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1.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(9): 812-816, 2019 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-31750823

RESUMO

Objective To investigate the effect of aldosterone (ALD) on the migration of rat hepatic stellate cells (HSC-T6) and its mechanism. Methods HSC-T6 cells were cultured and divided into control group (treated with medium only), ALD group (only 1 nmol/L ALD, 24 hours), spironolactone pre-treated group (a specific inhibitor of ALD receptor 10 nmol/L spironolactone at 1 hours before ALD treatment), Y27632 pre-treated group (a RhoA kinase inhibitor 10 nmol/L Y27632 at 1 hours before ALD treatment). A TranswellTM chamber system was used to observe the change of migration in the different groups. Changes in actin cytoskeletal organization were visualized by fluorescence staining using rhadamin-labeled phalloidin and fluorescence images were recorded using confocal microscopy. The levels of phosphorylated myosinlight chain (p-MLC) and phosphorylated moesin (p-moesin) in the RhoA/ROCK signaling pathway were evaluated by Western blotting in HSC-T6 cells. Results ALD treatment of HSC-T6 resulted in the enhancement of migration, but the effect of ALD-induced migration could be inhibited by spironolactone and Y27632. Stimulation of HSC-T6 with ALD induced a rapid morphological change conconmitant with a robust reorganization of actin cytoskeleton, while the morphological change was suppressed by spironolactone and Y27632. The effect of aldosterone on the activation of HSC migration was mediated by p-MLC and p-moesin protein expressions through the RhoA/ROCK signaling pathway. Spironolactone and Y27632 had the ability to block aldosterone-induced protein expressions in HSC-T6 cells. Conclusion ALD can induce the migration of activated HSC-T6 cells through the activation of the RhoA/ROCK signaling pathway.


Assuntos
Aldosterona/farmacologia , Movimento Celular , Células Estreladas do Fígado/efeitos dos fármacos , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Amidas , Animais , Células Cultivadas , Células Estreladas do Fígado/citologia , Piridinas , Ratos , Espironolactona
2.
Zhonghua Gan Zang Bing Za Zhi ; 27(8): 621-627, 2019 Aug 20.
Artigo em Chinês | MEDLINE | ID: mdl-31594080

RESUMO

Objective: To determine whether the anti-hepatic fibrosis effect of Fuzheng-Huayu formula is related to suppress autophagy in mice. Methods: C57 mice were randomly divided into normal group (N group) and model group. The model group was induced by intraperitoneal injection of carbon tetrachloride to induce liver fibrosis in mice, and the normal group was injected with equal volume of olive oil. After 1 week, the model group was randomly divided into model (M) group, rapamycin (Rapa) group, rapamycin plus chloroquine (Rapa+CQ) group, rapamycin plus salvianolic acid B (Rapa+Sal B) group, rapamycin plus Fuzheng -Huayu formula (Rapa+FZ) group. Each drug group was administered corresponding drugs by gavage on a daily basis, and N group and M group were given the equal amount of drinking water by gavage. After 5 weeks, the mice were sacrificed, and HE and Sirius red staining were used to observe the inflammation and collagen deposition on liver tissue in each group. The hydroxyproline content was determined by alkaline hydrolysis method. Western blotting was used to detect changes in the expression of autophagy in liver tissue and microtubule-associated protein 1 light chain 3II/I (LC3II/I), p62, α-smooth muscle actin (ɑ-SMA) and type I collagen expression. Immunofluorescence staining was used to observe the immunofluorescence localization of ɑ-SMA and LC3B in liver tissues of each group. ). A t-test was used to compare the two independent samples. LSD or Dunnett's T3 test were used to compare the mean of multiple samples. Results: There was no significant difference in N and M groups in terms of body weight. The body weight of the mice in each drug group decreased significantly (F = 14.041, P < 0.001). The liver/spleen /body weight ratios of each drug group and M group were significantly higher than the N group (F = 26.992, 6.589, P < 0.001). The expression of p62 protein in the liver tissue of mice in each drug group was lower than M group, and the difference between Rapa group and Rapa+Sal B group (F = 3.085, P = 0.039, 0.003) was statistically significant, while that of Rapa + Sal B group was lower. Compared with group M, the expression of LC3B II in Rapa group was significantly higher (F = 7.514, P = 0.01). Immunofluorescence staining showed that LC3B and α-SMA CO-stained cells were absent in the liver of mice in N group, and co-stained cells were found in the liver of mice in M group. The co-stained cells in the liver of mice in each drug group were significantly higher than M group, and the co-stained cells in Rapa+FZ group were fewer. Compared with the N group, the collagen deposition of M group and each drug group was significantly increased; the collagen deposition of each drug group was lower than that of the M group. There was no statistically significant difference between each drug group. Compared with N group (77.75 + 48.79), hydroxyproline in liver tissue of mice in M group was significantly increased (293.48 + 84.43) (F = 3.015, P = 0.005), and the content of hydroxyproline in liver tissue of mice in each drug group was lower than M group, but the difference was not statistically significant (F = 0.750, P = 0.573). Compared with the N group, the expressions of α-SMA and type I collagen in the M group were significantly increased (F = 27.718, 18.893, P < 0.01). The expression of α-SMA in Rapa group and Rapa+Sal B group was similar to M group, while Rapa + CQ group and Rapa + FZ group were significantly lower than Rapa group and M group (P < 0.01). The expression of type I collagen in Rapa + CQ group was significantly higher than Rapa group (P = 0.017), while the expression of type I collagen in Rapa + FZ group was significantly lower than M group (P = 0.013). Conclusion: Autophagy of hepatic stellate cells was observed in carbon tetrachloride-induced liver fibrosis model. Rapamycin can promote autophagy in hepatocytes and hepatic stellate cells. Fuzheng-Huayu formula and Salvianolic Acid B might antagonize the effect of rapamycin on autophagy.


Assuntos
Autofagia , Medicamentos de Ervas Chinesas/farmacologia , Células Estreladas do Fígado/citologia , Cirrose Hepática/tratamento farmacológico , Animais , Benzofuranos , Tetracloreto de Carbono , Cloroquina , Células Estreladas do Fígado/efeitos dos fármacos , Fígado , Cirrose Hepática/induzido quimicamente , Camundongos , Camundongos Endogâmicos C57BL , Distribuição Aleatória , Sirolimo
3.
J Agric Food Chem ; 67(36): 10245-10255, 2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31389238

RESUMO

Ginseng has been widely used as a functional food in the world because of its well-defined health benefits. Previous studies have confirmed that AD-1, a new ginsenoside derived from ginseng, can ameliorate thioacetamide-induced liver injury and fibrosis in mice. Simultaneously, amino acid supplementation is getting more attention as an important adjuvant therapy in the improvement of hepatopathy. The aim of this study was to conjugate AD-1 with several selected amino acids and investigate the cytotoxicity of the obtained conjugates in activated t-HSC/Cl-6 cells and normal human liver cells (LO2). Structure-activity relationships of conjugates and underlying mechanisms of the effect are also explored. The results indicated that conjugate 7c remarkably inhibited cell proliferation in activated t-HSC/Cl-6 cells (IC50 = 3.8 ± 0.4 µM) and appeared to be nontoxic to LO2. Besides, conjugate 7c had a relatively good plasma stability. Further study demonstrated that inducing S-phase arrest and activation of mitochondrial-mediated apoptosis were included in the mechanisms underlying the efficiency of conjugate 7c. These findings provided further insight into designing functional foods (ginsenoside and amino acid) for the application in prevention or improvement of liver fibrosis.


Assuntos
Aminoácidos/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ginsenosídeos/farmacologia , Células Estreladas do Fígado/citologia , Aminoácidos/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ginsenosídeos/química , Células Estreladas do Fígado/efeitos dos fármacos , Humanos , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos
4.
Cell Physiol Biochem ; 53(2): 301-322, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31343125

RESUMO

BACKGROUND/AIMS: Propolis is one of the most promising natural products, exhibiting not only therapeutic but also prophylactic actions. Propolis has several biological and pharmacological properties, including hepatoprotective activities. The present study aimed to investigate the underlying molecular mechanisms of propolis against CCl4-mediated liver fibrosis. METHODS: Three groups of male BALB/c mice (n=15/ group) were used: group 1 comprised control mice; groups 2 and 3 were injected with CCl4 for the induction of liver fibrosis. Group 3 was then orally supplemented with propolis (100 mg/kg body weight) for four weeks. Different techniques were used to monitor the antifibrotic effects of propolis, including histopathological investigations using H&E, Masson's trichrome and Sirius red staining; Western blotting; flow cytometry; and ELISA. RESULTS: We found that the induction of liver fibrosis by CCl4 was associated with a significant increase in hepatic collagen and α-smooth muscle actin (α-SMA) expression. Moreover, CCl4-treated mice also exhibited histopathological alterations in the liver architecture. Additionally, the liver of CCl4-treated mice exhibited a marked increase in proinflammatory signals, such as increased expression of HSP70 and increased levels of proinflammatory cytokines and ROS. Mechanistically, the liver of CCl4-treated mice exhibited a significant increase in the phosphorylation of AKT and mTOR; upregulation of the expression of BAX and cytochrome C; downregulation of the expression of Bcl2; a significant elevation in the levels of TGF-ß followed by increased phosphorylation of SMAD2; and a marked increase in the expression of P53 and iNOS. Interestingly, oral supplementation of CCl4-treated mice with propolis significantly abolished hepatic collagen deposition, abrogated inflammatory signals and oxidative stress, restored CCl4-mediated alterations in the signaling cascades, and hence repaired the hepatic architecture nearly to the normal architecture observed in the control mice. CONCLUSION: Our findings revealed the therapeutic potential and the underlying mechanisms of propolis against liver fibrosis.


Assuntos
Apoptose/efeitos dos fármacos , Cirrose Hepática Experimental/patologia , Própole/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Tetracloreto de Carbono/toxicidade , Citocinas/metabolismo , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico Sintase Tipo II/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Smad2/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/metabolismo
6.
Analyst ; 144(14): 4233-4240, 2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31210202

RESUMO

Hepatic stellate cells (HSCs), a major component of the tumor microenvironment in liver cancer, play important roles in cancer progression as well as drug resistance. Here, we presented a microchannel plate-based co-culture model that integrated Hepa1-6 tumor spheroids with JS-1 stellate cells in three-dimensional (3D) concave microwells to mimic the in vivo tumor microenvironment by recapitulating epithelial-mesenchymal transition and chemoresistance. The expression of epithelial-mesenchymal transition (EMT)-related markers and factors was analyzed using immunofluorescent staining and the changes in viability following exposure to different concentrations of paclitaxel were measured. Cell spheroids formed 3D tumor spheroids within 3 days. Culture conditions were optimized for Hepa1-6 cells and JS-1 cells, and their appropriate interactions were confirmed by reciprocal activation. JS-1 under co-culture showed a change in cellular morphology and an increased expression of α-SMA. The expression of EMT-related markers, such as vimentin and TGF-ß1, was higher in the co-cultured Hepa1-6 spheroids compared to that in mono-cultured spheroids. Following paclitaxel exposure, JS-1 cells showed significant changes in survival under both mono- and co-culture conditions, while Hepa1-6 presented negligible changes. The proposed microfluidic platform makes it possible to observe the positioned three-dimensional cell spheroids, which would be extensively used not only for well-organized spheroid creation, but also for better quantitative and qualitative understanding of the cell-cell interaction effect.


Assuntos
Técnicas de Cocultura/métodos , Células Estreladas do Fígado/metabolismo , Dispositivos Lab-On-A-Chip , Esferoides Celulares/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Técnicas de Cocultura/instrumentação , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Transição Epitelial-Mesenquimal/fisiologia , Células Estreladas do Fígado/efeitos dos fármacos , Neoplasias Hepáticas/metabolismo , Camundongos , Microfluídica/instrumentação , Microfluídica/métodos , Paclitaxel/farmacologia , Esferoides Celulares/efeitos dos fármacos , Microambiente Tumoral/fisiologia
7.
BMC Complement Altern Med ; 19(1): 138, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221141

RESUMO

BACKGROUND: Oxymatrine (OM), a quinolizidine alkaloid extracted from a herb Sophorae Flavescentis Radix, has been used to treat liver fibrotic diseases. However, the mechanism of its anti-fibrosis effects is still unclear. TGF-ß/Smad signaling and miR-195 have been proved to paly an important role in hepatic stellate cells (HSCs) activation and liver fibrosis. In this study, we investigated whether OM could inhibit HSCs activation through TGF-ß1/miR-195/Smads signaling or not. METHODS: First, the effects of OM on HSC-T6 in different concentrations and time points were tested by MTT assay. We choose three appropriate concentrations of OM as treatment concentrations in following experiment. By Quantitative Real-time PCR and Western Blot, then we investigated the effect of OM on miR-195, Smad7 and α-SMA's expressions to prove the correlation between OM and the TGF-ß1/miR-195/Smads signaling. Last, miR-195 mimic and INF-γ were used to investigate the relation between miR-195 and OM in HSC activation. RESULTS: Our results showed that the proliferation of HSC was significantly inhibited when OM concentration was higher than 200 µg/mL after 24 h, 100 µg/mL after 48 h and 10 µg/mL after 72 h. The IC50 of OM after 24, 48 and 72 h were 539, 454, 387 µg/mL respectively. OM could down-regulate miR-195 and α-SMA (P < 0.01), while up-regulate Smad7 (P < 0.05). In HSC-T6 cells transfected with miR-195 mimic and pretreated with OM, miR-195 and α-SMA were up-regulated (P < 0.05), and Smad7 was down-regulated (P < 0.05) . CONCLUSIONS: Given these results, OM could inhibit TGF-ß1 induced activation of HSC-T6 proliferation in a dose-dependent and time-dependent manner to some extent. We proved that OM inhibited HSC activation through down-regulating the expression of miR-195 and up-regulating Smad7.


Assuntos
Alcaloides/farmacologia , Células Estreladas do Fígado/efeitos dos fármacos , MicroRNAs/metabolismo , Extratos Vegetais/farmacologia , Quinolizinas/farmacologia , Proteína Smad7/metabolismo , Sophora/química , Fator de Crescimento Transformador beta1/metabolismo , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Humanos , Interferon gama/genética , Interferon gama/metabolismo , MicroRNAs/genética , Ratos , Transdução de Sinais/efeitos dos fármacos , Proteína Smad7/genética , Fator de Crescimento Transformador beta1/genética
8.
Gastroenterology ; 157(3): 793-806.e14, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31170413

RESUMO

BACKGROUND & AIMS: The role of aryl hydrocarbon receptor (AhR) in liver fibrosis is controversial because loss and gain of AhR activity both lead to liver fibrosis. The goal of this study was to investigate how the expression of AhR by different liver cell types, hepatic stellate cells (HSCs) in particular, affects liver fibrosis in mice. METHODS: We studied the effects of AhR on primary mouse and human HSCs, measuring their activation and stimulation of fibrogenesis using RNA-sequencing analysis. C57BL/6J mice were given the AhR agonists 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE); were given carbon tetrachloride (CCl4); or underwent bile duct ligation. We also performed studies in mice with disruption of Ahr specifically in HSCs, hepatocytes, or Kupffer cells. Liver tissues were collected from mice and analyzed by histology, immunohistochemistry, and immunoblotting. RESULTS: AhR was expressed at high levels in quiescent HSCs, but the expression decreased with HSC activation. Activation of HSCs from AhR-knockout mice was accelerated compared with HSCs from wild-type mice. In contrast, TCDD or ITE inhibited spontaneous and transforming growth factor ß-induced activation of HSCs. Mice with disruption of Ahr in HSCs, but not hepatocytes or Kupffer cells, developed more severe fibrosis after administration of CCl4 or bile duct ligation. C57BL/6J mice given ITE did not develop CCl4-induced liver fibrosis, whereas mice without HSC AhR given ITE did develop CCl4-induced liver fibrosis. In studies of mouse and human HSCs, we found that AhR prevents transforming growth factor ß-induced fibrogenesis by disrupting the interaction of Smad3 with ß-catenin, which prevents the expression of genes that mediate fibrogenesis. CONCLUSIONS: In studies of human and mouse HSCs, we found that AhR prevents HSC activation and expression of genes required for liver fibrogenesis. Development of nontoxic AhR agonists or strategies to activate AhR signaling in HSCs might be developed to prevent or treat liver fibrosis.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Senescência Celular , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Células Estreladas do Fígado/metabolismo , Cirrose Hepática Experimental/prevenção & controle , Fígado/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/agonistas , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proliferação de Células , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Regulação da Expressão Gênica , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/patologia , Indóis/farmacologia , Fígado/efeitos dos fármacos , Fígado/patologia , Cirrose Hepática Experimental/genética , Cirrose Hepática Experimental/metabolismo , Cirrose Hepática Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Receptores de Hidrocarboneto Arílico/agonistas , Receptores de Hidrocarboneto Arílico/deficiência , Receptores de Hidrocarboneto Arílico/genética , Transdução de Sinais , Proteína Smad3/metabolismo , Tiazóis/farmacologia , beta Catenina/metabolismo
9.
Fitoterapia ; 136: 104171, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31085309

RESUMO

Seven undescribed C21 steroids, namely cynanchin A-G, together with thirteen known analogues, were isolated from the roots of cynanchum otophyllum. Their structures were elucidated by 1D, 2D NMR and MS spectra, as well as chemical methods. Meanwhile, all of isolates were tested for their anti-hepatic fibrosis activity. Among them, compounds 4-6, 10-12 and 14-17 showed moderate or significant inhibitory effects for the proliferation of hepatic stellate cells (HSCs) induced by transforming growth factor-ß1 (TGF-ß1) in vitro.


Assuntos
Cynanchum/química , Células Estreladas do Fígado/efeitos dos fármacos , Raízes de Plantas/química , Esteroides/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , China , Humanos , Estrutura Molecular , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Esteroides/isolamento & purificação , Fator de Crescimento Transformador beta1
10.
Int J Mol Sci ; 20(10)2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31137784

RESUMO

BACKGROUND AND AIMS: Liver fibrosis is the excessive accumulation of extracellular matrix proteins, including collagen, which occurs in most types of chronic liver diseases. Advanced liver fibrosis results in cirrhosis, liver failure, and portal hypertension. Activated hepatic perivascular stellate cells, portal fibroblasts, and myofibroblasts of bone marrow origin have been identified as major collagen-producing cells in the injured liver. These cells are activated by fibrogenic cytokines, such as TGF-ß1. The inhibition of TGF-ß1 function or synthesis is a major target for the development of antifibrotic therapies. Our previous study showed that the water and ethanol extracts of Graptopetalum paraguayense (GP), a Chinese herbal medicine, can prevent dimethylnitrosamine (DMN)-induced hepatic inflammation and fibrosis in rats. METHODS: We used rat hepatic stellate HSC-T6 cells and a diethylnitrosamine (DEN)-induced rat liver injury model to test the potential mechanism of GP extracts and its fraction, HH-F3. RESULTS: We demonstrated that GP extracts and HH-F3 downregulated the expression levels of extracellular matrix (ECM) proteins and inhibited the proliferation and migration via suppression of the TGF-ß1 pathway in rat hepatic stellate HSC-T6 cells. Moreover, the HH-F3 fraction decreased hepatic collagen content and reduced plasma AST, ALT, and γ-GT activities in a DEN-induced rat liver injury model, suggesting that GP/HH-F3 has hepatoprotective effects against DEN-induced liver fibrosis. CONCLUSION: These findings indicate that GP/HH-F3 may be a potential therapeutic agent for the treatment of liver fibrosis. The inhibition of TGF-ß-mediated fibrogenesis may be a central mechanism by which GP/HH-F3 protects the liver from injury.


Assuntos
Crassulaceae/química , Cirrose Hepática/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Fator de Crescimento Transformador beta/metabolismo , Animais , Linhagem Celular , Colágeno/genética , Colágeno/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Humanos , Cirrose Hepática/metabolismo , Extratos Vegetais/farmacologia , Ratos , Ratos Wistar , Transdução de Sinais , Fator de Crescimento Transformador beta/genética
11.
Gastroenterology ; 157(3): 777-792.e14, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31078624

RESUMO

BACKGROUND & AIMS: We studied the role of interleukin 11 (IL11) signaling in the pathogenesis of nonalcoholic steatohepatitis (NASH) using hepatic stellate cells (HSCs), hepatocytes, and mouse models of NASH. METHODS: We stimulated mouse and human fibroblasts, HSCs, or hepatocytes with IL11 and other cytokines and analyzed them by imaging, immunoblot, and functional assays and enzyme-linked immunosorbent assays. Mice were given injections of IL11. Mice with disruption of the interleukin 11 receptor subunit alpha1 gene (Il11ra1-/-) mice and Il11ra1+/+ mice were fed a high-fat methionine- and choline-deficient diet (HFMCD) or a Western diet with liquid fructose (WDF) to induce steatohepatitis; control mice were fed normal chow. db/db mice were fed with methionine- and choline-deficient diet for 12 weeks and C57BL/6 NTac were fed with HFMCD for 10 weeks or WDF for 16 weeks. Some mice were given intraperitoneal injections of anti-IL11 (X203), anti-IL11RA (X209), or a control antibody at different timepoints on the diets. Livers and blood were collected; blood samples were analyzed by biochemistry and liver tissues were analyzed by histology, RNA sequencing, immunoblots, immunohistochemistry, hydroxyproline, and mass cytometry time of flight assays. RESULTS: HSCs incubated with cytokines produced IL11, resulting in activation (phosphorylation) of ERK and expression of markers of fibrosis. Livers of mice given injections of IL11 became damaged, with increased markers of fibrosis, hepatocyte cell death and inflammation. Following the HFMCD or WDF, livers from Il11ra1-/- mice had reduced steatosis, fibrosis, expression of markers of inflammation and steatohepatitis, compared to and Il11ra1+/+ mice on the same diets. Depending on the time of administration of anti-IL11 or anti-IL11RA antibodies to wild-type mice on the HFMCD or WDF, or to db/db mice on the methionine and choline-deficient diet, the antibodies prevented, stopped, or reversed development of fibrosis and steatosis. Blood samples from Il11ra1+/+ mice fed the WDF and given injections of anti-IL11 or anti-IL11RA, as well as from Il11ra1-/- mice fed WDF, had lower serum levels of lipids and glucose than mice not injected with antibody or with disruption of Il11ra1. CONCLUSIONS: Neutralizing antibodies that block IL11 signaling reduce fibrosis, steatosis, hepatocyte death, inflammation and hyperglycemia in mice with diet-induced steatohepatitis. These antibodies also improve the cardiometabolic profile of mice and might be developed for the treatment of NASH.


Assuntos
Anticorpos Neutralizantes/farmacologia , Hepatite/prevenção & controle , Subunidade alfa de Receptor de Interleucina-11/metabolismo , Interleucina-11/antagonistas & inibidores , Cirrose Hepática Experimental/prevenção & controle , Fígado/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Animais , Morte Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Hepatite/genética , Hepatite/metabolismo , Hepatite/patologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-11/metabolismo , Subunidade alfa de Receptor de Interleucina-11/deficiência , Subunidade alfa de Receptor de Interleucina-11/genética , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática Experimental/genética , Cirrose Hepática Experimental/metabolismo , Cirrose Hepática Experimental/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Transdução de Sinais/efeitos dos fármacos , Células THP-1
12.
Int J Nanomedicine ; 14: 2927-2944, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31118614

RESUMO

Background: Liver fibrosis is a chronic liver disease associated with an excessive accumulation of extracellualr matrix (ECM) proteins which ultimately lead to cirrohosis and hepatocellular carcinoma. Purpose: Liver fibrosis therapies that use combination approaches with the ability to affect multiple disease pathways have proven higher efficacies. This study aimed at optimizing and characterizing the co-encapsulation of pirfenidone (PF) and AMD3100 (AMD) into CXCR4-targeted combination liposomes (CTC liposome) for CXCR4 targeting, and the inhibition of major molecular culprits ie α-SMA, CXCR4, TGFß, and P-p38 involved in liver fibrosis in-vitro. Methods: The CTC liposomes were prepared using the thin-film hydration method. The concentration of encapsulated AMD and PF was measured by HPLC and UV spectrophotometry, respectively. Tramsmission electron microscopy (TEM) was used to determine the liposomal morphology. The CXCR4 targeting ability was determined by CXCR4 redistribution assay. Confocal microscopy and flowcytometry were used to determine the CXCR4 mediated cell uptake. The apoptosis inducing and protein downreguating ability of CTC liposomes were determined by apoptosis assay and western blot analysis, respectively. In-vivo biodistribution and Hoechst staining were used to confirm the feasibility of CTC liposome for the in-vivo applications and drug targeted accumulation, respectively. Results: The TEM studies revealed that CTC liposomes were spherical in shape. The cumulative release of AMD and PF from CTC liposome was 67% and 84%, respectively, at 48 h. Compared to the free drug counterparts, encapsulated drugs displayed higher cell viability. The CXCR4 redistribution assay confirmed the CXCR4 targeting and antagonistic ability of CTC liposomes. The CTC liposomes were internalized more effectively via caveolae-mediated endocytic pathways. CTC liposomes displayed aggressive apoptosis (87.3%) in TGFß-induced activated HSC-T6 cells suggesting a propensity to fibrosis regression. Also, CTC liposomes significantly reduced α-SMA (65%), CXCR4 (77%), TGFß (89%), and P-p38 (66%) expressions, better than free drugs. CTC@IR780 liposomes (CTC liposomes incorporating IR780 dye) were more accumulated in fibrotic livers compared to free IR780, as judged by in-vivo imaging, biodistribution analysis, and Hoechst staining. These findings suggest that this simple and stable CTC liposomal system holds a great promise for the treatment and prevention of liver fibrosis.


Assuntos
Sistemas de Liberação de Medicamentos , Células Estreladas do Fígado/patologia , Compostos Heterocíclicos/administração & dosagem , Cirrose Hepática/tratamento farmacológico , Piridonas/administração & dosagem , Receptores CXCR4/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Compostos Heterocíclicos/farmacologia , Compostos Heterocíclicos/uso terapêutico , Humanos , Lipossomos , Camundongos , Piridonas/farmacologia , Piridonas/uso terapêutico , Ratos , Receptores CXCR4/antagonistas & inibidores , Transdução de Sinais , Distribuição Tecidual/efeitos dos fármacos , Fator de Crescimento Transformador beta , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
13.
Life Sci ; 225: 20-28, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30928408

RESUMO

AIMS: Increasing nicotinamide adenine dinucleotide (NAD+) by Nicotinamide riboside (NR) provides protective benefits in multiple disorders. However, the role of NR on liver fibrosis is unclear. We performed in vivo and in vitro experiments to test the hepatic protective effects of NR against liver fibrosis and the underlying mechanisms. MATERIALS AND METHODS: Mice were injected with CCl4 to establish liver fibrosis model. NR was given by gavage to explore the hepatic protection of NR. LX-2 cells were given a TGF-ß stimulation ±â€¯NR, the activation of LX-2 cells and the acetylation of Smads were analyzed. To further confirm the role of Sirt1 on the protective pathway of NR, we knockdown Sirt1 in LX-2 cells. KEY FINDINGS: We found NR could prevent liver fibrosis and reverse the existing liver fibrosis. NR inhibited the activation of LX-2 cells induced by TGF-ß, activated Sirt1 and deacetylated Smad2/3. Sirt1 knockdown diminished the inhibiting effect of NR on LX-2 cells activation, and increased expressions of acetylated Smads. In conclusion, NR could prevent liver fibrosis via suppressing activation of hepatic stellate cells (HSCs). This protective effect was mediated by regulating the acetylation of Smads signaling pathway. SIGNIFICANCE: NR protected mice against liver fibrosis induced by CCl4. NR suppressed activation of hepatic stellate cells induced by TGF-ß. NR protects liver fibrosis via increasing the activity of Sirt1 and decreasing the expression of P300, resulting in the deacetylation of Smads in stellate cells.


Assuntos
Tetracloreto de Carbono/toxicidade , Células Estreladas do Fígado/efeitos dos fármacos , Cirrose Hepática/prevenção & controle , Niacinamida/análogos & derivados , Substâncias Protetoras/farmacologia , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Acetilação , Animais , Proteína p300 Associada a E1A/metabolismo , Regulação da Expressão Gênica , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Niacinamida/farmacologia , Sirtuína 1/metabolismo , Fator de Crescimento Transformador beta/metabolismo
14.
Molecules ; 24(7)2019 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-30986937

RESUMO

Silybin has been proposed as a treatment for nonalcoholic steatohepatitis (NASH). In this study, we assessed the effect of Silybin in a well-established in vitro coculture model of early-stage NASH. LX2 and Huh7 cells were exposed to free fatty acid (FFA) and Silybin as mono- or coculture (SCC). Cell viability, LX2 activation, collagen deposition, metalloproteinase 2 and 9 (MMP2-9) activity, and ROS generation were determined at 24, 96, and 144 h. Exposure to FFA induced the activation of LX2 as shown by the increase in cell viability and upregulation of collagen biosynthesis. Interestingly, while cotreatment with Silybin did not affect collagen production in LX2, a significant reduction was observed in SCC. MMP2-9 activity was reduced in FFA-treated Huh7 and SCC and cotreatment with Silybin induced a dose-dependent increase, while no effect was observed in LX2. Silybin also showed antioxidant properties by reducing the FFA-induced production of ROS in all the cell systems. Based on these data, Silybin exerts its beneficial effects by reducing LX2 proliferation and ROS generation. Moreover, MMP2-9 modulation in hepatocytes represents the driving mechanism for the net reduction of collagen in this NASH in vitro model, highlighting the importance of hepatic cells interplay in NASH development and resolution.


Assuntos
Colágeno/metabolismo , Fígado/metabolismo , Silibina/farmacologia , Linhagem Celular , Sobrevivência Celular , Técnicas de Cocultura , Ácidos Graxos não Esterificados , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fígado/efeitos dos fármacos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Espécies Reativas de Oxigênio/metabolismo
15.
BMC Cancer ; 19(1): 224, 2019 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-30866863

RESUMO

BACKGROUND: Our previous works have demonstrated that 8-bromo-7-methoxychrysin suppressed stemness of human hepatocellular carcinoma (HCC) cell line SMMC-7721 induced by condition medium from hepatic stellate cell line LX-2 that was activated by liver cancer stem-like cells (LCSCs). However, whether and whereby BrMC inhibits the stemness induced by co-culture of LCSCs and LX-2 cells remains to be investigated. METHODS: The second-generation spheres by sphere culture were identified and used as SMMC-7721-and MHCC97H-derived LCSLCs. SMMC-7721-and MHCC97-derived LCSCs/LX-2 cells transwell co-culture system was treated with BrMC and its lead compound chrysin. The concentrations of IL-6, IL-8, HGF and PDGF in condition medium from co-culture were measured by enzyme-linked immunosorbent assay (ELISA). The stemness of SMMC-7721 cells was evaluated by sphere formation assay and western blot analysis for expression levels of cancer stem cell markers (CD133 and CD44).The expression levels of cancer-associated fibroblast markers (FAP-α and α-SMA) were employed to evaluate pathologic activation of LX-2 cells. Addition of IL-6 and/or HGF or deletion of IL-6 and/or HGF was conducted to investigate the mechanisms for BrMC and chrysin treatment in SMMC-7721-derived LCSLCs co-cultured with LX-2cells. RESULTS: The co-culture of LCSLCs with LX-2 cells increased sphere formation capability as well as expression of CD133 and CD44 in SMMC-7721 cells, meanwhile, upregulated expression of FAP-α in LX-2 cells. ELISA indicated that the concentrations of IL-6 and HGF were significantly elevated in Co-CM than that of condition media from co-cultured SMMC-7721 cells/LX-2 cells. Treatment of BrMC and chrysin with co-cultures of SMMC-7721- and MHCC97H-derived LCSLCs and LX-2 cells effectively inhibited the above responses. Moreover, addition of IL-6 and/or HGF induced stemness of SMMC-7721 cells and activation of LX-2 cells, conversely, deletion of IL-6 and/or HGF suppressed those. Furthermore, the inhibitory effects of BrMC and chrysin on stemness of SMMC-7721 cells and activation of LX-2 cells were attenuated by addition of IL-6 or HGF, and enhanced by deletion of IL-6 or HGF. CONCLUSIONS: Our results suggest IL-6 and HGF may be the key communication molecules for the interaction between LCSLCs and HSCs, and BrMC and chrysin could block these effects and be the novel therapeutic candidates for HCC management.


Assuntos
Carcinoma Hepatocelular/metabolismo , Flavonoides/farmacologia , Células Estreladas do Fígado/metabolismo , Neoplasias Hepáticas/metabolismo , Células-Tronco Neoplásicas/metabolismo , Animais , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Feminino , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/patologia , Humanos , Interleucina-6/antagonistas & inibidores , Interleucina-6/metabolismo , Interleucina-8/antagonistas & inibidores , Interleucina-8/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
16.
World J Gastroenterol ; 25(9): 1067-1079, 2019 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-30862995

RESUMO

BACKGROUND: Studies show that the antifibrotic mechanism of taurine may involve its inhibition of the activation and proliferation of hepatic stellate cells (HSCs). Since the molecular mechanism of taurine-mediated antifibrotic activity has not been fully unveiled and is little studied, it is imperative to use "omics" methods to systematically investigate the molecular mechanism by which taurine inhibits liver fibrosis. AIM: To establish a network including transcriptomic and protein-protein interaction data to elucidate the molecular mechanism of taurine-induced HSC apoptosis. METHODS: We used microarrays, bioinformatics, protein-protein interaction (PPI) network, and sub-modules to investigate taurine-induced changes in gene expression in human HSCs (LX-2). Subsequently, all of the differentially expressed genes (DEGs) were subjected to gene ontology function and Kyoto encyclopedia of genes and genomes pathway enrichment analysis. Furthermore, the interactions of DEGs were explored in a human PPI network, and sub-modules of the DEGs interaction network were analyzed using Cytoscape software. RESULTS: A total of 635 DEGs were identified in taurine-treated HSCs when compared with the controls. Of these, 304 genes were statistically significantly up-regulated, and 331 down-regulated. Most of these DEGs were mainly located on the membrane and extracellular region, and are involved in the biological processes of signal transduction, cell proliferation, positive regulation of extracellular regulated protein kinases 1 (ERK1) and ERK2 cascade, extrinsic apoptotic signaling pathway and so on. Fifteen significantly enriched pathways with DEGs were identified, including mitogen-activated protein kinase (MAPK) signaling pathway, peroxisome proliferators-activated receptor signaling pathway, estrogen signaling pathway, Th1 and Th2 cell differentiation, cyclic adenosine monophosphate signaling pathway and so on. By integrating the transcriptomics and human PPI data, nine critical genes, including MMP2, MMP9, MMP21, TIMP3, KLF10, CX3CR1, TGFB1, VEGFB, and EGF, were identified in the PPI network analysis. CONCLUSION: Taurine promotes the apoptosis of HSCs via up-regulating TGFB1 and then activating the p38 MAPK-JNK-Caspase9/8/3 pathway. These findings enhance the understanding of the molecular mechanism of taurine-induced HSC apoptosis and provide references for liver disorder therapy.


Assuntos
Células Estreladas do Fígado/efeitos dos fármacos , Cirrose Hepática/tratamento farmacológico , Substâncias Protetoras/farmacologia , Mapas de Interação de Proteínas/efeitos dos fármacos , Taurina/farmacologia , Linhagem Celular , Perfilação da Expressão Gênica , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Cirrose Hepática/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Substâncias Protetoras/uso terapêutico , Taurina/uso terapêutico , Transcriptoma/efeitos dos fármacos
17.
Food Funct ; 10(4): 1974-1984, 2019 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-30889234

RESUMO

Hepatic stellate cells are liver-specific perivascular cells, identified as the major source of collagen in liver fibrosis, following their activation and conversion to myofibroblast-like cells. Lycopene is a carotenoid with biological activities and protective effects described in different pathologies, but little is known about its role in liver protection. We evaluated the influence of lycopene on the cell cycle and lipid metabolism and monitored the possible pathways involved in lycopene inhibition of stellate cell activation. Lycopene induced expression of the lipocyte phenotype, with an accumulation of fat droplets in cytoplasm, with high synthesis and turnover of phospholipids and triglycerides. Cell proliferation analysis showed that lycopene reduced the growth of GRX cells. Lycopene induced an arrest in the G0/G1 phase, followed by a decrease of cells in the G2/M phase, regardless of the concentration of lycopene used. Lycopene modulated relevant signaling pathways related to cholesterol metabolism, cellular proliferation, and lipid metabolism. Also, lycopene treatment increased the expression of RXR-α, RXR-ß, and PPARγ, important biomarkers of liver regeneration. These results show that lycopene was able to negatively modulate events related to the activation of hepatic stellate cells through mechanisms that involve changes in expression of cellular lipid metabolism factors, and suggest that this compound might provide a novel pharmacological approach for the prevention and treatment of fibrotic liver diseases.


Assuntos
Células Estreladas do Fígado/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Licopeno/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Humanos , Gotículas Lipídicas/metabolismo , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , Receptor X Retinoide alfa/genética , Receptor X Retinoide alfa/metabolismo , Receptor X Retinoide beta/genética , Receptor X Retinoide beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Triglicerídeos/metabolismo
18.
Cell Commun Signal ; 17(1): 11, 2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30744642

RESUMO

BACKGROUND: Contraction of hepatic stellate cells (HSCs) plays an important role in the pathogenesis of liver fibrosis by regulating sinusoidal blood flow and extracellular matrix remodeling. Here, we investigated how HSC contraction was affected by the natural compound oroxylin A, and elucidated the underlying mechanism. METHODS: Cell contraction and glycolysis were examined in cultured human HSCs and mouse liver fibrosis model upon oroxylin A intervention using diversified cellular and molecular assays, as well as genetic approaches. RESULTS: Oroxylin A limited HSC contraction associated with inhibiting myosin light chain 2 phosphorylation. Oroxylin A blocked aerobic glycolysis in HSCs evidenced by reduction in glucose uptake and consumption and lactate production. Oroxylin A also decreased extracellular acidification rate and inhibited the expression and activity of glycolysis rate-limiting enzymes (hexose kinase 2, phosphofructokinase 1 and pyruvate kinas type M2) in HSCs. Then, we identified that oroxylin A blockade of aerobic glycolysis contributed to inhibition of HSC contraction. Furthermore, oroxylin A inhibited the expression and activity of lactate dehydrogenase-A (LDH-A) in HSCs, which was required for oroxylin A blockade of glycolysis and suppression of contraction. Oral administration of oroxylin A at 40 mg/kg reduced liver injury and fibrosis, and inhibited HSC glycolysis and contraction in mice with carbon tetrachloride-induced hepatic fibrosis. However, adenovirus-mediated overexpression of LDH-A significantly counteracted the oroxylin A's effects in fibrotic mice. CONCLUSIONS: Blockade of aerobic glycolysis by oroxylin A via inhibition of LDH-A reduced HSC contraction and attenuated liver fibrosis, suggesting LDH-A as a promising target for intervention of hepatic fibrosis.


Assuntos
Flavonoides/farmacologia , Glicólise/efeitos dos fármacos , Células Estreladas do Fígado/enzimologia , L-Lactato Desidrogenase/antagonistas & inibidores , Aerobiose , Linhagem Celular , Células Estreladas do Fígado/efeitos dos fármacos , Humanos , L-Lactato Desidrogenase/metabolismo , Fígado/lesões , Fígado/patologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia
19.
Oxid Med Cell Longev ; 2019: 8148510, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30800209

RESUMO

Microenvironment plays a vital role in tumor progression; we focused on elucidating the role of hepatic stellate cells (HSCs) in hepatocarcinoma (HCC) aggressiveness and investigated the potential protective effect of curcumin on HSC-driven hepatocarcinoma angiogenesis and invasion. Our data suggest that HSCs increase HCC reactive oxygen species (ROS) production to upregulate hypoxia-inducible factor-1α (HIF-1α) expression to promote angiogenesis, epithelial to mesenchymal transition (EMT) process and invasion. And HSCs could secrete soluble factors, such as interleukin-6 (IL-6), vascular endothelial cell growth factor (VEGF), and stromal-derived factor-1 (SDF-1) to facilitate HCC progression. Curcumin could significantly suppress the above HSC-induced effects in HCC and could abrogate ROS and HIF-1α expression in HCC. HIF-1α or connective tissue growth factor (CTGF) knockdown could abolish the aforementioned curcumin affection. Moreover, CTGF is a downstream gene of HIF-1α. In addition, nuclear factor E2-related factor 2 (Nrf2) and glutathione (GSH) are involved in curcumin protection of HCC. These data indicate that curcumin may induce ROS scavenging by upregulating Nrf2 and GSH, thus inhibiting HIF-1α stabilization to suppress CTGF expression to exhibit its protection on HCC. Curcumin has a promising therapeutic effect on HCC. CTGF is responsible for curcumin-induced protection in HCC.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Fator de Crescimento do Tecido Conjuntivo/genética , Curcumina/uso terapêutico , Regulação para Baixo , Células Estreladas do Fígado/patologia , Neoplasias Hepáticas/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Quimiocina CXCL12/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Curcumina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Glutationa/metabolismo , Células Hep G2 , Células Estreladas do Fígado/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Interleucina-6/metabolismo , Neoplasias Hepáticas/patologia , Metaloproteinase 9 da Matriz/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Invasividade Neoplásica , Neovascularização Patológica/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo
20.
Biomater Sci ; 7(3): 1078-1087, 2019 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-30631870

RESUMO

Mesenchymal stem cells (MSCs) are known for their ability to repair liver damage. However, their therapeutic potential still needs to be enhanced. In the present study, we produced genome-edited MSCs that secrete interleukin 10 (IL-10) and evaluated their therapeutic potential in a liver fibrosis model. Multiple copies of the IL-10 gene were inserted into a safe harbor genomic locus in amniotic mesenchymal stem cells (AMMs) using transcription activator-like effector nucleases (TALENs). The IL-10 gene-edited AMMs (AMM/I) were characterized by reverse transcription PCR (RT-PCR), quantitative RT-PCR (qRT-PCR), and microarray. The effects of AMM/I-conditioned cell medium (CM) on the activation of hepatic stellate cells (HSC) were analyzed in vitro and in vivo therapeutic assays were performed on a mouse liver fibrosis model. The engineered AMM/I expressed high levels of IL-10. AMM/I-CM inhibited the activation of HSC (in vitro) and TNF-α expression of T cells/macrophage derived from fibrotic liver. In addition, human IL-10 was detected in the serum of the mice transplanted with AMM/I and transplantation of AMM/I significantly inhibited thioacetamide (TAA)-induced liver fibrosis and ameliorated abnormal liver function. Furthermore, a high number of human albumin-expressing AMM/I were successfully engrafted into the liver of recipient mice. Overall, genome-edited AMMs overexpressing anti-fibrotic IL-10 might be a promising alternative therapeutic option for the treatment of liver cirrhosis.


Assuntos
Interleucina-10/metabolismo , Cirrose Hepática/terapia , Transplante de Células-Tronco Mesenquimais , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Âmnio/citologia , Animais , Transdiferenciação Celular , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Modelos Animais de Doenças , Edição de Genes , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Humanos , Interleucina-10/análise , Cirrose Hepática/patologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/metabolismo
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