RESUMEN
BACKGROUND AND AIMS: Apoptosis Signal-regulating Kinase 1 (ASK1) is activated by various pathological stimuli and induces cell apoptosis through downstream p38 activation. We studied the effect of pharmacological ASK1 inhibition on cirrhosis and its sequelae using comprehensive preclinical in vivo and in vitro systems. APPROACH AND RESULTS: Short-term (4-6 wk) and long-term (24-44 wk) ASK1 inhibition using small molecule GS-444217 was tested in thioacetamide-induced and BALB/c. Mdr2-/- murine models of cirrhosis and HCC, and in vitro using primary hepatocyte cell death assays. Short-term GS-444217 therapy in both models strongly reduced phosphorylated p38, hepatocyte death, and fibrosis by up to 50%. Profibrogenic release of mitochondrial DAMP mitochondrial deoxyribonucleic acid from dying hepatocytes was blocked by ASK1 or p38 inhibition. Long-term (24 wk) therapy in BALBc.Mdr2 - / - model resulted in a moderate 25% reduction in bridging fibrosis, but not in net collagen deposition. Despite this, the development of cirrhosis was effectively prevented, with strongly reduced p21 + hepatocyte staining (by 72%), serum ammonia levels (by 46%), and portal pressure (average 6.07 vs. 8.53 mm Hg in controls). Extended ASK1 inhibition for 44 wk in aged BALB/c. Mdr2-/- mice resulted in markedly reduced tumor number and size by ~50% compared to the control group. CONCLUSIONS: ASK1 inhibition suppresses the profibrogenic release of mitochondrial deoxyribonucleic acid from dying hepatocytes in a p38-dependent manner and protects from liver fibrosis. Long-term ASK1 targeting resulted in diminished net antifibrotic effect, but the progression to liver cirrhosis and cancer in BALBc/ Mdr2- / - mice was effectively inhibited. These data support the clinical evaluation of ASK1 inhibitors in fibrotic liver diseases.
Asunto(s)
Progresión de la Enfermedad , Hepatocitos , Cirrosis Hepática , Neoplasias Hepáticas , MAP Quinasa Quinasa Quinasa 5 , Ratones Endogámicos BALB C , Proteínas Quinasas p38 Activadas por Mitógenos , Animales , MAP Quinasa Quinasa Quinasa 5/antagonistas & inhibidores , MAP Quinasa Quinasa Quinasa 5/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Ratones , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/patología , Cirrosis Hepática/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Masculino , Tioacetamida/toxicidad , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Modelos Animales de EnfermedadRESUMEN
Liver cirrhosis is a silent disease in humans and is experimentally induced by many drugs and toxins as thioacetamide (TAA) in particular, which is the typical model for experimental induction of hepatic fibrosis. Thus, the objective of the present study was to elucidate the possible protective effects of lactéol® forte (LF) and quercetin dihydrate (QD) against TAA-induced hepatic damage in male albino rats. Induction of hepatotoxicity was performed by TAA injection (200 mg/kg I/P, twice/ week) in rats. LF (1 × 109 CFU/rat 5 times/week) and QD (50 mg/kg 5 times/week) treated groups were administered concurrently with TAA injection (200 mg/kg I/P, twice/ week). The experimental treatments were conducted for 12 weeks. Hepatotoxicity was evaluated biochemically by measuring alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyl transferase (GGT) in the serum and histopathologically with the scoring of histopathological changes besides histochemical assessment of collagen by Masson's trichrome and immunohistochemical analysis for α-smooth muscle actin (α-SMA), Ki67 and caspase-3 expression in liver sections. Our results indicated that LF and QD attenuated some biochemical changes and histochemical markers in TAA-mediated hepatotoxicity in rats by amelioration of biochemical markers and collagen, α-SMA, Ki67 and caspase3 Immunoexpression. Additionally, LF and QD supplementation downregulated the proliferative, necrotic, fibroblastic changes, eosinophilic intranuclear inclusions, hyaline globules and Mallory-like bodies that were detected histopathologically in the TAA group. In conclusion, LF showed better hepatic protection than QD against TAA-induced hepatotoxicity in rats by inhibiting inflammatory reactions with the improvement of some serum hepatic transaminases, histopathological picture and immunohistochemical markers.
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Carbonato de Calcio , Enfermedad Hepática Inducida por Sustancias y Drogas , Lactosa , Quercetina , Humanos , Ratas , Masculino , Animales , Quercetina/farmacología , Tioacetamida/toxicidad , Antígeno Ki-67/metabolismo , Cirrosis Hepática/metabolismo , Hígado/metabolismo , Flavonoides/farmacología , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Colágeno/metabolismo , Estrés Oxidativo , Combinación de MedicamentosRESUMEN
BACKGROUND: Hepatic encephalopathy (HE) is closely associated with inflammatory responses. However, as a crucial regulator of the immune and inflammatory responses, the role of leucine-rich repeat kinase 2 (LRRK2) in the pathogenesis of HE remains unraveled. Herein, we investigated this issue in thioacetamide (TAA)-induced HE following acute liver failure (ALF). METHODS: TAA-induced HE mouse models of LRRK2 wild type (WT), LRRK2 G2019S mutation (Lrrk2G2019S) and LRRK2 knockout (Lrrk2-/-) were established. A battery of neurobehavioral experiments was conducted. The biochemical indexes and pro-inflammatory cytokines were detected. The prefrontal cortex (PFC), striatum (STR), hippocampus (HIP), and liver were examined by pathology and electron microscopy. The changes of autophagy-lysosomal pathway and activity of critical Rab GTPases were analyzed. RESULTS: The Lrrk2-/--HE model reported a significantly lower survival rate than the other two models (24% vs. 48%, respectively, p < 0.05), with no difference found between the WT-HE and Lrrk2G2019S-HE groups. Compared with the other groups, after the TAA injection, the Lrrk2-/- group displayed a significant increase in ammonium and pro-inflammatory cytokines, aggravated hepatic inflammation/necrosis, decreased autophagy, and abnormal phosphorylation of lysosomal Rab10. All three models reported microglial activation, neuronal loss, disordered vesicle transmission, and damaged myelin structure. The Lrrk2-/--HE mice presented no severer neuronal injury than the other genotypes. CONCLUSIONS: LRRK2 deficiency may exacerbate TAA-induced ALF and HE in mice, in which inflammatory response is evident in the brain and aggravated in the liver. These novel findings indicate a need of sufficient clinical awareness of the adverse effects of LRRK2 inhibitors on the liver.
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Encefalopatía Hepática , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Fallo Hepático Agudo , Ratones Noqueados , Tioacetamida , Animales , Ratones , Encefalopatía Hepática/patología , Encefalopatía Hepática/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Fallo Hepático Agudo/inducido químicamente , Fallo Hepático Agudo/patología , Fallo Hepático Agudo/genética , Ratones Endogámicos C57BL , Tioacetamida/toxicidadRESUMEN
BACKGROUND AND PURPOSE: Liver fibrosis is a wound-healing reaction which is the main cause of chronic liver diseases worldwide. The activated hepatic stellate cell (aHSC) is the main driving factor in the development of liver fibrosis. Inhibiting autophagy of aHSC can prevent the progression of liver fibrosis, but inhibiting autophagy of other liver cells has opposite effects. Hence, targeted inhibition of autophagy in aHSC is quite necessary for the treatment of liver fibrosis, which prompts us to explore the targeted delivery system of small molecule autophagy inhibitor hydroxychloroquine (HCQ) that can target aHSC and alleviate the liver fibrosis. METHODS: The delivery system of HCQ@retinol-liposome nanoparticles (HCQ@ROL-LNPs) targeting aHSC was constructed by the film dispersion and pH-gradient method. TGF-ß-induced HSC activation and thioacetamide (TAA)-induced liver fibrosis mice model were established, and the targeting ability and therapeutic effect of HCQ@ROL-LNPs in liver fibrosis were studied subsequently in vitro and in vivo. RESULTS: HCQ@ROL-LNPs have good homogeneity and stability. They inhibited the autophagy of aHSC selectively by HCQ and reduced the deposition of extracellular matrix (ECM) and the damage to other liver cells. Compared with the free HCQ and HCQ@LNPs, HCQ@ROL-LNPs had good targeting ability, showing enhanced therapeutic effect and low toxicity to other organs. CONCLUSION: Construction of HCQ@ROL-LNPs delivery system lays a theoretical and experimental foundation for the treatment of liver fibrosis and promotes the development of clinical therapeutic drugs for liver diseases.
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Autofagia , Células Estrelladas Hepáticas , Hidroxicloroquina , Cirrosis Hepática , Hidroxicloroquina/farmacología , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Animales , Autofagia/efectos de los fármacos , Ratones , Cirrosis Hepática/tratamiento farmacológico , Liposomas , Nanopartículas , Masculino , Modelos Animales de Enfermedad , Humanos , Tioacetamida , Ratones Endogámicos C57BLRESUMEN
Hepatic encephalopathy (HE) is a debilitating neurological disorder associated with liver failure and characterized by impaired brain function. Decade-long studies have led to significant advances in our understanding of HE; however, effective therapeutic management of HE is lacking, and HE continues to be a significant cause of morbidity and mortality in patients, underscoring the need for continued research into its pathophysiology and treatment. Accordingly, the present study provides a comprehensive overview aimed at elucidating the molecular underpinnings of HE and identifying potential therapeutic targets. A moderate-grade HE model was induced in rats using thioacetamide, which simulates the liver damage observed in patients, and its impact on cognitive function, neuronal arborization, and cellular morphology was also evaluated. We employed label-free LC-MS/MS proteomics to quantitatively profile hippocampal proteins to explore the molecular mechanism of HE pathogenesis; 2175 proteins were identified, 47 of which exhibited significant alterations in moderate-grade HE. The expression of several significantly upregulated proteins, such as FAK1, CD9 and Tspan2, was further validated at the transcript and protein levels, confirming the mass spectrometry results. These proteins have not been previously reported in HE. Utilizing Metascape, a tool for gene annotation and analysis, we further studied the biological pathways integral to brain function, including gliogenesis, the role of erythrocytes in maintaining blood-brain barrier integrity, the modulation of chemical synaptic transmission, astrocyte differentiation, the regulation of organ growth, the response to cAMP, myelination, and synaptic function, which were disrupted during HE. The STRING database further elucidated the proteinâprotein interaction patterns among the differentially expressed proteins. This study provides novel insights into the molecular mechanisms driving HE and paves the way for identifying novel therapeutic targets for improved disease management.
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Encefalopatía Hepática , Hipocampo , Proteoma , Ratas Sprague-Dawley , Animales , Hipocampo/metabolismo , Encefalopatía Hepática/metabolismo , Proteoma/metabolismo , Masculino , Ratas , Proteómica/métodos , Modelos Animales de Enfermedad , Espectrometría de Masas en Tándem , TioacetamidaRESUMEN
Iron overload has been recognized as a risk factor for liver disease; however, little is known about its pathological role in the modification of liver injury. The purpose of this study is to investigate the influence of iron overload on liver injury induced by two hepatotoxicants with different pathogenesis in rats. Rats were fed a control (Cont), 0.8% high-iron (0.8% Fe), or 1% high-iron diet (1% Fe) for 4 weeks and were then administered with saline, thioacetamide (TAA), or carbon tetrachloride (CCl4). Hepatic and systemic iron overload were seen in the 0.8% and 1% Fe groups. Twenty-four hours after administration, hepatocellular necrosis induced by TAA and hepatocellular necrosis, degeneration, and vacuolation induced by CCl4, as well as serum transaminase values, were exacerbated in the 0.8% and 1% Fe groups compared to the Cont group. On the other hand, microvesicular vacuolation induced by CCl4 was decreased in 0.8% and 1% Fe groups. Hepatocellular DNA damage was increased by iron overload in both models, whereas a synergistic effect of oxidative stress by excess iron and hepatotoxicant was only present in the CCl4 model. The data showed that dietary iron overload exacerbates TAA- and CCl4-induced acute liver injury with different mechanisms.
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Tetracloruro de Carbono , Enfermedad Hepática Inducida por Sustancias y Drogas , Sobrecarga de Hierro , Hígado , Tioacetamida , Animales , Tioacetamida/toxicidad , Ratas , Tetracloruro de Carbono/toxicidad , Masculino , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Hígado/efectos de los fármacos , Hígado/patología , Estrés Oxidativo/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Ratas Sprague-Dawley , Hierro/toxicidadRESUMEN
BACKGROUND: TGF-ß1 and SMAD3 are particularly pathogenic in the progression of renal fibrosis. AIM: This study aimed to evaluate the kidney protective potentials of silymarin (SM) and exosomes of mesenchymal stem cells against the nephrotoxin thioacetamide (TAA) in rats. METHODS: 32 female rats were randomly assigned into four groups: the control group, the TAA group, the TAA + SM group, and the TAA + Exosomes group. The kidney homogenates from all groups were examined for expression levels of TGF-ß receptors I and II using real-time PCR, expression levels of collagen type I and CTGF proteins using ELISA, and the expression levels of nuclear SMAD2/3/4, cytoplasmic SMAD2/3, and cytoplasmic SMAD4 proteins using the western blot technique. RESULTS: Compared to the control group, the injection of TAA resulted in a significant increase in serum levels of urea and creatinine, gene expression levels of TßRI and TßRII, protein expression levels of both collagen I and CTGF proteins, cytoplasmic SMAD2/3 complex, and nuclear SMAD2/3/4 (p-value < 0.0001), with significantly decreased levels of the co-SMAD partner, SMAD4 (p-value < 0.0001). Those effects were reversed considerably in both treatment groups, with the superiority of the exosomal treatment regarding the SMAD proteins and the expression levels of the TßRI gene, collagen I, and CTGF proteins returning to near-control values (p-value > 0.05). CONCLUSION: Using in vitro and in vivo experimental approaches, the research discovered a reno-protective role of silymarin and exosomes of BM-MSCs after thioacetamide-induced renal fibrosis in rats, with the advantage of exosomes.
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Exosomas , Enfermedades Renales , Silimarina , Ratas , Femenino , Animales , Factor de Crecimiento Transformador beta/metabolismo , Tioacetamida/toxicidad , Tioacetamida/metabolismo , Silimarina/farmacología , Exosomas/metabolismo , Fibrosis , Factor de Crecimiento Transformador beta1/metabolismo , Enfermedades Renales/patología , Colágeno Tipo I/metabolismo , Proteínas Smad/metabolismoRESUMEN
Chronic liver injury due to various etiological factors results in excess secretion and accumulation of extracellular matrix proteins, leading to scarring of liver tissue and ultimately to hepatic fibrosis. If left untreated, fibrosis might progress to cirrhosis and even hepatocellular carcinoma. Thymoquinone (TQ), an active compound of Nigella sativa, has been reported to exhibit antioxidant, anti-inflammatory and anticancer activities. Therefore, the effect of TQ against thioacetamide (TAA)-induced liver fibrosis was assessed in rats. Fibrosis was induced with intraperitoneal administration of TAA (250 mg/kg b.w.) twice a week for 5 weeks. TQ (20 mg/kg b.w.) and silymarin (50 mg/kg b.w.) were orally administered daily for 5 weeks separately in TAA administered groups. Liver dysfunction was reported by elevated liver enzymes, increased oxidative stress, inflammation and fibrosis upon TAA administration. Our study demonstrated that TQ inhibited the elevation of liver marker enzymes in serum. TQ administration significantly increased antioxidant markers, such as superoxide dismutase, catalase, glutathione, glutathione peroxidase and glutathione reductase in the liver tissue of rats. Further, TQ significantly attenuated liver fibrosis, as illustrated by the downregulation of TAA-induced interleukin-ß, tumour necrosis factor-α, inducible nitric oxide synthase and fibrosis markers like transforming growth factor-ß (TGF-ß), α-smooth muscle actin, collagen-1, Smad3 and 7. Therefore, these findings suggest that TQ has a promising hepatoprotective property, as indicated by its potential to effectively suppress TAA-induced liver fibrosis in rats by inhibiting oxidative stress and inflammation via TGF-ß/Smad signaling.
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Benzoquinonas , Neoplasias Hepáticas , Factor de Crecimiento Transformador beta1 , Ratas , Animales , Factor de Crecimiento Transformador beta1/metabolismo , Tioacetamida/toxicidad , Antioxidantes/metabolismo , Hígado/metabolismo , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/prevención & control , Factor de Crecimiento Transformador beta/metabolismo , Inflamación/metabolismo , Estrés Oxidativo , Neoplasias Hepáticas/metabolismoRESUMEN
Thioacetamide (TAA) within the liver generates hepatotoxic metabolites that can be induce hepatic fibrosis, similar to the clinical pathological features of chronic human liver disease. The potential protective effect of Albiflorin (ALB), a monoterpenoid glycoside found in Paeonia lactiflora Pall, against hepatic fibrosis was investigated. The mouse hepatic fibrosis model was induced with an intraperitoneal injection of TAA. Hepatic stellate cells (HSCs) were subjected to treatment with transforming growth factor-beta (TGF-ß), while lipopolysaccharide/adenosine triphosphate (LPS/ATP) was added to stimulate mouse peritoneal macrophages (MPMs), leading to the acquisition of conditioned medium. For TAA-treated mice, ALB reduced ALT, AST, HYP levels in serum or liver. The administration of ALB reduced histopathological abnormalities, and significantly regulated the expressions of nuclear receptor-related 1 protein (NURR1) and the P2X purinoceptor 7 receptor (P2×7r) in liver. ALB could suppress HSCs epithelial-mesenchymal transition (EMT), extracellular matrix (ECM) deposition, and pro-inflammatory factor level. ALB also remarkably up-regulated NURR1, inhibited P2×7r signaling pathway, and worked as working as C-DIM12, a NURR1 agonist. Moreover, deficiency of NURR1 in activated HSCs and Kupffer cells weakened the regulatory effect of ALB on P2×7r inhibition. NURR1-mediated inhibition of inflammatory contributed to the regulation of ALB ameliorates TAA-induced hepatic fibrosis, especially based on involving in the crosstalk of HSCs-macrophage. Therefore, ALB plays a significant part in the mitigation of TAA-induced hepatotoxicity this highlights the potential of ALB as a protective intervention for hepatic fibrosis.
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Células Estrelladas Hepáticas , Cirrosis Hepática , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Transducción de Señal , Tioacetamida , Animales , Tioacetamida/toxicidad , Células Estrelladas Hepáticas/efectos de los fármacos , Ratones , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Masculino , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Hidrocarburos Aromáticos con Puentes/farmacología , Ratones Endogámicos C57BL , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Transición Epitelial-Mesenquimal/efectos de los fármacosRESUMEN
Liver fibrosis affects approximately 800 million patients worldwide, with over 2 million deaths each year. Nevertheless, there are no approved medications for treating liver fibrosis. In this study, we investigated the impacts of ginkgetin on liver fibrosis and the underlying mechanisms. The impacts of ginkgetin on liver fibrosis were assessed in mouse models induced by thioacetamide or bile duct ligation. Experiments on human LX-2 cells and primary mouse hepatic stellate cells (HSCs) were performed to explore the underlying mechanisms, which were also validated in the mouse models. Ginkgetin significantly decreased hepatic extracellular matrix deposition and HSC activation in the fibrotic models induced by thioacetamide (TAA) and bile duct ligation (BDL). Beneficial effects also existed in inhibiting hepatic inflammation and improving liver function. In vitro experiments showed that ginkgetin markedly inhibited HSC viability and induced HSC apoptosis dose-dependently. Mechanistic studies revealed that the antifibrotic effects of ginkgetin depend on STAT1 activation, as the effects were abolished in vitro after STAT1 silencing and in vivo after inhibiting STAT1 activation by fludarabine. Moreover, we observed a meaningful cross-talk between HSCs and hepatocytes, in which IL-6, released by ginkgetin-induced apoptotic HSCs, enhanced hepatocyte proliferation by activating STAT3 signaling. Ginkgetin exhibits antifibrotic effects by inducing HSC apoptosis via STAT1 activation and enhances hepatocyte proliferation secondary to HSC apoptosis via the IL-6/STAT3 pathway.
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Biflavonoides , Células Estrelladas Hepáticas , Tioacetamida , Ratones , Animales , Humanos , Tioacetamida/metabolismo , Tioacetamida/farmacología , Tioacetamida/uso terapéutico , Interleucina-6/metabolismo , Cirrosis Hepática/tratamiento farmacológico , Modelos Animales de Enfermedad , Apoptosis , Hígado/metabolismo , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT1/farmacologíaRESUMEN
Although curcumin possesses anti-inflammatory, antioxidant, and cytoprotective qualities, its low absorption limits its medicinal uses. Before examining how curcumin influenced rats' liver fibrosis when thioacetamide (TAA) was produced, the current study employed nanoparticles (NPs) to improve curcumin bioavailability. Sixty mature rats were separated into six groups (Group 1, control; Group 2, curcumin; Group 3, curcumin nanoparticles; Group 4, TAA; Group 5, TAA + curcumin; Group 6, TAA + curcumin NPs). TAA administration caused considerable increases in serum liver enzymes associated with a remarkable depletion in the levels of albumin and total protein relative to the control. In addition, a significant elevation in malonaldehyde (MDA) level with a significant depletion in the antioxidant enzymes activity was detected. Also, TAA had a significant effect on the inflammation markers represented by the elevation in tumor necrosis factor (TNFα) and DNA damage. Administration of curcumin or curcumin NPs in TAA-intoxicated rats significantly (p < .001, p < .0001) alleviates liver injury by correcting antioxidant status, inflammatory markers, and oxidative stress. The results of comparing TAA-intoxicated rats treated with curcumin NPs to TAA-intoxicated rats treated with bulk curcumin revealed that the ameliorative effect of nanocurcumin was stronger. These observations concluded that nanoparticle formulation can increase curcumin bioavailability and solubility, enhancing its antioxidant and anti-inflammatory efficiency, resulting in greater potential against thioacetamide-induced hepatotoxicity in rats.
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Curcumina , Nanopartículas , Ratas , Animales , Antioxidantes/farmacología , Antioxidantes/metabolismo , Curcumina/farmacología , Curcumina/uso terapéutico , Tioacetamida/toxicidad , Hígado , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/metabolismo , Estrés Oxidativo , Antiinflamatorios/farmacología , Nanopartículas/uso terapéuticoRESUMEN
BACKGROUND: Acute liver failure (ALF) is an unpredictable and life-threatening critical illness. The pathological characteristic of ALF is massive necrosis of hepatocytes and lots of inflammatory cells infiltration which may lead to multiple organ failure. METHODS: Animals were divided into 3 groups, normal, thioacetamide (TAA, ALF model) and TAA + AGK2. Cultured L02 cells were divided into 5 groups, normal, TAA, TAA + mitofusin 2 (MFN2)-siRNA, TAA + AGK2, and TAA + AGK2 + MFN2-siRNA groups. The liver histology was evaluated with hematoxylin and eosin staining, inositol-requiring enzyme 1 (IRE1), activating transcription factor 6ß (ATF6ß), protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) and phosphorylated-PERK (p-PERK). C/EBP homologous protein (CHOP), reactive oxygen species (ROS), MFN2 and glutathione peroxidase 4 (GPX4) were measured with Western blotting, and cell viability and liver chemistry were also measured. Mitochondria-associated endoplasmic reticulum membranes (MAMs) were measured by immunofluorescence. RESULTS: The liver tissue in the ALF group had massive inflammatory cell infiltration and hepatocytes necrosis, which were reduced by AGK2 pre-treatment. In comparison to the normal group, apoptosis rate and levels of IRE1, ATF6ß, p-PERK, CHOP, ROS and Fe2+ in the TAA-induced ALF model group were significantly increased, which were decreased by AGK2 pre-treatment. The levels of MFN2 and GPX4 were decreased in TAA-induced mice compared with the normal group, which were enhanced by AGK2 pre-treatment. Compared with the TAA-induced L02 cell, apoptosis rate and levels of IRE1, ATF6ß, p-PERK, CHOP, ROS and Fe2+ were further increased and levels of MFN2 and GPX4 were decreased in the MFN2-siRNA group. AGK2 pre-treatment decreased the apoptosis rate and levels of IRE1, ATF6ß, p-PERK, CHOP, ROS and Fe2+ and enhanced the protein expression of MFN2 and GPX4 in MFN2-siRNA treated L02 cell. Immunofluorescence observation showed that level of MAMs was promoted in the AGK2 pre-treatment group when compared with the TAA-induced group in both mice and L02 cells. CONCLUSIONS: The data suggested that AGK2 pre-treatment had hepatoprotective role in TAA-induced ALF via upregulating the expression of MFN2 and then inhibiting PERK and ferroptosis pathway in ALF.
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Ferroptosis , Fallo Hepático Agudo , Ratones , Animales , Tioacetamida/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Fallo Hepático Agudo/inducido químicamente , Fallo Hepático Agudo/prevención & control , Transducción de Señal , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/efectos adversos , Proteínas Serina-Treonina Quinasas/metabolismo , Apoptosis , Necrosis , ARN Interferente Pequeño/efectos adversos , Estrés del Retículo Endoplásmico/genéticaRESUMEN
Animal studies are typically utilized to understand the complex mechanisms associated with toxicant-induced hepatotoxicity. Among the alternative approaches to animal studies, in vitro pooled human hepatocytes have the potential to capture population variability. Here, we examined the effect of the hepatotoxicant thioacetamide on pooled human hepatocytes, divided into five lots, obtained from forty diverse donors. For 24 h, pooled human hepatocytes were exposed to vehicle, 1.33 mM (low dose), and 12 mM (high dose) thioacetamide, followed by RNA-seq analysis. We assessed gene expression variability using heat maps, correlation plots, and statistical variance. We used KEGG pathways and co-expression modules to identify underlying physiological processes/pathways. The co-expression module analysis showed that the majority of the lots exhibited activation for the bile duct proliferation module. Despite lot-to-lot variability, we identified a set of common differentially expressed genes across the lots with similarities in their response to amino acid, lipid, and carbohydrate metabolism. We also examined efflux transporters and found larger lot-to-lot variability in their expression patterns, indicating a potential for alteration in toxicant bioavailability within the cells, which could in turn affect the gene expression patterns between the lots. Overall, our analysis highlights the challenges in using pooled hepatocytes to understand mechanisms of toxicity.
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Enfermedad Hepática Inducida por Sustancias y Drogas , Tioacetamida , Animales , Humanos , Tioacetamida/toxicidad , Hígado/metabolismo , Hepatocitos/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismoRESUMEN
Current data on the molecular mechanisms of liver fibrosis and cirrhosis fail to fully explain all stages of their development. Interactions between individual genes and signaling pathways are known to play an important role in their functions. However, data on their relationships are insufficient and often contradictory. For the first time, mRNA expression of Notch1, Notch2, Yap1, Tweak (Tnfsf12), Fn14 (Tnfrsf12a), Ang, Vegfa, Cxcl12 (Sdf), Nos2, and Mmp-9 was studied in detail at several stages of thioacetamide-induced liver fibrosis in Wistar rats. A factor analysis isolated three factors, which combined highly correlated target genes. The first factor included four genes: Cxcl12 (r = 0.829, p < 0.05), Tweak (r = 0.841, p < 0.05), Notch1 (r = 0.848, p < 0.05), and Yap1 (r = 0.921, p < 0.05). The second factor described the correlation between Mmp-9 (r = 0.791, p < 0.05) and Notch2 (r = 0.836, p < 0.05). The third factor included Ang (r = 0.748, p < 0.05) and Vegfa (r = 0.679, p < 0.05). The Nos2 and Fn14 genes were not included in any of the factors. The gene grouping by mRNA expression levels made it possible to assume a pathogenetic relationship between their products in the development of fibrotic changes due to liver toxicity.
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Quimiocina CXCL12 , Citocina TWEAK , ARN Mensajero , Ratas Wistar , Receptor Notch1 , Proteínas Señalizadoras YAP , Animales , Ratas , Proteínas Señalizadoras YAP/genética , Proteínas Señalizadoras YAP/metabolismo , Masculino , Receptor Notch1/genética , Receptor Notch1/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Citocina TWEAK/genética , Citocina TWEAK/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Regulación de la Expresión Génica , Cirrosis Hepática/genética , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Cirrosis Hepática/inducido químicamente , Tioacetamida/toxicidad , Receptor Notch2/genética , Receptor Notch2/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismoRESUMEN
Background and Objectives: TAA is potent hepatic/renal toxicant. Conversely, WGO is a potent dietary supplement with impressive antioxidant properties. Olmutinib is an apoptotic chemotherapy drug that does not harm the liver or kidney. This study investigated the impact of olmutinib and wheat germ oil (WGO) on Thioacetamide (TAA)-induced gene alterations in mice liver and kidney tissues. Materials and Methods: Adult male C57BL/6 mice were exposed to 0.3% TAA in drinking water for 14 days, followed by the oral administration of olmutinib (30 mg/kg) and WGO (1400 mg/kg) for 5 consecutive days. Treatment groups included the following: groups I (control), II (TAA-exposed), III (TAA + olmutinib), IV (TAA + WGO), and V (TAA + olmutinib + WGO). Results: The findings revealed that TAA exposure increased MKi67 and CDKN3 gene expression in liver and kidney tissues. Olmutinib treatment effectively reversed these TAA-induced effects, significantly restoring MKi67 and CDKN3 gene expression. WGO also reversed MKi67 effects in the liver but exhibited limited efficacy in reversing CDKN3 gene alterations induced by TAA exposures in both the liver and kidney. TAA exposure showed the tissue-specific expression of TP53, with decreased expression in the liver and increased expression in the kidney. Olmutinib effectively reversed these tissue-specific alterations in TP53 expression. While WGO treatment alone could not reverse the gene alterations induced by TAA exposure, the co-administration of olmutinib and WGO exhibited a remarkable potentiation of therapeutic effects in both the liver and kidney. The gene interaction analysis revealed 77.4% of physical interactions and co-localization between MKi67, CDKN3, and TP53 expressions. Protein-protein interaction networks also demonstrated physical interactions between MKi67, TP53, and CDKN3, forming complexes or signaling cascades. Conclusions: It was predicted that the increased expression of the MKi67 gene by TAA leads to the increase in TP53, which negatively regulates the cell cycle via increased CDKN3 expression in kidneys and the restoration of TP53 levels in the liver. These findings contribute to our understanding of the effects of olmutinib and WGO on TAA-induced gene expression changes and highlight their contrasting effects based on cell cycle alterations.
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Riñón , Hígado , Ratones Endogámicos C57BL , Tioacetamida , Animales , Ratones , Riñón/efectos de los fármacos , Riñón/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ciclo Celular/efectos de los fármacos , TriticumRESUMEN
BACKGROUND AND AIMS: Minimal hepatic encephalopathy (MHE) is a mild form of hepatic encephalopathy that lacks observable signs and symptoms. Nevertheless, MHE can cause neurocognitive dysfunction, although the neurobiological mechanisms are not fully understood. Here, the effects of hippocampal iron deposition on cognitive function and its role in MHE were investigated. MATERIALS AND METHODS: Eighteen rats were assigned to experimental and control groups. MHE was induced by thioacetamide. Spatial memory and exploratory behavior were assessed by the Morris water and elevated plus mazes. Hippocampal susceptibility was measured by quantitative susceptibility mapping, iron deposition in the hippocampus and liver by Prussian blue staining, and inflammatory cytokine and ferritin levels in the hippocampus were measured by ELISA. RESULTS: MHE rats showed impaired spatial memory and exploratory behavior (P < 0.05 for all parameters). The bilateral hippocampal susceptibility values were significantly raised in MHE rats, together with evidence of neuroinflammation (increased pro-inflammatory and reduced anti-inflammatory cytokine levels (all P < 0.05). Further analysis indicated good correlations between hippocampal susceptibility values with latency time and inflammatory cytokine levels in MHE but not in control rats. CONCLUSION: MHE induced by thioacetamide was associated with hippocampal iron deposition and inflammation, suggesting that iron overload may be an important driver of neuroinflammatory responses.
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Disfunción Cognitiva , Encefalopatía Hepática , Sobrecarga de Hierro , Ratas , Animales , Encefalopatía Hepática/complicaciones , Enfermedades Neuroinflamatorias , Tioacetamida , Disfunción Cognitiva/etiología , Inflamación/inducido químicamente , Inflamación/complicaciones , Citocinas , Sobrecarga de Hierro/complicaciones , HierroRESUMEN
Portal hypertension (PT) commonly occurs in cirrhosis. Nitric oxide (NO) imbalance contributes to PT via reduced soluble guanylyl cyclase (sGC) activation and cGMP production, resulting in vasoconstriction, endothelial cell dysfunction, and fibrosis. We assessed the effects of BI 685509, an NO-independent sGC activator, on fibrosis and extrahepatic complications in a thioacetamide (TAA)-induced cirrhosis and PT model. Male Sprague-Dawley rats received TAA twice-weekly for 15 weeks (300-150 mg/kg i.p.). BI 685509 was administered daily for the last 12 weeks (0.3, 1, and 3 mg/kg p.o.; n = 8-11 per group) or the final week only (Acute, 3 mg/kg p.o.; n = 6). Rats were anesthetized to measure portal venous pressure. Pharmacokinetics and hepatic cGMP (target engagement) were measured by mass spectrometry. Hepatic Sirius Red morphometry (SRM) and alpha-smooth muscle actin (αSMA) were measured by immunohistochemistry; portosystemic shunting was measured using colored microspheres. BI 685509 dose-dependently increased hepatic cGMP at 1 and 3 mg/kg (3.92 ± 0.34 and 5.14 ± 0.44 versus 2.50 ± 0.19 nM in TAA alone; P < 0.05). TAA increased hepatic SRM, αSMA, PT, and portosystemic shunting. Compared with TAA, 3 mg/kg BI 685509 reduced SRM by 38%, αSMA area by 55%, portal venous pressure by 26%, and portosystemic shunting by 10% (P < 0.05). Acute BI 685509 reduced SRM and PT by 45% and 21%, respectively (P < 0.05). BI 685509 improved hepatic and extrahepatic cirrhosis pathophysiology in TAA-induced cirrhosis. These data support the clinical investigation of BI 685509 for PT in patients with cirrhosis. SIGNIFICANCE STATEMENT: BI 685509 is an NO-independent sGC activator that was tested in a preclinical rat model of TAA-induced nodular, liver fibrosis, portal hypertension, and portal systemic shunting. BI 685509 reduced liver fibrosis, portal hypertension, and portal-systemic shunting in a dose-dependent manner, supporting its clinical assessment to treat portal hypertension in patients with cirrhosis.
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Hipertensión Portal , Cirrosis Hepática Experimental , Ratas , Masculino , Animales , Guanilil Ciclasa Soluble/farmacología , Tioacetamida/efectos adversos , Ratas Sprague-Dawley , Cirrosis Hepática Experimental/inducido químicamente , Cirrosis Hepática Experimental/tratamiento farmacológico , Hipertensión Portal/tratamiento farmacológico , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/complicaciones , Hígado , GMP CíclicoRESUMEN
BACKGROUND: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death, so we should be concerned and look for effective/less-harmful treatments than chemotherapeutics already clinically in application. Aspirin works well ''in conjunction'' with other therapies for HCC since aspirin can boost the sensitivity of anti-cancer activity. Vitamin C also was shown to have antitumor effects. In this study, we examined the anti-HCC activities of synergistic combination (aspirin and vitamin C) vs. doxorubicin on HCC-bearing rats and hepatocellular carcinoma (HepG-2) cells. METHODS: In vitro, we evaluated IC50 and selectivity index (SI) using HepG-2 and human lung fibroblast (WI-38) cell lines. In vivo, four rat groups were used: Normal, HCC (intraperitoneally (i.p.) administered 200 mg thioacetamide/kg/twice a week), HCC + DOXO (HCC-bearing rats i.p. administered 0.72 mg doxorubicin (DOXO)/rat/once a week), and HCC + Aspirin + Vit. C (i.p. administered vitamin C (Vit. C) 4 g/kg/day after day concomitant with aspirin 60 mg/kg/orally day after day). We evaluated biochemical factors [aminotransferases (ALT and AST), albumin, and bilirubin (TBIL) spectrophotometrically, caspase 8 (CASP8), p53, Bcl2 associated X protein (BAX), caspase 3 (CASP3), alpha-fetoprotein (AFP), cancer antigen 19.9 (CA19.9), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) using ELISA], and liver histopathologically. RESULTS: HCC induction was accompanied by significant time-dependent elevations in all measured biochemical parameters except the p53 level significantly declined. Liver tissue architecture organization appeared disturbed with cellular infiltration, trabeculae, fibrosis, and neovascularization. Following drug medication, all biochemical levels significantly reversed toward normal, with fewer signs of carcinogenicity in liver tissues. Compared to doxorubicin, aspirin & vitamin C therapy ameliorations were more appreciated. In vitro, combination therapy (aspirin & vitamin C) exhibited potent cytotoxicity (HepG-2 IC50 of 17.41 ± 1.4 µg/mL) and more excellent safety with a SI of 3.663. CONCLUSIONS: Based on our results, aspirin plus vitamin C can be considered reliable, accessible, and efficient synergistic anti-HCC medication.
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Ácido Ascórbico , Aspirina , Carcinoma Hepatocelular , Neoplasias Hepáticas , Animales , Humanos , Ratas , Ácido Ascórbico/uso terapéutico , Aspirina/uso terapéutico , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/tratamiento farmacológico , Doxorrubicina/uso terapéutico , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/tratamiento farmacológico , Tioacetamida/efectos adversos , Proteína p53 Supresora de Tumor/metabolismo , VitaminasRESUMEN
Osteoporosis, an age-related metabolic bone disease, is mainly caused by an imbalance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption. At present, there are many osteoporosis drugs that can promote bone formation or inhibit bone resorption. However, there were few therapeutic drugs that can simultaneously promote bone formation and inhibit bone resorption. Oridonin (ORI), a tetracyclic diterpenoid compound isolated from Rabdosia rubescens, has been proved to have anti-inflammatory, anti-tumor effects. However, little is known about the osteoprotective effect of oridonin. Thioacetamide (TAA) is a common organic compound with significant hepatotoxicity. Recent studies have found that there was a certain association between TAA and bone injury. In this work, we investigated the effect and mechanism of ORI on TAA-induced osteoclastogenesis and inhibition of osteoblast differentiation. The results showed that TAA could promote the osteoclastogenesis of RAW264.7 by promoting the MAPK/NF-κB pathway, and also promoted p65 nuclear translocation and activated intracellular ROS generation, and ORI can inhibit these effects to inhibit TAA-induced osteoclastogenesis. Moreover, ORI can also promote the osteogenic differentiation pathway and inhibit adipogenic differentiation of BMSCs to promote bone formation. In conclusion, our results revealed that ORI, as a potential therapeutic drug for osteoporosis, could protect against TAA-induced bone loss and TAA-inhibited bone formation.
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Resorción Ósea , Osteoporosis , Humanos , Osteogénesis , FN-kappa B/metabolismo , Tioacetamida/metabolismo , Tioacetamida/farmacología , Tioacetamida/uso terapéutico , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Osteoclastos/metabolismo , Resorción Ósea/metabolismo , Osteoporosis/inducido químicamente , Osteoporosis/tratamiento farmacológico , Diferenciación Celular , Ligando RANK/metabolismoRESUMEN
Upon chronic damage to the liver, multiple cytokines stimulate hepatic stellate cells (HSCs), causing the alterations of gene expression profiles and thus leading to HSC activation, a key step in liver fibrogenesis. Activated HSCs are the dominant contributors to liver fibrosis. Bromodomain containing protein 4 (BrD4), an important epigenetic reader, was demonstrated to concentrate on hundreds of enhancers associated with genes involved in multiple profibrotic pathways, thereby directing HSC activation and the fibrotic responses. The present studies were designed to examine the effect of transforming growth factor beta-1 (TGFß1), the most potent pro-fibrotic cytokine, on BrD4 expression in HSCs and, if so, elucidated the underlying mechanisms in vitro and in vivo. The experiments employed the heterogeneous TGFß1 knockout (TGFß1+/- ) mice, gene knockdown in vivo, and a model of thioacetamide (TAA)-induced liver injury. The results revealed that TGFß1 enhanced BrD4 expression in HSCs, which was mediated, at least, by Smad3 signaling and early-immediate gene Egr1 (early growth response-1). TGFß1-induced Smad3 signaling increased Egr1 expression and promoted Egr1 binding to BrD4 promoter at a site around -111 bp, promoting BrD4 expression. Egr1 knockdown reduced BrD4 expression in HSCs in a mouse model of TAA-induced liver injury and lessened liver fibrosis. Double fluorescence staining demonstrated a strong increase in BrD4 expression in activated HSCs in fibrotic areas of the human livers, paralleling the upregulation of p-Smad3 and Egr1. This research suggested novel molecular events underlying the roles of the master pro-fibrotic cytokine TGFß1 in HSC activation and liver fibrogenesis.