RESUMO
A molecular characterization of the main phytochemicals and antioxidant activity of Opuntia robusta (OR) fruit extract was carried out, as well as an evaluation of its hepatoprotective effect against diclofenac (DF)-induced acute liver injury was evaluated. Phenols, flavonoids and betalains were quantified, and antioxidant characterization was performed by means of the ABTSâ¢+, DPPH and FRAP assays. UPLC-QTOF-MS/MS was used to identify the main biocompounds present in OR fruit extract was carried out via. In the in vivo model, groups of rats were treated prophylactically with the OR fruit extract, betanin and N-acteylcysteine followed by a single dose of DF. Biochemical markers of oxidative stress (MDA and GSH) and relative gene expression of the inducible antioxidant response (Nrf2, Sod2, Hmox1, Nqo1 and Gclc), cell death (Casp3) and DNA repair (Gadd45a) were analyzed. Western blot analysis was performed to measure protein levels of Nrf2 and immunohistochemical analysis was used to assess caspase-3 activity in the experimental groups. In our study, the OR fruit extract showed strong antioxidant and cytoprotective capacity due to the presence of bioactive compounds, such as betalain and phenols. We conclude that OR fruit extract or selected components can be used clinically to support patients with acute liver injury.
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Liver fibrosis is a chronic disease associated with oxidative stress that has a great impact on the population mortality. Due to their antioxidant capacity, we evaluated the protective effect of Opuntia robusta fruit (Or) on liver fibrosis. A nutraceutical characterization of Or was performed and a model of fibrosis was induced with thioacetamide (TAA) in Wistar rats. Aminotransferases, reduced glutathione (GSH) and histopathology were evaluated. Or contained 436.5 ± 57 mg of Betacyanins equivalents/L., 793 mg of catechin equivalents (CAE)/100 g for flavonoids, 1118 mg of gallic acid equivalents (GAE)/100 g for total phenols, 141.14 mg/100 g for vitamin C and 429.9 µg/100 g for vitamin E. The antioxidant capacity of Or was: 2.27 mmol of Trolox® equivalents (TE)/L (DPPH), 62.2 ± 5.0 µmol TE/g (ABTSâ¢+), 80.2 ± 11.7 µmol TE/g (FRAP), 247.9 ± 15.6 µmol TE/g (AAPH) and 15.0% of H2O2 elimination. An increase (p < 0.05) of aminotransferases and a decrease (p < 0.05) of hepatic GSH was observed in the TAA group compared to the control and the concomitant groups. Histopathology showed changes in the normal architecture of the liver treated with TAA compared to the concomitant treatments. Or contains bioactive components with antioxidant capacity, which can reduce fibrotic liver damage.
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In recent years, knowledge on the biology and pathobiology of extracellular vesicles (EVs) has exploded. EVs are submicron membrane-bound structures secreted from different cell types containing a wide variety of bioactive molecules (e.g., proteins, lipids, and nucleic acids (coding and non-coding RNA) and mitochondrial DNA). EVs have important functions in cell-to-cell communication and are found in a wide variety of tissues and body fluids. Better delineation of EV structures and advances in the isolation and characterization of their cargo have allowed the diagnostic and therapeutic implications of these particles to be explored. In the field of liver diseases, EVs are emerging as key players in the pathogenesis of both nonalcoholic liver disease (NAFLD) and alcoholic liver disease (ALD), the most prevalent liver diseases worldwide, and their complications, including development of hepatocellular carcinoma. In these diseases, stressed/damaged hepatocytes release large quantities of EVs that contribute to the occurrence of inflammation, fibrogenesis, and angiogenesis, which are key pathobiological processes in liver disease progression. Moreover, the specific molecular signatures of released EVs in biofluids have allowed EVs to be considered as promising candidates to serve as disease biomarkers. Additionally, different experimental studies have shown that EVs may have potential for therapeutic use as a liver-specific delivery method of different agents, taking advantage of their hepatocellular uptake through interactions with specific receptors. In this review, we focused on the most recent findings concerning the role of EVs as new structures mediating autocrine and paracrine intercellular communication in both ALD and NAFLD, as well as their potential use as biomarkers of disease severity and progression. Emerging therapeutic applications of EVs in these liver diseases were also examined, along with the potential for successful transition from bench to clinic.
Assuntos
Vesículas Extracelulares/patologia , Hepatopatias Alcoólicas/patologia , Hepatopatias Alcoólicas/terapia , Hepatopatia Gordurosa não Alcoólica/patologia , Hepatopatia Gordurosa não Alcoólica/terapia , Biomarcadores/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Fígado/metabolismo , Fígado/patologia , Hepatopatias Alcoólicas/metabolismo , Modelos Biológicos , Hepatopatia Gordurosa não Alcoólica/metabolismoRESUMO
BACKGROUND: Obstructive sleep apnea syndrome (OSAS) is associated to intermittent hypoxia (IH) and is an aggravating factor of non-alcoholic fatty liver disease (NAFLD). We investigated the effects of hypoxia in both in vitro and in vivo models of NAFLD. METHODS: Primary rat hepatocytes treated with free fatty acids (FFA) were subjected to chemically induced hypoxia (CH) using the hypoxia-inducible factor-1 alpha (HIF-1α) stabilizer cobalt chloride (CoCl2). Triglyceride (TG) content, mitochondrial superoxide production, cell death rates, cytokine and inflammasome components gene expression and protein levels of cleaved caspase-1 were assessed. Also, Kupffer cells (KC) were treated with conditioned medium (CM) and extracellular vehicles (EVs) from hypoxic fat-laden hepatic cells. The choline deficient L-amino acid defined (CDAA)-feeding model used to assess the effects of IH on experimental NAFLD in vivo. RESULTS: Hypoxia induced HIF-1α in cells and animals. Hepatocytes exposed to FFA and CoCl2 exhibited increased TG content and higher cell death rates as well as increased mitochondrial superoxide production and mRNA levels of pro-inflammatory cytokines and of inflammasome-components interleukin-1ß, NLRP3 and ASC. Protein levels of cleaved caspase-1 increased in CH-exposed hepatocytes. CM and EVs from hypoxic fat-laden hepatic cells evoked a pro-inflammatory phenotype in KC. Livers from CDAA-fed mice exposed to IH exhibited increased mRNA levels of pro-inflammatory and inflammasome genes and increased levels of cleaved caspase-1. CONCLUSION: Hypoxia promotes inflammatory signals including inflammasome/caspase-1 activation in fat-laden hepatocytes and contributes to cellular crosstalk with KC by release of EVs. These mechanisms may underlie the aggravating effect of OSAS on NAFLD. [Abstract word count: 257].
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Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Hipóxia/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Hepatopatia Gordurosa não Alcoólica/genética , Apneia Obstrutiva do Sono/genética , Animais , Caspase 1/genética , Deficiência de Colina/genética , Deficiência de Colina/metabolismo , Deficiência de Colina/patologia , Cobalto/toxicidade , Modelos Animais de Doenças , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patologia , Ácidos Graxos não Esterificados/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Hipóxia/induzido quimicamente , Hipóxia/metabolismo , Hipóxia/patologia , Inflamassomos/genética , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Interleucina-1beta/genética , Células de Kupffer/metabolismo , Células de Kupffer/patologia , Camundongos , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Ratos , Apneia Obstrutiva do Sono/etiologia , Apneia Obstrutiva do Sono/metabolismo , Apneia Obstrutiva do Sono/patologia , Triglicerídeos/genéticaRESUMO
Therapy for nonalcoholic steatohepatitis (NASH) is limited. Andrographolide (ANDRO), a botanical compound, has a potent anti-inflammatory activity due to its ability to inhibit NF-κB. ANDRO has been also shown to inhibit the NLRP3 inflammasome, a relevant pathway in NASH. Our aim was to evaluate the effects of ANDRO in NASH and its influence on inflammasome activation in this setting. Thus, mice were fed a choline-deficient-amino-acid-defined (CDAA) diet with/without concomitant ANDRO administration (1 mg/kg, 3-times/week). Also, we assessed serum levels of alanine-aminotransferase (ALT), liver histology, hepatic triglyceride content (HTC) and hepatic expression of pro-inflammatory, pro-fibrotic and inflammasome genes. Inflammasome activation was also evaluated in fat-laden HepG2 cells. Our results showed that ANDRO administration decreased HTC and attenuated hepatic inflammation and fibrosis in CDAA-fed mice. ANDRO treatment determined a strong reduction in hepatic macrophage infiltration and reduced hepatic mRNA levels of both pro-inflammatory and pro-fibrotic genes. In addition, mice treated with ANDRO showed reduced expression of inflammasome genes. Finally, ANDRO inhibited LPS-induced interleukin-1ß expression through NF-κB inhibition in fat-laden HepG2 cells and inflammasome disassembly. In conclusion, ANDRO administration reduces inflammation and fibrosis in experimental NASH. Inflammasome modulation by a NF-κB-dependent mechanism may be involved in the therapeutic effects of ANDRO.
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Anti-Inflamatórios não Esteroides/administração & dosagem , Diterpenos/administração & dosagem , Inflamassomos/metabolismo , Inflamação/prevenção & controle , Hepatopatia Gordurosa não Alcoólica/complicações , Animais , Células Cultivadas , Fibrose/prevenção & controle , Inflamação/complicações , Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Ratos WistarRESUMO
Acetaminophen (APAP)-induced acute liver failure (ALF) is a serious health problem in developed countries. N-acetyl-L-cysteine (NAC), the current therapy for APAP-induced ALF, is not always effective, and liver transplantation is often needed. Opuntia spp. fruits are an important source of nutrients and contain high levels of bioactive compounds, including antioxidants. The aim of this study was to evaluate the hepatoprotective effect of Opuntia robusta and Opuntia streptacantha extracts against APAP-induced ALF. In addition, we analyzed the antioxidant activities of these extracts. Fruit extracts (800mg/kg/day, orally) were given prophylactically to male Wistar rats before intoxication with APAP (500 mg/kg, intraperitoneally). Rat hepatocyte cultures were exposed to 20mmol/LAPAP, and necrosis was assessed by LDH leakage. Opuntia robusta had signiï¬cantly higher levels of antioxidants than Opuntia streptacantha. Both extracts signiï¬cantly attenuated APAP-induced injury markers AST, ALT and ALP and improved liver histology. The Opuntia extracts reversed APAP-induced depletion of liver GSH and glycogen stores. In cultured hepatocytes, Opuntia extracts signiï¬cantly reduced leakage of LDH and cell necrosis, both prophylactically and therapeutically. Both extracts appeared to be superior to NAC when used therapeutically. We conclude that Opuntia extracts are hepatoprotective and can be used as a nutraceutical to prevent ALF.
Assuntos
Antioxidantes/farmacologia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Opuntia/química , Fitoterapia , Extratos Vegetais/farmacologia , Acetaminofen/efeitos adversos , Analgésicos não Narcóticos/efeitos adversos , Animais , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Suplementos Nutricionais , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Masculino , Ratos , Ratos WistarRESUMO
BACKGROUND: Therapeutic options to treat Non-alcoholic steatohepatitis (NASH) are limited. Mineralocorticoid receptor (MR) activation could play a role in hepatic fibrogenesis and its modulation could be beneficial for NASH. AIM: To investigate whether eplerenone, a specific MR antagonist, ameliorates liver damage in experimental NASH. METHODS: C57bl6 mice were fed a choline-deficient and amino acid-defined (CDAA) diet for 22 weeks with or without eplerenone supplementation. Serum levels of aminotransferases and aldosterone were measured and hepatic steatosis, inflammation and fibrosis scored histologically. Hepatic triglyceride content (HTC) and hepatic mRNA levels of pro-inflammatory pro-fibrotic, oxidative stress-associated genes and of MR were also assessed. RESULTS: CDAA diet effectively induced fibrotic NASH, and increased the hepatic expression of pro-inflammatory, pro-fibrotic and oxidative stress-associated genes. Hepatic MR mRNA levels significantly correlated with the expression of pro-inflammatory and pro-fibrotic genes and were significantly increased in hepatic stellate cells obtained from CDAA-fed animals. Eplerenone administration was associated to a reduction in histological steatosis and attenuation of liver fibrosis development, which was associated to a significant decrease in the expression of collagen-α1, collagen type III, alpha 1 and Matrix metalloproteinase-2. CONCLUSION: The expression of MR correlates with inflammation and fibrosis development in experimental NASH. Specific MR blockade with eplerenone has hepatic anti-steatotic and anti-fibrotic effects. These data identify eplerenone as a potential novel therapy for NASH. Considering its safety and FDA-approved status, human studies are warranted.