RESUMO
Cis-regulatory elements (CREs) are pivotal in orchestrating gene expression throughout diverse biological systems. Accurate identification and in-depth characterization of functional CREs are crucial for decoding gene regulation networks during cellular processes. In this study, we develop Kethoxal-Assisted Single-stranded DNA Assay for Transposase-Accessible Chromatin with Sequencing (KAS-ATAC-seq) to quantitatively analyze the transcriptional activity of CREs. A main advantage of KAS-ATAC-seq lies in its precise measurement of ssDNA levels within both proximal and distal ATAC-seq peaks, enabling the identification of transcriptional regulatory sequences. This feature is particularly adept at defining Single-Stranded Transcribing Enhancers (SSTEs). SSTEs are highly enriched with nascent RNAs and specific transcription factors (TFs) binding sites that define cellular identity. Moreover, KAS-ATAC-seq provides a detailed characterization and functional implications of various SSTE subtypes. Our analysis of CREs during mouse neural differentiation demonstrates that KAS-ATAC-seq can effectively identify immediate-early activated CREs in response to retinoic acid (RA) treatment. Our findings indicate that KAS-ATAC-seq provides more precise annotation of functional CREs in transcription. Future applications of KAS-ATAC-seq would help elucidate the intricate dynamics of gene regulation in diverse biological processes.
Assuntos
Fatores de Transcrição , Animais , Camundongos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica , Elementos Facilitadores Genéticos/genética , Cromatina/metabolismo , Cromatina/genética , Sítios de Ligação , Humanos , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação/métodos , Transposases/metabolismo , Transposases/genética , Elementos Reguladores de Transcrição , Tretinoína/farmacologia , Tretinoína/metabolismo , Regulação da Expressão Gênica , Diferenciação Celular/genética , Análise de Sequência de DNA/métodos , Sequências Reguladoras de Ácido Nucleico/genéticaRESUMO
Breast cancer (BC) is still one of the major issues in world health, especially for women, which necessitates innovative therapeutic strategies. In this study, we investigated the efficacy of retinoic acid derivatives as inhibitors of 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1), which plays a crucial role in the biosynthesis and metabolism of oestrogen and thereby influences the progression of BC and, the main objective of this investigation is to identify the possible drug candidate against BC through computational drug design approach including PASS prediction, molecular docking, ADMET profiling, molecular dynamics simulations (MD) and density functional theory (DFT) calculations. The result has reported that total eight derivatives with high binding affinity and promising pharmacokinetic properties among 115 derivatives. In particular, ligands 04 and 07 exhibited a higher binding affinity with values of -9.9 kcal/mol and -9.1 kcal/mol, respectively, than the standard drug epirubicin hydrochloride, which had a binding affinity of -8.2 kcal/mol. The stability of the ligand-protein complexes was further confirmed by MD simulations over a 100-ns trajectory, which included assessments of hydrogen bonds, root mean square deviation (RMSD), root mean square Fluctuation (RMSF), dynamic cross-correlation matric (DCCM) and principal component analysis. The study emphasizes the need for experimental validation to confirm the therapeutic utility of these compounds. This study enhances the computational search for new BC drugs and establishes a solid foundation for subsequent experimental and clinical research.
Assuntos
Neoplasias da Mama , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Humanos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Feminino , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ligantes , Simulação por Computador , Ligação Proteica , Tretinoína/metabolismo , Desenho de Fármacos , Antineoplásicos/farmacologia , Antineoplásicos/química , 17-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , 17-Hidroxiesteroide Desidrogenases/metabolismo , 17-Hidroxiesteroide Desidrogenases/química , Ligação de HidrogênioRESUMO
BACKGROUND: The Pharyngeal Endoderm (PE) is an extremely relevant developmental tissue, serving as the progenitor for the esophagus, parathyroids, thyroids, lungs, and thymus. While several studies have highlighted the importance of PE cells, a detailed transcriptional and epigenetic characterization of this important developmental stage is still missing, especially in humans, due to technical and ethical constraints pertaining to its early formation. RESULTS: Here we fill this knowledge gap by developing an in vitro protocol for the derivation of PE-like cells from human Embryonic Stem Cells (hESCs) and by providing an integrated multi-omics characterization. Our PE-like cells robustly express PE markers and are transcriptionally homogenous and similar to in vivo mouse PE cells. In addition, we define their epigenetic landscape and dynamic changes in response to Retinoic Acid by combining ATAC-Seq and ChIP-Seq of histone modifications. The integration of multiple high-throughput datasets leads to the identification of new putative regulatory regions and to the inference of a Retinoic Acid-centered transcription factor network orchestrating the development of PE-like cells. CONCLUSIONS: By combining hESCs differentiation with computational genomics, our work reveals the epigenetic dynamics that occur during human PE differentiation, providing a solid resource and foundation for research focused on the development of PE derivatives and the modeling of their developmental defects in genetic syndromes.
Assuntos
Diferenciação Celular , Endoderma , Epigênese Genética , Células-Tronco Embrionárias Humanas , Humanos , Endoderma/citologia , Endoderma/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Faringe/citologia , Faringe/metabolismo , Tretinoína/farmacologia , Tretinoína/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , CamundongosRESUMO
Human retinal organoids have become indispensable tools for retinal disease modeling and drug screening. Despite its versatile applications, the long timeframe for their differentiation and maturation limits the throughput of such research. Here, we successfully shortened this timeframe by accelerating human retinal organoid development using unique pharmacological approaches. Our method comprised three key steps: 1) a modified self-formed ectodermal autonomous multizone (SEAM) method, including dual SMAD inhibition and bone morphogenetic protein 4 treatment, for initial neural retinal induction; 2) the concurrent use of a Sonic hedgehog agonist SAG, activin A, and all-trans retinoic acid for rapid retinal cell specification; and 3) switching to SAG treatment alone for robust retinal maturation and lamination. The generated retinal organoids preserved typical morphological features of mature retinal organoids, including hair-like surface structures and well-organized outer layers. These features were substantiated by the spatial immunostaining patterns of several retinal cell markers, including rhodopsin and L/M opsin expression in the outermost layer, which was accompanied by reduced ectopic cone photoreceptor generation. Importantly, our method required only 90 days for retinal organoid maturation, which is approximately two-thirds the time necessary for other conventional methods. These results indicate that thoroughly optimized pharmacological interventions play a pivotal role in rapid and precise photoreceptor development during human retinal organoid differentiation and maturation. Thus, our present method may expedite human retinal organoid research, eventually contributing to the development of better treatment options for various degenerative retinal diseases.
Assuntos
Ativinas , Diferenciação Celular , Proteínas Hedgehog , Organoides , Retina , Transdução de Sinais , Tretinoína , Humanos , Ativinas/farmacologia , Ativinas/metabolismo , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/citologia , Proteínas Hedgehog/metabolismo , Tretinoína/farmacologia , Retina/metabolismo , Retina/citologia , Retina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismoRESUMO
A limited number of accessible and representative models of human trophoblast cells currently exist for the study of placentation. Current stem cell models involve either a transition through a naïve stem cell state or precise dynamic control of multiple growth factors and small-molecule cues. Here, we demonstrated that a simple five-day treatment of human induced pluripotent stem cells with two small molecules, retinoic acid (RA) and Wnt agonist CHIR 99021 (CHIR), resulted in rapid, synergistic upregulation of CDX2. Transcriptomic analysis of RA + CHIR-treated cells showed high similarity to primary trophectoderm cells. Multipotency was verified via further differentiation towards cells with syncytiotrophoblast or extravillous trophoblast features. RA + CHIR-treated cells were also assessed for the established criteria defining a trophoblast cell model, and they possess all the features necessary to be considered valid. Collectively, our data demonstrate a facile, scalable method for generating functional trophoblast-like cells in vitro to better understand the placenta.
Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Tretinoína , Trofoblastos , Humanos , Trofoblastos/efeitos dos fármacos , Trofoblastos/metabolismo , Trofoblastos/citologia , Tretinoína/farmacologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Piridinas/farmacologia , Feminino , Fator de Transcrição CDX2/metabolismo , Fator de Transcrição CDX2/genética , Pirimidinas/farmacologia , Gravidez , Modelos Biológicos , Células CultivadasRESUMO
Acne vulgaris is a common dermatological diagnosis observed in pediatric patients with skin of color, often resulting in scarring, keloid formation, and post-inflammatory hyperpigmentation, significantly impacting their quality of life. This exploratory retrospective chart review included 77 black pediatric patients seen at a tertiary care center for acne vulgaris between 2018 and 2023. We analyzed demographics, acne descriptors, and treatment modalities. The most common acne morphology was comedonal acne (83.6%), with 71% of the patients being female. Significant age differences were observed particularly for acne at the chin and overall face. Treatment regimens commonly prescribed included combinations of adapalene and benzoyl peroxide (22%), topical antibiotics, tretinoin, and benzoyl peroxide (34%). Given the higher risk of sequelae for patients with darker skin, it is crucial to address their unique treatment needs. This study highlights the distinctive characteristics of acne in black pediatric patients and calls for further research to enhance our understanding and treatment of this population. Limitations include the lack of direct patient interactions and reliance on chart data. Further studies are needed to compare acne presentation in skin of color of other populations, refining our knowledge of acne clinical presentation, complications, and treatment modalities for diverse patient populations.
Assuntos
Acne Vulgar , Antibacterianos , Negro ou Afro-Americano , Fármacos Dermatológicos , Humanos , Acne Vulgar/tratamento farmacológico , Feminino , Criança , Masculino , Estudos Retrospectivos , Adolescente , Fármacos Dermatológicos/uso terapêutico , Antibacterianos/uso terapêutico , Peróxido de Benzoíla/uso terapêutico , Tretinoína/uso terapêutico , Fatores EtáriosRESUMO
A 43-year-old man with pancytopenia was diagnosed with acute promyelocytic leukemia (APL). On the first day of induction therapy with all-trans retinoic acid (ATRA) alone, he presented with high fever and was found to have coronavirus disease 2019 (COVID-19) infection by SARS-CoV2 antigen test. While it is generally recommended to delay treatment for APL patients with COVID-19 unless urgent APL treatment is required, this patient needed to continue treatment due to APL-induced disseminated intravascular coagulation (DIC). Considering the challenge of distinguishing between differentiation syndrome (DS) and COVID-19 exacerbation, the ATRA dosage was reduced to 50%. The patient was able to continue treatment without development of DS or exacerbation of DIC, leading to his recovery from COVID-19 and remission of APL.
Assuntos
COVID-19 , Leucemia Promielocítica Aguda , Indução de Remissão , Tretinoína , Humanos , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/complicações , Tretinoína/administração & dosagem , Tretinoína/uso terapêutico , Masculino , Adulto , COVID-19/complicações , Resultado do Tratamento , Coagulação Intravascular Disseminada/tratamento farmacológico , Coagulação Intravascular Disseminada/etiologiaRESUMO
Cleft palate is the most common facial birth defect worldwide. It is caused by environmental factors or genetic mutations. Environmental factors such as pharmaceutical exposure in women are known to induce cleft palate. The aim of the present study was to investigate the protective effect of Sasa veitchii extract against medicine-induced inhibition of proliferation of human embryonic palatal mesenchymal cells. We demonstrated that all-trans-retinoic acid inhibited human embryonic palatal mesenchymal cell proliferation in a dose-dependent manner, whereas dexamethasone treatment had no effect on cell proliferation. Cotreatment with Sasa veitchii extract repressed all-trans-retinoic acid-induced toxicity in human embryonic palatal mesenchymal cells. We found that cotreatment with Sasa veitchii extract protected all-trans-retinoic acid-induced cyclin D1 downregulation in human embryonic palatal mesenchymal cells. Furthermore, Sasa veitchii extract suppressed all-trans-retinoic acid-induced miR-4680-3p expression. Additionally, the expression levels of the genes that function downstream of the target genes ( ERBB2 and JADE1 ) of miR-4680-3p in signaling pathways were enhanced by cotreatment with Sasa veitchii extract and all-trans-retinoic acid compared to all-trans-retinoic acid treatment. These results suggest that Sasa veitchii extract suppresses all-trans-retinoic acid-induced inhibition of cell proliferation via modulation of miR-4680-3p expression.
Assuntos
Proliferação de Células , Fissura Palatina , Palato , Extratos Vegetais , Tretinoína , Humanos , Tretinoína/farmacologia , Proliferação de Células/efeitos dos fármacos , Palato/efeitos dos fármacos , Palato/embriologia , Palato/citologia , Extratos Vegetais/farmacologia , MicroRNAs/metabolismo , MicroRNAs/genética , MicroRNAs/efeitos dos fármacos , Ciclina D1/metabolismo , Ciclina D1/genética , Células Cultivadas , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
SH-SY5Y is a human neuroblastoma cell line that can be differentiated into several neuronal phenotypes, depending on culture conditions. For this reason, this cell line has been widely used as an in vitro model of neurodegenerative conditions, such as Parkinson's disease (PD). However, most studies published to date used fetal bovine serum (FBS) as culture medium supplement for SH-SY5Y cell differentiation. We report on the testing of human platelet lysate (hPL) as a culture medium supplement to support SH-SY5Y cell culture. Both standard hPL and a fibrinogen-depleted hPL (FD-hPL) formulation, which does not require the addition of anticoagulants to culture media, promoted an increase in SH-SY5Y cell proliferation in comparison to FBS, without compromising metabolic activity. SH-SY5Y cells cultured in hPL or FD-hPL also displayed a higher number of neurite extensions and stained positive for MAP2 and synaptophysin, in the absence of differentiation stimuli; reducing hPL or FD-hPL concentration to 1% v/v did not affect cell proliferation or metabolic activity. Furthermore, following treatment with retinoic acid (RA) and further stimulation with brain-derived neurotrophic factor (BDNF) and nerve growth factor beta (NGF-ß), the percentage of SH-SY5Y cells stained positive for dopaminergic neuronal differentiation markers (tyrosine hydroxylase [TH] and Dopamine Transporter [DAT]) was higher in hPL or FD-hPL than in FBS, and gene expression of dopaminergic markers TH, DAT, and DR2 was also detected. Overall, the data herein presented supports the use of hPL to differentiate SH-SY5Y cells into a neuronal phenotype with dopaminergic features, and the adoption of FD-hPL as a fully xenogeneic free alternative to FBS to support the use of SH-SY5Y cells as a neurodegeneration model.
Assuntos
Plaquetas , Técnicas de Cultura de Células , Diferenciação Celular , Proliferação de Células , Neurônios Dopaminérgicos , Neuroblastoma , Humanos , Proliferação de Células/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Linhagem Celular Tumoral , Plaquetas/metabolismo , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/citologia , Técnicas de Cultura de Células/métodos , Meios de Cultura/química , Meios de Cultura/farmacologia , Tretinoína/farmacologia , FenótipoRESUMO
Nuclear factor-erythroid-2-related factor-2 (NRF-2) is a cellular resistance protein to oxidants. We investigated the effect of exogenous all-trans retinoic acid (ATRA) on the antioxidant system and NRF-2 in mice kidneys under hyperoxia-induced oxidative stress. Mice were divided into four groups. Daily, two groups were given either peanut-oil/dimethyl sulfoxide (PoDMSO) mixture or 50 mg/kg ATRA. Oxidative stress was induced by hyperoxia in the remaining groups. They were treated with PoDMSO or ATRA as described above, following hyperoxia (100% oxygen) for 72 h. NRF-2 and active-caspase-3 levels, lipid peroxidation (LPO), activities of antioxidant enzymes, xanthine oxidase (XO), paraoxonase1 (PON1), lactate dehydrogenase (LDH), tissue factor (TF), and prolidase were assayed in kidneys. Hyperoxia causes kidney damage induced by oxidative stress and apoptosis. Increased LPO, LDH, TF, and XO activities and decreased PON1 and prolidase activities contributed to kidney damage in hyperoxic mice. After hyperoxia, increases in the activities of antioxidant enzymes and NRF-2 level could not prevent this damage. ATRA attenuated damage via its oxidative stress-lowering effect. The decreased LDH and TF activities increased PON1 and prolidase activities, and normalized antioxidant statuses are indicators of the positive effects of ATRA. We recommend that ATRA can be used as a renoprotective agent against oxidative stress induced-kidney damage.
Assuntos
Apoptose , Fator 2 Relacionado a NF-E2 , Estresse Oxidativo , Tretinoína , Animais , Estresse Oxidativo/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Camundongos , Tretinoína/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Masculino , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Hiperóxia/metabolismo , Hiperóxia/tratamento farmacológico , Antioxidantes/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacosRESUMO
Opsins are a class of transmembrane proteins encoded by opsin genes, and they play a variety of functional roles. Short wavelength-sensitive opsin 2 (sws2), one of the five classes of visual opsin genes, mainly senses blue light. Previous research has indicated that sws2 is essential for melanocyte formation in fish; however, its specific role in skin color differentiation remains to be elucidated. Here, we identified the sws2 gene in a prized reef-dwelling fish, Plectropomus leopardus. The full-length P. leopardus sws2 gene encodes a protein consisting of 351 amino acids, and exhibits substantial homology with other fish species. The expression of the sws2 gene was widespread across P. leopardus tissues, with high expression in eye and skin tissues. Through immunohistochemistry and in situ hybridization analyses, we discovered that the sws2 gene was primarily localized in the rod and cone cells of the retina, and epidermal cells of the skin. Furthermore, dsRNA interference was used for sws2 gene knockdown in living P. leopardus to elucidate its function in skin color differentiation. Black-color-related genes, melanin contents, and tyrosinase activity in the skin significantly decreased after sws2 knockdown (p < 0.05), but red-color-related genes and carotenoid and lutein contents significantly increased (p < 0.05). Retinoic acid injection produced the opposite results. Our results suggested that the sws2 gene influences P. leopardus skin color regulation by affecting vitamin synthesis and melanin-related gene expression levels. This study establishes a foundation for elucidating the molecular mechanisms by which sws2 regulates melanocyte formation in fish skin.
Assuntos
Melaninas , Pele , Tretinoína , Animais , Melaninas/biossíntese , Melaninas/metabolismo , Tretinoína/metabolismo , Pele/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Pigmentação da Pele/genética , Opsinas/metabolismo , Opsinas/genética , Regulação da Expressão GênicaRESUMO
Acute myeloid leukemia (AML) is characterized by the abnormal proliferation and differentiation arrest of myeloid progenitor cells. The clinical treatment of AML remains challenging. Promoting AML cell differentiation is a valid strategy, but effective differentiation drugs are lacking for most types of AML. In this study, we generated Tg(drl:hoxa9) zebrafish, in which hoxa9 overexpression was driven in hematopoietic cells and myeloid differentiation arrest was exhibited. Using Tg(drl:hoxa9) embryos, we performed chemical screening and identified four FDA-approved drugs, ethacrynic acid, khellin, oxcarbazepine, and alendronate, that efficiently restored myeloid differentiation. The four drugs also induced AML cell differentiation, with ethacrynic acid being the most effective. By an RNA-seq analysis, we found that during differentiation, ethacrynic acid activated the IL-17 and MAPK signaling pathways, which are known to promote granulopoiesis. Furthermore, we found that ethacrynic acid enhanced all-trans retinoic acid (ATRA)-induced differentiation, and both types of signaling converged on the IL-17/MAPK pathways. Inhibiting the IL-17/MAPK pathways impaired ethacrynic acid and ATRA-induced differentiation. In addition, we showed that ethacrynic acid is less toxic to embryogenesis and less disruptive to normal hematopoiesis than ATRA. Thus, the combination of ethacrynic acid and ATRA may have broader clinical applications. In conclusion, through zebrafish-aided screening, our study identified four drugs that can be repurposed to induce AML differentiation, thus providing new agents for AML therapy.
Assuntos
Diferenciação Celular , Leucemia Mieloide Aguda , Peixe-Zebra , Animais , Peixe-Zebra/embriologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/metabolismo , Diferenciação Celular/efeitos dos fármacos , Humanos , Embrião não Mamífero/efeitos dos fármacos , Tretinoína/farmacologia , Ácido Etacrínico/farmacologia , Antineoplásicos/farmacologiaRESUMO
Activation of pregnane X receptor (PXR) by xenobiotics has been associated with metabolic diseases. This study aimed to reveal the impact of PXR activation on hepatic metabolome and explore novel mechanisms underlying PXR-mediated lipid metabolism disorder in the liver. Wild-type and PXR-deficient male C57BL/6 mice were used as in vivo models, and hepatic steatosis was induced by pregnenolone-16α-carbonitrile, a typical rodent PXR agonist. Metabolomic analysis of liver tissues showed that PXR activation led to significant changes in metabolites involved in multiple metabolic pathways previously reported, including lipid metabolism, energy homeostasis, and amino acid metabolism. Moreover, the level of hepatic all-trans retinoic acid (ATRA), the main active metabolite of vitamin A, was significantly increased by PXR activation, and genes involved in ATRA metabolism exhibited differential expression following PXR activation or deficiency. Consistent with previous research, the expression of downstream target genes of peroxisome proliferator-activated receptor α (PPARα) was decreased. Analysis of fatty acids by Gas Chromatography-Mass Spectrometer further revealed changes in polyunsaturated fatty acid metabolism upon PXR activation, suggesting inhibition of PPARα activity. Taken together, our findings reveal a novel metabolomic signature of hepatic steatosis induced by PXR activation in mice.
Assuntos
Ácidos Graxos Insaturados , Fígado Gorduroso , Fígado , Metabolômica , Camundongos Endogâmicos C57BL , PPAR alfa , Receptor de Pregnano X , Tretinoína , Animais , Masculino , Receptor de Pregnano X/metabolismo , Receptor de Pregnano X/genética , Tretinoína/metabolismo , Fígado/metabolismo , Fígado/efeitos dos fármacos , Fígado Gorduroso/metabolismo , Fígado Gorduroso/induzido quimicamente , Ácidos Graxos Insaturados/metabolismo , PPAR alfa/metabolismo , PPAR alfa/genética , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Camundongos Knockout , Carbonitrila de Pregnenolona/farmacologia , Modelos Animais de DoençasRESUMO
OBJECTIVES: The imbalance between apoptosis and proliferation in fibroblast-like synoviocytes (FLSs) plays a key role in the pathogenesis of rheumatoid arthritis (RA). This study aims to investigate the potential of all-trans retinoic acid (ATRA) as a supplementary therapeutic agent alongside methotrexate (MTX) for RA, by examining its ability to inhibit synovial cell proliferation and enhance apoptosis through the ROS-JNK signalling pathway. METHODS: The viability, apoptosis, and autophagy levels of human rheumatoid arthritis fibroblast-like synovial cells (HFLS-RA) were evaluated, while ROS generation was measured through the DCFH-DA fluorescence microplate assay. Western blotting was used to analyse the expression levels of JNK signalling pathway-related proteins. To assess therapeutic potential in vivo, a collagen-induced arthritis (CIA) model was established in Wistar rats. RESULTS: Small doses of MTX did not significantly affect the viability of HFLS-RAs or induce apoptosis. However, when ATRA was added to the treatment, the therapy markedly inhibited cell proliferation and induced apoptosis and excessive autophagy. Mechanistically, ATRA activated the ROS/JNK signalling pathway in HFLS-RAs. ROS scavengers and JNK inhibitors significantly attenuated ATRA-induced apoptosis and autophagy. In vivo, the combination therapy demonstrated a remarkable enhancement of the anti-arthritic efficacy in CIA rats. CONCLUSIONS: The ability of ATRA to inhibit proliferation in RA FLSs through autophagy and apoptosis underscores its potential as a supplementary therapeutic agent alongside MTX for RA, particularly when compared to the limited impact of MTX on these processes. This combined strategy holds promise for enhancing therapeutic outcomes and warrants further investigation in the management of RA.
Assuntos
Apoptose , Artrite Experimental , Artrite Reumatoide , Autofagia , Proliferação de Células , Metotrexato , Ratos Wistar , Espécies Reativas de Oxigênio , Sinoviócitos , Tretinoína , Tretinoína/farmacologia , Apoptose/efeitos dos fármacos , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/patologia , Artrite Reumatoide/metabolismo , Metotrexato/farmacologia , Autofagia/efeitos dos fármacos , Animais , Humanos , Artrite Experimental/tratamento farmacológico , Artrite Experimental/patologia , Artrite Experimental/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sinoviócitos/efeitos dos fármacos , Sinoviócitos/patologia , Sinoviócitos/metabolismo , Proliferação de Células/efeitos dos fármacos , Quimioterapia Combinada , Antirreumáticos/farmacologia , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/patologia , Membrana Sinovial/metabolismo , Masculino , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Ratos , Linhagem CelularRESUMO
BACKGROUND: Keloid is a disease characterized by proliferation of fibrous tissue after the healing of skin tissue, which seriously affects the daily life of patients. However, the clinical treatment of keloids still has limitations, that is, it is not effective in controlling keloids, resulting in a high recurrence rate. Thus, it is urgent to identify new signatures to improve the diagnosis and treatment of keloids. METHOD: Bulk RNA seq and scRNA seq data were downloaded from the GEO database. First, we used WGCNA and MEGENA to co-identify keloid/immune-related DEGs. Subsequently, we used three machine learning algorithms (Randomforest, SVM-RFE, and LASSO) to identify hub immune-related genes of keloid (KHIGs) and investigated the heterogeneous expression of KHIGs during fibroblast subpopulation differentiation using scRNA-seq. Finally, we used HE and Masson staining, quantitative reverse transcription-PCR, western blotting, immunohistochemical, and Immunofluorescent assay to investigate the dysregulated expression and the mechanism of retinoic acid in keloids. RESULTS: In the present study, we identified PTGFR, RBP5, and LIF as KHIGs and validated their diagnostic performance. Subsequently, we constructed a novel artificial neural network molecular diagnostic model based on the transcriptome pattern of KHIGs, which is expected to break through the current dilemma faced by molecular diagnosis of keloids in the clinic. Meanwhile, the constructed IG score can also effectively predict keloid risk, which provides a new strategy for keloid prevention. Additionally, we observed that KHIGs were also heterogeneously expressed in the constructed differentiation trajectories of fibroblast subtypes, which may affect the differentiation of fibroblast subtypes and thus lead to dysregulation of the immune microenvironment in keloids. Finally, we found that retinoic acid may treat or alleviate keloids by inhibiting RBP5 to differentiate pro-inflammatory fibroblasts (PIF) to mesenchymal fibroblasts (MF), which further reduces collagen secretion. CONCLUSION: In summary, the present study provides novel immune signatures (PTGFR, RBP5, and LIF) for keloid diagnosis and treatment, and identifies retinoic acid as potential anti-keloid drugs. More importantly, we provide a new perspective for understanding the interactions between different fibroblast subtypes in keloids and the remodeling of their immune microenvironment.
Assuntos
Queloide , RNA-Seq , Queloide/genética , Queloide/diagnóstico , Queloide/patologia , Queloide/imunologia , Queloide/tratamento farmacológico , Humanos , Transcriptoma/genética , Perfilação da Expressão Gênica , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/imunologia , Redes Reguladoras de Genes , Tretinoína/farmacologia , Tretinoína/uso terapêutico , Análise de Célula Única/métodos , Diferenciação Celular/genética , Análise de Sequência de RNA/métodos , Aprendizado de Máquina , Análise da Expressão Gênica de Célula ÚnicaRESUMO
Spermatogenesis in mammalian testes is essential for male fertility, ensuring a continuous supply of mature sperm. The testicular microenvironment finely tunes this process, with retinoic acid, an active metabolite of vitamin A, serving a pivotal role. Retinoic acid is critical for various stages, including the differentiation of spermatogonia, meiosis in spermatogenic cells, and the production of mature spermatozoa. Vitamin A deficiency halts spermatogenesis, leading to the degeneration of numerous germ cells, a condition reversible with retinoic acid supplementation. Although retinoic acid can restore fertility in some males with reproductive disorders, it does not work universally. Furthermore, high doses may adversely affect reproduction. The inconsistent outcomes of retinoid treatments in addressing infertility are linked to the incomplete understanding of the molecular mechanisms through which retinoid signaling governs spermatogenesis. In addition to the treatment of male reproductive disorders, the role of retinoic acid in spermatogenesis also provides new ideas for the development of male non-hormone contraceptives. This paper will explore three facets: the synthesis and breakdown of retinoic acid in the testes, its role in spermatogenesis, and its application in male reproduction. Our discussion aims to provide a comprehensive reference for studying the regulatory effects of retinoic acid signaling on spermatogenesis and offer insights into its use in treating male reproductive issues.
Assuntos
Espermatogênese , Tretinoína , Masculino , Espermatogênese/efeitos dos fármacos , Tretinoína/metabolismo , Tretinoína/farmacologia , Humanos , Animais , Reprodução/efeitos dos fármacos , Testículo/metabolismo , Testículo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Infertilidade Masculina/metabolismo , Espermatozoides/metabolismo , Espermatozoides/efeitos dos fármacosRESUMO
The transformation of acute promyelocytic leukemia (APL) from the most fatal to the most curable subtype of acute myeloid leukemia (AML), with long-term survival exceeding 90%, has represented one of the most exciting successes in hematology and in oncology. APL is a paradigm for oncoprotein-targeted cure.APL is caused by a 15/17 chromosomal translocation which generates the PML-RARA fusion protein and can be cured by the chemotherapy-free approach based on the combination of two therapies targeting PML-RARA: retinoic acid (RA) and arsenic. PML-RARA is the key driver of APL and acts by deregulating transcriptional control, particularly RAR targets involved in self-renewal or myeloid differentiation, also disrupting PML nuclear bodies. PML-RARA mainly acts as a modulator of the expression of specific target genes: genes whose regulatory elements recruit PML-RARA are not uniformly repressed but also may be upregulated or remain unchanged. RA and arsenic trioxide directly target PML-RARA-mediated transcriptional deregulation and protein stability, removing the differentiation block at promyelocytic stage and inducing clinical remission of APL patients.
Assuntos
Leucemia Promielocítica Aguda , Proteínas de Fusão Oncogênica , Tretinoína , Humanos , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patologia , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Tretinoína/uso terapêutico , Tretinoína/farmacologia , Trióxido de Arsênio/uso terapêutico , Trióxido de Arsênio/farmacologia , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Antineoplásicos/uso terapêutico , Antineoplásicos/farmacologia , Arsenicais/uso terapêutico , Arsenicais/farmacologia , Óxidos/uso terapêutico , Óxidos/farmacologia , AnimaisRESUMO
Purpose: Abnormalities in aquaporins are implicated in the pathological progression of dry eye syndrome. Retinoic acid (RA) regulates cellular proliferation, differentiation, and apoptosis in the cornea, thereby being associated with dry eye disease (DED). The objective of this study is to explore the underlying mechanisms responsible for RA metabolic abnormalities in corneas lacking aquaporin 5 (AQP5). Methods: Dry eye (DE) models were induced via subcutaneous scopolamine hydrobromide. Aqp5 knockout (Aqp5-/-) mice and DE mice were utilized to assess corneal epithelial alterations. Tear secretion, goblet cell counts, and corneal punctate defects were evaluated. The impact of Aqp5 on RA-related enzymes and receptors was investigated using pharmacological RA or SR (A JunB inhibitor), a transcription factor JunB inhibitor, treatment in mouse corneal epithelial cells (CECs), or human corneal epithelial cells (HCECs). The HCECs and NaCl-treated HCECs underwent quantitative real-time PCR (qRT-PCR), immunofluorescent, Western blot, and TUNEL assays. The regulation of transcription factor JunB on Aldh1a1 was explored via ChIP-PCR. Results: Aqp5 and Aldh1a1 were reduced in both CECs of DE mice and NaCl-induced HCECs. Aqp5-/- mice exhibited DE phenotype and reduced Aldh1a1. RA treatment reduced apoptosis, promoted proliferation, and improved the DE phenotype in Aqp5-/- mice. JunB enrichment in the Aldh1a1 promoter was identified by ChIP-PCR. SR significantly increased Aldh1a1 expression, Ki67, and ΔNp63-positive cells, and decreased TUNEL-positive cells in CECs and HCECs. Conclusions: Our findings demonstrated the downregulation of Aqp5 expression and aberrant RA metabolism in DE conditions. Knockout of Aqp5 resulted in reduced production of RA through activation of JunB, subsequently leading to the manifestation of DE symptoms.
Assuntos
Apoptose , Aquaporina 5 , Modelos Animais de Doenças , Síndromes do Olho Seco , Camundongos Knockout , Tretinoína , Animais , Aquaporina 5/genética , Aquaporina 5/biossíntese , Aquaporina 5/metabolismo , Síndromes do Olho Seco/metabolismo , Síndromes do Olho Seco/patologia , Síndromes do Olho Seco/genética , Camundongos , Tretinoína/farmacologia , Epitélio Corneano/metabolismo , Epitélio Corneano/patologia , Reação em Cadeia da Polimerase em Tempo Real , Camundongos Endogâmicos C57BL , Western Blotting , Humanos , Células Cultivadas , Lágrimas/metabolismo , Marcação In Situ das Extremidades Cortadas , Regulação da Expressão Gênica , Proliferação de CélulasRESUMO
BACKGROUND: When massive necrosis occurs in acute liver failure (ALF), rapid expansion of HSCs called liver progenitor cells (LPCs) in a process called ductular reaction is required for survival. The underlying mechanisms governing this process are not entirely known to date. In ALF, high levels of retinoic acid (RA), a molecule known for its pleiotropic roles in embryonic development, are secreted by activated HSCs. We hypothesized that RA plays a key role in ductular reaction during ALF. METHODS: RNAseq was performed to identify molecular signaling pathways affected by all-trans retinoid acid (atRA) treatment in HepaRG LPCs. Functional assays were performed in HepaRG cells treated with atRA or cocultured with LX-2 cells and in the liver tissue of patients suffering from ALF. RESULTS: Under ALF conditions, activated HSCs secreted RA, inducing RARα nuclear translocation in LPCs. RNAseq data and investigations in HepaRG cells revealed that atRA treatment activated the WNT-ß-Catenin pathway, enhanced stemness genes (SOX9, AFP, and others), increased energy storage, and elevated the expression of ATP-binding cassette transporters in a RARα nuclear translocation-dependent manner. Further, atRA treatment-induced pathways were confirmed in a coculture system of HepaRG with LX-2 cells. Patients suffering from ALF who displayed RARα nuclear translocation in the LPCs had significantly better MELD scores than those without. CONCLUSIONS: During ALF, RA secreted by activated HSCs promotes LPC activation, a prerequisite for subsequent LPC-mediated liver regeneration.
Assuntos
Falência Hepática Aguda , Células-Tronco , Tretinoína , Humanos , Tretinoína/farmacologia , Células-Tronco/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Fígado/efeitos dos fármacos , Receptor alfa de Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico/metabolismo , Técnicas de Cocultura , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismoRESUMO
Alzheimer's disease (AD) is a neurodegenerative disease that causes physical damage to neuronal connections, leading to brain atrophy. This disruption of synaptic connections results in mild to severe cognitive impairments. Unfortunately, no effective treatment is currently known to prevent or reverse the symptoms of AD. The aim of this study was to investigate the effects of three synthetic peptides, i.e., KLVFF, RGKLVFFGR and RIIGL, on an AD in vitro model represented by differentiated SH-SY5Y neuroblastoma cells exposed to retinoic acid (RA) and brain-derived neurotrophic factor (BDNF). The results demonstrated that RIIGL peptide had the least significant cytotoxic activity to normal SH-SY5Y while exerting high cytotoxicity against the differentiated cells. The mechanism of RIIGL peptide in the differentiated SH-SY5Y was investigated based on changes in secretory proteins compared to another two peptides. A total of 380 proteins were identified, and five of them were significantly detected after treatment with RIIGL peptide. These secretory proteins were found to be related to microtubule-associated protein tau (MAPT) and amyloid-beta precursor protein (APP). RIIGL peptide acts on differentiated SH-SY5Y by regulating amyloid-beta formation, neuron apoptotic process, ceramide catabolic process, and oxidative phosphorylation and thus has the potentials to treat AD.