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1.
Int J Nanomedicine ; 15: 7995-8018, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33116514

RESUMO

Purpose: Zein/phospholipid composite nanoparticles (CNPs) were developed as a delivery platform for gallic acid (GA), a polyphenolic compound with reported preclinical antifibrotic activities. However, the therapeutic applicability of GA is hampered owing to its low bioavailability and rapid clearance. Accordingly, we developed GA-loaded CNPs. The effect of their size, surface charge and targeting strategies was investigated and optimized, with the aim of enhancing their ability to deliver GA to the activated hepatic stellate cells (aHSCs) in order to suppress hepatic fibrosis progression. Methods: Different CNP systems were prepared and characterized with regard to their particle size, zeta potential, and GA entrapment efficiency (EE%). Also, they were statistically optimized via response surface methodology. The optimized systems were investigated with regard to their in vitro GA release, in vitro efficacy on aHSCs, and in vivo biodistribution in healthy rats. Results: The GA-loaded cationic CNPs coupled with vitamin A (GA-CACNP/VA; 192 nm) showed high GA EE% (60% w/w), highest cellular internalization via active targeting, and more selective hepatic distribution, relative to free GA solution, GA-loaded anionic, and GA-loaded cationic systems. Furthermore, GA-CACNP/VA markedly triggered the apoptosis of aHSCs, repressed collagen deposition, and inhibited HSCs' activation to a lesser extent. Conclusion: The GA-CACNP/VA was shown to be a promising candidate for specific and controlled delivery of GA to aHSCs, which may provide an effective antifibrotic therapeutic approach.


Assuntos
Portadores de Fármacos/química , Ácido Gálico/química , Células Estreladas do Fígado/metabolismo , Nanopartículas/química , Fosfolipídeos/química , Vitamina A/química , Zeína/química , Animais , Apoptose/efeitos dos fármacos , Disponibilidade Biológica , Ácido Gálico/metabolismo , Ácido Gálico/farmacocinética , Ácido Gálico/farmacologia , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/efeitos dos fármacos , Masculino , Tamanho da Partícula , Ratos , Propriedades de Superfície , Distribuição Tecidual
2.
Zhongguo Ying Yong Sheng Li Xue Za Zhi ; 36(3): 273-278, 2020 May.
Artigo em Chinês | MEDLINE | ID: mdl-32981285

RESUMO

Objective: To observe the effects of propofol on the activation of hepatic stellate cell line HSC2-T6 induced by transforming growth factor-beta 1 (TGF-ß1) and explore its possible mechanism.Methods: The cells were divided into control group, TGF-ß1 group, propofol group, TGF-ß1 + propofol group, rapamycin group, TGF-ß1 + propofol + rapamycin group. Cells were treated with rapamycin (5 µmol/L) for 1 hour, propofol (100 µmol/L) for 1 hour, then TGF-ß1 (5 ng/ml) was added to co-culture for 24 hours. Cell proliferation was measured by MTT assay. The concentrations of hyaluronic acid (HA), collagen IV (IV-C) and laminin (LN) in the supernatant of cell culture medium were measured by ELISA. The ultrastructure of cells was observed by transmission electron microscopy. The expressions of alpha-smooth muscle actin (α-SMA), mammalian rapamycin target protein (mTOR), phosphorylated mTOR (p-mTOR) and the autophagy related gene Beclin 1, LC3 and p62 were measured by Western blot. Results: Compared with control group, cell proliferation, the expression of α-SMA, the concentrations of HA, IV-C and LN in culture supernatant, the number of autophages, the expressions of Beclin-1 and LC3-II, the ratio of LC3-II/LC3-I in HSC2-T6 cells were increased significantly, while the expression of p-mTOR, the ratio of p-mTOR/mTOR and the expression of p62 protein were decreased significantly in TGF-ß1 group (All P<0.05). Compared with TGF-ß1 group, cell proliferation, the expression of α-SMA, the concentrations of HA, IV-C and LN in culture supernatant, the number of autophages, the expressions of Beclin-1 and LC3-II, the ratio of LC3-II/LC3-I in HSC2-T6 cells in TGF-ß1 group were decreased significantly, and the expression of p-mTOR, the ratio of p-mTOR/mTOR and expression of p62 protein were increased significantly in TGF-ß1 + propofol group (All P<0.05). Conclusion: Propofol inhibits the activation of hepatic stellate cells induced by TGF-beta 1, and its mechanism involves the mTOR-autophagy pathway.


Assuntos
Autofagia , Células Estreladas do Fígado , Propofol , Fator de Crescimento Transformador beta1 , Animais , Autofagia/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Hipnóticos e Sedativos/farmacologia , Propofol/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Fator de Crescimento Transformador beta1/farmacologia
3.
PLoS One ; 15(7): e0236992, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32735603

RESUMO

We wanted to investigate whether Isoniazid (INH) can directly stimulate activation of hepatic stellate cells (HSCs) and enhance production of collagen. Treatment of human hepatic stellate cell line LX2 with or without 5µM INH for 24 to 72 hours was performed to look into content of cytochrome P450 2E1 (CYP2E1), activity of NADPH oxidase (NOX) and intracellular oxidative stress. Protein level as well as mRNA expression of alpha smooth muscle actin (α-SMA) and collagen1A1 (COL1A1) were assessed by western blot and real time PCR. In some experiments pyrazole (PY) was pre-treated to LX2 cells to induce CYP2E1 prior to INH treatment. CYP2E1 level as well as NOX activity was gradually increased with INH treatment in LX2 cells till 72 hours. Following 72 hours of INH exposure, intracellular glutathione (GSH) level was found to be reduced compared to control (p<0.01) and showed expression of α-SMA, indicating activation of HSC. We could not found any change in collagen expression in this experimental study. Pyrazole (PY) pre-treatment to LX2 cells caused significant increase in cellular CYP2E1 content associated with increase of NOX, intracellular reactive oxygen species (ROS), and expression of α-SMA and collagen1 after INH exposure. CYP2E1 is present in insignificant amount in HSCs and INH treatment could not induce collagen expression, although altered cellular oxidant levels was observed. But in LX2 cells when CYP2E1 was over-expressed by PY, INH administration provokes oxidative stress mediated stellate cells activation along with collagen type I expression.


Assuntos
Colágeno Tipo I , Citocromo P-450 CYP2E1 , Isoniazida/farmacologia , Linhagem Celular , Colágeno Tipo I/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Citocromo P-450 CYP2E1/efeitos dos fármacos , Citocromo P-450 CYP2E1/metabolismo , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Humanos , NADPH Oxidases/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos
4.
Toxicol Lett ; 333: 22-32, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32721574

RESUMO

HSCs (hepatic stellate cells) contribute to the excessive extracellular matrix (ECM) deposition, inflammatory pathways and crucial cell-cell interactions in hepatic disease leading to fibrosis. P2x7R is considered a potential orchestrater in liver fibrosis. For this reason, this work explored the role of P2x7R in liver fibrosis and the mechanism by which P2x7R in macrophages promotes fibrogenesis. In a model of liver fibrosis induced by administration of thioacetamide (TAA), inhibition of P2x7R with its selective inhibitor A438079 reversed TAA-induced liver damage and fibrosis. The mechanism was linked to inhibition of P2x7R-NLRP3 inflammasome activation and thereby infiltration of macrophages and neutrophils into the liver. This result indicated that the P2x7R-TLR4-NLRP3 axis is involved in the process of TGF-ß-mediated ECM deposition in HSCs. Ectopic overexpression of P2x7R lowered the threshold of extracellular matrix (ECM) deposition and maintained HSCs in an activated state. The culture medium of THP-1 macrophages stimulated by LPS/ATP aggravated ECM deposition in HSCs by activating P2x7R. Additionally, IL-1ß secreted by LPS / ATP activated macrophages amplified fibrosis. These data indicate that P2x7R plays a key regulative role in the activation and maintenance of HSCs promoted by macrophages. Thus, pharmacological inhibition of P2x7R could be a potential therapeutic mechanism to treat human liver fibrosis.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Células Estreladas do Fígado/efeitos dos fármacos , Cirrose Hepática/metabolismo , Macrófagos Peritoneais/efeitos dos fármacos , Receptores Purinérgicos P2X7/metabolismo , Animais , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Doença Hepática Induzida por Substâncias e Drogas/patologia , Modelos Animais de Doenças , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Retroalimentação Fisiológica , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Humanos , Cirrose Hepática/patologia , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Antagonistas do Receptor Purinérgico P2X/farmacologia , Piridinas/farmacologia , Tetrazóis/farmacologia , Tioacetamida/toxicidade
5.
PLoS One ; 15(6): e0234038, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32492075

RESUMO

Extracellular adenosine triphosphate (eATP) released by damaged cells, and its purinergic receptors, comprise a crucial signaling network after injury. Purinergic receptor P2X7 (P2RX7), a major driver of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation and IL-1ß processing, has been shown to play a role in liver injury in murine diet- and chemically-induced liver injury models. It is unclear, however, whether P2RX7 plays a role in non-alcoholic steatohepatitis (NASH) and which cell type is the main target of P2RX7 pharmacological inhibition. Here, we report that P2RX7 is expressed by infiltrating monocytes and resident Kupffer cells in livers from NASH-affected individuals. Using primary isolated human cells, we demonstrate that P2RX7 expression in CD14+ monocytes and Kupffer cells primarily mediates IL-1ß release. In addition, we show that pharmacological inhibition of P2RX7 in monocytes and Kupffer cells, blocks IL-1ß release, reducing hepatocyte caspase 3/7 activity, IL-1ß-mediated CCL2 and CCL5 chemokine gene expression and secretion, and hepatic stellate cell (HSC) procollagen secretion. Consequently, in a chemically-induced nonhuman primate model of liver fibrosis, treatment with a P2RX7 inhibitor improved histological characteristics of NASH, protecting from liver inflammation and fibrosis. Taken together, these findings underscore the critical role of P2RX7 in the pathogenesis of NASH and implicate P2RX7 as a promising therapeutic target for the management of this disease.


Assuntos
Inflamação/prevenção & controle , Cirrose Hepática/tratamento farmacológico , Antagonistas do Receptor Purinérgico P2X/uso terapêutico , Receptores Purinérgicos P2X7/metabolismo , Animais , Caspase 3/metabolismo , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Inflamação/patologia , Interleucina-1beta/metabolismo , Macrófagos do Fígado/citologia , Macrófagos do Fígado/efeitos dos fármacos , Macrófagos do Fígado/metabolismo , Lipopolissacarídeos/farmacologia , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Macaca fascicularis , Masculino , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Pró-Colágeno/metabolismo , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/genética
6.
Artigo em Inglês | MEDLINE | ID: mdl-32471116

RESUMO

Phthalates are often added to plastic products to increase their flexibility. Di-(2-ethylhexyl) phthalate (DEHP) is one of the most common plasticizers. Previously, a major incident involving phthalate-contaminated foodstuffs occurred, where phthalates were deliberately added to foodstuffs as a substitute for emulsifiers, resulting in a threat to public health. DEHP exposure can cause liver damage and further lead to cancer; however, the effects of long-term exposure to low-dose DEHP on hepatic stellate cells (HSCs) and on liver fibrosis are still unclear. In this study, we showed that chronic exposure to low-dose DEHP results in an accumulation of cholesterol in HSCs by disturbing the cholesterol metabolism and enhancing endogenous cholesterol synthesis. In addition, long-term exposure to low-dose DEHP reduces the sensitivity of HSCs to platelet-derived growth factor BB (PDGF-BB)-induced proliferation by blocking the MAPK pathway. Dysfunction of mitochondrial respiration and induction of caspase 3/PARP-dependent apoptosis were observed in HSCs following chronic, low-dose exposure. The carbon tetrachloride (CCl4)-induced liver fibrosis mouse model showed that long-term administration of DEHP significantly promoted liver damage, inflammatory infiltration, cholesterol accumulation, and deposition of hepatic collagen. In conclusion, long-term exposure to low-dose DEHP may perturb the cholesterol metabolism in HSCs and accelerate liver damage and fibrosis.


Assuntos
Colesterol/metabolismo , Dietilexilftalato , Células Estreladas do Fígado/efeitos dos fármacos , Cirrose Hepática/patologia , Animais , Dietilexilftalato/toxicidade , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/induzido quimicamente , Camundongos , Plastificantes/toxicidade
7.
Clin Sci (Lond) ; 134(7): 853-869, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32227122

RESUMO

Angiotensin II (Ang II) has been reported to aggravate hepatic fibrosis by inducing NADPH oxidase (NOX)-dependent oxidative stress. Alamandine (ALA) protects against fibrosis by counteracting Ang II via the MAS-related G-protein coupled (MrgD) receptor, though the effects of alamandine on hepatic fibrosis remain unknown. Autophagy activated by reactive oxygen species (ROS) is a novel mechanism of hepatic fibrosis. However, whether autophagy is involved in the regulation of Ang II-induced hepatic fibrosis still requires investigation. We explored the effect of alamandine on hepatic fibrosis via regulation of autophagy by redox balance modulation. In vivo, alamandine reduced CCl4-induced hepatic fibrosis, hydrogen peroxide (H2O2) content, protein levels of NOX4 and autophagy impairment. In vitro, Ang II treatment elevated NOX4 protein expression and ROS production along with up-regulation of the angiotensin converting enzyme (ACE)/Ang II/Ang II type 1 receptor (AT1R) axis. These changes resulted in the accumulation of impaired autophagosomes in hepatic stellate cells (HSCs). Treatment with NOX4 inhibitor VAS2870, ROS scavenger N-acetylcysteine (NAC), and NOX4 small interfering RNA (siRNA) inhibited Ang II-induced autophagy and collagen synthesis. Alamandine shifted the balance of renin-angiotensin system (RAS) toward the angiotensin converting enzyme 2 (ACE2)/alamandine/MrgD axis, and inhibited both Ang II-induced ROS and autophagy activation, leading to attenuation of HSCs migration or collagen synthesis. In summary, alamandine attenuated liver fibrosis by regulating autophagy induced by NOX4-dependent ROS.


Assuntos
Antioxidantes/farmacologia , Autofagia/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Células Estreladas do Fígado/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Cirrose Hepática Experimental/prevenção & controle , Fígado/efeitos dos fármacos , NADPH Oxidase 4/metabolismo , Oligopeptídeos/farmacologia , Animais , Tetracloreto de Carbono , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Colágeno/metabolismo , Células Estreladas do Fígado/enzimologia , Células Estreladas do Fígado/ultraestrutura , Fígado/enzimologia , Fígado/ultraestrutura , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/enzimologia , Cirrose Hepática Experimental/patologia , Masculino , Proteínas do Tecido Nervoso/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Peptidil Dipeptidase A/metabolismo , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas-G/efeitos dos fármacos , Receptores Acoplados a Proteínas-G/metabolismo , Sistema Renina-Angiotensina/efeitos dos fármacos , Transdução de Sinais
8.
Zhongguo Zhong Yao Za Zhi ; 45(3): 631-635, 2020 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-32237523

RESUMO

This paper was aimed to observe the interventional effect of Sedum sarmentosum total flavanones on hepatic fibrosis and its possible mechanism through the subcutaneous injection of CCl_4 in rats. Sixty male SD rats were randomly divided into normal control group, model group, low-dose, medium-dose, high-dose S. sarmentosum total flavanones groups(100, 200, 400 mg·kg~(-1)) and silymarin group(200 mg·kg~(-1)). The model of liver fibrosis was established by subcutaneous injection of rats with 40% CCl_4. After the modeling, the drug groups were intragastrically administered with corresponding drugs once a day for consecutively five weeks, while the normal group and the model group were given 0.9% sodium chloride solution during the same period. After the experiment, the general conditions of rats and the pathological changes of liver tissues were observed, and the contents of serum ALT, AST, HA and LN were measured. Besides, the expressions of the protein and relevant mRNA of Smad2/3, Smad4 and α-SMA in rats were detected. Compared with model group, S. sarmentosum total flavanones could significantly increase the rats' body weight, inhibit the increase of liver and spleen index in rats of liver fibrosis, reduce the levels of ALT, AST, HA and LN, and alleviate pathological changes. Meanwhile, compared with the model group, the protein expressions of Smad2/3, Smad4 and α-SMA as well as relevant mRNA expressions in S. sarmentosum total flavanones group were obviously decreased, while Smad7 expression was markedly increased. As a result, S. sarmentosum total flavanones could significantly alleviate CCl_4-induced liver fibrosis, and its anti-hepatic fibrosis mechanism may be related to intervention with Smads pathway, so as to inhibit the activation of HSC.


Assuntos
Medicamentos de Ervas Chinesas/uso terapêutico , Flavanonas/uso terapêutico , Células Estreladas do Fígado/efeitos dos fármacos , Cirrose Hepática/tratamento farmacológico , Sedum/química , Proteínas Smad/metabolismo , Animais , Tetracloreto de Carbono , Fígado , Cirrose Hepática/induzido quimicamente , Masculino , Ratos , Ratos Sprague-Dawley , Transdução de Sinais
9.
Toxicology ; 440: 152475, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32344006

RESUMO

OBJECTIVES: 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. MATERIALS AND METHODS: 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. RESULTS: 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 CCl4-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. CONCLUSION: 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.


Assuntos
Moléculas de Adesão Celular/antagonistas & inibidores , Células Estreladas do Fígado/efeitos dos fármacos , Sesquiterpenos/farmacologia , Fator de Transcrição RelA/antagonistas & inibidores , Animais , Intoxicação por Tetracloreto de Carbono/patologia , Intoxicação por Tetracloreto de Carbono/prevenção & controle , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Contagem de Células , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/patologia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Regulação para Baixo/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Proteínas Recombinantes
10.
Lab Invest ; 100(8): 1042-1056, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32291390

RESUMO

Sparc/osteonectin, cwcv, and kazal-like domain proteoglycan 1 (SPOCK1) is a matricellular protein which regulates cell proliferation, invasion, and survival but the function of SPOCK1 in liver fibrosis is obscure. In this study, we found that SPOCK1 expression increased significantly in fibrotic liver tissues and activated primary rat hepatic stellate cells (R-HSCs). SPOCK1 co-localized with α-smooth muscle actin (α-SMA) in the cytoplasm. Mechanistically, we found platelet-derived growth factor-BB (PDGF-BB) induced SPOCK1 expression by activating the PI3K/Akt/forkhead box M1 (FoxM1) signaling pathway. Intracellular SPOCK1 downregulation decreased the HSC activation, proliferation, and migration induced by PDGF-BB. Furthermore, intracellular SPOCK1 overexpression or recombinant SPOCK1 treatment promoted HSC activation, proliferation, and migration by activating the PI3K/Akt signaling pathway. Co-immunoprecipitation, double immunofluorescence staining indicated that SPOCK1 interacted with integrin α5ß1, and neutralization of integrin α5ß1 significantly reduced the role of recombinant SPOCK1 in HSCs. In vivo HSC-specific SPOCK1 knockdown following lentivirus administration dramatically ameliorated thioacetamide (TAA)-induced collagen deposition in rat livers. Collectively, our study indicates that SPOCK1 is crucial for hepatic fibrosis and it might be a promising therapeutic target.


Assuntos
Becaplermina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Cirrose Hepática/genética , Proteínas/metabolismo , Proteoglicanas/genética , Animais , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Estreladas do Fígado/metabolismo , Humanos , Integrina alfa5beta1/metabolismo , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Cirrose Hepática/metabolismo , Masculino , Fosfatidilinositol 3-Quinases/metabolismo , Proteoglicanas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
11.
Zhonghua Gan Zang Bing Za Zhi ; 28(2): 135-140, 2020 Feb 20.
Artigo em Chinês | MEDLINE | ID: mdl-32164064

RESUMO

Objective: To investigate the mechanism of occurrence and development of zinc-alpha-2-glycoprotein (AZGP1) in the activated hepatic stellate cells (HSCs) and liver fibrosis. Methods: The activated human hepatic stellate cell line LX2 was induced by the stimulation of transforming growth factor - ß1 to construct carbon tetrachloride liver fibrosis mice model. The situation expression of AZGP1 in liver cells and tissues were observed. Plasmid transfection method was used to detect the activation, proliferation, apoptotic functions and changes in related factors of LX2 cells, respectively, after the overexpression and inhibition of AZGP1expression. Univariate analysis of variance was used for multiple group comparison. Results: The results of immunofluorescence staining showed that AZGP1 protein was decreased and α-smooth muscle actin was increased in the activated LX2 cells, and the two were negatively correlated. AZGP1 gene and protein were significantly under-expressed in activated LX2 cells and liver tissues of mice with carbon tetrachloride liver fibrosis. Collagen I, matrix metalloproteinase-2, and α-smooth muscle actin genes and proteins were significantly down-regulated in LX2 cells after over-expression of AZGP1. Cell fluorescence showed that AZGP1-overexpressing cells were activated and α-smooth muscle actin protein was reduced. In addition, the proliferative activity and G1/S-specific cyclin D1 protein of LX2 cells were significantly reduced after overexpression of AZGP1, while cell cycle experiments showed that the proportion of cells overexpressing AZGP1 was significantly increased in the G0/G1 phase, and the proportion of S phase was significantly reduced. AZGP1 had no significant effect on the apoptosis of LX2 cells. Conclusion: AZGP1 can reverse liver fibrosis by inhibiting the activation and proliferation of hepatic stellate cells, and thereby overexpression of AZGP1 is expected to become a new target for liver fibrosis treatment.


Assuntos
Tetracloreto de Carbono/toxicidade , Proliferação de Células/efeitos dos fármacos , Glicoproteínas/metabolismo , Células Estreladas do Fígado/efeitos dos fármacos , Actinas , Animais , Glicoproteínas/genética , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Cirrose Hepática , Metaloproteinase 2 da Matriz , Camundongos , Zinco
12.
J Nat Med ; 74(3): 513-524, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32193805

RESUMO

Liver fibrosis is a pathological manifestation induced by chronic liver injury and may cause cirrhosis and liver cancer with the chronic progression of fibrosis. During the onset and progression of liver fibrosis, the activation of hepatic stellate cells (HSCs) is the core mechanism for the secretion of many extracellular matrices to induce fibrosis. Lignans are reportedly the main effective components of Schisandra chinensis with good anti-fibrosis effects. In this study, we compared the inhibiting effects of the seven lignan components from S. chinensis on HSC activation. We found that the seven lignans inhibited the activation of human HSCs (LX-2) in various degrees. Among all lignans, schisanhenol showed the best effect in inhibiting the activation of LX-2 with a dose-effect relationship. Sal also inhibited the phosphorylations of Smad1, Smad2, Smad3, extracellular regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), p38, and nuclear transcription factor-κB (NF-κB), as well as downregulated Smad4. All these findings suggested that schisanhenol may ameliorate liver fibrosis by inhibiting the transforming growth factor ß (TGF-ß)/Smad and mitogen-activated protein kinase (MAPK) signaling pathways. Remarkably, schisanhenol may be a potential anti-liver fibrosis drug and warrants further research.


Assuntos
Ciclo-Octanos/farmacologia , Células Estreladas do Fígado/metabolismo , Lignanas/farmacologia , Cirrose Hepática/prevenção & controle , Compostos Policíclicos/farmacologia , Schisandra/química , Linhagem Celular , Frutas/química , Células Estreladas do Fígado/efeitos dos fármacos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Cirrose Hepática/patologia , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Smad1/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
13.
Mol Cell Biochem ; 467(1-2): 107-116, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32108279

RESUMO

Hepatic stellate cells (HSCs) are known to play a key role in the progression of liver fibrosis by producing excessive extracellular matrix (ECM). Matrix metalloproteinases (MMPs) belong to a family of endopeptidases, which have a well-established role in the degradation of ECM. Our study suggests that, besides the degradation of the extracellular matrix, matrix metalloproteinase-8 (MMP-8) has a non-canonical role in activating the quiescent HSCs to myofibroblasts by regulating the expression of Col1A1 and αSMA. We have identified that MMP-8 secreted from macrophages as a response to LPS stimulation activates HSCs via ERK1/2-dependent pathway. In addition to this, we determined that MMP-8 may regulate the homodimerization of c-Jun in LX-2 cells, during the trans-differentiation process from quiescent HSC to activate myofibroblasts. Macrophage-released MMP-8 plays a master role in activating the dormant HSCs to activate myofibroblasts through the Erk-mediated pathway and Jun cellular translocation leading to liver fibrosis. Significance MMP-8 can be used as a therapeutic target against liver fibrosis.


Assuntos
Células Estreladas do Fígado/citologia , Lipopolissacarídeos/efeitos adversos , Sistema de Sinalização das MAP Quinases , Metaloproteinase 8 da Matriz/metabolismo , Técnicas de Cultura de Células , Transdiferenciação Celular , Colágeno Tipo I/metabolismo , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Humanos , Miofibroblastos/citologia , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Multimerização Proteica , Proteínas Proto-Oncogênicas c-jun/química , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Células THP-1
14.
Molecules ; 25(4)2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-32054011

RESUMO

Twenty 9O-substituted palmatine derivatives were prepared and tested for their biological effect against collagen α1 (I) (COL1A1) promotor in human hepatic stellate LX-2 cells. The structure-activity relationship (SAR) indicated that the introduction of a benzyl motif on the 9O atom was favorable for activity. Among them, compound 6c provided the highest inhibitory effect against COL1A1 with an IC50 value of 3.98 µM, and it also dose-dependently inhibited the expression of fibrogenic COL1A1, α-soomth muscle actin (α-SMA), matrix metalloprotein 2 (MMP2) in both mRNA and protein levels, indicating extensive inhibitory activity against fibrogenesis. A further primary mechanism study indicated that it might repress the hepatic fibrogenesis via inhibiting both canonical transforming growth factor-beta 1 (TGF-ß1)/Smads and non-canonical janus-activated kinase 1 (JAK1)/singal transducer and activator of transcription 3 (STAT3) signaling pathways. Additionally, 6c owned a high safety profile with the LD50 value of over 1000 mg·kg-1 in mice. These results identified palmatine derivatives as a novel class of anti-fibrogenic agents, and provided powerful information for further structure optimization.


Assuntos
Alcaloides de Berberina/química , Alcaloides de Berberina/farmacologia , Colágeno Tipo I/antagonistas & inibidores , Janus Quinase 1/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Relação Dose-Resposta a Droga , Expressão Gênica , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Humanos , Modelos Biológicos , Estrutura Molecular , Relação Estrutura-Atividade
15.
Nat Commun ; 11(1): 240, 2020 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-31932588

RESUMO

Farnesoid X receptor (FXR) is a promising target for nonalcoholic steatohepatitis (NASH) and fibrosis. Although various FXR agonists have shown anti-fibrotic effects in diverse preclinical animal models, the response rate and efficacies in clinical trials were not optimum. Here we report that prophylactic but not therapeutic administration of obeticholic acid (OCA) prevents hepatic stellate cell (HSC) activation and fibrogenesis. Activated HSCs show limited response to OCA and other FXR agonists due to enhanced FXR SUMOylation. SUMOylation inhibitors rescue FXR signaling and thereby increasing the efficacy of OCA against HSC activation and fibrosis. FXR upregulates Perilipin-1, a direct target gene of FXR, to stabilize lipid droplets and thereby prevent HSC activation. Therapeutic coadministration of OCA and SUMOylation inhibitors drastically impedes liver fibrosis induced by CCl4, bile duct ligation, and more importantly NASH. In conclusion, we propose a promising therapeutic approach by combining SUMOylation inhibitors and FXR agonists for liver fibrosis.


Assuntos
Cirrose Hepática/tratamento farmacológico , Receptores Citoplasmáticos e Nucleares/agonistas , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/antagonistas & inibidores , Sumoilação , Animais , Células Cultivadas , Ácido Quenodesoxicólico/administração & dosagem , Ácido Quenodesoxicólico/análogos & derivados , Ácido Quenodesoxicólico/farmacologia , Modelos Animais de Doenças , Quimioterapia Combinada , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/patologia , Humanos , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/patologia , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/patologia , Perilipina-1/genética , Perilipina-1/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ativação Transcricional/efeitos dos fármacos , Resultado do Tratamento
16.
Chem Biol Interact ; 317: 108945, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31935363

RESUMO

Liver fibrosis is a common pathological consequence of liver injury, which increases liver failure-related morbidity and mortality. Hence, anti-fibrotic treatment is urgently needed. Oxidative stress plays a pivotal role in the progression of liver fibrosis. Thus, targeting ROS may be an effective strategy for liver fibrosis treatment. In this study, we investigated four benzoquinones derivatives, including 5-isopropyl-2-methyl-1,4-benzoquinone (TQ), 2-tert-butyl-1,4-benzoquinone (tBu-Q) 2,5-dimethyl-p-benzoquinone (Dime-Q) and p-benzoquinone (Ph-Q), as well as the evaluation of their antioxidant activity and anti-fibrotic effects on activated hepatic stellate cells and TAA-induced mice. Electrochemical analysis showed that all compounds possessed antioxidant property. The result was first confirmed by in vitro experiments, which revealed potential anti-fibrotic activity of all four compounds at the cellular level. Benzoquinone derivatives act as ROS-scavenging molecules, which modulated the TLR4-CD14 signaling pathway to inhibit the expression of procaspase-1 and IL-1ß in cells, induced apoptosis via a mitochondrial pathway by upregulating the ratio of Bax/Bcl-2 and by activating caspase-3, as well as inhibited the expression of the anti-apoptotic proteins FLIP and XIAP in activated LX-2 cells. In addition, a TAA (Thioacetamide)-induced mouse model was used to further validate the results. Treatment with benzoquinone derivatives significantly decreased the levels of liver injury markers and lipid peroxidation caused by excessive ROS, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), and malondialdehyde (MDA). Moreover, treatment with benzoquinone derivatives significantly inhibited extracellular matrix (ECM) deposition and downregulated the mRNA and protein expression of liver fibrosis markers, such as collagen I, alpha-smooth muscle actin (α-SMA), and TIMP-1. In summary, these results indicate that benzoquinone derivatives may act as potential therapeutic drugs against liver fibrosis.


Assuntos
Antioxidantes/farmacologia , Benzoquinonas/farmacologia , Células Estreladas do Fígado/efeitos dos fármacos , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/tratamento farmacológico , Tioacetamida/toxicidade , Actinas/genética , Actinas/metabolismo , Animais , Biomarcadores , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/metabolismo , Inflamação/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Estresse Oxidativo , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo
17.
Mol Med Rep ; 21(2): 667-674, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31974596

RESUMO

The aim of the present study was to investigate whether class C1 decoy oligodeoxynucleotides (ODNs) can inhibit the expression of pro­fibrotic genes associated with rat hepatic stellate cell (HSC) activation and hepatic fibrosis. Luciferase reporter assays were performed to test the promoter activities of transforming growth factor (TGF)­ß and its downstream target genes following transfection of decoy ODNs and plasmids into HSC­T6 cells, and western blot assays were performed to measure the protein expression of those genes following decoy ODN transfection. Class C1 decoy ODNs were confirmed to inhibit the promoter activity of TGF­ß and its downstream target genes, such as type 1 collagen (COLI)α1, tissue inhibitor of metalloproteinases (TIMP)1 and α­smooth muscle actin by Gaussia luciferase reporter assay, and to further downregulate the expression of TGF­ß, SMAD3, COLIα1 and TIMP1 by western blotting in activated HSC­T6 cells. In conclusion, class C1 decoy ODNs inhibited pro­fibrotic gene expression in rat HSCS by downregulating TGF­ß signaling.


Assuntos
Regulação da Expressão Gênica , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/genética , Oligodesoxirribonucleotídeos/uso terapêutico , Animais , Linhagem Celular , Colágeno Tipo I/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Oligodesoxirribonucleotídeos/farmacologia , Regiões Promotoras Genéticas/genética , Ratos , Proteína Smad3/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Fatores de Transcrição HES-1/genética , Fatores de Transcrição HES-1/metabolismo , Fator de Crescimento Transformador beta/metabolismo
18.
Gastroenterology ; 158(6): 1728-1744.e14, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31982409

RESUMO

BACKGROUND & AIMS: Development of liver fibrosis is associated with activation of quiescent hepatic stellate cells (HSCs) into collagen type I-producing myofibroblasts (activated HSCs). Cessation of liver injury often results in fibrosis resolution and inactivation of activated HSCs/myofibroblasts into a quiescent-like state (inactivated HSCs). We aimed to identify molecular features of phenotypes of HSCs from mice and humans. METHODS: We performed studies with LratCre, Ets1-floxed, Nf1-floxed, Pparγ-floxed, Gata6-floxed, Rag2-/-γc-/-, and C57/Bl6 (control) mice. Some mice were given carbon tetrachloride (CCl4) to induce liver fibrosis, with or without a peroxisome proliferator-activated receptor-γ (PPARγ) agonist. Livers from mice were analyzed by immunohistochemistry. Quiescent, activated, and inactivated HSCs were isolated from livers of Col1α1YFP mice and analyzed by chromatin immunoprecipitation and sequencing. Human HSCs were isolated from livers denied for transplantation. We compared changes in gene expression patterns and epigenetic modifications (histone H3 lysine 4 dimethylation and histone H3 lysine 27 acetylation) in primary mouse and human HSCs. Transcription factors were knocked down with small hairpin RNAs in mouse HSCs. RESULTS: Motif enrichment identified E26 transcription-specific transcription factors (ETS) 1, ETS2, GATA4, GATA6, interferon regulatory factor (IRF) 1, and IRF2 transcription factors as regulators of the mouse and human HSC lineage. Small hairpin RNA-knockdown of these transcription factors resulted in increased expression of genes that promote fibrogenesis and inflammation, and loss of HSC phenotype. Disruption of Gata6 or Ets1, or Nf1 or Pparγ (which are regulated by ETS1), increased the severity of CCl4-induced liver fibrosis in mice compared to control mice. Only mice with disruption of Gata6 or Pparγ had defects in fibrosis resolution after CCl4 administration was stopped, associated with persistent activation of HSCs. Administration of a PPARγ agonist accelerated regression of liver fibrosis after CCl4 administration in control mice but not in mice with disruption of Pparγ. CONCLUSIONS: Phenotypes of HSCs from humans and mice are regulated by transcription factors, including ETS1, ETS2, GATA4, GATA6, IRF1, and IRF2. Activated mouse and human HSCs can revert to a quiescent-like, inactivated phenotype. We found GATA6 and PPARγ to be required for inactivation of human HSCs and regression of liver fibrosis in mice.


Assuntos
Fator de Transcrição GATA6/metabolismo , Células Estreladas do Fígado/patologia , Cirrose Hepática Experimental/patologia , Proteína Proto-Oncogênica c-ets-1/metabolismo , Animais , Tetracloreto de Carbono/toxicidade , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Fator de Transcrição GATA6/genética , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células Estreladas do Fígado/efeitos dos fármacos , Humanos , Cirrose Hepática Experimental/induzido quimicamente , Camundongos , Camundongos Transgênicos , Miofibroblastos/patologia , PPAR gama/agonistas , PPAR gama/genética , Cultura Primária de Células , Proteína Proto-Oncogênica c-ets-1/genética
19.
Am J Physiol Gastrointest Liver Physiol ; 318(3): G504-G517, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31928221

RESUMO

Activation of hepatic stellate cells (HSCs), characterized by development of a robust actin cytoskeleton and expression of abundant extracellular matrix (ECM) proteins, such as type 1 collagen (COL.1), is a central cellular and molecular event in liver fibrosis. It has been demonstrated that HSCs express both myocardin and myocardin-related transcription factor-A (MRTF-A). However, the biological effects of myocardin and MRTF-A on HSC activation and liver fibrosis, as well as the molecular mechanism under the process, remain unclear. Here, we report that myocardin and MRTF-A's expression and nuclear accumulation are prominently increased during the HSC activation process, accompanied by robust activation of actin cytoskeleton dynamics. Targeting myocardin and MRTF-A binding and function with a novel small molecule, CCG-203971, led to dose-dependent inhibition of HSC actin cytoskeleton dynamics and abrogated multiple functional features of HSC activation (i.e., HSC contraction, migration and proliferation) and decreased COL.1 expression in vitro and liver fibrosis in vivo. Mechanistically, blocking the myocardin and MRTF-A nuclear translocation pathway with CCG-203971 directly inhibited myocardin/MRTF-A-mediated serum response factor (SRF), and Smad2/3 activation in the COL.1α2 promoter and indirectly abrogated actin cytoskeleton-dependent regulation of Smad2/3 and Erk1/2 phosphorylation and their nuclear accumulation. Finally, there was no effect of CCG-203971 on markers of inflammation, suggesting a direct effect of the compound on HSCs and liver fibrosis. These data reveal that myocardin and MRTF-A are two important cotranscriptional factors in HSCs and represent entirely novel therapeutic pathways that might be targeted to treat liver fibrosis.NEW & NOTEWORTHY Myocardin and myocardin-related transcription factor-A (MRTF-A) are upregulated in activated hepatic stellate cells (HSCs) in vitro and in vivo, closely associated with robustly increased actin cytoskeleton remodeling. Targeting myocardin and MRTF-A by CCG-203971 leads to actin cytoskeleton-dependent inhibition of HSC activation, reduced cell contractility, impeded cell migration and proliferation, and decreased COL.1 expression in vitro and in vivo. Dual expression of myocardin and MRTF-A in HSCs may represent novel therapeutic targets in liver fibrosis.


Assuntos
Citoesqueleto de Actina/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Células Estreladas do Fígado/metabolismo , Cirrose Hepática Experimental/metabolismo , Fígado/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/patologia , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/patologia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/patologia , Fígado/efeitos dos fármacos , Fígado/patologia , Cirrose Hepática Experimental/genética , Cirrose Hepática Experimental/patologia , Cirrose Hepática Experimental/prevenção & controle , Masculino , Camundongos Endogâmicos BALB C , Ácidos Nipecóticos/farmacologia , Proteínas Nucleares/genética , Fosforilação , Ratos Sprague-Dawley , Transdução de Sinais , Proteínas Smad Reguladas por Receptor/metabolismo , Fatores de Tempo , Transativadores/genética , Fatores de Transcrição/genética , Regulação para Cima
20.
J Biochem Mol Toxicol ; 34(1): e22413, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31714634

RESUMO

Hepatic diseases leading to fibrosis affect millions of individuals worldwide and are a major public health challenge. Although, there have been many advances in understanding hepatic fibrogenesis, an effective therapy remains elusive. Studies focus primarily on activation of the hepatic stellate cells (HSCs), the principal fibrogenic cells in the liver; however, fewer numbers of studies have examined molecular mechanisms that deactivate HSC, controlling the profibrogenic phenotype. In the present study, we evaluated cellular and molecular actions of the chemical triclosan (TCS) in reverting activated HSCs to a quiesced phenotype. We demonstrated that the inhibition of the enzyme fatty acid synthase by TCS in activated HSCs promotes survival of the cells and triggers cellular and molecular changes that promote cellular phenotypic reversion, offering potentially new therapeutic directions.


Assuntos
Inibidores da Síntese de Ácidos Graxos/farmacologia , Células Estreladas do Fígado/efeitos dos fármacos , Triclosan/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ácido Graxo Sintases/antagonistas & inibidores , Células Estreladas do Fígado/citologia , Humanos
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