RESUMEN
BACKGROUND AND AIMS: TGF-ß induces multiple structural and functional changes in quiescent HSCs, including an increase in proliferation, mitochondrial mass, and matrix deposition. HSC transdifferentiation requires significant bioenergetic capacity, and it is not known how TGF-ß-mediated transcriptional upregulation is coordinated with the bioenergetic capacity of HSCs. APPROACH AND RESULTS: Mitochondria are key bioenergetic organelles, and here, we report that TGF-ß induces release of mitochondrial DNA (mtDNA) from healthy HSCs through voltage-dependent anion channels (VDACs), with the formation of an mtDNA-CAP on the external mitochondrial membrane. This stimulates organization of cytosolic cyclic GMP-AMP synthase (cGAS) onto the mtDNA-CAP and subsequent activation of the cGAS-STING-IRF3 pathway. TGF-ß is unable to induce conversion of HSCs from a quiescent to a transdifferentiated phenotype in the absence of mtDNA, VDAC, or stimulator of interferon genes (STING). Transdifferentiation by TGF-ß is blocked by a STING inhibitor, which also reduces liver fibrosis prophylactically and therapeutically. CONCLUSIONS: We have identified a pathway that requires the presence of functional mitochondria for TGF-ß to mediate HSC transcriptional regulation and transdifferentiation and therefore provides a key link between bioenergetic capacity of HSCs and signals for transcriptional upregulation of genes of anabolic pathways.
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ADN Mitocondrial , Células Estrelladas Hepáticas , Proteínas de la Membrana , Factor de Crecimiento Transformador beta , Humanos , Diferenciación Celular/genética , Diferenciación Celular/fisiología , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Células Estrelladas Hepáticas/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Nucleotidiltransferasas/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
This study tested the ability of a fermented soy product to induce tumor cell toxicity and to assess if this was due to fermentation of soy, and to the genistein content. Four cancer cell lines were cultured without additive, with fermented soy (Q-CAN® PLUS), nonfermented soy, or genistein, and cell viability was examined at 24 h, 48 h, and 72 h. The sensitivity of the cell lines to apoptosis by Q-CAN PLUS was tested with the Annexin V assay. All cell lines demonstrated a dose and time response reduction in tumor cell viability with exposure to Q-CAN PLUS (IC50 at 24 h 3.8 mg/mL to 9 mg/mL). Unfermented soy did not show reduction in viability of any cell line within the same concentration range. The IC50 of genistein for each of the cell lines was significantly greater than for Q-CAN PLUS. All four tumor cell lines demonstrated apoptosis in response to Q-CAN PLUS. Q-CAN PLUS reduces viability and increases apoptosis of cancer cells in a concentration- and fermentation-dependent manner. Taking into consideration the IC50 of genistein and the concentration of genistein in Q-CAN PLUS, the genistein content of Q-CAN PLUS is not responsible for the majority reduction in tumor cell viability. This suggests that fermentation of soy results in the production of metabolites that reduce cancer cell viability and induce cellular apoptosis, and play a major role in addition to any effects produced by their genistein content.
Asunto(s)
Isoflavonas , Neoplasias , Apoptosis , Línea Celular Tumoral , Supervivencia Celular , Genisteína/farmacología , Isoflavonas/farmacología , Neoplasias/tratamiento farmacológico , Glycine maxRESUMEN
Subcellular machinery of NLRP3 is essential for inflammasome assembly and activation. However, the stepwise process and mechanistic basis of NLRP3 engagement with organelles remain unclear. Herein, we demonstrated glycogen synthase kinase 3ß (GSK3ß) as a molecular determinant for the spatiotemporal dynamics of NLRP3 inflammasome activation. Using live cell multispectral time-lapse tracking acquisition, we observed that upon stimuli NLRP3 was transiently associated with mitochondria and subsequently recruited to the Golgi network (TGN) where it was retained for inflammasome assembly. This occurred in relation to the temporal contact of mitochondria to Golgi apparatus. NLRP3 stimuli initiate GSK3ß activation with subsequent binding to NLRP3, facilitating NLRP3 recruitment to mitochondria and transition to TGN. GSK3ß activation also phosphorylates phosphatidylinositol 4-kinase 2 Α (PI4k2A) in TGN to promote sustained NLRP3 oligomerization. Our study has identified the interplay between GSK3ß signaling and the organelles dynamics of NLRP3 required for inflammasome activation and opens new avenues for therapeutic intervention.
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Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , 1-Fosfatidilinositol 4-Quinasa , Glucógeno Sintasa Quinasa 3 beta , Aparato de Golgi/metabolismo , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismoRESUMEN
N6-methyladenosine (m6A) RNA modification is a fundamental determinant of mRNA metabolism, but its role in innate immunity-driven non-alcoholic fatty liver disease (NAFLD) and obesity is not known. Here, we show that myeloid lineage-restricted deletion of the m6A "writer" protein Methyltransferase Like 3 (METTL3) prevents age-related and diet-induced development of NAFLD and obesity in mice with improved inflammatory and metabolic phenotypes. Mechanistically, loss of METTL3 results in the differential expression of multiple mRNA transcripts marked with m6A, with a notable increase of DNA Damage Inducible Transcript 4 (DDIT4) mRNA level. In METTL3-deficient macrophages, there is a significant downregulation of mammalian target of rapamycin (mTOR) and nuclear factor κB (NF-κB) pathway activity in response to cellular stress and cytokine stimulation, which can be restored by knockdown of DDIT4. Taken together, our findings identify the contribution of METTL3-mediated m6A modification of Ddit4 mRNA to macrophage metabolic reprogramming in NAFLD and obesity.
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Adenosina/análogos & derivados , Macrófagos/inmunología , Metiltransferasas/fisiología , Células Mieloides/inmunología , Enfermedad del Hígado Graso no Alcohólico/patología , Obesidad/patología , ARN Mensajero/química , Adenosina/química , Animales , Metilación de ADN , Femenino , Inmunidad Innata , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/genética , FN-kappa B/metabolismo , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Obesidad/etiología , Obesidad/metabolismo , ARN Mensajero/genéticaRESUMEN
Soy-based beverages are well recognized for their rich nutritional contents and positive health benefits. However, there is little information regarding the composition of various commercially available soy-based beverages and uncertainty among patients regarding the utility of fermented soy products. Current study evaluates the health benefits of QCAN® Plus-an easily available fermented soy drink. This study was performed in lean (n = 10) and obese (n = 10) subjects. The subjects were observed during pre-soy (weeks -2, -1, and 0), on-soy (weeks 1, 2, 3, and 4), and post-soy (weeks 6, 8, 10, and 12) periods. The serum samples during these visits were subjected to lipid profile analysis and multiplex assay for cytokines. The results revealed that total cholesterol and low-density lipoprotein (LDL) cholesterol levels were significantly reduced in both lean and obese individuals during on-soy (P ≤ .05). Furthermore, cytokines such as platelet-derived growth factor (PDGF) AA and AB/BB were significantly lowered on-soy compared with pre-soy (P ≤ .05) in lean subjects and PDGF AA, IL-1RA, and GMCSF were significantly reduced on-soy (P ≤ .05) in obese subjects. In addition, a qualitative and quantitative analysis of the Q-CAN Plus by a third-party laboratory confirmed its chemical and microbial safety. Our preliminary study on Q-CAN Plus ensures its safety for consumption and highlights its hypolipidemic and suppressive effect on certain cytokines. These observations and relevant studies in future might guide clinicians in future to consider Q-CAN Plus as a therapeutic nutritional supplement.
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Colesterol/sangre , Citocinas/sangre , Alimentos Fermentados , Lípidos/sangre , Adulto , HDL-Colesterol/sangre , LDL-Colesterol/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Obesidad , Glycine max/química , Adulto JovenRESUMEN
The cardiac glycoside digoxin was identified as a potent suppressor of pyruvate kinase isoform 2-hypoxia-inducible factor-α (PKM2-HIF-1α) pathway activation in liver injury mouse models via intraperitoneal injection. We have assessed the therapeutic effects of digoxin to reduce nonalcoholic steatohepatitis (NASH) by the clinically relevant oral route in mice and analyzed the cellular basis for this effect with differential involvement of liver cell subsets. C57BL/6J male mice were placed on a high-fat diet (HFD) for 10 wk and started concurrently with the gavage of digoxin (2.5, 0.5, 0.125 mg/kg twice a week) for 5 wk. Digoxin significantly reduced HFD-induced hepatic damage, steatosis, and liver inflammation across a wide dosage range. The lowest dose of digoxin (0.125 mg/kg) showed significant protective effects against liver injury and sterile inflammation. Consistently, digoxin attenuated HIF-1α sustained NLRP3 inflammasome activation in macrophages. We have reported for the first time that PKM2 is upregulated in hepatocytes with hepatic steatosis, and digoxin directly improved hepatocyte mitochondrial dysfunction and steatosis. Mechanistically, digoxin directly bound to PKM2 and inhibited PKM2 targeting HIF-1α transactivation without affecting PKM2 enzyme activation. Thus, oral digoxin showed potential to therapeutically inhibit liver injury in NASH through the regulation of PKM2-HIF-1α pathway activation with involvement of multiple cell types. Because of the large clinical experience with oral digoxin, this may have significant clinical applicability in human NASH.NEW & NOTEWORTHY This study is the first to assess the therapeutic efficacy of oral digoxin on nonalcoholic steatohepatitis (NASH) in a high-fat diet (HFD) mouse model and to determine the divergent of cell type-specific effects. Oral digoxin reduced liver damage, steatosis, and inflammation in HFD mice. Digoxin attenuated hypoxia-inducible factor (HIF)-1α axis-sustained inflammasome activity in macrophages and hepatic oxidative stress response in hepatocytes via the regulation of PKM2-HIF-1α axis pathway activation. Oral digoxin may have significant clinical applicability in human NASH.
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Digoxina/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Hepatocitos/enzimología , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Piruvato Quinasa/antagonistas & inhibidores , Activación Transcripcional/efectos de los fármacos , Animales , Línea Celular , Dieta Alta en Grasa , Hepatitis/patología , Hepatocitos/efectos de los fármacos , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Estrés Oxidativo/efectos de los fármacos , Piruvato Quinasa/metabolismoRESUMEN
Drug repurposing has been an emerging therapeutic strategy, which involves exploration of a new therapeutic approach for the use of an existing drug. Glibenclamide (Gli) is an anti-diabetic sulfonylurea drug extensively used for the treatment of type-2 diabetes, it has also been shown to possess anti-proliferative effect against several types of tumors. The present study was executed to understand the mechanisms underlying the interaction of Gli with DNA under physiological conditions. The binding mechanism of Gli with DNA was scrutinized by UV-vis absorption spectroscopy and fluorescence emission spectroscopy. The conformational changes and electrochemical properties were analyzed by circular dichroism spectroscopy and cyclic voltammetry. Isothermal titration calorimetry was employed to examine the thermodynamic changes and molecular docking technique used to analyze the interaction mode of Gli with DNA. The spectroscopic studies revealed that Gli interacts with DNA through groove binding mode. Further, isothermal titration calorimetry depicted a stronger mode of interaction favorably groove-binding. Recently, systemic combination therapy has shown significant promise in inhibiting multiple targets simultaneously yielding high therapeutic competence with lesser side effects. With this concern, we intended to study the combined cytotoxicity of Gli with doxorubicin (Dox). The results of MTT assay and acridine orange (AO)/ethidium bromide (EtBr) staining showed synergistic cytotoxicity of Gliâ¯+â¯Dox combination on HepG2 & A549 cells. The present study documents the intricate mechanism of Gli-DNA interaction and delivers a multifaceted access for chemotherapy by Gliâ¯+â¯Dox combination.
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Antineoplásicos/farmacología , Citotoxinas/farmacología , Doxorrubicina/farmacología , Gliburida/farmacología , Células A549 , Calorimetría/métodos , Línea Celular Tumoral , Dicroismo Circular/métodos , ADN/efectos de los fármacos , Sinergismo Farmacológico , Fluorescencia , Células Hep G2 , Humanos , Simulación del Acoplamiento Molecular/métodos , Espectrometría de Fluorescencia/métodos , TermodinámicaRESUMEN
Neutrophil is a significant contributor to ischemia reperfusion (IR) induced liver tissue damage. However, the exact role of neutrophils in IR induced innate immune activation and liver damage is not quite clear. Our study sheds light on the role of chronic oxidative stress end products in worsening the IR inflammatory process by neutrophil recruitment and activation following liver surgery. We employed specific inhibitors for molecular targets-NOX2 (NADPH oxidase 2) and P38 MAPK (Mitogen activated protein kinase) signal to counteract neutrophil activation and neutrophil extracellular trap (NET) release induced liver damage in IR injury. We found that acrolein initiated neutrophil chemotaxis and induced NET release both in vitro and in vivo. Acrolein exposure caused NET induced nuclear and mitochondrial damage in HepG2 cells as well as aggravated the IR injury in rat liver. Pretreatment with F-apocynin and naringin, efficiently suppressed acrolein induced NET release in vitro. Notably, it suppressed the expression of inflammatory cytokines, P38MAPK-ERK activation, and apoptotic signals in rat liver exposed to acrolein and subjected to IR. Moreover, this combination effectively attenuated acrolein induced NET release and hepatic IR injury. In the current study we have shown that the acrolein accumulation in liver due to chronic stress, is responsible for neutrophil recruitment and its activation leading to NET induced liver damage during surgery. Our study shows that therapeutic targeting of NOX2 and P38MAPK signaling in patients with chronic hepatic disorders would improve post operative hepatic function and survival.
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Inhibidores Enzimáticos/uso terapéutico , Trampas Extracelulares/metabolismo , Hígado/irrigación sanguínea , NADPH Oxidasa 2/antagonistas & inhibidores , Daño por Reperfusión/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Acetofenonas/farmacología , Acetofenonas/uso terapéutico , Acroleína , Animales , Antioxidantes/metabolismo , Apoptosis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Quimiotaxis/efectos de los fármacos , Daño del ADN , Inhibidores Enzimáticos/farmacología , Flavanonas/farmacología , Flavanonas/uso terapéutico , Células Hep G2 , Histonas/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Hígado/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , NADPH Oxidasa 2/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Neutrófilos/ultraestructura , Peroxidasa/metabolismo , Ratas Wistar , Estallido Respiratorio/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Drawbacks and limitations of recently available therapies to hepatocellular cancer (HCC) devoted the scientist to focus on emerging new strategies. ZnO nanoparticles (ZnONPs) based chemotherapeutics has been emanating as a promising approach to maximize therapeutic synergy facilitating the discovery of novel multitargeted combinations. In the present study we conjugated ZnONPs with ferulic acid (ZnONPs-FAC) characterized by computational, spectroscopic and microscopic techniques. In vitro anticancer potential has been evaluated by assessing cell viability, morphology, ROS generation, mitochondrial membrane permeability, comet assay, immunofluorescent staining of 8-OHdG, Ki67 and γ-H2AX, cell cycle analysis and western blot analysis and in vivo anticancer potential against DEN induced HCC was analyzed by histopathological and immunohistochemical methods. The results revealed that ZnONPs-FAC induces cell death through apoptosis and can suppress the DEN-induced HCC. Our study documents therapeutic potential of nanoparticle conjugated with phytochemicals, suggesting a new platform for combinatorial chemotherapy.