Rats repeatedly injected with CCl4 and cells stimulated with lipopolysaccharide were used as in vivo and in vitro models for liver fibrosis, respectively. The viability of LX-2 cells was evaluated with MTT assay. Relative messenger RNA (mRNA) expression of representative extracellular matrix (ECM) components was detected with real-time quantitative polymerase chain reaction (RT-qPCR). Moreover, total and phosphorylation levels of ECM proteins and pathway-related proteins were detected with western blotting. Immunofluorescent staining was used to show the nuclear translocation of nuclear factor kappa b (NF-kappaB) p65. Hematoxylin & eosin (H&E) and Masson trichrome staining and immunohistochemistry were performed to evaluate the extent of liver fibrosis. The levels of alanine transaminase (ALT), aspartate transaminase (AST), gamma-glutamyl transpeptidase (GGT), Hyp, tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) were tested with an enzyme-linked immunosorbent assay. In addition, 7.0T micro-magnetic resonance imaging (micro-MRI) was used to evaluate the severity of hepatic damage. RESULTS The Zi Qi decoction inhibited lipopolysaccharide-mediated upregulation of mRNA and protein levels of representative ECM proteins both in vivo and in vitro. The Zi Qi decoction also suppressed activation of the Toll-like receptor 4 (TLR4)-related NF-kappaB signaling pathway and subsequently inhibited the nuclear translocation of activated NF-kappaB. Moreover, another TLR4 downstream pathway, mitogen-activated protein kinase (MAPK), was simultaneously restrained. The results of liver pathology and MRI in rat models also suggested the efficacy of the Zi Qi decoction in attenuating liver damage. CONCLUSIONS The Zi Qi decoction inhibited liver fibrosis by inhibiting the TLR4-related NF-kB and MAPK signaling pathways and preventing activation of HSCs.
Assuntos
Misturas Complexas/uso terapêutico , Medicamentos de Ervas Chinesas/uso terapêutico , Células Estreladas do Fígado/fisiologia , Cirrose Hepática/terapia , Fígado/metabolismo , Alanina Transaminase/metabolismo , Animais , Aspartato Aminotransferases/metabolismo , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Fígado/patologia , Sistema de Sinalização das MAP Quinases , Masculino , Medicina Tradicional Chinesa , NF-kappa B/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor 4 Toll-Like/metabolismoRESUMO
Hepatic stellate cell (HSC) activation is an essential event during liver fibrogenesis. Phosphatase and tension homolog deleted on chromosome 10 (PTEN) is a negative regulator of this process. DNA methyltransferase 1 (DNMT1), which catalyzes DNA methylation and subsequently leads to the transcriptional repression of PTEN, is selectively induced in myofibroblasts from diseased livers. Sennoside A (SA), a major purgative constituent of senna and the Chinese herb rhubarb, is widely used in China and other Asian countries as an irritant laxative. SA is reported to improve hepatic steatosis. However, the effect and mechanism of SA on liver fibrosis remain largely unknown. We recently identified a novel strategy for protecting liver fibrosis via epigenetic modification by targeting DNMT1. A Surface Plasmon Resonance (SPR) assay first reported that SA could directly bind DNMT1 and inhibit its activity. Administration of SA significantly prevented liver fibrosis, as evidenced by the dramatic downregulation of α-smooth muscle actin (α-SMA) and type I collagen alpha-1 (Col1α1) protein levels in a CCl4 -induced mouse hepatic fibrosis model and in TGF-ß1-activated HSC-T6 cells, in vivo and in vitro. SA decreased the expression of Cyclin D1, CDK, and C-myc, indicating that SA may inhibit the activation and proliferation of TGF-ß1-induced HSC-T6. Moreover, SA significantly promoted the expression of PTEN and remarkably inhibited the expression of p-AKT and p-ERK in vitro. Blocking PTEN or overexpressing DNMT1 could reduce the effect of SA on liver fibrosis. These data suggest that SA directly binds and inhibits the activity and that attenuated DNMT1-mediated PTEN hypermethylation caused the loss of PTEN expression, followed by the inhibition of the AKT and ERK pathways and prevented the development of liver fibrosis. Hence, SA might be employed as a promising natural supplement for liver fibrosis drug therapy.
Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA , Células Estreladas do Fígado/efeitos dos fármacos , Cirrose Hepática/tratamento farmacológico , PTEN Fosfo-Hidrolase/genética , Senosídeos/farmacologia , Actinas/genética , Actinas/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Ciclina D1/genética , Ciclina D1/metabolismo , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/fisiologia , Cirrose Hepática/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos C57BL , PTEN Fosfo-Hidrolase/metabolismo , Ligação Proteica , Senosídeos/uso terapêutico , Transdução de Sinais , Fator de Crescimento Transformador beta/farmacologiaRESUMO
It has been shown that both nilotinib as a tyrosine kinase inhibitor, and atorvastatin as a rho-kinase inhibitor, have antifibrotic effects. Therefore, considering the relationship between these two pathways, this study aimed to investigate the effects of their co-treatment against hepatic stellate cells (HSCs) activation and liver fibrosis. For this purpose, the activation of HSCs coincided with these therapies. Also, liver fibrosis by carbon tetrachloride (CCl4 ) was induced in male Wistar rats and treated simultaneously with these compounds. The expression of alpha-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), Ras homolog gene family, and member A (RhoA)/Rho-associated protein kinase (ROCK) in HSCs were measured. The expression of transforming growth factor beta-1 (TGF-ß1), its receptor (TßRII), CTGF, and platelets derived growth factor (PDGF), in the livers, were also investigated, all by real-time PCR and western blot analysis. Also, histopathologic and immunohistochemical evaluations were performed to evaluate changes in liver fibrosis during treatment. The results indicated the down-regulation of RhoA/ROCK, CTGF, and α-SMA, and inhibition of the HSCs activation toward myofibroblasts. The results also showed that the combined use of atorvastatin and nilotinib has significantly higher inhibitory effects. The antifibrotic effects of atorvastatin and nilotinib co-administration were also observed by histopathologic and immunohistochemical observations, and inhibiting the expression of TGF-ß1, TßRII, CTGF, and PDGF. Taken together, this study revealed that co-administration of nilotinib-atorvastatin has novel antifibrotic effects, by inhibiting RhoA/ROCK, and CTGF pathway. Therefore, the importance of the common pathway of RhoA/ROCK and CTGF, in reducing fibrosis may almost be concluded.
Assuntos
Atorvastatina/administração & dosagem , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Cirrose Hepática/prevenção & controle , Inibidores de Proteínas Quinases/administração & dosagem , Pirimidinas/administração & dosagem , Animais , Atorvastatina/farmacologia , Tetracloreto de Carbono , Células Cultivadas , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/fisiologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Fígado/efeitos dos fármacos , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Masculino , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Ratos , Ratos Wistar , Resultado do TratamentoRESUMO
Liver cirrhosis is the result of an uncontrolled fibrogenetic process, due to the activation and subsequent differentiation into myofibroblasts of the hepatic stellate cells (HSC). It is known that HSC express adrenoreceptors (AR), and the use of AR antagonists protects experimental animals from cirrhosis. However, several studies suggest that the toxicity generated by metabolism of these antagonists would hinder its use in cirrhotic patients. In addition, liver fibrosis may be associated with a decrease of the antioxidant response of the nuclear factor erythroid 2-related factor 2 (Nrf-2) and the overregulation of the proinflammatory pathway of nuclear factor kappa B (NF-κB). Therefore, in the present work, the capacity of doxazosin (α1 antagonist), carvedilol (nonselective beta-adrenoceptor blocker with alpha 1-blocking properties), and curcumin (antioxidant and anti-inflammatory compound) to reverse liver cirrhosis and studying the possible modulation of Nrf-2 and NF-κB were evaluated. Hamsters received CCl4 for 20 weeks, and then treatments were immediately administered for 4 weeks more. The individual administration of doxazosin or carvedilol showed less ability to reverse cirrhosis in relation to concomitantly curcumin administration. However, the best effect was the combined effect of doxazosin, carvedilol, and curcumin, reversing liver fibrosis and decreasing the amount of collagen I (Sirius red stain) without affecting the morphology of hepatocytes (hematoxylin and eosin stain), showing normal hepatic function (glucose, albumin, AST, ALT, total bilirubin, and total proteins). In addition, carvedilol treatment and the combination of doxazosin with curcumin increased Nrf-2/NF-κB mRNA ratio and its protein expression in the inflammatory cells in the livers, possibly as another mechanism of hepatoprotection. Therefore, these results suggest for the first time that α/ß adrenergic blockers with curcumin completely reverse hepatic damage, possibly as a result of adrenergic antagonism on HSC and conceivably by the increase of Nrf-2/NF-κB mRNA ratio.
Assuntos
Antagonistas Adrenérgicos alfa/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Curcumina/uso terapêutico , Células Estreladas do Fígado/fisiologia , Cirrose Hepática/tratamento farmacológico , Fígado/patologia , Miofibroblastos/fisiologia , Animais , Tetracloreto de Carbono , Carvedilol/uso terapêutico , Diferenciação Celular , Cricetinae , Modelos Animais de Doenças , Doxazossina/uso terapêutico , Sinergismo Farmacológico , Quimioterapia Combinada , Fibrose , Humanos , Fígado/efeitos dos fármacos , Cirrose Hepática/induzido quimicamente , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismoRESUMO
OBJECTIVE: To investigate the effects of Shengmai Injection (, SMI) on the proliferation, apoptosis and N-myc downstream-regulated gene 2 (NDRG2, a tumour suppressor gene) expression in varying densities of human hepatic stellate cells LX-2. METHODS: LX-2 cells were cultured in vitro. Then, cells were plated in 96-well plates at an approximate density of 2.5×104 cells/mL and cultured for 48, 72, 96 or 120 h followed by the application of different concentrations of SMI (0.6, 1.2, 2.4, 4.8 or 6 µL/mL). Cell proliferation was measured after an additional 24 or 48 h using the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of SMI on different cell growth states (cultured for 48, 72, 96, or 120 h) were observed by light microscopy at 24 h after treatment. When the cells reached 80% conflfluence, apoptosis was detected by flflow cytometry after 24 h. Lastly, LX-2 cells were treated with different concentrations of SMI and extracted with protein lysis buffer. The levels of NDRG2 were measured by Western blot. RESULTS: When the LX-2 cells grew for 48, 72, 96 and 120 h, 4.8 and 6 µL/mL of SMI significantly inhibited cell proliferation at 24 and 48 h after treatment (P<0.05). And 2.4 µL/mL of SMI also inhibited cell proliferation at 24 h after treatment when cell growth for 48 h (P<0.05) and at 48 h after treatment when cell growth for 72, 96 and 120 h (P<0.05). The NDRG2 expression level in the LX-2 cell was significantly increased when treated with SMI at concentrations of 1.2, 2.4, 4.8 or 6 µL/mL (P<0.05). CONCLUSION: The inhibitory effects of SMI on the proliferation of LX-2 cells were related to not only concentration dependent but also cell density. In addition, SMI (2.4, 4.8 and 6 µL/mL) could accelerate apoptosis in LX-2 cells, and the mechanism might be associated with NDRG2 over-expression.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Células Estreladas do Fígado/efeitos dos fármacos , Cirrose Hepática/tratamento farmacológico , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Combinação de Medicamentos , Células Estreladas do Fígado/fisiologia , Humanos , Injeções , Proteínas Supressoras de Tumor/genéticaRESUMO
Stevia has been shown to prevent oxidative stress and inflammation in carbon tetrachlorideinduced cirrhosis models. This study aimed to investigate the ability of an aqueous extract of stevia (AES) to prevent thioacetamide (TAA)induced cirrhosis in rats and to explore its mechanism of action. Liver cirrhosis was established by administering TAA (200 mg/kg by i.p. injections three times a week for 10 weeks); AES was administered (100 mg/kg by gavage daily) during the TAA treatment. Liver damage and fibrosis were evaluated, and the profibrotic pathways were analyzed by western blotting and immunohistochemistry. TAA increased nuclear factor kappa B (NFκB) and proinflammatory cytokine production, as well as the malondialdehyde and 4hydroxynonenal levels, whereas the glutathione/glutathione disulfide and nuclear factorE2related factor 2 (Nrf2) levels were decreased. Moreover, TAA increased collagen production, hepatic stellate cell (HSC) activation, and expression of profibrogenic mediators. TAAtreated rats that had been exposed to Mn2+ exhibited altered striatal dopamine turnover, indicating hepatic encephalopathy. AES partially or completely prevented all of these effects. AES showed antioxidant, antiinflammatory, and antifibrotic properties, probably because of its capacity to induce Nrf2 expression, reduce NFκB expression, and block several profibrogenic signaling pathways, subsequently inhibiting HSC activation and preventing fibrosis and dopamine turnover.
Assuntos
Células Estreladas do Fígado/efeitos dos fármacos , Cirrose Hepática Experimental/prevenção & controle , Fator 2 Relacionado a NF-E2/fisiologia , NF-kappa B/fisiologia , Extratos Vegetais/uso terapêutico , Proteína Smad7/fisiologia , Stevia , Fator de Crescimento Transformador beta/fisiologia , Animais , Células Estreladas do Fígado/fisiologia , Cirrose Hepática Experimental/induzido quimicamente , Masculino , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , TioacetamidaRESUMO
Liver fibrosis is a reversible pathophysiological process correlated with intense repair and cicatrization mechanisms, and its end-stage cirrhosis is responsible for high morbidity and mortality worldwide. Interestingly, the use of natural products as a realistic option for the treatment of liver fibrosis has broadly been accepted. Oroxylin A, a safe and natural product, shows a wide range of pharmacological activities such as anti-inflammatory, anti-oxidant, and anti-tumor properties. However, the effects of Oroxylin A on liver fibrosis remain poorly understood. In the present study, we sought to determine the effect of Oroxylin A on carbon tetrachloride (CCl4)-induced liver fibrosis, and to further examine the molecular mechanisms. We found that treatment with Oroxylin A markedly decreased the level of liver injury markers, alkaline phosphatase (ALP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT), in a dose dependent manner. Moreover, Oroxylin A treatment remarkably inhibited extracellular matrix (ECM) deposition, and significantly down-regulated the mRNA and protein expression of liver fibrosis markers including α1(I)collagen, fibronectin, alpha-smooth muscle actin (α-SMA), PDGF-ßR, and TGF-ßR1 in CCl4-induced murine model of liver fibrosis. Furthermore, experimental results in vitro showed that Oroxylin A treatment reduced the mRNA and protein expression of HSC activation markers, α-SMA, desmin, α1 (I) collagen, fibronectin, TGF-ß, and TNF-α, in a dose dependent manner. Attractively, Oroxylin A treatment also markedly up-regulated the expression of autophagy makers, LC3-B, Atg3, Atg4, Atg5, Beclin1/Atg6, Atg7, Atg9, ATG12, and Atg14, and apparently reduced the expression of autophagy substrate p62 in both CCl4-induced murine model of liver fibrosis and PDGF-BB-treated HSCs. Importantly, inhibition of autophagy by specific inhibitor 3-methyladenine (3-MA) completely abolished Oroxylin A-induced anti-fibrosis effect, indicating that activation of autophagy was required for Oroxylin A to alleviate liver fibrosis. Overall, these results provide novel implications to reveal the molecular mechanism of Oroxylin A-induced anti-fibrosis properties, by which points to the possibility of using Oroxylin A for the treatment of liver fibrosis.
Assuntos
Anti-Inflamatórios/uso terapêutico , Flavonoides/uso terapêutico , Células Estreladas do Fígado/fisiologia , Cirrose Hepática/tratamento farmacológico , Fígado/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Autofagia , Tetracloreto de Carbono , Células Cultivadas , Colágeno Tipo I/metabolismo , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/metabolismo , Células Estreladas do Fígado/efeitos dos fármacos , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos ICR , Terapia de Alvo Molecular , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Scutellaria baicalensis/imunologiaRESUMO
Glaucocalyxin A (GLA) is a biologically active ent-kauranoid diterpenoid isolated from Rabdosia japonica var. glaucocalyx. A large number of studies have shown that GLA possesses important pharmacological activities, such as anti-inflammatory, antitumor, antifibrosis, and antiplatelet activities. However, the role of GLA in the pathogenesis of liver fibrosis remains undefined. Therefore, the aim of this study was to investigate the effects of GLA on hepatic stellate cells (HSCs) activation/proliferation and migration in vitro and its possible mechanism in liver fibrosis. HSCs were incubated with different concentrations of GLA in the presence or absence of TGF-ß1 for 24 h. Cell proliferation and migration were evaluated by the MTT assay and Transwell migration assay, respectively. Intracellular reactive oxygen species (ROS) production was measured using 2',7'-dichlorodihydrofluorescin diacetate (DCFH-DA). Western blot was used to detect the expression levels of α-smooth-muscle actin (α-SMA), collagen-I, p-Smad2, Smad2, p-Smad3, and Smad3. Our results demonstrated that GLA significantly inhibited the proliferation and migration of HSCs, and suppressed the expression of extracellular matrix in TGF-ß1-stimulated HSC-T6 cells. In addition, pretreatment with GLA markedly suppressed TGF-ß1-induced ROS level in HSC-T6 cells. Furthermore, GLA greatly inhibited the phosphorylation levels of Smad2 and Smad3 in TGF-ß1-stimulated HSC-T6 cells. Taken together, these findings indicated that GLA inhibits the proliferation and activation of HSC-T6 cells, at least in part, through the TGF-ß1/Smad signaling pathway. Therefore, GLA may be a promising potential therapeutic agent for liver fibrosis.
Assuntos
Diterpenos do Tipo Caurano/farmacologia , Células Estreladas do Fígado/fisiologia , Transdução de Sinais , Linhagem Celular , Movimento Celular , Proliferação de Células , Avaliação Pré-Clínica de Medicamentos , Matriz Extracelular/metabolismo , Células Estreladas do Fígado/efeitos dos fármacos , Humanos , Cirrose Hepática/tratamento farmacológico , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/fisiologiaRESUMO
Carapax Trionycis is used as a traditional Chinese medicine with a long history of clinical application in China, and it represents an essential medication used for liver fibrosis treatment. Previous studies demonstrated that Carapax Trionycis extracts protect liver against fibrosis in CCL4-induced animal models. This study investigated the anti-fibrotic molecular mechanisms exerted by Carapax Trionycis extracts with molecular weight less than 6 KD (CT6) in rat hepatic stellate cell line HSC-T6 activated by TGF-ß1. HSC-T6 cells induced by TGF-ß1 were used to evaluate CT6 anti-fibrotic effect in vitro. CCK8 was used to evaluate cell viability and CT6 effect on HSC-T6 proliferation. ELISA was performed to detect the presence of inflammatory cytokines. Western blot and q-PCR were performed to explore the molecular mechanisms. Our data demonstrated that CT6 did not clearly affect cell viability but suppressed TGF-ß1-induced HSC-T6 proliferation. Collagen I and α-smooth muscle actin (α-SMA) protein levels were decreased by CT6 in TGF-ß1-induced HSC-T6, followed by the inhibition of TIMP1, TIMP2 and TGF-ß1/Smad pathway. Furthermore, CT6 decreased Jun D and p-p65 protein levels, down-regulated Tgf-ß1, Tnf-α, Il-1ß, Il-6 mRNA and TNF-α, IL-1ß and IL-6 expression in TGF-ß1-treated HSC-T6. These results suggested that CT6 inhibited HSC-T6 activation induced by TGF-ß1, indicating the potential therapeutic effect of these extracts against liver fibrosis.
Assuntos
Produtos Biológicos , Regulação da Expressão Gênica/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , NF-kappa B/metabolismo , Proteínas Smad/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular , Células Estreladas do Fígado/fisiologia , Cirrose Hepática/prevenção & controle , NF-kappa B/genética , Ratos , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/genética , Fator de Crescimento Transformador beta1RESUMO
In patients with hepatocellular carcinoma (HCC) receiving sorafenib, drug resistance is common. HCC develops in a microenvironment enriched with extracellular matrix proteins including laminin (Ln)-332, produced by hepatic stellate cells (HSCs). Ln-332 is the ligand of α3ß1 and α6ß4 integrins, differently expressed on the HCC cell surface, that deliver intracellular pathways. The aim of this study was to investigate the effect of Ln-332 on sorafenib's effectiveness. HCC cells were challenged with sorafenib in the presence of Ln-332 and of HSC conditioned medium (CM). Sorafenib impaired HCC cell proliferation and induced apoptosis. HSC-CM or Ln-332 inhibited sorafenib's effectiveness in HCC cells expressing both α3ß1 and α6ß4. Inhibiting α3 but not α6 integrin subunit using blocking antibodies or small interfering RNA abrogated the protection induced by Ln-332 and HSC-CM. Hep3B cells expressing α6ß4 but lacking the α3 integrin were insensitive to Ln-332 and HSC-CM protective effects. Hep3B α3-positive, but not wild-type and scramble transfected, cells acquired protection by sorafenib when plated on Ln-332-CM or HSCs. Sorafenib dephosphorylated focal adhesion kinase (FAK) and extracellular signal-regulated kinases 1/2, whereas Ln-332 and HSC-CM partially restored the pathways. Silencing FAK, but not extracellular signal-regulated kinases 1/2, abrogated the protection induced by Ln-332 and HSC-CM, suggesting a specific role for FAK. Sorafenib down-regulated total FAK, inducing its proteasomal degradation, while Ln-332 and HSC-CM promoted the escape of FAK from ubiquitination, probably inducing a preferential membrane localization. CONCLUSION: This study unveils a novel mechanism of sorafenib resistance depending on the α3ß1/Ln-332 axis and requiring FAK ubiquitination, providing new insights into personalizing therapy for patients with HCC. (Hepatology 2016;64:2103-2117).
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Proteína-Tirosina Quinases de Adesão Focal/fisiologia , Células Estreladas do Fígado/fisiologia , Integrina alfa3/fisiologia , Laminina/fisiologia , Neoplasias Hepáticas/tratamento farmacológico , Niacinamida/análogos & derivados , Compostos de Fenilureia/uso terapêutico , Ubiquitinação , Humanos , Niacinamida/uso terapêutico , Sorafenibe , Células Tumorais CultivadasRESUMO
Elevated levels of the transcriptional regulators Yes-associated protein (YAP) and transcriptional coactivators with PDZ-binding motif (TAZ), key effectors of the Hippo pathway, have been shown to play essential roles in controlling liver cell fate and the activation of hepatic stellate cells (HSCs). The dietary intake of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) has been positively associated with a number of health benefits including prevention and reduction of cardiovascular diseases, inflammation and cancers. However, little is known about the impact of ω-3 PUFAs on liver fibrosis. In this study, we used CCl4-induced liver fibrosis mouse model and found that YAP/TAZ is over-expressed in the fibrotic liver and activated HSCs. Fish oil administration to the model mouse attenuates CCl4-induced liver fibrosis. Further study revealed that ω-3 PUFAs down-regulate the expression of pro-fibrogenic genes in activated HSCs and fibrotic liver, and the down-regulation is mediated via YAP, thus identifying YAP as a target of ω-3 PUFAs. Moreover, ω-3 PUFAs promote YAP/TAZ degradation in a proteasome-dependent manner. Our data have identified a mechanism of ω-3 PUFAs in ameliorating liver fibrosis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ácidos Graxos Ômega-3/administração & dosagem , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/fisiologia , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/patologia , Fosfoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Aciltransferases , Animais , Proteínas de Ciclo Celular , Modelos Animais de Doenças , Regulação para Baixo , Fígado/patologia , Masculino , Camundongos Endogâmicos BALB C , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Resultado do Tratamento , Proteínas de Sinalização YAPRESUMO
Liver fibrosis represents a frequent event following chronic insult to trigger wound healing responses in the liver. Activation of hepatic stellate cells (HSCs), which is a pivotal event during liver fibrogenesis, is accompanied by enhanced expressions of a series of marker proteins and pro-fibrogenic signaling molecules. Artemisinin, a powerful antimalarial medicine, is extracted from the Chinese herb Artemisia annua L., and can inhibit the proliferation of cancer cells. Dihydroartemisinin (DHA), the major active metabolite of artemisinin, is able to attenuate lung injury and fibrosis. However, the effect of DHA on liver fibrosis remains unclear. The aim of this study was to investigate the effect of DHA on bile duct ligation-induced injury and fibrosis in rats. DHA improved the liver histological architecture and attenuated collagen deposition in the fibrotic rat liver. Experiments in vitro showed that DHA inhibited the proliferation of HSCs and arrested the cell cycle at the S checkpoint by altering several cell-cycle regulatory proteins. Moreover, DHA reduced the protein expressions of a-SMA, α1 (I) collagen and fibronectin, being associated with interference of the platelet-derived growth factor ß receptor (PDGF-ßR)-mediated ERK pathway. These data collectively revealed that DHA relieved liver fibrosis possibly by targeting HSCs via the PDGF-ßR/ERK pathway. DHA may be a therapeutic antifibrotic agent for the treatment of hepatic fibrosis.
Assuntos
Anti-Inflamatórios/uso terapêutico , Artemisininas/uso terapêutico , Células Estreladas do Fígado/efeitos dos fármacos , Fígado/efeitos dos fármacos , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Artemisia annua/imunologia , Ductos Biliares/cirurgia , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fibrose , Células Estreladas do Fígado/fisiologia , Humanos , Fígado/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Medicina Tradicional Chinesa , Ratos , Ratos Sprague-DawleyRESUMO
Hepatic stellate cells (HSCs) are universally acknowledged to play a stimulative role in the pathogenesis of hepatic fibrosis and portal hypertension. HSCs when activated in response to liver injury are characterized with many changes, with HSC contraction being the most common cause of portal hypertension. Previous studies have shown that dihydroartemisinine (DHA) is a potential antifibrotic natural product by inducing HSC apoptosis, whereas the role of DHA in regulating HSC contraction and the mechanisms involved remain a riddle. Recent studies have emphasized on the importance of farnesoid X receptor (FXR) and sphingosine-1-phosphate receptor 2 (S1PR2) in controlling cell contractility. This study showed that DHA strongly induced the mRNA and protein expression of FXR in LX-2 cells in a dose- and time-dependent manner and inhibited HSC activation, implying a conceivable impact of DHA on HSC contraction. The gel contraction assays and fluorescence staining of actin cytoskeleton verified that DHA dose-dependently limited contraction of collagen lattices and reorganization of actin stress fibers in LX-2 cells. DHA also decreased the phosphorylation of myosin light chain that is responsible for the contractile force of HSCs. Furthermore, gain- or loss-of-function analyses exhibited a FXR- and S1PR2-dependent mechanism of inhibiting HSC contraction by DHA, and DHA decreased S1PR2 expression by modulating FXR activation. Subsequent work revealed that inhibition of both Ca(2+) -dependent and Ca(2+) -sensitization signaling transductions contributed to DHA-induced HSC relaxation. In summary, these findings suggest that DHA could restrict HSC contraction through modulating FXR/S1PR2 pathway-mediated Ca(2+) -dependent and Ca(2+) -sensitization signaling. Our discoveries make DHA a potential candidate for portal hypertension. © 2016 IUBMB Life 68(5):376-387, 2016.
Assuntos
Artemisininas/farmacologia , Células Estreladas do Fígado/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Células Estreladas do Fígado/efeitos dos fármacos , Humanos , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Transdução de SinaisRESUMO
As a frequent event following chronic insult, liver fibrosis triggers wound healing reactions, with extracellular matrix components accumulated in the liver. During liver fibrogenesis, activation of hepatic stellate cells (HSCs) is the pivotal event. Fibrosis regression can feasibly be treated through pharmacological induction of HSC apoptosis. Herein we showed that dihydroartemisinin (DHA) improved liver histological architecture, decreased hepatic enzyme levels, and inhibited HSCs activation in the fibrotic rat liver. DHA also induced apoptosis of HSCs in such liver, as demonstrated by reduced distribution of α-SMA-positive cells and the presence of high number of cleaved-caspase-3-positive cells in vivo, as well as by down-regulation of Bcl-2 and up-regulation of Bax. In addition, in vitro experiments showed that DHA significantly inhibited HSC proliferation and led to dramatic morphological alterations in HSCs. we found that DHA disrupted mitochondrial functions and led to activation of caspase cascades in HSCs. Mechanistic investigations revealed that DHA induced HSC apoptosis through disrupting the phosphoinositide 3-kinase (PI3K)/Akt pathway and that PI3K specific inhibitor LY294002 mimicked the pro-apoptotic effect of DHA. DHA is a promising candidate for the prevention and treatment of liver fibrosis.
Assuntos
Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Artemisininas/farmacologia , Células Estreladas do Fígado/fisiologia , Cirrose Hepática/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Animais , Ductos Biliares/patologia , Sobrevivência Celular , Avaliação Pré-Clínica de Medicamentos , Células Estreladas do Fígado/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Fosfatidilinositol 3-Quinases/metabolismo , Fator de Crescimento Derivado de Plaquetas/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-DawleyRESUMO
OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is closely linked to obesity and constitutes part of the metabolic syndrome, which have been associated with low serum vitamin D (VD). Due to known crosstalk between VD and transforming growth factor (TGF)-ß signalling, VD has been proposed as an antifibrotic treatment. DESIGN: We evaluated the association between VD, the vitamin D receptor (VDR) and liver fibrosis in primary human hepatic stellate cells (phHSC) and 106 morbidly obese patients with NAFLD. RESULTS: Treating phHSC with VD ameliorated TGF-ß-induced fibrogenesis via both VDR-dependent and VDR-independent mechanisms. Reduction of fibrogenic response was abolished in cells homozygous for GG at the A1012G single nucleotide polymorphisms within the VDR gene. Compared with healthy livers, NAFLD livers expressed higher levels of VDR mRNA and VDR fragments. VDR mRNA was lower in patients homozygous for GG at A1012G and expression of pro-fibrogenic genes was higher in patients carrying the G allele. CONCLUSIONS: VD may be an antifibrotic treatment option early in the onset of fibrosis in specific genotypes for VDR. Known polymorphisms of the VDR may influence the response to VD treatment.
Assuntos
Células Estreladas do Fígado/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Receptores de Calcitriol/fisiologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Vitamina D/farmacologia , Adulto , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Regulação da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes/métodos , Células Estreladas do Fígado/fisiologia , Humanos , Fígado/metabolismo , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Obesidade Mórbida/complicações , Obesidade Mórbida/metabolismo , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/genética , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Transdução de Sinais/fisiologia , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta/fisiologia , Vitamina D/sangue , Adulto JovemRESUMO
UNLABELLED: Liver fibrosis and its endstage, cirrhosis, represent a major public health problem worldwide. Activation of hepatic stellate cells (HSCs) is a central event in hepatic fibrosis. However, the proteins that control HSC activation are incompletely understood. Here we show that (6aS, 10S, 11aR, 11bR, 11cS)-10-methylamino-dodecahydro-3a, 7a-diaza-benzo [de]anthracene-8-thione (MASM) exhibits potent inhibitory activity against liver fibrosis in vitro and in vivo associated with the reduction of Akt phosphorylation. Furthermore, ribosomal protein S5 (RPS5) was identified as a direct target of MASM, which stabilized RPS5 in cultured HSCs and in the liver of experimental animals after dimethylnitrosamine (DMN) or bile duct ligation (BDL). Functional studies revealed that RPS5 could prevent HSC activation. RPS5 overexpression in HSCs resulted in Akt dephosphorylation at both Ser473 and Thr308, and led to subsequent dephosphorylation of GSK3ß or P70S6K. Progression of DMN- and BDL-induced hepatic fibrosis was aggravated by Rps5 knockdown and alleviated by RPS5 overexpression, which correlated with the modulation of Akt phosphorylation and HSC number in the fibrotic livers. Moreover, RPS5 was substantially reduced in the transdifferentiated HSCs, experimental fibrotic livers, and human cirrhosis samples. CONCLUSION: These results demonstrate that RPS5 is implicated in hepatic fibrogenesis and may represent a promising target for potential therapeutic intervention in liver fibrotic diseases.
Assuntos
Alcaloides/farmacologia , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/patologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/patologia , Quinolizinas/farmacologia , Proteínas Ribossômicas/fisiologia , Adenoviridae/genética , Animais , Linhagem Celular Transformada , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Células Estreladas do Fígado/fisiologia , Cirrose Hepática/fisiopatologia , Masculino , Medicina Tradicional Chinesa/métodos , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/patologia , Miofibroblastos/fisiologia , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Proteínas Ribossômicas/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , MatrinasRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Liver fibrosis is the result of long-term liver damage and the wound-healing process, in which the hepatic stellate cell (HSC) plays a crucial role during fibrogenesis. The liver sinusoidal endothelial cell (LSEC) is a liver-resident scavenger, contributing to sinusoidal remodeling, HSC activation and liver fibrosis. Lipopolysaccharide (LPS) causes an inflammatory reaction associated with portal circulation and LSECs signaling. Scutellariae radix, the root of Scutellaria baicalensis Georgi, is a Chinese herb widely used for liver diseases. However, its effect on LSEC activation and HSC migration in liver fibrosis has not been investigated yet. AIM OF THIS STUDY: LPS-induced rat LSEC (rLSEC) activation was used as a model to screen and explore the active components of Scutellariae radix. The anti-fibrotic effect of Scutellariae radix on rLSEC activation and rHSC migration was further investigated. MATERIALS AND METHODS: LPS-induced rLSEC mRNA expression, including VEGF, VEGFR, MCP-1, and TGF-ß1, were examined by real-time PCR analyses. MCP-1 protein levels were measured by an ELISA kit. rLSEC conditioned medium on rHSC migration was measured by wound-healing assay and transwell chemoattraction assay. RESULTS: Results showed LPS-induced rLSEC activation with upregulated MCP-1 mRNA and protein expressions, and that rLSEC-condition medium enhanced rHSC migration. Both baicalein and wogonin from the active subfraction significantly reduced MCP-1 expression, but only baicalein markedly inhibited rHSC migration in rLSEC conditioned medium. CONCLUSION: This study demonstrated that Scutellariae radix attenuates LPS-induced rLSEC activation and HSC migration with downregulation of MCP-1 expression. The results provide supporting evidence that Scutellariae radix may be beneficial for the amelioration of liver fibrosis.
Assuntos
Movimento Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Flavonoides/farmacologia , Células Estreladas do Fígado/efeitos dos fármacos , Extratos Vegetais/farmacologia , Scutellaria baicalensis , Animais , Movimento Celular/fisiologia , Quimiocina CCL2/genética , Células Endoteliais/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células Estreladas do Fígado/fisiologia , Lipopolissacarídeos , Fígado/citologia , Raízes de Plantas , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Fator de Crescimento Transformador beta1/genética , Fator A de Crescimento do Endotélio Vascular/genética , CicatrizaçãoRESUMO
BACKGROUND & AIMS: Proliferation of liver progenitor cells (LPCs) is associated with inflammation and fibrosis in chronic liver diseases. However, how inflammation and fibrosis affect LPCs remains obscure. METHODS: We examined the role of interferon (IFN)-γ, an important pro-inflammatory and anti-fibrotic cytokine, in LPC expansion in HBV-infected patients and in mice challenged with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)- or choline-deficient, ethionine-supplemented (CDE) diet as well as in primary LPCs and LPC cell line. RESULTS: The CK19 staining scores correlated with inflammation and fibrosis grades in the livers from 110 HBV-infected patients. Nine-month IFN-γ treatment decreased LPC numbers, inflammation, and fibrosis in these HBV-infected patients. Similarly, a two-week IFN-γ treatment also decreased LPC activation in DDC-treated mice. Disruption of IFN-γ or its signaling components (e.g., IFNGR, STAT1, and IRF-1) increased LPC proliferation and liver fibrosis in DDC-fed mice. In contrast, deletion of IFN-γ did not increase, but rather slightly reduced LPC proliferation in CDE-fed mice. In vitro, IFN-γ attenuated proliferation of the LPC cell line BMOL and of primary LPCs from wild type mice, but not STAT1(-/-) or IRF-1(-/-) mice. Furthermore, co-culture assays suggest that IFN-γ can indirectly promote LPC proliferation via the activation of macrophages but attenuate it via the inhibition of hepatic stellate cells. CONCLUSIONS: IFN-γ inhibits LPC expansion via the direct inhibition of LPC proliferation and indirect attenuation of liver fibrosis in the DDC model, but it may also enhance LPC expansion via the promotion of inflammation in the CDE model; thereby playing dual roles in regulating LPC proliferation in vivo.
Assuntos
Células-Tronco Adultas/citologia , Hepatite B Crônica/patologia , Hepatócitos/citologia , Interferon gama/fisiologia , Células-Tronco Adultas/efeitos dos fármacos , Células-Tronco Adultas/fisiologia , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/patologia , Células Estreladas do Fígado/fisiologia , Hepatite B Crônica/fisiopatologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/fisiologia , Humanos , Interferon gama/deficiência , Interferon gama/farmacologia , Cirrose Hepática/patologia , Cirrose Hepática/fisiopatologia , Cirrose Hepática/prevenção & controle , Ativação de Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Knockout , Piridinas/administração & dosagem , Piridinas/toxicidade , Transdução de Sinais/efeitos dos fármacosRESUMO
UNLABELLED: Activation of hepatic stellate cells (HSCs) is crucial to the development of fibrosis in nonalcoholic fatty liver disease. Quiescent HSCs contain lipid droplets (LDs), whose depletion upon activation induces a fibrogenic gene program. Here we show that liver fatty acid-binding protein (L-Fabp), an abundant cytosolic protein that modulates fatty acid (FA) metabolism in enterocytes and hepatocytes, also modulates HSC FA utilization and in turn regulates the fibrogenic program. L-Fabp expression decreased 10-fold following HSC activation, concomitant with depletion of LDs. Primary HSCs isolated from L-FABP(-/-) mice contain fewer LDs than wild-type (WT) HSCs, and exhibit up-regulated expression of genes involved in HSC activation. Adenoviral L-Fabp transduction inhibited activation of passaged WT HSCs and increased both the expression of prolipogenic genes and also augmented intracellular lipid accumulation, including triglyceride and FA, predominantly palmitate. Freshly isolated HSCs from L-FABP(-/-) mice correspondingly exhibited decreased palmitate in the free FA pool. To investigate whether L-FABP deletion promotes HSC activation in vivo, we fed L-FABP(-/-) and WT mice a high-fat diet supplemented with trans-fatty acids and fructose (TFF). TFF-fed L-FABP(-/-) mice exhibited reduced hepatic steatosis along with decreased LD abundance and size compared to WT mice. In addition, TFF-fed L-FABP(-/-) mice exhibited decreased hepatic fibrosis, with reduced expression of fibrogenic genes, compared to WT mice. CONCLUSION: L-FABP deletion attenuates both diet-induced hepatic steatosis and fibrogenesis, despite the observation that L-Fabp paradoxically promotes FA and LD accumulation and inhibits HSC activation in vitro. These findings highlight the importance of cell-specific modulation of hepatic lipid metabolism in promoting fibrogenesis in nonalcoholic fatty liver disease. (Hepatology 2013).
Assuntos
Proteínas de Ligação a Ácido Graxo/metabolismo , Fígado Gorduroso/metabolismo , Células Estreladas do Fígado/fisiologia , Hepatócitos/metabolismo , Metabolismo dos Lipídeos , Animais , Gorduras na Dieta/efeitos adversos , Modelos Animais de Doenças , Ácidos Graxos/metabolismo , Fígado Gorduroso/etiologia , Feminino , Fibrose , Frutose/efeitos adversos , Técnicas de Transferência de Genes , Lipogênese , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Perilipina-5 , Proteínas/metabolismo , Triglicerídeos/metabolismoRESUMO
Both oxidative stress and endotoxins mediated immunological reactions play a major role in the progression of alcoholic hepatic fibrosis. Ascorbic acid has been reported to reduce alcohol-induced toxicity and ascorbic acid levels are reduced in alcoholics. Hence, we investigated the hepatoprotective action of ascorbic acid in the reversal of alcohol-induced hepatic fibrosis in male guinea pigs (n = 36), and it was compared with the animals abstenting from alcohol treatment. In comparison with the alcohol abstention group, there was a reduction in the activities of toxicity markers and levels of lipid and protein peroxidation products, expression of α-SMA, caspase-3 activity and mRNA levels of CYP2E1, TGF-ß(1), TNF-α and α(1)(I) collagen in liver of the ascorbic acid-supplemented group. The ascorbic acid content in liver was significantly reduced in the alcohol-treated guinea pigs. But it was reversed to normal level in the ascorbic acid-supplemented group. The anti-fibrotic action of ascorbic acid in the rapid regression of alcoholic liver fibrosis may be attributed to decrease in the oxidative stress, hepatic stellate cells activation, cytotoxicity and mRNA expression of fibrotic genes CYP2E1, TGF-ß(1), TNF-α and α(1) (I) collagen in hepatic tissues.