RESUMEN
Histone deacetylase 6 (HDAC6) belongs to the class IIb group of the histone deacetylase family, which participates in remodelling of various tissues. Herein, we sought to examine the potential regulation of HDAC6 in cardiac remodelling post-infarction. Experimental myocardial infarction (MI) was created in HDAC6-deficient (HDAC6-/-) mice and wild-type (HADC6+/+) by left coronary artery ligation. At days 0 and 14 post-MI, we evaluated cardiac function, morphology and molecular endpoints of repair and remodelling. At day 14 after surgery, the ischemic myocardium had increased levels of HADC6 gene and protein of post-MI mice compared to the non-ischemic myocardium of control mice. As compared with HDAC6-/--MI mice, HADC6 deletion markedly improved infarct size and cardiac fibrosis as well as impaired left ventricular ejection fraction and left ventricular fraction shortening. At the molecular levels, HDAC6-/- resulted in a significant reduction in the levels of the transforming growth factor-beta 1 (TGF-ß1), phosphor-Smad-2/3, collagen I and collagen III proteins and/or in the ischemic cardiac tissues. All of these beneficial effects were reproduced by a pharmacological inhibition of HADC6 in vivo. In vitro, hypoxic stress increased the expressions of HADC6 and collagen I and III gene; these alterations were significantly prevented by the HADC6 silencing and TubA loading. These findings indicated that HADC6 deficiency resists ischemic injury by a reduction of TGF-ß1/Smad2/3 signalling activation, leading to decreased extracellular matrix production, which reduces cardiac fibrosis and dysfunction, providing a potential molecular target in the treatment of patients with MI.
Asunto(s)
Fibrosis , Histona Desacetilasa 6 , Infarto del Miocardio , Transducción de Señal , Proteína Smad2 , Proteína smad3 , Factor de Crecimiento Transformador beta1 , Remodelación Ventricular , Animales , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Infarto del Miocardio/genética , Factor de Crecimiento Transformador beta1/metabolismo , Proteína Smad2/metabolismo , Ratones , Histona Desacetilasa 6/metabolismo , Histona Desacetilasa 6/genética , Proteína smad3/metabolismo , Proteína smad3/genética , Miocardio/metabolismo , Miocardio/patología , Ratones Noqueados , Masculino , Ratones Endogámicos C57BL , Modelos Animales de EnfermedadRESUMEN
OBJECTIVE: To investigate the mechanism by which conbercept reverses transforming growth factor-ß2 (TGF-ß2)-induced epithelial-mesenchymal transition (EMT) in human lens epithelial cells (HLECs). METHODS: Cultured HLEC SRA01/04 cells were treated with TGF-ß2, conbercept, or both, and the changes in cell proliferation, apoptosis, and migration were observed using MTT assay, flow cytometry, scratch assay, and Transwell assay. Western blotting and qRT-PCR were used to detect the changes in the expression of EMT-related epithelial cell markers (E-Cadherin, α-SMA, and Snail), extracellular matrix components, and genes related to the TGF-ß/Smad signaling pathway. RESULTS: Conbercept significantly reduced TGF-ß2-induced EMT of SRA01/04 cells, decreased the expression levels of mesenchymal and extracellular matrix markers α-SMA, Snail, collagen I, collagen IV, and FN1, and upregulated the protein and mRNA expressions of E-cadherin (P <0.05). Transwell assay showed significantly lower cell migration ability in TGF-ß2+conbercept group than in TGF-ß2 group (P <0.05). Conbercept also inhibited the increase in Smad2/3 phosphorylation levels in HLEC-SRA01/04 cells with TGF-ß2-induced EMT (P <0.01). CONCLUSION: Conbercept inhibits TGF-ß2 induced EMT by downregulating the expression of pSmad2/3 in TGF-ß/Smad signaling pathway, indicating a potential therapeutic strategy against visual loss induced by posterior capsule opacification.
Asunto(s)
Proliferación Celular , Células Epiteliales , Transición Epitelial-Mesenquimal , Cristalino , Transducción de Señal , Proteínas Smad , Factor de Crecimiento Transformador beta2 , Humanos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Factor de Crecimiento Transformador beta2/metabolismo , Cristalino/citología , Cristalino/metabolismo , Proteínas Smad/metabolismo , Proliferación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Cadherinas/metabolismo , Apoptosis/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Línea Celular , Proteína Smad2/metabolismoRESUMEN
Myelodysplastic syndrome and acute myeloid leukemia (AML) belong to a continuous disease spectrum of myeloid malignancies with poor prognosis in the relapsed/refractory setting necessitating novel therapies. Natural killer (NK) cells from patients with myeloid malignancies display global dysfunction with impaired killing capacity, altered metabolism, and an exhausted phenotype at the single-cell transcriptomic and proteomic levels. In this study, we identified that this dysfunction was mediated through a cross-talk between NK cells and myeloid blasts necessitating cell-cell contact. NK cell dysfunction could be prevented by targeting the αvß-integrin/TGF-ß/SMAD pathway but, once established, was persistent because of profound epigenetic reprogramming. We identified BATF as a core transcription factor and the main mediator of this NK cell dysfunction in AML. Mechanistically, we found that BATF was directly regulated and induced by SMAD2/3 and, in turn, bound to key genes related to NK cell exhaustion, such as HAVCR2, LAG3, TIGIT, and CTLA4. BATF deletion enhanced NK cell function against AML in vitro and in vivo. Collectively, our findings reveal a previously unidentified mechanism of NK immune evasion in AML manifested by epigenetic rewiring and inactivation of NK cells by myeloid blasts. This work highlights the importance of using healthy allogeneic NK cells as an adoptive cell therapy to treat patients with myeloid malignancies combined with strategies aimed at preventing the dysfunction by targeting the TGF-ß pathway or BATF.
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Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Epigénesis Genética , Células Asesinas Naturales , Leucemia Mieloide Aguda , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/inmunología , Humanos , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/inmunología , Animales , Factor de Crecimiento Transformador beta/metabolismo , Transducción de Señal , Ratones , Reprogramación Celular , Proteína smad3/metabolismo , Proteína Smad2/metabolismoRESUMEN
Allergic asthma is an important public health problem and is a complicated respiratory sickness that is characterized by bronchial inflammation, bronchoconstriction, and breathlessness. Asthma is orchestrated by type 2 immune response and remodeling is one of the important outputted problem in chronic asthma. Thymol is a naturally occurring monocyclic phenolic, it has a series of biological properties, and its immunomodulatory and anti-remodeling effects on allergic asthma were evaluated. The OVA-LPS-induced asthmatic mice were treated with thymol. Methacholine challenge test, eosinophil count, and levels of IL-4, IL-5, IL-13, and IL-33 in bronchoalveolar lavage fluid, total and OVA-specific IgE levels in serum, remodeling factors, gene expression of TGF-ß, Smad2, Smad3, and lung histopathology were done. Treatment with thymol could control AHR, eosinophil percentage levels of Th2 cytokines and Igs, remodeling factors, expression of TGF-ß, Smad2 and Smad3 genes, inflammation, goblet cell hyperplasia, and mucus production in asthmatic mice. Thymol can control asthma pathogens and related remodeling and fibrosis bio-factors and can be a potential treatment of asthma.
Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias) , Asma , Modelos Animales de Enfermedad , Ratones Endogámicos BALB C , Transducción de Señal , Proteína smad3 , Timol , Factor de Crecimiento Transformador beta , Animales , Timol/farmacología , Asma/inmunología , Asma/tratamiento farmacológico , Remodelación de las Vías Aéreas (Respiratorias)/efectos de los fármacos , Remodelación de las Vías Aéreas (Respiratorias)/inmunología , Proteína smad3/metabolismo , Ratones , Factor de Crecimiento Transformador beta/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Citocinas/metabolismo , Femenino , Ovalbúmina/inmunología , Líquido del Lavado Bronquioalveolar/inmunología , Líquido del Lavado Bronquioalveolar/citología , Eosinófilos/inmunología , Eosinófilos/efectos de los fármacos , Humanos , Inmunoglobulina E/inmunología , Inmunoglobulina E/sangre , Proteína Smad2/metabolismoRESUMEN
Epithelial ovarian cancer is the deadliest gynecologic malignancy, characterized by high metastasis. Transforming growth factor-ß1 (TGF-ß1) drives epithelial- mesenchymal transformation (EMT), a key process in tumor metastasis. Tumor necrosis factor-α-induced protein 8 (TNFAIP8)-like 2 (TIPE2) acts as a negative regulator of innate and adaptive immunity and involves in various cancers. However, its relationship with TGF-ß1 in ovarian cancer and its role in reversing TGF-ß1-induced EMT remain unclear. This study examined TIPE2 mRNA and protein expression using quantitative RT-PCR (qRT-PCR), western blot and immunohistochemistry. The effects of TIPE2 overexpression and knockdown on the proliferation, migration and invasion of epithelial ovarian cancer cells were assessed through 5-ethynyl-2-deoxyuridine, colony-forming, transwell migration and invasion assays. The relationship between TIPE2 and TGF-ß1 was investigated using qRT-PCR and enzyme-linked immunosorbent assay, while the interaction between TIPE2 and Smad2 was identified via co-immunoprecipitation. The results revealed that TIPE2 protein was significantly down-regulated in epithelial ovarian cancer tissues and correlated with the pathological type of tumor, patients' age, tumor differentiation degree and FIGO stage. TIPE2 and TGF-ß1 appeared to play an opposite role to each other during the progression of human ovarian cancer cells. Furthermore, TIPE2 inhibited the metastasis and EMT of ovarian cancer cells by combining with Smad2 in vitro or in an intraperitoneal metastasis model. Consequently, these findings suggest that TIPE2 plays a crucial inhibitory role in ovarian cancer metastasis by modulating the TGF-ß1/Smad2/EMT signaling pathway and may serve as a potential target for ovarian cancer, providing important direction for future diagnostic and therapeutic strategies.
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Carcinoma Epitelial de Ovario , Movimiento Celular , Transición Epitelial-Mesenquimal , Péptidos y Proteínas de Señalización Intracelular , Neoplasias Ováricas , Proteína Smad2 , Factor de Crecimiento Transformador beta1 , Proteína Smad2/metabolismo , Proteína Smad2/genética , Humanos , Femenino , Factor de Crecimiento Transformador beta1/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Carcinoma Epitelial de Ovario/metabolismo , Carcinoma Epitelial de Ovario/patología , Carcinoma Epitelial de Ovario/genética , Neoplasias Ováricas/patología , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/genética , Línea Celular Tumoral , Animales , Ratones , Invasividad Neoplásica , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Ratones Desnudos , Ratones Endogámicos BALB C , Transducción de SeñalRESUMEN
Liver fibrosis is a pathological process that endangers human health, for which effective treatments remain elusive to date. Paeoniflorin (PAE), a pineane-type monoter penoid compound from the traditional Chinese medicine PaeoniaeRubra Radix, and metformin (MET), an oral biguanide hypoglycemic agent, both demonstrate anti-inflammatory and hepatoprotective effects. In current work, we first discovered that the combined treatment of PAE and MET synergistically inhibited the progression of liver fibrosis in two different animal models: therapeutic and preventive. This therapeutic effect is evidenced by a reduction in the expression levels of liver fibrosis markers and an improvement in histopathological characteristics. Mechanistic exploration further revealed that this combination therapy downregulated the expression of TGF-ß1 and p-Smad2, while upregulating Smad7 expression in both models. Importantly, we also found that this combinatorial approach significantly reduced hepatotoxicity and nephrotoxicity in both models. Our findings suggest an effective combination therapy for liver fibrosis and provide the possibility of therapeutic improvement for patients with liver fibrosis.
Asunto(s)
Sinergismo Farmacológico , Glucósidos , Cirrosis Hepática , Metformina , Monoterpenos , Animales , Monoterpenos/farmacología , Monoterpenos/uso terapéutico , Monoterpenos/administración & dosificación , Glucósidos/farmacología , Glucósidos/uso terapéutico , Glucósidos/administración & dosificación , Metformina/farmacología , Metformina/uso terapéutico , Metformina/administración & dosificación , Ratones , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/patología , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/metabolismo , Masculino , Progresión de la Enfermedad , Ratones Endogámicos C57BL , Quimioterapia Combinada , Factor de Crecimiento Transformador beta1/metabolismo , Hígado/efectos de los fármacos , Hígado/patología , Hígado/metabolismo , Proteína Smad2/metabolismo , Modelos Animales de EnfermedadRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Yajieshaba (YJSB), approved by the Yunnan Provincial Food and Drug Administration in 2008, are known for their anti-inflammatory, antiviral, and pro-apoptotic properties, effectively treating Hepatic fibrosis (HF). However, its mechanism of action remains unclear. AIM OF THE STUDY: The objective of this investigation is to explore how YJSB influences the TGF-ß1/Smad signaling pathway as a strategy for reducing HF. METHODS: The establishment of a HF model in mice involved ligation of the common bile duct, followed by administration of YJSB. Body and liver weights were measured, and the liver index calculated. Serum levels of ALT, AST, ALP, TBA, and TBIL were assessed using colorimetric methods. Additionally, liver homogenates were analyzed for PIIINP, Col-IV, LN, HA, and Hyp, as well as TGF-ß1 activity, using ELISA. Histological analyses of liver sections, stained with H&E, Ag, and Masson's trichrome, were performed to examine inflammation and the accumulation of collagen and reticular fibers. These studies aimed to elucidate the pharmacodynamic effects of YJSB on HF in mice with bile duct obstruction. The target pathways of YJSB were preliminarily identified through immunofluorescence detection of TGF-ß1, P-Smad2L, P-Smad2C, P-Smad3L, P-Smad3C, and Smad4 proteins. In vitro experiments included the induction of hepatic stellate cell (HSC-T6) activation by H2O2. A cell injury model was established for HSC-T6, and the CCK-8 assay was used to determine the optimal YJSB concentration and treatment duration. After pirfenidone (PFD) administration, which inhibits the TGF-ß1/Smad pathway, the effects of YJSB on HSC-T6 cell proliferation were observed. ELISA assays quantified Col-III, α-SMA, and Col-I in cell lysates to assess YJSB's impact on collagen synthesis in HSC-T6 cells. Western blot analysis was performed to assess the protein levels within the TGF-ß1/Smad signaling cascade. RESULTS: In the HF mouse model, administration of YJSB notably augmented the body weight and reduced the liver index. Concurrently, there was an elevation in serum concentrations of ALP, AST, ALT, TBA, and TBIL. Similarly, in the liver homogenates of HF mice, increases were observed in the levels of HA, PIIINP, Col-IV, LN, Hyp, and TGF-ß1. Histological assessments using H&E, Ag, and Masson stains indicated a substantial diminution in liver tissue damage. Through immunofluorescence analysis, it was discerned that YJSB modulated the expression of TGF-ß1, P-Smad2L, P-Smad2C, and P-Smad3L downwards, while elevating P-Smad3C and Smad4 protein expressions. Additional investigations revealed a significant reduction in α-SMA, Col-I, and Col-III levels in cell culture fluids, suggesting a decrease in collagen synthesis and a protective role against cellular damage. Western blot analyses demonstrated that the TGF-ß1/Smad pathway inhibitor, PFD, acted in synergy with YJSB, enhancing its regulatory effects on this pathway, decreasing levels of TGF-ß1, P-Smad2L, P-Smad2C, P-Smad3L, and promoting the expression of P-Smad3C. CONCLUSIONS: YJSB demonstrates a pharmacodynamic effect against HF, enhancing liver functionality and effectively mitigating the damage associated with bile duct obstruction. The proposed action mechanism of YJSB involves modulation of the TGF-ß1/Smad signaling pathway. Research indicates that YJSB might play a role in suppressing the movement, programmed cell death, and activation of HSC-T6, potentially decelerating the advancement of hepatic fibrosis.
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Colestasis , Células Estrelladas Hepáticas , Peróxido de Hidrógeno , Cirrosis Hepática , Transducción de Señal , Factor de Crecimiento Transformador beta1 , Animales , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Factor de Crecimiento Transformador beta1/metabolismo , Ratones , Masculino , Peróxido de Hidrógeno/metabolismo , Colestasis/metabolismo , Colestasis/patología , Colestasis/tratamiento farmacológico , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Transducción de Señal/efectos de los fármacos , Medicamentos Herbarios Chinos/farmacología , Línea Celular , Modelos Animales de Enfermedad , Hígado/efectos de los fármacos , Hígado/patología , Hígado/metabolismo , Proteínas Smad/metabolismo , Ratones Endogámicos C57BL , Proteína Smad2/metabolismoRESUMEN
Differentiation of cardiac fibroblasts to myofibroblasts is necessary for matrix remodeling and fibrosis in heart failure. We previously reported that mitochondrial calcium signaling drives α-ketoglutarate-dependent histone demethylation, promoting myofibroblast formation. Here we investigate the role of ATP-citrate lyase (ACLY), a key enzyme for acetyl-CoA biosynthesis, in histone acetylation regulating myofibroblast fate and persistence in cardiac fibrosis. We show that inactivation of ACLY prevents myofibroblast differentiation and reverses myofibroblasts towards quiescence. Genetic deletion of Acly in post-activated myofibroblasts prevents fibrosis and preserves cardiac function in pressure-overload heart failure. TGFß stimulation enhances ACLY nuclear localization and ACLY-SMAD2/3 interaction, and increases H3K27ac at fibrotic gene loci. Pharmacological inhibition of ACLY or forced nuclear expression of a dominant-negative ACLY mutant prevents myofibroblast formation and H3K27ac. Our data indicate that nuclear ACLY activity is necessary for myofibroblast differentiation and persistence by maintaining histone acetylation at TGFß-induced myofibroblast genes. These findings provide targets to prevent and reverse pathological fibrosis.
Asunto(s)
ATP Citrato (pro-S)-Liasa , Diferenciación Celular , Fibrosis , Histonas , Miofibroblastos , Proteína Smad2 , Miofibroblastos/metabolismo , Miofibroblastos/efectos de los fármacos , ATP Citrato (pro-S)-Liasa/metabolismo , ATP Citrato (pro-S)-Liasa/genética , Animales , Fibrosis/metabolismo , Diferenciación Celular/efectos de los fármacos , Histonas/metabolismo , Proteína Smad2/metabolismo , Proteína Smad2/genética , Acetilación/efectos de los fármacos , Núcleo Celular/metabolismo , Núcleo Celular/efectos de los fármacos , Proteína smad3/metabolismo , Proteína smad3/genética , Células Cultivadas , Cromatina/metabolismo , Ratones Noqueados , Factor de Crecimiento Transformador beta/metabolismo , Modelos Animales de Enfermedad , Transducción de Señal , Ratones Endogámicos C57BL , Masculino , Ratones , Regulación de la Expresión Génica/efectos de los fármacosRESUMEN
Chronic Kidney Disease (CKD) has emerged as a global public health concern, with its primary pathological basis being Renal Fibrosis (RF), crucial to halt its progression to End-Stage Renal Disease (ESRD). However, effective treatment options are currently lacking. Therefore, exploring the mechanisms of RF, identifying drug targets and diagnostic biomarkers are important. In this study, we identified ADAMTS16 as a newly expressed regulatory factor highly expressed in renal fibrosis tissue. ADAMTS16 interacts with latency-associated peptide (LAP)-transforming growth factor (TGF)-ß, leading to the activation of TGF-ß. Loss of ADAMTS16 expression effectively reduces TGF-ß-dependent transcription activity. Furthermore, the use of RRFR tetrapeptide derived from ADAMTS16 can activate the TGF-ß/Smad signaling axis, promoting RF. In summary, ADAMTS16 is induced in the progression of CKD, interacting with LAP-TGF-ß and potentially activating SMAD2/3. Therefore, targeting ADAMTS16 may serve as a crucial new strategy to alleviate RF and treat CKD patients.
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Proteínas ADAMTS , Fibrosis , Transducción de Señal , Factor de Crecimiento Transformador beta , Animales , Masculino , Ratones , Proteínas ADAMTS/metabolismo , Riñón/patología , Riñón/metabolismo , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Proteínas Smad/metabolismo , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND: Pulmonary fibrosis (PF) is a progressive lung disease caused by previous acute lung injury (ALI), but there is currently no satisfactory therapy available. Aerosol inhalation of medicine is an effective way for treating PF. Total ginsenosides (TG) shows potential for the treatment of ALI and PF, but the effects of inhaled TG remain unclear. PURPOSE: To determine the therapeutic effects of TG in ALI and PF, to assess the superiority of the inhaled form of TG over the routine form, and to clarify the mechanism of action of inhaled TG. METHODS: Ultrahigh-performance liquid chromatography coupled with Q-Exactive Orbitrap mass spectrometry (UPLC-QE-MS) was applied to determine the chemoprofile of TG. A mouse model of ALI and PF was established to evaluate the effects of inhaled TG by using bronchoalveolar lavage fluid (BALF) analysis, histopathological observation, hydroxyproline assay, and immunohistochemical analysis. Primary mouse lung fibroblasts (MLF) and human lung fibroblast cell line (HFL1) were applied to determine the in vitro effects and mechanism of TG by using cell viability assay, quantitative real time PCR (qPCR) assay, and western blot (WB) analysis. RESULTS: The UPLC-QE-MS results revealed the main types of ginsenosides in TG, including Re (14.15 ± 0.42%), Rd (8.42 ± 0.49%), Rg1 (6.22 ± 0.42%), Rb3 (3.28 ± 0.01%), Rb2 (3.09 ± 0.00%), Rc (2.33 ± 0.01%), Rg2 (2.09 ± 0.04%), Rb1 (1.43 ± 0.24%), and Rf (0.13 ± 0.06%). Inhaled TG, at dosages of 10, 20, and 30 mg/kg significantly alleviated both ALI and PF in mice. Analyses of BALF and HE staining revealed that TG modulated the levels of IFN-γ, IL-1ß, and TGF-ß1, reduced inflammatory cell infiltration, and restored the alveolar architecture of the lung tissues. Furthermore, HE and Masson's trichrome staining demonstrated that TG markedly decreased fibroblastic foci and collagen fiber deposition, evidenced by the reduction of blue-stained collagen fibers. Hydroxyproline assay and immunohistochemical analyses indicated that TG significantly decreased hydroxyproline level and down-regulated the expression of Col1a1, Col3a1, and α-sma. The inhaled administration of TG demonstrated enhanced efficacy over the oral route when comparable doses were used. Additionally, inhaled TG showed superior safety and therapeutic profiles compared to pirfenidone, as evidenced by a CCK8 assay, which confirmed that TG concentrations ranging from 20 to 120 µg/ml were non-cytotoxic. qPCR and WB analyses revealed that TG, at concentrations of 25, 50, and 100 µg/ml, significantly suppressed the phosphorylation of smad2 induced by TGF-ß1 and down-regulated the expression of fibrotic genes and proteins, including α-sma, Col1a1, Col3a1, and FN1, suggesting an anti-fibrotic mechanism mediated by the smad2 signaling pathway. In vitro, TG's safety and efficacy were also found to be superior to those of pirfenidone. CONCLUSIONS: This study demonstrates, for the first time, the therapeutic efficacy of inhaled TG in treating ALI and PF. Inhaled TG effectively inhibits inflammation and reduces collagen deposition, with a particular emphasis on its role in modulating the Smad2 signaling pathway, which is implicated in the anti-fibrotic mechanism of TG. The study also highlights the superiority of inhaled TG over the oral route and its favorable safety profile in comparison to pirfenidone, positioning it as an ideal alternative for ALI and PF therapy.
Asunto(s)
Lesión Pulmonar Aguda , Ginsenósidos , Fibrosis Pulmonar , Transducción de Señal , Proteína Smad2 , Ginsenósidos/farmacología , Ginsenósidos/administración & dosificación , Animales , Lesión Pulmonar Aguda/tratamiento farmacológico , Fibrosis Pulmonar/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Masculino , Ratones , Proteína Smad2/metabolismo , Humanos , Administración por Inhalación , Líquido del Lavado Bronquioalveolar/química , Ratones Endogámicos C57BL , Pulmón/efectos de los fármacos , Pulmón/patología , Modelos Animales de Enfermedad , Fibroblastos/efectos de los fármacos , Aerosoles , Línea Celular , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Tumor endothelial marker 1 (TEM1), an activated mesenchymal cell marker, is implicated in tissue remodeling and repair. Herein, we investigated the role and therapeutic implications of TEM1 in abdominal aortic aneurysm (AAA), a potentially life-threatening aortic disease characterized by vascular inflammation and matrix turnover. Characterization of human AAA revealed increased TEM1 expression derived mainly from medial vascular smooth muscle cells (VSMCs) and adventitial fibroblasts. Bioinformatics analysis demonstrated the association between TEM1-expressing VSMCs and fibroblasts and collagen gene expression. Consistently, collagen content and TEM1 expressed by VSMCs and fibroblasts were increased during CaCl2-induced AAA formation in mice. TEM1 silencing in VSMCs and fibroblasts inhibited transforming growth factor-ß1-induced phenotypic change, SMAD2 phosphorylation, and COL1A1 gene expression. Also, Tem1 deficiency reduced collagen synthesis and exacerbated CaCl2-induced AAA formation in mice without disturbing elastin destruction and inflammatory responses. In contrast, rTEM1 promoted phenotypic change and COL1A1 gene expression through SMAD2 phosphorylation in VSMCs and fibroblasts. Treatment with rTEM1 enhanced collagen synthesis, attenuated elastin fragmentation, and inhibited CaCl2-induced and angiotensin II-infused AAA formation. In summary, TEM1 in resident stromal cells regulates collagen synthesis to counteract aortic wall failure during AAA formation. Matrix integrity restored by rTEM1 treatment may hold therapeutic potential against AAA.
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Aneurisma de la Aorta Abdominal , Animales , Humanos , Masculino , Ratones , Aneurisma de la Aorta Abdominal/metabolismo , Fibroblastos/metabolismo , Ratones Endogámicos C57BL , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteína Smad2/metabolismoRESUMEN
This study investigates whether hAFSCs can improve bladder function in partial bladder outlet obstruction (pBOO) rats by targeting specific cellular pathways. Thirty-six female rats were divided into sham and pBOO groups with and without hAFSCs single injection into the bladder wall. Cystometry, inflammation/hypoxia, collagen/fibrosis/gap junction proteins, and smooth muscle myosin/muscarinic receptors were examined at 2 and 6 weeks after pBOO or sham operation. In pBOO bladders, significant increases in peak voiding pressure and residual volume stimulated a significant upregulation of inflammatory and hypoxic factors, TGF-ß1 and Smad2/3. Collagen deposition proteins, collagen 1 and 3, were significantly increased, but bladder fibrosis markers, caveolin 1 and 3, were significantly decreased. Gap junction intercellular communication protein, connexin 43, was significantly increased, but the number of caveolae was significantly decreased. Markers for the smooth muscle phenotype, myosin heavy chain 11 and guanylate-dependent protein kinase, as well as M2 muscarinic receptors, were significantly increased in cultured detrusor cells. However, hAFSCs treatment could significantly ameliorate bladder dysfunction by inactivating the TGFß-Smad signaling pathway, reducing collagen deposition, disrupting gap junctional intercellular communication, and modifying the expressions of smooth muscle myosin and caveolae/caveolin proteins. The results support the potential value of hAFSCs-based treatment of bladder dysfunction in BOO patients.
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Conexina 43 , Obstrucción del Cuello de la Vejiga Urinaria , Vejiga Urinaria , Animales , Obstrucción del Cuello de la Vejiga Urinaria/metabolismo , Obstrucción del Cuello de la Vejiga Urinaria/patología , Femenino , Ratas , Vejiga Urinaria/metabolismo , Vejiga Urinaria/fisiopatología , Vejiga Urinaria/patología , Conexina 43/metabolismo , Trasplante de Células Madre/métodos , Transducción de Señal , Ratas Sprague-Dawley , Proteína Smad2/metabolismo , Modelos Animales de Enfermedad , Uniones Comunicantes/metabolismo , Colágeno/metabolismoRESUMEN
Atrial fibrosis is associated with the occurrence of atrial fibrillation (AF) and regulated by the transforming growth factor-ß1 (TGF-ß1)/Smad2/3 signalling pathway. Unfortunately, the mechanisms of regulation of TGF-ß1/Smad2/3-induced atrial fibrosis and vulnerability to AF remain still unknown. Previous studies have shown that sirtuin3 (SIRT3) sulfhydration has strong anti-fibrotic effects. We hypothesised that SIRT3 sulfhydration inhibits angiotensin II (Ang-II)-induced atrial fibrosis via blocking the TGF-ß1/Smad2/3 signalling pathway. In this study, we found that SIRT3 expression was decreased in the left atrium of patients with AF compared to that in those with sinus rhythm (SR). In vitro, SIRT3 knockdown by small interfering RNA significantly expanded Ang-II-induced atrial fibrosis and TGF-ß1/Smad2/3 signalling pathway activation, whereas supplementation with Sodium Hydrosulfide (NaHS, exogenous hydrogen sulfide donor and sulfhydration agonist) and SIRT3 overexpression using adenovirus ameliorated Ang-II-induced atrial fibrosis. Moreover, we observed suppression of the TGF-ß1/Smad2/3 pathway when Ang-II was combined with NaHS treatment, and the effect of this co-treatment was consistent with that of Ang-II combined with LY3200882 (Smad pathway inhibitor) on reducing atrial fibroblast proliferation and cell migration in vitro. Supplementation with dithiothreitol (DTT, a sulfhydration inhibitor) and adenovirus SIRT3 shRNA blocked the ameliorating effect of NaHS and AngII co-treatment on atrial fibrosis in vitro. Finally, continued treatment with NaHS in rats ameliorated atrial fibrosis and remodelling, and further improved AF vulnerability induced by Ang-II, which was reversed by DTT and adenovirus SIRT3 shRNA, suggesting that SIRT3 sulfhydration might be a potential therapeutic target in atrial fibrosis and AF.
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Angiotensina II , Fibrilación Atrial , Fibrosis , Atrios Cardíacos , Sulfuro de Hidrógeno , Transducción de Señal , Sirtuina 3 , Proteína Smad2 , Proteína smad3 , Factor de Crecimiento Transformador beta1 , Angiotensina II/farmacología , Sirtuina 3/metabolismo , Sirtuina 3/genética , Fibrilación Atrial/metabolismo , Fibrilación Atrial/patología , Fibrilación Atrial/prevención & control , Factor de Crecimiento Transformador beta1/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína smad3/metabolismo , Animales , Proteína Smad2/metabolismo , Atrios Cardíacos/efectos de los fármacos , Atrios Cardíacos/metabolismo , Atrios Cardíacos/patología , Sulfuro de Hidrógeno/farmacología , Sulfuro de Hidrógeno/metabolismo , Masculino , Humanos , Ratas , Ratas Sprague-Dawley , Femenino , Persona de Mediana Edad , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacosRESUMEN
Transforming growth factor-ß (TGF-ß) is a pleiotropic cytokine that modulates a wide variety of cellular responses by regulating target gene expression. It principally transmits signals via receptor-activated transcription factors Smad2 and Smad3, which form trimeric complexes with Smad4 upon activation and regulate gene expression by binding to genomic DNA. Here, we examined the mechanisms by which TGF-ß regulates the transcription of target genes in a cell context-dependent manner by screening a double-stranded DNA oligonucleotide library for DNA sequences bound to endogenous activated Smad complexes. Screening was performed by cyclic amplification of selected targets (CASTing) using an anti-Smad2/3 antibody and nuclear extracts isolated from three cell lines (A549, HepG2, and HaCaT) stimulated with TGF-ß. The preference of the activated Smad complexes for conventional Smad-binding motifs such as Smad-binding element (SBE) and CAGA motifs was different in HepG2 than in the other two cell lines, which may indicate the distinct composition of the activated Smad complexes. Several transcription factor-binding motifs other than SBE or CAGA, including the Fos/Jun-binding motifs, were detected in the enriched sequences. Reporter assays using sequences containing these transcription factor-binding motifs together with Smad-binding motifs indicated that some of the motifs may be involved in cell type-dependent transcriptional activation by TGF-ß. The results suggest that the CASTing method is useful for elucidating the molecular basis of context-dependent Smad signaling.
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ADN , Transducción de Señal , Factor de Crecimiento Transformador beta , Humanos , Factor de Crecimiento Transformador beta/metabolismo , Células Hep G2 , ADN/metabolismo , Unión Proteica , Proteína smad3/metabolismo , Proteína Smad2/metabolismo , Células A549 , Células HaCaT , Proteínas Smad/metabolismoRESUMEN
The liver is innervated by primary sensory nerve fibres releasing the neuropeptide calcitonin gene-related peptide (CGRP). Elevated plasma levels of CGRP have been found in patients with liver fibrosis or cirrhosis. We hypothesised that signalling of CGRP and its receptors might regulate liver fibrosis and propose a novel potential target for the treatment. In this study, hepatic expression of CGRP and its receptor component, the receptor activity-modifying protein 1 (RAMP1), was dramatically increased in diseased livers of patients. In a murine liver fibrosis model, deficiency of RAMP1 resulted in attenuated fibrogenesis characterized by less collagen deposition and decreased activity of hepatic stellate cells (HSC). Mechanistically, activity of the TGFß1 signalling core component Smad2 was severely impaired in the absence of RAMP1, and Yes-associated protein (YAP) activity was found to be diminished in RAMP1-deficient liver parenchyma. In vitro, stimulation of the HSC line LX-2 cells with CGRP induces TGFß1 production and downstream signalling as well as HSC activation documented by increased α-SMA expression and collagen synthesis. We further demonstrate in LX-2 cells that CGRP promotes YAP activation and its nuclear translocation subsequent to TGFß1/Smad2 signals. These data support a promotive effect of CGRP signalling in liver fibrosis via stimulation of TGFß1/Smad2 and YAP activity.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina , Células Estrelladas Hepáticas , Cirrosis Hepática , Proteína 1 Modificadora de la Actividad de Receptores , Transducción de Señal , Proteína Smad2 , Factor de Crecimiento Transformador beta1 , Proteínas Señalizadoras YAP , Péptido Relacionado con Gen de Calcitonina/metabolismo , Péptido Relacionado con Gen de Calcitonina/genética , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Cirrosis Hepática/genética , Animales , Factor de Crecimiento Transformador beta1/metabolismo , Proteína 1 Modificadora de la Actividad de Receptores/metabolismo , Proteína 1 Modificadora de la Actividad de Receptores/genética , Humanos , Proteína Smad2/metabolismo , Proteína Smad2/genética , Ratones , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/patología , Proteínas Señalizadoras YAP/metabolismo , Proteínas Señalizadoras YAP/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Masculino , Ratones Endogámicos C57BL , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Ratones NoqueadosRESUMEN
BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a long-term, progressive, and irreversible pulmonary interstitial disease. The activation of Smad family member 2 (Smad2) and Smad3 transcription factors by transforming growth factor ß-1 (TGF-ß1) is a critical event in the pathogenesis of IPF. However, there is still a lack of understanding regarding the molecular mechanisms governing Smad2 and Smad3 proteins. Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that plays a vital role in regulating protein stability within cells. However, its regulation of the TGF-ß signaling pathway and its significance in IPF remain undiscovered. This study aims to clarify the function of USP7 in the TGF-ß signaling pathway, while simultaneously exploring the specific molecular mechanisms involved. Additionally, this study seeks to evaluate the therapeutic potential of targeted USP7 inhibitors in IPF, thereby providing novel insights for the diagnosis and management of IPF. METHODS: We first detected the expression of USP7 in lung tissues of mice with Bleomycin (BLM)-induced pulmonary fibrosis and in Beas-2B cells treated with or without TGF-ß1 through Western blot analysis. Subsequently, we explored the influence of USP7 on fibrotic processes and the TGF-ß1 signaling pathway, utilizing in vitro and in vivo studies. Finally, we assessed the effectiveness of USP7-specific inhibitors in an IPF murine model. RESULTS: In the present study, USP7 was found to de-ubiquitinate Smad2 and Smad3, consequently increasing their stability and promoting the TGF-ß1-induced production of profibrotic proteins including α-smooth muscle actin (α-SMA) and fibronectin 1 (FN-1). Inhibition or knockdown of USP7 resulted in decreased levels of Smad2 and Smad3 proteins, leading to reduced expression of FN-1, Collagen Type I Alpha 1 Chain (Col1A1), and α-SMA induced by TGF-ß1 in human pulmonary epithelial cells. These findings demonstrate that overexpression of USP7 reduces Smad2/3 ubiquitination, whereas inhibition or knockdown of USP7 enhances their ubiquitination. USP7 is abundantly expressed in IPF lungs. The expressions of USP7, Smad2, and Smad3 were upregulated in bleomycin-induced lung injury. The USP7 inhibitor P22077 reduced the expression of FN-1 and type I collagen as well as Smad2/3 and collagen deposition in lung tissue in a model of pulmonary fibrosis induced by bleomycin. CONCLUSIONS: This study demonstrates that USP7 promotes TGF-ß1 signaling by stabilizing Smad2 and Smad3. The contribution of USP7 to the progression of IPF indicates it may be a viable treatment target.
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Bleomicina , Transducción de Señal , Proteína Smad2 , Proteína smad3 , Factor de Crecimiento Transformador beta1 , Peptidasa Específica de Ubiquitina 7 , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Proteína smad3/metabolismo , Peptidasa Específica de Ubiquitina 7/metabolismo , Peptidasa Específica de Ubiquitina 7/genética , Ratones , Transducción de Señal/efectos de los fármacos , Humanos , Proteína Smad2/metabolismo , Bleomicina/toxicidad , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Fibrosis Pulmonar/inducido químicamente , Ubiquitinación , Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/patología , Fibrosis Pulmonar Idiopática/inducido químicamente , Fibrosis Pulmonar Idiopática/genética , Masculino , Ratones Endogámicos C57BL , Línea Celular , Pulmón/patología , Pulmón/metabolismo , Modelos Animales de EnfermedadRESUMEN
We previously reported pulmonary arterial remodelling and active endothelial-to-mesenchymal transition (EndMT) in smokers and patients with early chronic obstructive pulmonary disease (COPD). In the present study, we aimed to evaluate the role of different drivers of EndMT. Immunohistochemical staining for EndMT drivers, TGF-ß1, pSMAD-2/3, SMAD-7, and ß-catenin, was performed on lung resections from 46 subjects. Twelve were non-smoker-controls (NC), six normal lung function smokers (NLFS), nine patients with small-airway diseases (SAD), nine mild-moderate COPD-current smokers (COPD-CS) and ten COPD-ex-smokers (COPD-ES). Histopathological measurements were done using Image ProPlus softwarev7.0. We observed lower levels of total TGF-ß1 (P<0.05) in all smoking groups than in the non-smoking control (NC). Across arterial sizes, smoking groups exhibited significantly higher (P<0.05) total and individual layer pSMAD-2/3 and SMAD-7 than in the NC group. The ratio of SAMD-7 to pSMAD-2/3 was higher in COPD patients compared with NC. Total ß-catenin expression was significantly higher in smoking groups across arterial sizes (P<0.05), except for COPD-ES and NLFS groups in small and medium arteries, respectively. Increased total ß-catenin was positively correlated with total S100A4 in small and medium arteries (r = 0.35, 0.50; P=0.02, 0.01, respectively), with Vimentin in medium arteries (r = 0.42, P=0.07), and with arterial thickness of medium and large arteries (r = 0.34, 0.41, P=0.02, 0.01, respectively). This is the first study uncovering active endothelial SMAD pathway independent of TGF-ß1 in smokers, SAD, and COPD patients. Increased expression of ß-catenin indicates its potential interaction with SMAD pathway, warranting further research to identify the deviation of this classical pathway.
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Arteria Pulmonar , Enfermedad Pulmonar Obstructiva Crónica , Fumar , Factor de Crecimiento Transformador beta1 , beta Catenina , Humanos , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/patología , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , beta Catenina/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Masculino , Femenino , Persona de Mediana Edad , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Arteria Pulmonar/fisiopatología , Fumar/efectos adversos , Anciano , Proteína Smad2/metabolismo , Transición Epitelial-Mesenquimal , Proteína smad7/metabolismo , Fumadores , Estudios de Casos y Controles , Proteína smad3/metabolismo , Adulto , Transición Endotelial-MesenquimatosaRESUMEN
Acute respiratory distress syndrome (ARDS) occurs as an acute onset condition, and patients present with diffuse alveolar damage, refractory hypoxemia, and non-cardiac pulmonary edema. ARDS progresses through an initial exudative phase, an inflammatory phase, and a final fibrotic phase. Pirfenidone, a powerful anti-fibrotic agent, is known as an agent that inhibits the progression of fibrosis in idiopathic pulmonary fibrosis. In this study, we studied the treatment efficiency of pirfenidone on lipopolysaccharide (LPS) and bleomycin-induced ARDS using rats. The ARDS rat model was created by the intratracheal administration of 3 mg/kg LPS of and 3 mg/kg of bleomycin dissolved in 0.2 mL of normal saline. The pirfenidone treatment group was administered 100 or 200 mg/kg of pirfenidone dissolved in 0.5 mL distilled water orally 10 times every 2 days for 20 days. The administration of LPS and bleomycin intratracheally increased lung injury scores and significantly produced pro-inflammatory cytokines. ARDS induction increased the expressions of transforming growth factor (TGF)-ß1/Smad-2 signaling factors. Additionally, matrix metalloproteinase (MMP)-9/tissue inhibitor of metalloproteinase (TIMP)-1 imbalance occurred, resulting in enhanced fibrosis-related factors. Treatment with pirfenidone strongly suppressed the expressions of TGF-ß1/Smad-2 signaling factors and improved the imbalance of MMP-9/TIMP-1 compared to the untreated group. These effects led to a decrease in fibrosis factors and pro-inflammatory cytokines, promoting the recovery of damaged lung tissue. These results of this study showed that pirfenidone administration suppressed inflammation and fibrosis in the ARDS animal model. Therefore, pirfenidone can be considered a new early treatment for ARDS.
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Bleomicina , Lipopolisacáridos , Piridonas , Síndrome de Dificultad Respiratoria , Transducción de Señal , Animales , Piridonas/farmacología , Piridonas/uso terapéutico , Síndrome de Dificultad Respiratoria/tratamiento farmacológico , Síndrome de Dificultad Respiratoria/metabolismo , Síndrome de Dificultad Respiratoria/patología , Síndrome de Dificultad Respiratoria/inducido químicamente , Transducción de Señal/efectos de los fármacos , Ratas , Masculino , Bleomicina/efectos adversos , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Proteína Smad2/metabolismo , Ratas Sprague-Dawley , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/patología , Modelos Animales de Enfermedad , Metaloproteinasa 9 de la Matriz/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Pulmón/patología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Proteínas Smad/metabolismoRESUMEN
Hepatic stellate cell (HSC) activation is the essential pathological process of liver fibrosis (LF). The molecular mechanisms regulating HSC activation and LF are incompletely understood. Here, we explored the effect of transcription factor SRY-related high mobility group box 7 (SOX7) on HSC activation and LF, and the underlying molecular mechanism. We found the expression levels of SOX7 were decreased in human and mouse fibrotic livers, particularly at the fibrotic foci. SOX7 was also downregulated in primary activated HSCs and TGF-ß1 stimulated LX-2 cells. SOX7 knockdown promoted activation and proliferation of LX-2 cells while inhibiting their apoptosis. On the other hand, overexpression of SOX7 suppressed the activation and proliferation of HSCs. Mechanistically, SOX7 attenuates HSC activation and LF by decreasing the expression of ß-catenin and phosphorylation of Smad2 and Smad3 induced by TGF-ß1. Furthermore, overexpression of SOX7 using AAV8-SOX7 mouse models ameliorated the extent of LF in response to CCl4 treatment in vivo. Collectively, SOX7 suppressed HSC activation and LF. Targeting SOX7, therefore, could be a potential novel strategy to protect against LF.
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Células Estrelladas Hepáticas , Cirrosis Hepática , Factores de Transcripción SOXF , Células Estrelladas Hepáticas/metabolismo , Animales , Cirrosis Hepática/metabolismo , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Ratones , Humanos , Masculino , Factores de Transcripción SOXF/metabolismo , Factores de Transcripción SOXF/genética , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/genética , Proliferación Celular , Ratones Endogámicos C57BL , beta Catenina/metabolismo , beta Catenina/genética , Apoptosis , Proteína Smad2/metabolismo , Proteína Smad2/genética , Línea Celular , Proteína smad3/metabolismo , Proteína smad3/genéticaRESUMEN
Metabolic dysfunction-associated fatty liver disease (MAFLD) is a disease that causes an abnormal accumulation of fat in the liver, triggering inflammation and fibrosis, the mechanism of which is not fully understood and for which there is a lack of specific drug therapy. Far-infrared radiation (FIR) has demonstrated evident therapeutic efficacy across various diseases, and novel nanomaterial graphene patches can emit it through electric heating. This study aimed to investigate the potential protective effects of FIR against MAFLD. Mice were fed with a MCD diet to mimic MAFLD progression, and histopathology analysis, biochemical analysis, RT-qPCR, and Western blotting analysis were performed to assess the effect of FIR on MAFLD in vivo. The effect of FIR treatment on MAFLD in vitro was investigated by biochemical analysis and gene expression profiling of hepatocytes. Mice subjected to the MCD diet and treated with FIR exhibited reduced hepatic lipid deposition, inflammation, fibrosis and liver damage. The therapeutic effect exerted by FIR in mice may be caused by the enhancement of AMPK phosphorylation and inhibition of the TGFß1-SMAD2/3 pathway. Besides, FIR intervention alleviated MAFLD in hepatocytes in vitro and the results were verified by gene expression profiling. Our results revealed a promising potential of FIR as a novel therapeutic approach for MAFLD.