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1.
Platelets ; 35(1): 2347331, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38722091

RESUMO

Platelet-rich plasma (PRP) holds promise as a therapeutic modality for wound healing; however, immediate utilization encounters challenges related to volume, concentration, and consistency. Cryopreservation emerges as a viable solution, preserving PRP's bioactive components and extending its shelf life. This study explores the practicality and efficacy of cryopreserved platelet-rich plasma (cPRP) in wound healing, scrutinizing both cellular mechanisms and clinical implications. Fresh PRP and cPRP post freeze-thaw underwent assessment in macrophage, fibroblast, and endothelial cell cultures. The impact of cPRP on active component release and cell behavior pertinent to wound healing was evaluated. Varied concentrations of cPRP (1%, 5%, 10%) were examined for their influence on cell polarization, migration, and proliferation. The results showed minimal changes in cPRP's IL-1ß levels, a slight decrease in PDGF-BB, and superior effects on macrophage M2 polarization and fibroblast migration, while no statistical significance was observed in endothelial cell angiogenesis and proliferation. Remarkably, 5% PRP exhibited the most significant stimulation among all cPRP concentrations, notably impacting cell proliferation, angiogenesis, and migration. The discussion underscores that cPRP maintains platelet phenotype and function over extended periods, with 5% cPRP offering the most favorable outcomes, providing a pragmatic approach for cold storage to extend post-thaw viability and amplify therapeutic effects.


What is the context? Platelet-rich plasma (PRP) is a potential bioactive material for wound healing, but using it immediately faces issues like volume, concentration, and consistency.Low-temperature freezing is a method employed to preserve PRP. However, the current understanding of the effects of the freezing-thawing process on the components of PRP and its impact on cells relevant to wound healing remains unclear.What is new? This study explores the feasibility and effectiveness of using cryopreserved PRP at −80°C for promoting wound healing. This research stands out for its focus on cellular responses and practical implications in therapeutic contexts.To understand their distinct impact on different cell types relevant to wound healing, the study meticulously examined various final concentrations of cPRP (1%, 5%, 10%).The study identified the superior effects of 5% cPRP on crucial cellular activities, notably in cell polarization, proliferation, angiogenesis, and migration.What is the impact? Low-temperature freezing can be considered an effective method for PRP preservation.Some bioactive components in cPRP exhibit subtle changes; however, these changes result in better effects on certain cell types related to healing.The study illustrates that all concentrations of cPRP effectively enhance cell proliferation, migration, and differentiation, emphasizing the comparable efficacy of cryopreserved PRP to non-cryopreserved PRP.


Assuntos
Criopreservação , Plasma Rico em Plaquetas , Cicatrização , Plasma Rico em Plaquetas/metabolismo , Humanos , Criopreservação/métodos , Proliferação de Células , Movimento Celular , Fibroblastos/metabolismo
2.
Molecules ; 29(9)2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38731556

RESUMO

Red rice, a variety of pigmented grain, serves dual purposes as both a food and medicinal resource. In recent years, we have witnessed an increasing interest in the dermatological benefits of fermented rice extracts, particularly their whitening and hydrating effects. However, data on the skincare advantages derived from fermenting red rice with Aspergillus oryzae remain sparse. This study utilized red rice as a substrate for fermentation by Aspergillus oryzae, producing a substance known as red rice Aspergillus oryzae fermentation (RRFA). We conducted a preliminary analysis of RRFA's composition followed by an evaluation of its skincare potential through various in vitro tests. Our objective was to develop a safe and highly effective skincare component for potential cosmetic applications. RRFA's constituents were assessed using high-performance liquid chromatography (HPLC), Kjeldahl nitrogen determination, the phenol-sulfuric acid method, and enzyme-linked immunosorbent assay (ELISA). We employed human dermal fibroblasts (FB) to assess RRFA's anti-aging and antioxidative properties, immortalized keratinocytes (HaCaT cells) and 3D epidermal models to examine its moisturizing and reparative capabilities, and human primary melanocytes (MCs) to study its effects on skin lightening. Our findings revealed that RRFA encompasses several bioactive compounds beneficial for skin health. RRFA can significantly promote the proliferation of FB cells. And it markedly enhances the mRNA expression of ECM-related anti-aging genes and reduces reactive oxygen species production. Furthermore, RRFA significantly boosts the expression of Aquaporin 3 (AQP3), Filaggrin (FLG), and Hyaluronan Synthase 1 (HAS1) mRNA, alongside elevating moisture levels in a 3D epidermal model. Increases were also observed in the mRNA expression of Claudin 1 (CLDN1), Involucrin (IVL), and Zonula Occludens-1 (ZO-1) in keratinocytes. Additionally, RRFA demonstrated an inhibitory effect on melanin synthesis. Collectively, RRFA contains diverse ingredients which are beneficial for skin health and showcases multifaceted skincare effects in terms of anti-aging, antioxidant, moisturizing, repairing, and whitening capabilities in vitro, highlighting its potential for future cosmetic applications.


Assuntos
Aspergillus oryzae , Fermentação , Proteínas Filagrinas , Oryza , Aspergillus oryzae/metabolismo , Oryza/química , Oryza/metabolismo , Humanos , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Queratinócitos/metabolismo , Queratinócitos/efeitos dos fármacos , Células HaCaT , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Melanócitos/metabolismo , Melanócitos/efeitos dos fármacos , Higiene da Pele/métodos , Pele/metabolismo
3.
Molecules ; 29(9)2024 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-38731597

RESUMO

Fibrosis is a ubiquitous pathology, and prior studies have indicated that various artemisinin (ART) derivatives (including artesunate (AS), artemether (AM), and dihydroartemisinin (DHA)) can reduce fibrosis in vitro and in vivo. The medicinal plant Artemisia annua L. is the natural source of ART and is widely used, especially in underdeveloped countries, to treat a variety of diseases including malaria. A. afra contains no ART but is also antimalarial. Using human dermal fibroblasts (CRL-2097), we compared the effects of A. annua and A. afra tea infusions, ART, AS, AM, DHA, and a liver metabolite of ART, deoxyART (dART), on fibroblast viability and expression of key fibrotic marker genes after 1 and 4 days of treatment. AS, DHA, and Artemisia teas reduced fibroblast viability 4 d post-treatment in up to 80% of their respective controls. After 4 d of treatment, AS DHA and Artemisia teas downregulated ACTA2 up to 10 fold while ART had no significant effect, and AM increased viability by 10%. MMP1 and MMP3 were upregulated by AS, 17.5 and 32.6 fold, respectively, and by DHA, 8 and 51.8 fold, respectively. ART had no effect, but A. annua and A. afra teas increased MMP3 5 and 16-fold, respectively. Although A. afra tea increased COL3A1 5 fold, MMP1 decreased >7 fold with no change in either transcript by A. annua tea. Although A. annua contains ART, it had a significantly greater anti-fibrotic effect than ART alone but was less effective than A. afra. Immunofluorescent staining for smooth-muscle α-actin (α-SMA) correlated well with the transcriptional responses of drug-treated fibroblasts. Together, proliferation, qPCR, and immunofluorescence results show that treatment with ART, AS, DHA, and the two Artemisia teas yield differing responses, including those related to fibrosis, in human dermal fibroblasts, with evidence also of remodeling of fibrotic ECM.


Assuntos
Artemisia , Artemisininas , Fibroblastos , Fibrose , Humanos , Artemisininas/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Artemisia/química , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Sobrevivência Celular/efeitos dos fármacos , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/genética , Actinas/metabolismo , Actinas/genética , Artesunato/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Artemeter/farmacologia , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/patologia
4.
BMC Immunol ; 25(1): 31, 2024 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-38734625

RESUMO

BACKGROUND: Thyroid eye disease (TED) is an inflammatory process involving lymphocyte-mediated immune response and orbital tissue damage. The anti-insulin-like growth factor-1 receptor (IGF-1R) antibodies produced by B lymphocytes are involved in the activation of orbital fibroblasts and the inflammatory process of orbital tissue damage in TED. The purpose of this study was to explore the role of IGF-1R in the mechanistic connection between orbital fibroblasts and B lymphocytes in TED. METHODS: Orbital fibroblasts sampled from orbital connective tissues and peripheral B lymphocytes isolated from peripheral blood, which were obtained from 15 patients with TED and 15 control patients, were co-cultured at a ratio of 1:20. The level of IGF-1R expression in orbital fibroblasts was evaluated by flow cytometry and confocal microscopy. Transient B lymphocyte depletion was induced with anti-CD20 monoclonal antibody rituximab, while the IGF-1R pathway was blocked by the IGF-1R binding protein. The expression levels of interleukin-6 (IL-6) and regulated upon activation, normal T cell expressed and secreted (RANTES) in the co-culture model were quantified via ELISA. RESULTS: IGF-1R expression was significantly elevated in TED orbital fibroblasts compared to that of controls. A 24-h co-culture of orbital fibroblasts with peripheral B lymphocytes induced elevated expression levels of IL-6 and RANTES in each group (TED patients and controls), with the highest levels occurring in TED patients (T + T group). Rituximab and IGF-1R binding protein significantly inhibited increased levels of IL-6 and RANTES in the co-culture model of TED patients. CONCLUSIONS: IGF-1R may mediate interaction between orbital fibroblasts and peripheral B lymphocytes; thus, blocking IGF-1R may reduce the local inflammatory response in TED. Rituximab-mediated B lymphocyte depletion played a role in inhibiting inflammatory responses in this in vitro co-culture model, providing a theoretical basis for the clinical application of anti-CD20 monoclonal antibodies in TED.


Assuntos
Linfócitos B , Técnicas de Cocultura , Fibroblastos , Oftalmopatia de Graves , Receptor IGF Tipo 1 , Humanos , Oftalmopatia de Graves/metabolismo , Oftalmopatia de Graves/imunologia , Fibroblastos/metabolismo , Receptor IGF Tipo 1/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Feminino , Masculino , Pessoa de Meia-Idade , Adulto , Rituximab/farmacologia , Rituximab/uso terapêutico , Órbita/metabolismo , Órbita/imunologia , Depleção Linfocítica , Interleucina-6/metabolismo , Células Cultivadas , Quimiocina CCL5/metabolismo , Comunicação Celular , Idoso
5.
Sci Rep ; 14(1): 10610, 2024 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-38719857

RESUMO

Histone lysine methylation is thought to play a role in the pathogenesis of rheumatoid arthritis (RA). We previously reported aberrant expression of the gene encoding mixed-lineage leukemia 1 (MLL1), which catalyzes methylation of histone H3 lysine 4 (H3K4), in RA synovial fibroblasts (SFs). The aim of this study was to elucidate the involvement of MLL1 in the activated phenotype of RASFs. SFs were isolated from synovial tissues obtained from patients with RA or osteoarthritis (OA) during total knee joint replacement. MLL1 mRNA and protein levels were determined after stimulation with tumor necrosis factor α (TNFα). We also examined changes in trimethylation of H3K4 (H3K4me3) levels in the promoters of RA-associated genes (matrix-degrading enzymes, cytokines, and chemokines) and the mRNA levels upon small interfering RNA-mediated depletion of MLL1 in RASFs. We then determined the levels of H3K4me3 and mRNAs following treatment with the WD repeat domain 5 (WDR5)/MLL1 inhibitor MM-102. H3K4me3 levels in the gene promoters were also compared between RASFs and OASFs. After TNFα stimulation, MLL1 mRNA and protein levels were higher in RASFs than OASFs. Silencing of MLL1 significantly reduced H3K4me3 levels in the promoters of several cytokine (interleukin-6 [IL-6], IL-15) and chemokine (C-C motif chemokine ligand 2 [CCL2], CCL5, C-X-C motif chemokine ligand 9 [CXCL9], CXCL10, CXCL11, and C-X3-C motif chemokine ligand 1 [CX3CL1]) genes in RASFs. Correspondingly, the mRNA levels of these genes were significantly decreased. MM-102 significantly reduced the promoter H3K4me3 and mRNA levels of the CCL5, CXCL9, CXCL10, and CXCL11 genes in RASFs. In addition, H3K4me3 levels in the promoters of the IL-6, IL-15, CCL2, CCL5, CXCL9, CXCL10, CXCL11, and CX3CL1 genes were significantly higher in RASFs than OASFs. Our findings suggest that MLL1 regulates the expression of particular cytokines and chemokines in RASFs and is associated with the pathogenesis of RA. These results could lead to new therapies for RA.


Assuntos
Artrite Reumatoide , Quimiocinas , Citocinas , Fibroblastos , Histona-Lisina N-Metiltransferase , Histonas , Proteína de Leucina Linfoide-Mieloide , Membrana Sinovial , Humanos , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Artrite Reumatoide/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/genética , Fibroblastos/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteína de Leucina Linfoide-Mieloide/genética , Citocinas/metabolismo , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Histonas/metabolismo , Quimiocinas/metabolismo , Quimiocinas/genética , Regulação da Expressão Gênica , Fator de Necrose Tumoral alfa/metabolismo , Regiões Promotoras Genéticas , Feminino , Masculino , Células Cultivadas , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Osteoartrite/metabolismo , Osteoartrite/patologia , Osteoartrite/genética , Idoso
6.
Physiol Rep ; 12(9): e16032, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38720166

RESUMO

INPP4A has been shown to be involved in the regulation of cell proliferation and apoptosis of multiple cell types including fibroblasts. Previous reports from our group have demonstrated the role of inositol polyphosphate 4-phosphatase Type I A (INPP4A) in these functions. Though existing evidences suggest a critical role for INPP4A in the maintenance of lung homeostasis, its role in chronic lung diseases is relatively under explored. In the current study, we made an attempt to understand the regulation of INPP4A in idiopathic pulmonary fibrosis (IPF). Through integration of relevant INPP4A gene expression data from public repositories with our results from in vitro experiments and mouse models, we show that INPP4A is altered in IPF. Interestingly, the direction of the change is dependent both on the disease stage and the region of the lung used. INPP4A was found to be upregulated when analyzed in lung sample representative of the whole lung, but was downregulated in the fibrotic regions of the lung. Similarly, INPP4A was found to be high, compared to controls, only in the early stage of the disease. Though the observed increase in INPP4A was found to be negatively correlated to physiological indices, FVC, and DLCO, of lung function, treatment with anti-INPP4A antibody worsened the condition in bleomycin treated mice. These contrasting results taken together are suggestive of a nuanced regulation of INPP4A in IPF which is dependent on the disease stage, cellular state and extent of fibrosis in the lung region being analyzed.


Assuntos
Fibrose Pulmonar Idiopática , Monoéster Fosfórico Hidrolases , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Fibrose Pulmonar Idiopática/genética , Animais , Humanos , Monoéster Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/genética , Camundongos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos Endogâmicos C57BL , Fibroblastos/metabolismo , Feminino
8.
Front Immunol ; 15: 1323410, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38726004

RESUMO

Background: Huntingtin-interacting protein-1 (HIP1) is a new arthritis severity gene implicated in the regulation of the invasive properties of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). These invasive properties of FLS strongly correlate with radiographic and histology damage in patients with RA and rodent models of arthritis. While HIP1 has several intracellular functions, little is known about its binding proteins, and identifying them has the potential to expand our understanding of its role in cell invasion and other disease-contributing phenotypes, and potentially identify new targets for therapy. Methods: FLS cell lines from arthritic DA (highly invasive) and from arthritis-protected congenic rats R6 (minimally invasive), which differ in an amino-acid changing HIP1 SNP, were cultured and lysed, and proteins were immunoprecipitated with an anti-HIP1 antibody. Immunoprecipitates were analyzed by mass spectrometry. Differentially detected (bound) proteins were selected for functional experiments using siRNA knockdown in human RA FLS to examine their effect in cell invasiveness, adhesion, cell migration and proliferation, and immunofluorescence microscopy. Results: Proteins detected included a few known HIP1-binding proteins and several new ones. Forty-five proteins differed in levels detected in the DA versus R6 congenic mass spectrometry analyses. Thirty-two of these proteins were knocked down and studied in vitro, with 10 inducing significant changes in RA FLS phenotypes. Specifically, knockdown of five HIP1-binding protein genes (CHMP4BL1, COPE, KIF1C, YWHAG, and YWHAH) significantly decreased FLS invasiveness. Knockdown of KIF1C also reduced RA FLS migration. The binding of four selected proteins to human HIP1 was confirmed. KIF1C colocalized with lamellipodia, and its knockdown prevented RA FLS from developing an elongated morphology with thick linearized actin fibers or forming polarized lamellipodia, all required for cell mobility and invasion. Unlike HIP1, KIF1C knockdown did not affect Rac1 signaling. Conclusion: We have identified new HIP1-binding proteins and demonstrate that 10 of them regulate key FLS phenotypes. These HIP1-binding proteins have the potential to become new therapeutic targets and help better understand the RA FLS pathogenic behavior. KIF1C knockdown recapitulated the morphologic changes previously seen in the absence of HIP1, but did not affect the same cell signaling pathway, suggesting involvement in the regulation of different processes.


Assuntos
Artrite Reumatoide , Fibroblastos , Cinesinas , Fenótipo , Sinoviócitos , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Artrite Reumatoide/genética , Humanos , Animais , Sinoviócitos/metabolismo , Sinoviócitos/patologia , Cinesinas/genética , Cinesinas/metabolismo , Ratos , Fibroblastos/metabolismo , Movimento Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo
9.
J Nanobiotechnology ; 22(1): 246, 2024 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-38735970

RESUMO

Excessive production of reactive oxygen species (ROS) and inflammation are the key problems that impede diabetic wound healing. In particular, dressings with ROS scavenging capacity play a crucial role in the process of chronic wound healing. Herein, Zr-based large-pore mesoporous metal-organic frameworks (mesoMOFs) were successfully developed for the construction of spatially organized cascade bioreactors. Natural superoxide dismutase (SOD) and an artificial enzyme were spatially organized in these hierarchical mesoMOFs, forming a cascade antioxidant defense system, and presenting efficient intracellular and extracellular ROS scavenging performance. In vivo experiments demonstrated that the SOD@HMUiO-MnTCPP nanoparticles (S@M@H NPs) significantly accelerated diabetic wound healing. Transcriptomic and western blot results further indicated that the nanocomposite could inhibit fibroblast senescence and ferroptosis as well as the stimulator of interferon genes (STING) signaling pathway activation in macrophages mediated by mitochondrial oxidative stress through ROS elimination. Thus, the biomimetic multi-enzyme cascade catalytic system with spatial ordering demonstrated a high potential for diabetic wound healing, where senescence, ferroptosis, and STING signaling pathways may be potential targets.


Assuntos
Inflamação , Estruturas Metalorgânicas , Espécies Reativas de Oxigênio , Cicatrização , Cicatrização/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Animais , Estruturas Metalorgânicas/química , Estruturas Metalorgânicas/farmacologia , Camundongos , Superóxido Dismutase/metabolismo , Porosidade , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Células RAW 264.7 , Masculino , Ferroptose/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Diabetes Mellitus Experimental , Nanopartículas/química , Humanos , Antioxidantes/farmacologia , Nanocompostos/química , Proteínas de Membrana
10.
Biochem Biophys Res Commun ; 716: 150020, 2024 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-38692011

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive disease with high mortality rates. It has been shown that pirfenidone (PFD) and nintedanib (Ofev) can slow down the decline in lung function of IPF patients, but their efficacy remains suboptimal. Some studies have suggested that the combination of PFD and Ofev may yield promising results. However, there is a lack of research on the combined application of these two medications in the treatment of IPF. A mouse model of bleomycin-induced (BLM) pulmonary fibrosis was established to investigate the impact of combination therapy on pulmonary fibrosis of mice. The findings demonstrated a significant reduction in lung tissue damage in mice treated with the combination therapy. Subsequent transcriptome analysis identified the differential gene secreted phosphoprotein 1 (SPP1), which was found to be associated with macrophages and fibroblasts based on multiple immunofluorescence staining results. Analysis of a phosphorylated protein microarray indicated that SPP1 plays a regulatory role in macrophages and fibroblasts via the AKT pathway. Consequently, the regulation of macrophages and fibroblasts in pulmonary fibrosis by the combination of PFD and Ofev is mediated by SPP1 through the AKT pathway, potentially offering a novel therapeutic option for IPF patients. Further investigation into the targeting of SPP1 for the treatment of pulmonary fibrosis is warranted.


Assuntos
Fibroblastos , Indóis , Macrófagos , Camundongos Endogâmicos C57BL , Osteopontina , Proteínas Proto-Oncogênicas c-akt , Piridonas , Animais , Piridonas/farmacologia , Piridonas/uso terapêutico , Indóis/farmacologia , Indóis/uso terapêutico , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Osteopontina/metabolismo , Osteopontina/genética , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Fibrose Pulmonar Idiopática/induzido quimicamente , Transdução de Sinais/efeitos dos fármacos , Antifibróticos/farmacologia , Antifibróticos/uso terapêutico , Masculino , Quimioterapia Combinada , Bleomicina
11.
Clin Transl Med ; 14(5): e1660, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38764260

RESUMO

BACKGROUND: Human dermal fibroblasts (HDFs) are essential in the processes of skin ageing and wound healing. However, the underlying mechanism of HDFs in skin healing of the elderly has not been well defined. This study aims to elucidate the mechanisms of HDFs senescence and how senescent HDFs affect wound healing in aged skin. METHODS: The expression and function of sperm equatorial segment protein 1 (SPESP1) in skin ageing were evaluated via in vivo and in vitro experiments. To delve into the potential molecular mechanisms by which SPESP1 influences skin ageing, a combination of techniques was employed, including proteomics, RNA sequencing, immunoprecipitation, chromatin immunoprecipitation and liquid chromatography-mass spectrometry analyses. Clearance of senescent cells by dasatinib plus quercetin (D+Q) was investigated to explore the role of SPESP1-induced senescent HDFs in wound healing. RESULTS: Here, we define the critical role of SPESP1 in ameliorating HDFs senescence and retarding the skin ageing process. Mechanistic studies demonstrate that SPESP1 directly binds to methyl-binding protein, leading to Decorin demethylation and subsequently upregulation of its expression. Moreover, SPESP1 knockdown delays wound healing in young mice and SPESP1 overexpression induces wound healing in old mice. Notably, pharmacogenetic clearance of senescent cells by D+Q improved wound healing in SPESP1 knockdown skin. CONCLUSIONS: Taken together, these findings reveal the critical role of SPESP1 in skin ageing and wound healing, expecting to facilitate the development of anti-ageing strategies and improve wound healing in the elderly.


Assuntos
Senescência Celular , Fibroblastos , Cicatrização , Animais , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Camundongos , Senescência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Envelhecimento da Pele/efeitos dos fármacos , Humanos , Quercetina/farmacologia , Masculino
12.
Elife ; 132024 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-38767331

RESUMO

Wound infections are highly prevalent and can lead to delayed or failed healing, causing significant morbidity and adverse economic impacts. These infections occur in various contexts, including diabetic foot ulcers, burns, and surgical sites. Enterococcus faecalis is often found in persistent non-healing wounds, but its contribution to chronic wounds remains understudied. To address this, we employed single-cell RNA sequencing (scRNA-seq) on infected wounds in comparison to uninfected wounds in a mouse model. Examining over 23,000 cells, we created a comprehensive single-cell atlas that captures the cellular and transcriptomic landscape of these wounds. Our analysis revealed unique transcriptional and metabolic alterations in infected wounds, elucidating the distinct molecular changes associated with bacterial infection compared to the normal wound healing process. We identified dysregulated keratinocyte and fibroblast transcriptomes in response to infection, jointly contributing to an anti-inflammatory environment. Notably, E. faecalis infection prompted a premature, incomplete epithelial-mesenchymal transition in keratinocytes. Additionally, E. faecalis infection modulated M2-like macrophage polarization by inhibiting pro-inflammatory resolution in vitro, in vivo, and in our scRNA-seq atlas. Furthermore, we discovered macrophage crosstalk with neutrophils, which regulates chemokine signaling pathways, while promoting anti-inflammatory interactions with endothelial cells. Overall, our findings offer new insights into the immunosuppressive role of E. faecalis in wound infections.


If wounds get infected, they heal much more slowly, sometimes leading to skin damage and other complications, including disseminated infections or even amputation. Infections can happen in many types of wounds, ranging from ulcers in patients with diabetes to severe burns. If infections are not cleared quickly, the wounds can become 'chronic' and are unable to heal without intervention. Enterococcus faecalis is a type of bacteria that normally lives in the gut. Within that environment, in healthy people, it is not harmful. However, if it comes into contact with wounds ­ particularly diabetic ulcers or the site of a surgery ­ it can cause persistent infections and prevent healing. Although researchers are beginning to understand how E. faecalis initially colonises wounds, the biological mechanisms that transform these infections into chronic wounds are still largely unknown. Celik et al. therefore set out to investigate exactly how E. faecalis interferes with wound healing. To do this, Celik et al. looked at E. faecalis-infected wounds in mice and compared them to uninfected ones. Using a genetic technique called single-cell RNA sequencing, Celik et al. were able to determine which genes were switched on in individual skin and immune cells at the site of the wounds. This in turn allowed the researchers to determine how those cells were behaving in both infected and uninfected conditions. The experiments revealed that when E. faecalis was present in wounds, several important cell types in the wounds did not behave normally. For example, although the infected skin cells still underwent a change in behaviour required for healing (called an epithelial-mesenchymal transition), the change was both premature and incomplete. In other words, the skin cells in infected wounds started changing too early and did not finish the healing process properly. E. faecalis also changed the way macrophages and neutrophils worked within the wounds. These are cells in our immune system that normally promote inflammation, a process involved in both uninfected wounds or during infections and is a key part of wound healing when properly controlled. In the E. faecalis-infected wounds, these cells' inflammatory properties were suppressed, making them less helpful for healing. These results shed new light on how E. faecalis interacts with skin cells and the immune system to disrupt wound healing. Celik et al. hope that this knowledge will allow us to find new ways to target E. faecalis infections, and ultimately develop treatments to help chronic wounds heal better and faster.


Assuntos
Enterococcus faecalis , Infecções por Bactérias Gram-Positivas , Queratinócitos , Cicatrização , Enterococcus faecalis/fisiologia , Enterococcus faecalis/genética , Animais , Camundongos , Infecções por Bactérias Gram-Positivas/microbiologia , Queratinócitos/microbiologia , Queratinócitos/metabolismo , Macrófagos/microbiologia , Macrófagos/metabolismo , Macrófagos/imunologia , Modelos Animais de Doenças , Infecção dos Ferimentos/microbiologia , Transcriptoma , Camundongos Endogâmicos C57BL , Análise de Célula Única , Transição Epitelial-Mesenquimal/genética , Masculino , Fibroblastos/microbiologia , Fibroblastos/metabolismo
13.
PLoS One ; 19(5): e0303789, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38768102

RESUMO

Mucopolysaccharidosis type I (MPS I) is an inherited lysosomal disease caused by lowered activity of the enzyme alpha-L-iduronidase (IDUA). Current therapeutic options show limited efficacy and do not treat some important aspects of the disease. Therefore, it may be advantageous to identify strategies that could improve the efficacy of existing treatments. Pharmacological chaperones are small molecules that protect proteins from degradation, and their use in combination with enzyme replacement therapy (ERT) has been proposed as an alternative therapeutic strategy. Using the SEE-Tx® proprietary computational drug discovery platform, a new allosteric ligand binding cavity in IDUA was identified distal from the active site. Virtual high-throughput screening of approximately 5 million compounds using the SEE-Tx® docking platform identified a subset of small molecules that bound to the druggable cavity and functioned as novel allosteric chaperones of IDUA. Experimental validation by differential scanning fluorimetry showed an overall hit rate of 11.4%. Biophysical studies showed that one exemplary hit molecule GT-01803 bound to (Kd = 22 µM) and stabilized recombinant human IDUA (rhIDUA) in a dose-dependent manner. Co-administration of rhIDUA and GT-01803 increased IDUA activity in patient-derived fibroblasts. Preliminary in vivo studies have shown that GT-01803 improved the pharmacokinetic (PK) profile of rhIDUA, increasing plasma levels in a dose-dependent manner. Furthermore, GT-01803 also increased IDUA enzymatic activity in bone marrow tissue, which benefits least from standard ERT. Oral bioavailability of GT-01803 was found to be good (50%). Overall, the discovery and validation of a novel allosteric chaperone for rhIDUA presents a promising strategy to enhance the efficacy of existing treatments for MPS I. The compound's ability to increase rhIDUA activity in patient-derived fibroblasts and its good oral bioavailability underscore its potential as a potent adjunct to ERT, particularly for addressing aspects of the disease less responsive to standard treatment.


Assuntos
Iduronidase , Mucopolissacaridose I , Iduronidase/metabolismo , Iduronidase/genética , Mucopolissacaridose I/tratamento farmacológico , Humanos , Regulação Alostérica/efeitos dos fármacos , Animais , Camundongos , Terapia de Reposição de Enzimas/métodos , Descoberta de Drogas , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Estabilidade Enzimática , Simulação de Acoplamento Molecular
14.
Elife ; 132024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38771186

RESUMO

Current studies on cultured meat mainly focus on the muscle tissue reconstruction in vitro, but lack the formation of intramuscular fat, which is a crucial factor in determining taste, texture, and nutritional contents. Therefore, incorporating fat into cultured meat is of superior value. In this study, we employed the myogenic/lipogenic transdifferentiation of chicken fibroblasts in 3D to produce muscle mass and deposit fat into the same cells without the co-culture or mixture of different cells or fat substances. The immortalized chicken embryonic fibroblasts were implanted into the hydrogel scaffold, and the cell proliferation and myogenic transdifferentiation were conducted in 3D to produce the whole-cut meat mimics. Compared to 2D, cells grown in 3D matrix showed elevated myogenesis and collagen production. We further induced fat deposition in the transdifferentiated muscle cells and the triglyceride content could be manipulated to match and exceed the levels of chicken meat. The gene expression analysis indicated that both lineage-specific and multifunctional signalings could contribute to the generation of muscle/fat matrix. Overall, we were able to precisely modulate muscle, fat, and extracellular matrix contents according to balanced or specialized meat preferences. These findings provide new avenues for customized cultured meat production with desired intramuscular fat contents that can be tailored to meet the diverse demands of consumers.


Assuntos
Transdiferenciação Celular , Galinhas , Fibroblastos , Carne , Animais , Fibroblastos/metabolismo , Fibroblastos/citologia , Tecido Adiposo/citologia , Células Musculares/citologia , Desenvolvimento Muscular , Proliferação de Células , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Carne in vitro
15.
Int J Mol Sci ; 25(9)2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38732100

RESUMO

The use of temporary resin for provisional restorations is a fundamental step to maintain the position of prepared teeth, to protect the pulpal vitality and the periodontal health as well as the occlusion. The present study aimed at evaluating the biological effects of two resins used in dentistry for temporary restorations, Coldpac (Yates Motloid) and ProTemp 4™ (3M ESPE ™), and their eluates, in an in vitro model of human gingival fibroblasts (hGFs). The activation of the inflammatory pathway NFκB p65/NLRP3/IL-1ß induced by the self-curing resin disks was evaluated by real-time PCR, Western blotting and immunofluorescence analysis. The hGFs adhesion on resin disks was investigated by means of inverted light microscopy and scanning electron microscopy (SEM). Our results suggest that hGF cells cultured in adhesion and with eluate derived from ProTemp 4™ (3M ESPE ™) resin evidenced a downregulation in the expression of the inflammatory mediators such as NFκB p65, NLRP3 and IL-1ß compared to the cells cultured with Coldpac (Yates Motloid) after 24 h and 1 week of culture. Furthermore, the cells cultured with ProTemp 4™ (3M ESPE ™) after 24 h and 1 week of culture reported a higher cell viability compared to the cells cultured with Coldpac (Yates Motloid), established by MTS cell analysis. Similar results were obtained when hGFs were placed in culture with the eluate derived from ProTemp 4™ (3M ESPE ™) resin which showed a higher cell viability compared to the cells cultured with eluate derived from Coldpac (Yates Motloid). These results highlighted the lower pro-inflammatory action and improved cell biocompatibility of ProTemp 4™ (3M ESPE ™), suggesting a better performance in terms of cells-material interaction.


Assuntos
Resinas Compostas , Fibroblastos , Gengiva , Interleucina-1beta , Polimetil Metacrilato , Humanos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Gengiva/citologia , Resinas Compostas/farmacologia , Resinas Compostas/química , Polimetil Metacrilato/química , Polimetil Metacrilato/farmacologia , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Células Cultivadas , Fator de Transcrição RelA/metabolismo , Adesão Celular/efeitos dos fármacos
16.
Life Sci Alliance ; 7(7)2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38719752

RESUMO

Septins are cytoskeletal proteins that participate in cell adhesion, migration, and polarity establishment. The septin subunit SEPT9 directly interacts with the single LIM domain of epithelial protein lost in neoplasm (EPLIN), an actin-bundling protein. Using a human SEPT9 KO fibroblast cell line, we show that cell adhesion and migration are regulated by the interplay between both proteins. The low motility of SEPT9-depleted cells could be partly rescued by increased levels of EPLIN. The normal organization of actin-related filopodia and stress fibers was directly dependent on the expression level of SEPT9 and EPLIN. Increased levels of SEPT9 and EPLIN enhanced the size of focal adhesions in cell protrusions, correlating with stabilization of actin bundles. Conversely, decreased levels had the opposite effect. Our work thus establishes the interaction between SEPT9 and EPLIN as an important link between the septin and the actin cytoskeleton, influencing cell adhesion, motility, and migration.


Assuntos
Adesão Celular , Movimento Celular , Fibroblastos , Adesões Focais , Proteínas com Domínio LIM , Septinas , Humanos , Septinas/metabolismo , Septinas/genética , Movimento Celular/genética , Fibroblastos/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas com Domínio LIM/genética , Adesões Focais/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/genética , Pseudópodes/metabolismo , Citoesqueleto de Actina/metabolismo , Linhagem Celular , Actinas/metabolismo , Fibras de Estresse/metabolismo
17.
J Transl Med ; 22(1): 440, 2024 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-38720358

RESUMO

PURPOSE: To explore the impact of microRNA 146a (miR-146a) and the underlying mechanisms in profibrotic changes following glaucoma filtering surgery (GFS) in rats and stimulation by transforming growth factor (TGF)-ß1 in rat Tenon's capsule fibroblasts. METHODS: Cultured rat Tenon's capsule fibroblasts were treated with TGF-ß1 and analyzed with microarrays for mRNA profiling to validate miR-146a as the target. The Tenon's capsule fibroblasts were then respectively treated with lentivirus-mediated transfection of miR-146a mimic or inhibitor following TGF-ß1 stimulation in vitro, while GFS was performed in rat eyes with respective intraoperative administration of miR-146a, mitomycin C (MMC), or 5-fluorouracil (5-FU) in vivo. Profibrotic genes expression levels (fibronectin, collagen Iα, NF-KB, IL-1ß, TNF-α, SMAD4, and α-smooth muscle actin) were determined through qPCR, Western blotting, immunofluorescence staining and/or histochemical analysis in vitro and in vivo. SMAD4 targeting siRNA was further used to treat the fibroblasts in combination with miR-146a intervention to confirm its role in underlying mechanisms. RESULTS: Upregulation of miR-146a reduced the proliferation rate and profibrotic changes of rat Tenon's capsule fibroblasts induced by TGF-ß1 in vitro, and mitigated subconjunctival fibrosis to extend filtering blebs survival after GFS in vivo, where miR-146a decreased expression levels of NF-KB-SMAD4-related genes, such as fibronectin, collagen Iα, NF-KB, IL-1ß, TNF-α, SMAD4, and α-smooth muscle actin(α-SMA). Additionally, SMAD4 is a key target gene in the process of miR-146a inhibiting fibrosis. CONCLUSIONS: MiR-146a effectively reduced TGF-ß1-induced fibrosis in rat Tenon's capsule fibroblasts in vitro and in vivo, potentially through the NF-KB-SMAD4 signaling pathway. MiR-146a shows promise as a novel therapeutic target for preventing fibrosis and improving the success rate of GFS.


Assuntos
Fibroblastos , Fibrose , Cirurgia Filtrante , Glaucoma , MicroRNAs , Ratos Sprague-Dawley , Animais , MicroRNAs/metabolismo , MicroRNAs/genética , Glaucoma/patologia , Glaucoma/genética , Cirurgia Filtrante/efeitos adversos , Fibroblastos/metabolismo , Masculino , Cápsula de Tenon/metabolismo , Cápsula de Tenon/patologia , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismo , Ratos , Proteína Smad4/metabolismo , Proteína Smad4/genética , NF-kappa B/metabolismo , Mitomicina/farmacologia , Mitomicina/uso terapêutico , Regulação da Expressão Gênica
18.
Int J Rheum Dis ; 27(5): e15174, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38720423

RESUMO

OBJECTIVES: This study investigates the role of TNF-induced protein 3 (TNFAIP3) and CCAAT/enhancer-binding protein ß (C/EBPß) in alveolar macrophages (AMs) of patients with systemic sclerosis-associated interstitial lung disease (SSc-ILD) and their influence on pulmonary fibrosis. METHODS: Transfection of HEK293T cells and AMs with plasmids carrying TNFAIP3 and C/EBPß was performed, followed by co-culturing AMs with pulmonary fibroblasts. Immunoblotting analysis was then utilized to assess the expression of TNFAIP3, C/EBPß, and collagen type 1 (Col1). Quantitative PCR analysis was conducted to quantify the mRNA levels of C/EBPß, IL-10, and TGF-ß1. STRING database analysis, and immunoprecipitation assays were employed to investigate the interactions between TNFAIP3 and C/EBPß. RESULTS: TNFAIP3 expression was significantly reduced in SSc-ILD AMs, correlating with increased Col1 production in fibroblasts. Overexpression of TNFAIP3 inhibited this pro-fibrotic activity. Conversely, C/EBPß expression was elevated in SSc-ILD AMs, and its reduction through TNFAIP3 restoration decreased pro-fibrotic cytokines IL-10 and TGFß1 levels. Protein-protein interaction studies confirmed the regulatory relationship between TNFAIP3 and C/EBPß. CONCLUSIONS: This study highlights the important role of TNFAIP3 in regulating pulmonary fibrosis in SSc-ILD by modulating C/EBPß expression in AMs. These findings suggest that targeting TNFAIP3 could be a potential therapeutic strategy for managing SSc-ILD patients.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT , Técnicas de Cocultura , Fibroblastos , Doenças Pulmonares Intersticiais , Macrófagos Alveolares , Escleroderma Sistêmico , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Fibroblastos/metabolismo , Células HEK293 , Interleucina-10/metabolismo , Interleucina-10/genética , Pulmão/metabolismo , Pulmão/patologia , Doenças Pulmonares Intersticiais/metabolismo , Doenças Pulmonares Intersticiais/etiologia , Macrófagos Alveolares/metabolismo , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/etiologia , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/complicações , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Adulto , Idoso
19.
BMC Infect Dis ; 24(1): 483, 2024 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-38730352

RESUMO

BACKGROUND: Monkeypox (Mpox) is an important human pathogen without etiological treatment. A viral-host interactome study may advance our understanding of molecular pathogenesis and lead to the discovery of suitable therapeutic targets. METHODS: GEO Expression datasets characterizing mRNA profile changes in different host responses to poxviruses were analyzed for shared pathway identification, and then, the Protein-protein interaction (PPI) maps were built. The viral gene expression datasets of Monkeypox virus (MPXV) and Vaccinia virus (VACV) were used to identify the significant viral genes and further investigated for their binding to the library of targeting molecules. RESULTS: Infection with MPXV interferes with various cellular pathways, including interleukin and MAPK signaling. While most host differentially expressed genes (DEGs) are predominantly downregulated upon infection, marked enrichments in histone modifiers and immune-related genes were observed. PPI analysis revealed a set of novel virus-specific protein interactions for the genes in the above functional clusters. The viral DEGs exhibited variable expression patterns in three studied cell types: primary human monocytes, primary human fibroblast, and HeLa, resulting in 118 commonly deregulated proteins. Poxvirus proteins C6R derived protein K7 and K7R of MPXV and VACV were prioritized as targets for potential therapeutic interventions based on their histone-regulating and immunosuppressive properties. In the computational docking and Molecular Dynamics (MD) experiments, these proteins were shown to bind the candidate small molecule S3I-201, which was further prioritized for lead development. RESULTS: MPXV circumvents cellular antiviral defenses by engaging histone modification and immune evasion strategies. C6R-derived protein K7 binding candidate molecule S3I-201 is a priority promising candidate for treating Mpox.


Assuntos
Interações Hospedeiro-Patógeno , Monkeypox virus , Vaccinia virus , Proteínas Virais , Humanos , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vaccinia virus/genética , Vaccinia virus/metabolismo , Células HeLa , Monkeypox virus/genética , Mpox/virologia , Mapas de Interação de Proteínas , Perfilação da Expressão Gênica , Simulação de Acoplamento Molecular , Poxviridae/genética , Poxviridae/metabolismo , Fibroblastos/virologia , Fibroblastos/metabolismo
20.
Int J Mol Sci ; 25(9)2024 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-38731817

RESUMO

MCPH1 has been identified as the causal gene for primary microcephaly type 1, a neurodevelopmental disorder characterized by reduced brain size and delayed growth. As a multifunction protein, MCPH1 has been reported to repress the expression of TERT and interact with transcriptional regulator E2F1. However, it remains unclear whether MCPH1 regulates brain development through its transcriptional regulation function. This study showed that the knockout of Mcph1 in mice leads to delayed growth as early as the embryo stage E11.5. Transcriptome analysis (RNA-seq) revealed that the deletion of Mcph1 resulted in changes in the expression levels of a limited number of genes. Although the expression of some of E2F1 targets, such as Satb2 and Cdkn1c, was affected, the differentially expressed genes (DEGs) were not significantly enriched as E2F1 target genes. Further investigations showed that primary and immortalized Mcph1 knockout mouse embryonic fibroblasts (MEFs) exhibited cell cycle arrest and cellular senescence phenotype. Interestingly, the upregulation of p19ARF was detected in Mcph1 knockout MEFs, and silencing p19Arf restored the cell cycle and growth arrest to wild-type levels. Our findings suggested it is unlikely that MCPH1 regulates neurodevelopment through E2F1-mediated transcriptional regulation, and p19ARF-dependent cell cycle arrest and cellular senescence may contribute to the developmental abnormalities observed in primary microcephaly.


Assuntos
Pontos de Checagem do Ciclo Celular , Senescência Celular , Inibidor p16 de Quinase Dependente de Ciclina , Camundongos Knockout , Microcefalia , Animais , Camundongos , Senescência Celular/genética , Microcefalia/genética , Microcefalia/metabolismo , Microcefalia/patologia , Pontos de Checagem do Ciclo Celular/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Fibroblastos/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo
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