RESUMEN
Hepatic stellate cells (HSCs) play a central role in the progression of liver fibrosis by producing extracellular matrices. The development of drugs to suppress liver fibrosis has been hampered by the lack of human quiescent HSCs (qHSCs) and an appropriate in vitro model that faithfully recapitulates HSC activation. In the present study, we developed a culture system to generate qHSC-like cells from human-induced pluripotent stem cells (hiPSCs) that can be converted into activated HSCs in culture. To monitor the activation process, a red fluorescent protein (RFP) gene was inserted in hiPSCs downstream of the activation marker gene actin alpha 2 smooth muscle (ACTA2). Using qHSC-like cells derived from RFP reporter iPSCs, we screened a repurposing chemical library and identified therapeutic candidates that prevent liver fibrosis. Hence, hiPSC-derived qHSC-like cells will be a useful tool to study the mechanism of HSC activation and to identify therapeutic agents.
Asunto(s)
Técnicas de Cultivo de Célula , Ciclo Celular , Descubrimiento de Drogas , Células Estrelladas Hepáticas/citología , Células Madre Pluripotentes Inducidas/citología , Modelos Biológicos , Animales , Evaluación Preclínica de Medicamentos , Perfilación de la Expresión Génica , Células Estrelladas Hepáticas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Masculino , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Liver fibrosis and fibrosis-related hepatocarcinogenesis are a rising cause for morbidity and death worldwide. Although transforming growth factor-ß (TGF-ß) is a critical mediator of chronic liver fibrosis, targeting TGF-ß isoforms and receptors lead to unacceptable side effect. This study was designed to explore the antifibrotic effect of Compound kushen injection (CKI), an approved traditional Chinese medicine formula, via a therapeutic strategy of rebalancing TGF-ß/Smad7 signaling. METHODS: A meta-analysis was performed to evaluate CKI intervention on viral hepatitis-induced fibrosis or cirrhosis in clinical randomized controlled trials (RCTs). Mice were given carbon tetrachloride (CCl4 ) injection or methionine-choline deficient (MCD) diet to induce liver fibrosis, followed by CKI treatment. We examined the expression of TGF-ß/Smad signaling and typical fibrosis-related genes in hepatic stellate cells (HSCs) and fibrotic liver tissues by qRT-PCR, Western blotting, RNA-seq, immunofluorescence, and immunohistochemistry. RESULTS: Based on meta-analysis results, CKI improved the liver function and relieved liver fibrosis among patients. In our preclinical studies by using two mouse models, CKI treatment demonstrated promising antifibrotic effects and postponed hepatocarcinogenesis with improved liver function and histopathologic features. Mechanistically, we found that CKI inhibited HSCs activation by stabilizing the interaction of Smad7/TGF-ßR1 to rebalance Smad2/Smad3 signaling, and subsequently decreased the extracellular matrix formation. Importantly, Smad7 depletion abolished the antifibrotic effect of CKI in vivo and in vitro. Moreover, matrine, oxymatrine, sophocarpine, and oxysophocarpine were identified as material basis responsible for the antifibrosis effect of CKI. CONCLUSIONS: Our results unveil the approach of CKI in rebalancing TGF-ß/Smad7 signaling in HSCs to protect against hepatic fibrosis and hepatocarcinogenesis in both preclinical and clinical studies. Our study suggests that CKI can be a candidate for treatment of hepatic fibrosis and related oncogenesis.
Asunto(s)
Medicamentos Herbarios Chinos/farmacología , Transducción de Señal/efectos de los fármacos , Proteína smad7/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Tetracloruro de Carbono/toxicidad , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Línea Celular , Medicamentos Herbarios Chinos/uso terapéutico , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/metabolismo , Humanos , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Masculino , Medicina Tradicional China , Metaanálisis como Asunto , Ratones , Ratones Endogámicos C57BL , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteína smad7/antagonistas & inhibidores , Proteína smad7/genéticaRESUMEN
This research aimed to evaluate the antihepatic fibrosis effect and explore the mechanism of Qiwei Qinggan Powder (QGS-7) in vivo and in vitro. Carbon tetrachloride (CCl4)-treated rats and hepatic stellate cells (HSCs) were used. QGS-7 treatment significantly improved the liver function of rats as indicated by decreased serum enzymatic activities of alanine aminotransferase, aspartate transaminase, and alkaline phosphatase. Meanwhile, the hydroxyproline of liver was significantly decreased. Histopathological results indicated that QGS-7 alleviated liver damage and reduced the formation of fibrosis septa. Moreover, QGS-7 significantly attenuated expressions of Alpha smooth muscle actin, Collagen I, Janus kinase 2 (JAK2), phosphorylation-JAK2, signal transducer and activator of transcription 3 (STAT3), phosphorylation-STAT3 in the rat hepatic fibrosis model. QGS-7 inhibited HSC proliferation and promoted it apoptosis. QGS-7 may affect hepatic fibrosis through JAK2/STAT3 signaling pathway so as to play an antihepatic fibrosis role.
Asunto(s)
Intoxicación por Tetracloruro de Carbono/tratamiento farmacológico , Cirrosis Hepática/tratamiento farmacológico , Medicina Tradicional Mongoliana , Animales , Intoxicación por Tetracloruro de Carbono/metabolismo , Proliferación Celular/efectos de los fármacos , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/metabolismo , Hidroxiprolina/metabolismo , Janus Quinasa 2/metabolismo , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/metabolismo , Pruebas de Función Hepática , Mongolia , Fosforilación , Polvos , Ratas , Factor de Transcripción STAT3/metabolismoRESUMEN
Liver fibrosis (LF) mortality rate is approximately 2 million per year. Irrespective of the etiology of LF, a key element in its development is the transition of hepatic stellate cells (HSCs) from a quiescent phenotype to a myofibroblast-like cell with the production of fibrotic proteins. It is necessary to define optimal isolation and culturing conditions for good HSCs yield and proper phenotype preservation for studying the activation of HSCs in vitro. In the present study, the optimal conditions of HSC isolation and culture were examined to maintain the HSC's undifferentiated phenotype. HSCs were isolated from Balb/c mice liver using Nycodenz, 8, 9.6, and 11%. The efficiency of the isolation procedure was evaluated by cell counting and purity determination by flow cytometry. Quiescent HSCs were cultured in test media supplemented with different combinations of fetal bovine serum (FBS), glutamine (GLN), vitamin A (vitA), insulin, and glucose. The cells were assessed at days 3 and 7 of culture by evaluating the morphology, proliferation using cell counting kit-8, lipid storage using Oil Red O (ORO) staining, expression of a-smooth muscle actin, collagen I, and lecithin-retinol acyltransferase by qRT-PCR and immunocytochemistry (ICC). The results showed that Nycodenz, at 9.6%, yielded the best purity and quantity of HSCs. Maintenance of HSC undifferentiated phenotype was achieved optimizing culturing conditions (serum-free Dulbecco's Modified Eagle's Medium (DMEM) supplemented with glucose (100 mg/dl), GLN (0.5 mM), vitA (100 µM), and insulin (50 ng/ml)) with a certain degree of proliferation allowing their perpetuation in culture. In conclusion, we have defined optimal conditions for HSCs isolation and culture.
Asunto(s)
Células Estrelladas Hepáticas/citología , Animales , Células Cultivadas , Medios de Cultivo , Yohexol/análisis , Ratones , Ratones Endogámicos BALB CRESUMEN
Activated hepatic stellate cells (HSCs) are primarily responsible for the accumulation of extracellular matrix substances during the development of liver fibrosis. It has been shown that n-3 polyunsaturated fatty acids (PUFAs) can prevent liver fibrosis development. However, the underlying mechanisms of action need further investigation. The objective of this study was to determine the regulatory roles of fatty acids (FAs) on the expression of profibrogenic genes in HSCs with the elucidation of mechanisms. LX-2 cells and primary human and mouse HSCs were treated with palmitic acid, oleic acid, linoleic acid, α-linolenic acid, eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) to determine their effect on profibrogenic gene expression upon the activation by transforming growth factor ß1 (TGFß1). PUFAs significantly suppressed TGFß1-induced expression of profibrogenic genes in LX-2 and primary human HSCs with n-3 being more potent than n-6 PUFAs. However, PUFAs did not inhibit the phosphorylation and nuclear translocation of SMA- and MAD-related protein in primary human HSCs. Furthermore, PUFAs did not alter the profibrogenic gene expression in primary mouse HSCs. The inhibitory effect of EPA and DHA on TGFß1-induced profibrogenic gene expression was diminished by peroxisome proliferator-activated receptor gamma (PPARG) knockdown, although chemical inhibition of PPARγ did not elicit a similar result. The results suggest that n-3 PUFAs possess the most potent protective effects against TGFß1-induced profibrogenic gene expression, which is, at least in part, PPARγ-dependent in HSCs.
Asunto(s)
Ácidos Grasos Omega-3/metabolismo , Células Estrelladas Hepáticas/metabolismo , PPAR gamma/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Línea Celular , Células Cultivadas , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células Estrelladas Hepáticas/citología , Humanos , Cirrosis Hepática/genética , Cirrosis Hepática/metabolismo , Ratones , Ratones Endogámicos C57BL , PPAR gamma/genética , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
Hepatic fibrosis is characterized by the abnormal deposition of extracellular matrix (ECM) proteins. During hepatic fibrogenesis, hepatic stellate cell (HSC) activation followed by chronic injuries is considered a key event in fibrogenesis, and activated HSCs are known to comprise approximately 90% of ECM-producing myofibroblasts. Here, we demonstrated that (-)-catechin-7-O-ß-d-apiofuranoside (C7A) significantly inhibited HSC activation via blocking the signal transducer and activator of transcription 3 (STAT3) signaling pathway. This is the first study to show the hepatic protective effects of C7A with possible mechanisms in vitro and in vivo. In our bioactivity screening, we figured out that the EtOH extract of Ulmusdavidiana var. japonica root barks, which have been used as a Korean traditional medicine, inhibited collagen synthesis in HSCs. Four catechins isolated from the EtOAc fraction of the EtOH extract were compared with each other in terms of reduction in collagen, which is considered as a marker of hepatic protective effects, and C7A showed the strongest inhibitory effects on HSC activation in protein and qPCR analyses. As a possible mechanism, we investigated the effects of C7A on the STAT3 signaling pathway, which is known to activate HSCs. We found that C7A inhibited phosphorylation of STAT3 and translocation of STAT3 to nucleus. C7A also inhibited expressions of MMP-2 and MMP-9, which are downstream genes of STAT3 signaling. Anti-fibrotic effects of C7A were evaluated in a thioacetamide (TAA)-induced liver fibrosis model, which indicated that C7A significantly inhibited ECM deposition through inhibiting STAT3 signaling. C7A decreased serum levels of aspartate amino transferase and alanine transaminase, which were markedly increased by TAA injection. Moreover, ECM-associated proteins and mRNA expression were strongly suppressed by C7A. Our study provides the experimental evidence that C7A has inhibitory effects on HSC activation after live injury and has preventive and therapeutic potentials for the management of hepatic fibrosis.
Asunto(s)
Catequina/administración & dosificación , Células Estrelladas Hepáticas/citología , Factor de Transcripción STAT3/metabolismo , Ulmus/química , Animales , Catequina/química , Catequina/farmacología , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Humanos , Masculino , Fosforilación , Corteza de la Planta/química , Extractos Vegetales/química , Transporte de Proteínas/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
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 (É-SMA) and type I collagen expression. Immunofluorescence staining was used to observe the immunofluorescence localization of É-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.
Asunto(s)
Autofagia , Medicamentos Herbarios Chinos/farmacología , Células Estrelladas Hepáticas/citología , Cirrosis Hepática/tratamiento farmacológico , Animales , Benzofuranos , Tetracloruro de Carbono , Cloroquina , Células Estrelladas Hepáticas/efectos de los fármacos , Hígado , Cirrosis Hepática/inducido químicamente , Ratones , Ratones Endogámicos C57BL , Distribución Aleatoria , SirolimusRESUMEN
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.
Asunto(s)
Aminoácidos/farmacología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ginsenósidos/farmacología , Células Estrelladas Hepáticas/citología , Aminoácidos/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Ginsenósidos/química , Células Estrelladas Hepáticas/efectos de los fármacos , Humanos , Puntos de Control de la Fase S del Ciclo Celular/efectos de los fármacosRESUMEN
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.
Asunto(s)
Apoptosis/efectos de los fármacos , Cirrosis Hepática Experimental/patología , Própolis/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Tetracloruro de Carbono/toxicidad , Citocinas/metabolismo , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática Experimental/inducido químicamente , Cirrosis Hepática Experimental/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteína Smad2/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
OBJECTIVE: Liver fibrosis is part of the non-alcoholic fatty liver disease (NAFLD) spectrum, which currently has no approved pharmacological treatment. In this study, we investigated whether supplementation of nicotinamide riboside (NR), a nicotinamide adenine dinucleotide (NAD+) precursor, can reduce the development of liver fibrosis in a diet-induced mouse model of liver fibrosis. METHODS: Male C57BL/6â¯J mice were fed a low-fat control (LF), a high-fat/high-sucrose/high-cholesterol control (HF) or a HF diet supplemented with NR at 400â¯mg/kg/day (HF-NR) for 20â¯weeks. Features of liver fibrosis were assessed by histological and biochemical analyses. Whole-body energy metabolism was also assessed using indirect calorimetry. Primary mouse and human hepatic stellate cells were used to determine the anti-fibrogenic effects of NR in vitro. RESULTS: NR supplementation significantly reduced body weight of mice only 7â¯weeks after mice were on the supplementation, but did not attenuate serum alanine aminotransferase levels, liver steatosis, or liver inflammation. However, NR markedly reduced collagen accumulation in the liver. RNA-Seq analysis suggested that the expression of genes involved in NAD+ metabolism is altered in activated hepatic stellate cells (HSCs) compared to quiescent HSCs. NR inhibited the activation of HSCs in primary mouse and human HSCs. Indirect calorimetry showed that NR increased energy expenditure, likely by upregulation of ß-oxidation in skeletal muscle and brown adipose tissue. CONCLUSION: NR attenuated HSC activation, leading to reduced liver fibrosis in a diet-induced mouse model of liver fibrosis. The data suggest that NR may be developed as a potential preventative for human liver fibrosis.
Asunto(s)
Hígado/efectos de los fármacos , Niacinamida/análogos & derivados , Enfermedad del Hígado Graso no Alcohólico/patología , Animales , Peso Corporal/efectos de los fármacos , Colágeno/metabolismo , Dieta Alta en Grasa , Suplementos Dietéticos , Modelos Animales de Enfermedad , Metabolismo Energético/efectos de los fármacos , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/metabolismo , Humanos , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , NAD/metabolismo , Niacinamida/administración & dosificación , Niacinamida/farmacología , Enfermedad del Hígado Graso no Alcohólico/prevención & control , Compuestos de PiridinioRESUMEN
The activation of hepatic stellate cells (HSCs) is a key event in the development of hepatic fibrosis caused by arsenic. However, it is unclear how arsenic induces the activation of HSCs. In the present study, we found that arsenic trioxide (As2O3) induced liver tissue damage, stimulated autophagy and HSCs activation, and increased collagen accumulation in the liver of mice. Supplemented with taurine (Tau) attenuated the changes mentioned above caused by As2O3. In human hepatic stellate cell line LX-2â¯cells, we found that As2O3-induced activation of HSCs was autophagy-dependent, and we found that peroxisome proliferator activated receptors alpha (PPARα) played an important role in arsenic-induced HSCs activation. In addition, inhibiting autophagy and PPARα alleviated the activation of HSCs and lipid droplet loss induced by As2O3. Moreover, we found that Tau alleviated As2O3-induced elevation of autophagy and PPARα expression, and activation of the HSCs. Our results indicated that autophagy was regulated by PPARα and was involved in lipid droplet loss during the activation of HSCs. Tau alleviated As2O3-induced HSCs activation by inhibiting the PPARα/autophagy pathway. These findings give an innovative insight into the association of PPARα, autophagy, the activation of HSCs and hepatic fibrosis induced by As2O3.
Asunto(s)
Trióxido de Arsénico/toxicidad , Autofagia/efectos de los fármacos , PPAR alfa/metabolismo , Taurina/farmacología , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
Sea buckthorn ( Hippophae rhamnoides L.) is a berry bearing multiple nutritional properties. In this study, 46 compounds were isolated from sea buckthorn berries. Preliminary data showed that the components, C13, C15, and C32, exhibited profound inhibitory effect on the activation of hepatic stellate cells (HSCs) induced by transforming growth factor-ß (TGF-ß) and decreased the levels of inflammatory factors. Furthermore, these compounds over-regulated the proteins of DNA damage signaling pathway and alpha-smooth muscle actin (α-SMA). Moreover, active components of sea buckthorn berry (ACSB) treatment attenuated fibrosis development in rats after bile duct ligation (BDL), reducing liver injury and inflammation, and reviving liver function in a dose-dependent manner. Moreover, ACSB down-regulated the expression of α-SMA, while over-regulating the DNA damage signaling pathway and the related genes. These suggest that ACSB inhibit DNA repair of HSCs, make them in a damaged state, inhibit the expression of TGF-ß, and induce apoptosis.
Asunto(s)
Células Estrelladas Hepáticas/efectos de los fármacos , Hippophae/química , Cirrosis Hepática/tratamiento farmacológico , Extractos Vegetales/química , Extractos Vegetales/farmacología , Actinas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Frutas/química , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/metabolismo , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Cirrosis Hepática/metabolismo , Cirrosis Hepática/fisiopatología , Masculino , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Berberine (BBR) is an isoquinoline alkaloid extracted from the roots, rhizomes and stems of coptis. Liver fibrosis is a worldwide health problem with no established therapy until now. The aim of our study is to investigate the efficacy of BBR on hepatic fibrosis induced in rats and to uncover other mechanisms. Rats were injected with thioacetamide (TAA) (200â¯mg/kg, i.p) twice per week for 6 weeks to induce fibrosis. Treated groups were gavaged with BBR (50â¯mg/kg/day, p.o) simultaneously with TAA injection. Hepatic antioxidant enzymes (catalase, SOD, GPx) were assessed in hepatic homogenate. Their activities were attenuated by TAA injection and elevated by BBR administration. Additionally, serum IL-6 and mRNA levels of IL-1ß, IL-6, IL-10 and IFN-γ were evaluated as inflammatory markers. Our results showed that BBR suppressed the inflammation induced by TAA injection. Tissue expression of α-SMA (marker of activated HSCs), TGF-ß1 and fibronectin were measured by immunohistochemistry as well as mRNA expressions of TGF-ß1 and fibronectin were quantified as fibrotic markers. The collagen deposition in hepatic tissues was assessed by Masson's trichome staining. BBR significantly alleviated TGF-ß1 production, decreased collagen and fibronectin deposition and consequently attenuated hepatic fibrogenesis. Akt pathway controls cell survival, proliferation, migration and adhesion. The relative phosphorylation of Akt was determined in hepatic homogenates that was increased with TAA injection and decreased by BBR treatment. Inhibition of Akt pathway has been linked to the intrinsic pathway of apoptosis. Caspase-3, caspase-9, Bcl-2 and Bax were quantified as apoptotic markers using qPCR and also caspase-3 by immunohistochemistry. BBR-treated rats showed an increase in the expression of apoptotic markers. Moreover, BBR-treated rats showed restoration of normal liver lobular architecture as shown by H&E staining. In conclusion, BBR is a potential therapeutic candidate for liver fibrosis owing to its antioxidant and anti-inflammatory activities.
Asunto(s)
Antiinflamatorios/uso terapéutico , Antioxidantes/uso terapéutico , Berberina/uso terapéutico , Cirrosis Hepática/prevención & control , Actinas/genética , Actinas/metabolismo , Animales , Antiinflamatorios/farmacología , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Berberina/farmacología , Colágeno/metabolismo , Citocinas/genética , Citocinas/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/patología , Masculino , Estrés Oxidativo/efectos de los fármacos , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Sprague-Dawley , Tioacetamida/toxicidad , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Sugarcane (Saccharum officinarum L.), which is one of the most important sources of sugar, is also rich in polyphenolic compounds. In this study, polyphenols from sugarcane were extracted, and the dominant component was characterized quantitatively via HPLC to be (-)-epicatechin. Fibrosis occurs in many organs and is associated with severe tissue damage. Liver fibrosis is the excessive accumulation of extracellular matrix proteins and advanced liver fibrosis, resulting in cirrhosis, liver failure and portal hypertension. Thus, the prevention and treatment of liver fibrosis is urgent. Therefore, we further investigated the protective effect of sugarcane polyphenol extract (SPE) on carbon tetrachloride-induced liver fibrosis in rats and observed that SPE (20 or 50 mg kg-1) improved the serum GOT (glutamic oxaloacetic transferase) and GPT (glutamic pyruvate transaminase) levels and decreased the expression of α-smooth muscle actin (α-SMA) in liver tissues. The mechanistic study showed that in transforming growth factor ß1(TGF-ß1)-induced hepatic stellate cells (HSCs), SPE attenuated the phosphorylation of p38 and JNK1/2 and down-regulated the expression of α-SMA. Collectively, SPE mitigated carbon tetrachloride-induced liver fibrosis in rats and its mechanism may be related to the p38 and JNK signalling pathways.
Asunto(s)
Cirrosis Hepática/tratamiento farmacológico , MAP Quinasa Quinasa 4/metabolismo , Extractos Vegetales/administración & dosificación , Polifenoles/administración & dosificación , Saccharum/química , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Alanina Transaminasa/metabolismo , Animales , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/efectos de los fármacos , Humanos , Hígado/efectos de los fármacos , Hígado/enzimología , Cirrosis Hepática/enzimología , Cirrosis Hepática/genética , MAP Quinasa Quinasa 4/genética , Masculino , Fosforilación/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
BACKGROUND: Our previous study showed that during in vitro experiments changes in calcium concentration were associated with apoptosis. We presumed that the calcium ion might play a role as intermediate messenger for apoptosis-related genes. No such evidence has been reported in the literature. Here, we investigate the effect of calcium ionophore A23187 on the apoptosis of rat hepatic stellate cells (HSCs) stimulated by transforming growth factor-ß1 (TGF-ß1) to explore the mechanism of apoptosis through the endoplasmic reticulum stress pathway. METHODS: The apoptotic rate was determined using flow cytometry. The changes in Ca2+ level in HSCs were examined with laser confocal microscopy. The expressions of caspase-12 GRP78 and caspase-9 were assayed via western blot. RESULTS: The respective apoptosis rates for the blank group, the TGF-ß1 group and the TGF-ß1 + low, medium and high dose calcium ionophore A23187 groups were 3.40 ± 0.10%, 1.76 ± 0.12%, 5.86 ± 0.31%, 11.20 ± 0.48% and 15.08 ± 0.75%, with significant differences between the groups (p < 0.05). The concentration of Ca2+and the expression of the GRP78, caspase-9 and caspase-12 proteins significantly increased with increasing calcium ionophore A23187 doses (p < 0.05). CONCLUSION: Calcium ionophore A23187 increased intracellular Ca2+ and activated endoplasmic reticulum stress, which promoted HSC apoptosis.
Asunto(s)
Apoptosis , Calcimicina/farmacología , Ionóforos de Calcio/farmacología , Células Estrelladas Hepáticas/metabolismo , Factor de Crecimiento Transformador beta1/fisiología , Animales , Calcio/metabolismo , Caspasas/metabolismo , Línea Celular , Proteínas de Choque Térmico/metabolismo , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/efectos de los fármacos , RatasRESUMEN
Eucommia ulmoides is an important traditional Chinese medicine and has been used as a tonic with a long history. Aucubin is an active component extracted from Eucommia ulmoides, which has liver-protection effects. However the mechanisms are still unclear. To investigate the inhibitory effects and the underlying mechanisms of aucubin on TGF-ß1-induced activation of hepatic stellate cells and ECM deposition, Human hepatic stellate cells (LX-2 cells) were incubated with TGF-ß1 to evaluate the anti-fibrotic effect of aucubin. Western blot was used to investigate the expression of α-SMA, Col I, Col III, MMP-2 and TIMP-1. ROS production was monitored using DCFH-DA probe, and NOX4 expression was detected by Real-time PCR. Results indicated that TGF-ß1 stimulated the activation and ECM deposition of LX-2 cells. Compared with the control group, aucubin and aucubigenin both reduced the protein expression of α-SMA, Col I, Col III and MMP-2 in LX-2 cells. Aucubin and aucubigenin also suppressed the generation of ROS and down-regulated the NOX4 mRNA expression. Taken together, aucubin and aucubigenin both inhibit the activation and ECM deposition of LX-2 cells activated by TGF-ß1. Aucubin and aucubigenin are potential therapeutic candidate drugs for liver fibrosis.
Asunto(s)
Células Estrelladas Hepáticas/efectos de los fármacos , Glucósidos Iridoides/farmacología , Factor de Crecimiento Transformador beta1/farmacología , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/metabolismo , Humanos , NADPH Oxidasa 4 , NADPH Oxidasas/genética , Piranos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Diallyl trisulfide (DATS), a major garlic derivative, inhibits cell proliferation and triggers apoptosis in a variety of cancer cell lines. However, the effects of DATS on hepatic stellate cells (HSCs) remain unknown. The aim of this study was to analyze the effects of DATS on cell proliferation and apoptosis, as well as the protein expression profile in rat HSCs. Rat HSCs were treated with or without 12 and 24 µg/mL DATS for various time intervals. Cell proliferation and apoptosis were determined using tetrazolium dye (MTT) colorimetric assay, bromodeoxyuridine (5-bromo-2'-deoxyuridine; BrdU) assay, Hoechst 33342 staining, electroscopy, and flow cytometry. Protein expression patterns in HSCs were systematically studied using 2-dimensional electrophoresis and mass spectrometry. DATS inhibited cell proliferation and induced apoptosis of HSCs in a time-dependent manner. We observed clear morphological changes in apoptotic HSCs and dramatically increased annexin V-positive - propidium iodide negative apoptosis compared with the untreated control group. Twenty-one significant differentially expressed proteins, including 9 downregulated proteins and 12 upregulated proteins, were identified after DATS administration, and most of them were involved in apoptosis. Our results suggest that DATS is an inducer of apoptosis in HSCs, and several key proteins may be involved in the molecular mechanism of apoptosis induced by DATS.
Asunto(s)
Compuestos Alílicos/farmacología , Apoptosis/efectos de los fármacos , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/efectos de los fármacos , Proteómica , Sulfuros/farmacología , Animales , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ajo/química , Regulación de la Expresión Génica/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , RatasRESUMEN
Saikosaponin a (SSa) is one of the main active components of Bupleurum falcatum. It is commonly used to treat liver injury and fibrosis in traditional Chinese medicine. Our previous study showed that SSa induces apoptosis and inhibits the proliferation of rat hepatic stellate cell (HSC) line HSC-T6. The aim of the present study was to elucidate the mechanism of SSa-mediated apoptosis. Rat HSC cell line HSC-T6 and human HSC cell line LX-2 were used in this study. SSa triggered cell death mainly by apoptosis, as indicated by the typical morphological changes, sub-G1 phase of cell cycle increase, and activation of the caspase-9/caspase-3 cascade. In addition, SSa-induced apoptosis was partially inhibited by the caspase-3 inhibitor Z-DEVD-FMK, suggesting an involvement of caspase-3 dependent and independent pathways. Moreover, SSa upregulated pro-apoptotic proteins [BAK, Bcl-2-associated death promoter (BAD), and p53 upregulated modulator of apoptosis (PUMA)] and downregulated anti-apoptotic proteins (Bcl-2). In the mitochondria, SSa triggered the translocation of BAX and BAK from the cytosol to the outer membrane, resulting in a reduction of mitochondrial functions and membrane potential and subsequent release of apoptotic factors. Therefore, this study demonstrates that SSa induces apoptosis through the intrinsic mitochondrial-dependent pathway in HSCs.
Asunto(s)
Apoptosis/efectos de los fármacos , Células Estrelladas Hepáticas/citología , Mitocondrias/metabolismo , Ácido Oleanólico/análogos & derivados , Saponinas/farmacología , Animales , Apoptosis/genética , Bupleurum , Caspasa 3/metabolismo , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Células Cultivadas , Ácido Oleanólico/farmacología , Ratas , Estimulación QuímicaRESUMEN
The increased proliferation of activated hepatic stellate cells (HSCs) is associated with hepatic fibrosis and excessive extracellular matrix (ECM)-protein production. We examined the inhibitory effects of the Monascus purpureus-fermented metabolites, ankaflavin and monascin (15 and 30 µM), on the Akt/nuclear factor (NF)-κB and p38 mitogen-activated protein kinase (MAPK) signaling pathways in HSC-T6 (activated hepatic stellate cell line). Ankaflavin and monascin (30 µM) induced apoptosis and significantly inhibited cell growth (cell viabilities: 80.2 ± 5.43% and 62.8 ± 8.20%, respectively, versus control cells; P < 0.05). Apoptosis and G1 phase arrest (G1 phase percentages: 76.1 ± 2.85% and 79.9 ± 1.80%, respectively, versus control cells 65.9 ± 4.94%; P < 0.05) correlated with increased p53 and p21 levels and caspase 3 activity and decreased cyclin D1 and Bcl-2-family protein levels (P < 0.05, all cases). The apoptotic effects of ankaflavin and monascin were HSC-T6-specific, suggesting their potential in treating liver fibrosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Flavinas/farmacología , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/efectos de los fármacos , Compuestos Heterocíclicos con 3 Anillos/farmacología , Cirrosis Hepática/fisiopatología , Monascus/química , Extractos Vegetales/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Humanos , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/genética , Cirrosis Hepática/metabolismo , Masculino , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
OBJECTIVE: To investigate the inhibitory effect of Panax notoginseng saponins (PNS) on liver fibrosis and explore the underlying mechanisms. METHODS: Carbon tetrachloride (CCl4)-treated rats and hepatic stellate cells (HSCs) were used. The effect of PNS on CCl4-induced liver fibrosis was studied with histochemical and biochemical analysis. Transforming growth factor (TGF)-ß1, α-smooth muscle actin (α-SMA), and collagen I mRNA expression were determined by reverse transcripwhile, the protein expression levels of α-SMA, collagen I, phosphorylation-Janus activated kinase signal transducer (p-Jak2)/Jak2, and phosphorylation-activator of transcription (p-Stat)3/Stat3 were determined by immunohistochemistry and/or immunoblotting. RESULTS: PNS treatment significantly improved the liver function of rats as indicated by decreased serum enzymatic activities of alanine aminotransferase and aspartate aminotransferase. Histopathological results indicated that PNS alleviated liver damage and reduced the formation of fibrous septa. Moreover, PNS significantly decreased liver hydroxyproline and significantly attenuated expressions of collagen I, α-SMA, TGF-ß1, p-Jak2 / Jak2, and p-Stat3/Stat3 in the rat liver fibrosis model and HSCs. CONCLUSION: PNS can relieve liver fibrosis by modulating Jak2/Stat3 signaling transduction pathway, which may be one of its mechanisms to suppress hepatic fibrosis.