RESUMO
OBJECTIVE: For treatment of medication-related osteonecrosis of the jaw, one proposed approach is the use of a topical agent to block entry of these medications in oral soft tissues. We tested the ability of phosphonoformic acid (PFA), an inhibitor of bisphosphonate entry through certain sodium-dependent phosphate contransporters (SLC20A1, 20A2, 34A1-3) as well as Dynasore, a macropinocytosis inhibitor, for their abilities to prevent zoledronate-induced (ZOL) death in human gingival fibroblasts (HGFs). METHODOLOGY: MTT assay dose-response curves were performed to determine non-cytotoxic levels of both PFA and Dynasore. In the presence of 50 µM ZOL, optimized PFA and Dynasore doses were tested for their ability to restore HGF viability. To determine SLC expression in HGFs, total HGF RNA was subjected to quantitative real-time RT-PCR. Confocal fluorescence microscopy was employed to see if Dynasore inhibited macropinocytotic HGF entry of AF647-ZOL. Endosomal acidification in the presence of Dynasore was measured by live cell imaging utilizing LysoSensor Green DND-189. As a further test of Dynasore's ability to interfere with ZOL-containing endosomal maturation, perinuclear localization of mature endosomes containing AF647-ZOL or TRITC-dextran as a control were assessed via confocal fluorescence microscopy with CellProfiler™ software analysis of the resulting photomicrographs. RESULTS: 0.5 mM PFA did not rescue HGFs from ZOL-induced viability loss at 72 hours while 10 and 30 µM geranylgeraniol did partially rescue. HGFs did not express the SLC transporters as compared to the expression in positive control tissues. 10 µM Dynasore completely prevented ZOL-induced viability loss. In the presence of Dynasore, AF647-ZOL and FITC-dextran co-localized in endosomes. Endosomal acidification was inhibited by Dynasore and perinuclear localization of both TRITC-dextran- and AF647-ZOL-containing endosomes was inhibited by 30 µM Dynasore. CONCLUSION: Dynasore prevents ZOL-induced viability loss in HGFs by partially interfering with macropinocytosis and by inhibiting the endosomal maturation pathway thought to be needed for ZOL delivery to the cytoplasm.
Assuntos
Sobrevivência Celular , Difosfonatos , Endossomos , Fibroblastos , Gengiva , Hidrazonas , Imidazóis , Ácido Zoledrônico , Ácido Zoledrônico/farmacologia , Humanos , Fibroblastos/efeitos dos fármacos , Gengiva/efeitos dos fármacos , Gengiva/citologia , Difosfonatos/farmacologia , Imidazóis/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Hidrazonas/farmacologia , Células Cultivadas , Fatores de Tempo , Reação em Cadeia da Polimerase em Tempo Real , Conservadores da Densidade Óssea/farmacologia , Reprodutibilidade dos Testes , Microscopia Confocal , Relação Dose-Resposta a Droga , Pinocitose/efeitos dos fármacosRESUMO
Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal fibrotic lung disease. While recent studies have suggested the potential efficacy of tyrosine kinase inhibitors in managing IPF, masitinib, a clinically used tyrosine kinase inhibitor, has not yet been investigated for its efficacy in fibrotic lung diseases. In a previous study on an in vitro neurodegenerative model, we demonstrated the synergistic antitoxic and antioxidant effects of masitinib combined with cromolyn sodium, an FDA-approved mast cell stabilizer. This study aims to investigate the anti-fibrotic and antioxidant effects of the masitinib-cromolyn sodium combination in an in vitro model of pulmonary fibrosis. Fibroblast cell cultures treated with bleomycin and/or hydrogen peroxide (H2O2) were subjected to masitinib and/or cromolyn sodium, followed by assessments of cell viability, morphological and apoptotic nuclear changes, triple-immunofluorescence labeling, and total oxidant/antioxidant capacities, besides ratio of Bax and Bcl-2 mRNA expressions as an indication of apoptosis. The combined treatment of masitinib and cromolyn sodium effectively prevented the fibroblast myofibroblast transition, a hallmark of fibrosis, and significantly reduced bleomycin / H2O2-induced apoptosis and oxidative stress. This study is the first to demonstrate the additive anti-fibrotic, cell-protective, and antioxidant effects of the masitinib-cromolyn sodium combination in an in vitro fibrosis model, suggesting its potential as an innovative therapeutic approach for pulmonary fibrosis. Combination therapy may be more advantageous in that both drugs could be administered in lower doses, exerting less side effects, and at the same time providing diverse mechanisms of action simultaneously.
Assuntos
Antioxidantes , Apoptose , Benzamidas , Bleomicina , Cromolina Sódica , Fibroblastos , Miofibroblastos , Estresse Oxidativo , Piperidinas , Piridinas , Tiazóis , Antioxidantes/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Benzamidas/farmacologia , Piridinas/farmacologia , Apoptose/efeitos dos fármacos , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Piperidinas/farmacologia , Cromolina Sódica/farmacologia , Animais , Tiazóis/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Sinergismo Farmacológico , Humanos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/patologia , Células Cultivadas , Camundongos , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/patologiaRESUMO
Aging is a risk factor for various human disorders, including cancer. Current literature advocates that the primary principles of aging depend on the endogenous stress-induced DNA damage caused by reactive oxygen species 50 Hz low-frequency magnetic field was suggested to induce DNA damage and chromosomal instability. NF-kB, activated by DNA damage, is upregulated in age-related cancers and inhibition of NF-kB results in aging-related delayed pathologies. Metformin (Met), an NF-kB inhibitor, significantly reduces both NF-kB activation and expression in aging and cancer. This in vitro study, therefore, was set out to assess the effects of 5mT MF in 50 Hz frequency and Met treatment on the viability and proliferation of aged mouse NIH/3T3 fibroblasts and expression of RELA/p65, matrix metalloproteinases MMP2 and MMP9, and E-cadherin (CDH1) genes. The trypan blue exclusion assay was used to determine cell viability and the BrdU incorporation assay to determine cell proliferation. The MMP-2/9 protein analysis was carried out by immunocytochemistry, NF-kB activity by ELISA and the expressions of targeted genes by qRT-PCR methods. Four doses of Met (500 uM, 1 mM, 2 mM and 10 mM) suppressed both the proliferation and viability of fibroblasts exposed to the MF in a dose-dependent pattern, and the peak inhibition was recorded at the 10 mM dose. Met reduced the expression of NF-kB, and MMP2/9, elevated CDH1 expression and suppressed NF-kB activity. These findings suggest that Met treatment suppresses the carcinogenic potential of 50 Hz MFs in aged mouse fibroblasts, possibly through modulation of NF-kB activation and epithelial-mesenchymal transition modulation.
Assuntos
Proliferação de Células , Sobrevivência Celular , Fibroblastos , Campos Magnéticos , Metformina , NF-kappa B , Animais , Metformina/farmacologia , Camundongos , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Células NIH 3T3 , NF-kappa B/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Carcinogênese/patologia , Fator de Transcrição RelA/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/genética , Caderinas/metabolismo , Caderinas/genética , Senescência Celular/efeitos dos fármacosRESUMO
BACKGROUND: Boswellic acids (BAs) showed promising effects in cancer treatment, immune response regulation, and anti-inflammatory therapy. We aimed to assess the roles of alpha-BA (α-BA) in treating acute wound healing. METHODS: In vivo wound-healing models were established to evaluate the therapeutic effects of α-BA. Cell assays were conducted to assess the impact of α-BA on cellular biological functions. Western blot analysis was employed to validate the potential mechanisms of action of α-BA. RESULTS: Animal models indicated that wound healing was notably accelerated in the α-BA group compared to the control group (P < 0.01). Hematoxylin and eosin (HE) staining and enzyme-linked immunosorbent assay (ELISA) assay preliminarily suggested that α-BA may accelerate wound healing by inhibiting excessive inflammatory reactions and increasing the protein levels of growth factors. Cell function experiments demonstrated that α-BA suppressed the proliferation and migration ability of human hypertrophic scar fibroblasts (HSFBs), thereby favoring wound healing. Additionally, α-BA exerted a significant impact on cell cycle progression. Mechanistically, the protein levels of key genes in nuclear factor kappa beta (NF-κB) signaling pathway, including cyclin D1, p65, IκBα, and p-IκBα, were downregulated by α-BA. CONCLUSIONS: α-BA demonstrated the ability to counteract the abnormal proliferation of skin scar tissues, consequently expediting wound healing. These findings suggest its potential for development as a new agent for treating acute wound healing.
Assuntos
Proliferação de Células , NF-kappa B , Transdução de Sinais , Triterpenos , Cicatrização , Triterpenos/farmacologia , Cicatrização/efeitos dos fármacos , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Humanos , Proliferação de Células/efeitos dos fármacos , Masculino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Movimento Celular/efeitos dos fármacos , Cicatriz Hipertrófica/tratamento farmacológico , Cicatriz Hipertrófica/metabolismo , Cicatriz Hipertrófica/patologia , CamundongosRESUMO
Chronic wounds represent a significant global health concern, statistically impacting 1-2% of the population in developed countries throughout their lifetimes. These wounds cause considerable discomfort for patients and necessitate substantial expenditures of time and resources for treatment. Among the emerging therapeutic approaches, medicated dressings incorporating bioactive molecules, including natural compounds, are particularly promising. Hence, the objective of this study was to develop novel antimicrobial dressings for wound treatment. Specifically, polycaprolactone membranes were manufactured using the electrospinning technique and subsequently coated with natural polyelectrolytes (chitosan as a polycation and a mixture of manuka honey with essential oils nanoemulsions as a polyanion) employing the Layer-by-Layer assembly technique. Physico-chemical and morphological characterization was conducted through QCM-D, FTIR-ATR, XPS, and SEM analyses. The results from SEM and QCM-D demonstrated successful layer deposition and coating formation. Furthermore, FTIR-ATR and XPS analyses distinguished among different coating compositions. The coated membranes were tested in the presence of fibroblast cells, demonstrating biocompatibility and expression of genes coding for VEGF, COL1, and TGF-ß1, which are associated with the healing process (assessed through RT-qPCR analysis). Finally, the membranes exhibited excellent antibacterial activity against both Staphylococcus aureus and Pseudomonas aeruginosa, with higher bacterial strain inhibition observed when cinnamon essential oil nanoemulsion was incorporated. Taken together, these results demonstrate the potential application of nanocoated membranes for biomedical applications, such as wound healing.
Assuntos
Mel , Óleos Voláteis , Poliésteres , Cicatrização , Óleos Voláteis/farmacologia , Óleos Voláteis/química , Cicatrização/efeitos dos fármacos , Poliésteres/química , Humanos , Antibacterianos/farmacologia , Antibacterianos/química , Membranas Artificiais , Leptospermum/química , Bandagens , Staphylococcus aureus/efeitos dos fármacos , Quitosana/química , Quitosana/farmacologia , Fibroblastos/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Polieletrólitos/químicaRESUMO
Sodium fluoride-polyvinyl alcohol (NaF-PVA) tape was developed to deliver fluoride to teeth by adding fluoride to polymer tape. Previous studies have demonstrated that tapes are effective and have antimicrobial properties. This study aimed to evaluate the cytotoxicity of two fluoride-releasing adhesive tapes. We investigated two polyvinyl alcohol (PVA) tapes: (i) a fluoride-PVA (F-PVA) tape, and (ii) a pullulan-incorporated F-PVA (PF-PVA) tape. The cytotoxicity test was conducted on human gingival fibroblasts (HGF) and human periodontal ligament (PDL) cells. Using an adhesive tape containing fluoride, we performed the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on these cells. Genetic analysis of the cells was performed to conduct a stability test on humans. In the MTT assay, PF-PVA had 66% greater cytotoxicity than control by PDL and 69% by HGF. F-PVA showed less cytotoxicity than PF-PVA by 29% in PDL and 33% in HGF. Gene ontology (GO) analysis and gene set enrichment analysis (GSEA) were performed as gene expression analyses. GO analysis indicated that PF-PVA displayed more expression changes of genes related to cytotoxicity than F-PVA. In addition, GSEA found more inflammatory response associations in PF-PVA than in F-PVA. MTT and genetic testing yielded comparable results.
Assuntos
Fibroblastos , Gengiva , Ligamento Periodontal , Fluoreto de Sódio , Humanos , Fibroblastos/efeitos dos fármacos , Gengiva/citologia , Gengiva/efeitos dos fármacos , Ligamento Periodontal/efeitos dos fármacos , Ligamento Periodontal/citologia , Álcool de Polivinil , Células Cultivadas , Teste de Materiais , Sobrevivência Celular/efeitos dos fármacosRESUMO
Systemic sclerosis (SSc), also known as scleroderma, is an autoimmune-driven connective tissue disorder that results in fibrosis of the skin and internal organs such as the lung. Fibroblasts are known as the main effector cells involved in the progression of SSc through the induction of extracellular matrix (ECM) proteins and myofibroblast differentiation. Here, we demonstrate that 4'-(cyclopropylmethyl)-N2-4-pyridinyl-[4,5'-bipyrimidine]-2,2'-diamine (PIK-III), known as class III phosphatidylinositol 3-kinase (PIK3C3/VPS34) inhibitor, exerts potent antifibrotic effects in human dermal fibroblasts (HDFs) by attenuating transforming growth factor-beta 1 (TGF-ß1)-induced ECM expression, cell contraction and myofibroblast differentiation. Unexpectedly, neither genetic silencing of PIK3C3 nor other PIK3C3 inhibitors (e.g., SAR405 and Autophinib) were able to mimic PIK-III-mediated antifibrotic effect in dermal fibroblasts, suggesting that PIK-III inhibits fibroblast activation through another signaling pathway. We identified that PIK-III effectively inhibits p38 activation in TGF-ß1-stimulated dermal fibroblasts. Finally, PIK-III administration significantly attenuated dermal and lung fibrosis in bleomycin-injured mice.
Assuntos
Fibroblastos , Fibrose , Proteínas Quinases p38 Ativadas por Mitógeno , Animais , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Humanos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Camundongos , Escleroderma Sistêmico/patologia , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/genética , Bleomicina , Fator de Crescimento Transformador beta1/metabolismo , Pirimidinas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Piridinas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Pele/patologia , Pele/metabolismo , Pele/efeitos dos fármacos , Pulmão/patologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismoRESUMO
Melatonin has been reported to regulate circadian rhythms and have anti-inflammatory characteristics in various inflammatory autoimmune diseases, but its effects in diseases-associated muscle atrophy remain controversial. This study is aimed to determine the evidence of melatonin in rheumatoid arthritis (RA)-related pathological muscle atrophy. We used initially bioinformatics results to show that melatonin regulated significantly the correlation between pro-inflammation and myogenesis in RA synovial fibroblasts (RASF) and myoblasts. The conditioned medium (CM) from melatonin-treated RASF was incubated in myoblasts with growth medium and differentiated medium to investigate the markers of pro-inflammation, atrophy, and myogenesis. We found that melatonin regulated RASF CM-induced pathological muscle pro-inflammation and atrophy in myoblasts and differentiated myocytes through NF-κB signaling pathways. We also showed for the first time that miR-30c-1-3p is negatively regulated by three inflammatory cytokines in human RASF, which is associated with murine-differentiated myocytes. Importantly, oral administration with melatonin in a collagen-induced arthritis (CIA) mouse model also significantly improved arthritic swelling, hind limb grip strength as well as pathological muscle atrophy. In conclusion, our study is the first to demonstrate not only the underlying mechanism whereby melatonin decreases pro-inflammation in RA-induced pathological muscle atrophy but also increases myogenesis in myoblasts and differentiated myocytes.
Assuntos
Artrite Reumatoide , Fibroblastos , Melatonina , Músculo Esquelético , Melatonina/farmacologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Artrite Reumatoide/tratamento farmacológico , Humanos , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Animais , Camundongos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/efeitos dos fármacos , Inflamação/metabolismo , Inflamação/patologia , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Membrana Sinovial/efeitos dos fármacos , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Artrite Experimental/tratamento farmacológico , Masculino , Mioblastos/metabolismo , Mioblastos/efeitos dos fármacos , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Atrofia Muscular/tratamento farmacológico , Camundongos Endogâmicos DBARESUMO
The burgeoning incidence of thyroid cancer globally necessitates a deeper understanding of its etiological factors. Emerging research suggests a link to environmental contaminants, notably perfluoroalkyl carboxylates (PFACs). This study introduces a novel biomaterial-based approach for modeling thyroid cancer and assesses PFAC exposure-related health risks. This biomaterial-centric methodology enabled a realistic simulation of long-term, low-dose PFAC exposure, yielding critical insights into their carcinogenic potential. Initially, the no observed adverse effect level concentration of 10 µM for four different PFACs, determined using cytotoxicity tests in 2D cell cultures, was employed with thyroid cancer organoids. Specifically, these organoids were exposed to 10 µM of PFACs, refreshed every 3 days over a period of 21 days. The impact of these PFACs on the organoids was assessed using western blotting and immunofluorescence, complemented by high-content screening imaging. This evaluation focused on thyroid-specific biomarkers, epithelial-mesenchymal transition markers, and the proliferation marker Ki-67. Findings indicated significant alterations in these markers, particularly with long-chain PFACs, suggesting an increased risk of thyroid cancer progression and metastasis upon prolonged exposure. This research advances our understanding of thyroid cancer pathology within the context of environmental health risks by investigating the effects of low-dose, long-term exposure to PFACs on human thyroid cancer organoids. The findings reveal the potential carcinogenic risk associated with these substances, emphasizing the urgent need for stricter regulatory controls.
Assuntos
Matriz Extracelular , Fibroblastos , Fluorocarbonos , Organoides , Neoplasias da Glândula Tireoide , Humanos , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/induzido quimicamente , Fluorocarbonos/toxicidade , Organoides/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Ácidos Carboxílicos/toxicidade , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Poluentes Ambientais/toxicidadeRESUMO
Drug-induced gene expression profiles can identify potential mechanisms of toxicity. We focus on obtaining signatures for cardiotoxicity of FDA-approved tyrosine kinase inhibitors (TKIs) in human induced-pluripotent-stem-cell-derived cardiomyocytes, using bulk transcriptomic profiles. We use singular value decomposition to identify drug-selective patterns across cell lines obtained from multiple healthy human subjects. Cellular pathways affected by cardiotoxic TKIs include energy metabolism, contractile, and extracellular matrix dynamics. Projecting these pathways to published single cell expression profiles indicates that TKI responses can be evoked in both cardiomyocytes and fibroblasts. Integration of transcriptomic outlier analysis with whole genomic sequencing of our six cell lines enables us to correctly reidentify a genomic variant causally linked to anthracycline-induced cardiotoxicity and predict genomic variants potentially associated with TKI-induced cardiotoxicity. We conclude that mRNA expression profiles when integrated with publicly available genomic, pathway, and single cell transcriptomic datasets, provide multiscale signatures for cardiotoxicity that could be used for drug development and patient stratification.
Assuntos
Cardiotoxicidade , Perfilação da Expressão Gênica , Miócitos Cardíacos , Inibidores de Proteínas Quinases , Transcriptoma , Humanos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/toxicidade , Perfilação da Expressão Gênica/métodos , Cardiotoxicidade/genética , Cardiotoxicidade/etiologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Linhagem Celular , Análise de Célula Única/métodos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismoRESUMO
Modern research has shown that Cucurbitacin B (Cu B) possesses various biological activities such as liver protection, anti-inflammatory, and anti-tumor effects. However, the majority of research has primarily concentrated on its hepatoprotective effects, with limited attention devoted to exploring its potential impact on the prostate. Our research indicates that Cu B effectively inhibits the proliferation of human prostate stromal cells (WPMY-1) and fibroblasts (HPRF), while triggering apoptosis in prostate cells. When treated with 100 nM Cu B, the apoptosis rates of WPMY-1 and HPRF cells reached 51.73 ± 5.38% and 26.83 ± 0.40%, respectively. In addition, the cell cycle assay showed that Cu B had a G2/M phase cycle arrest effect on WPMY-1 cells. Based on RNA-sequencing analysis, Cu B might inhibit prostate cell proliferation via the p53 signaling pathway. Subsequently, the related gene and protein expression levels were measured using quantitative real-time PCR (RT-qPCR), immunocytochemistry (ICC), and enzyme-linked immunosorbent assays (ELISA). Our results mirrored the regulation of tumor protein p53 (TP53), mouse double minute-2 (MDM2), cyclin D1 (CCND1), and thrombospondin 1 (THBS1) in Cu B-induced prostate cell apoptosis. Altogether, Cu B may inhibit prostate cell proliferation and correlate to the modulation of the p53/MDM2 signaling cascade.
Assuntos
Apoptose , Proliferação de Células , Proteínas Proto-Oncogênicas c-mdm2 , Transdução de Sinais , Triterpenos , Proteína Supressora de Tumor p53 , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Humanos , Proliferação de Células/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Triterpenos/farmacologia , Masculino , Apoptose/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Próstata/efeitos dos fármacos , Próstata/metabolismo , Próstata/citologia , Linhagem CelularRESUMO
This study aimed to investigate the regulatory mechanism of Linggui Zhugan Decoction(LGZGD)-medicated serum on the fibrosis of cardiac fibroblasts(CFs) and the protein expression of the Wnt/ß-catenin signaling pathway. Blank serum and LGZGD-medicated serum were prepared, and primary CFs were isolated and cultured using trypsin-collagenase digestion and differential adhesion method. Immunofluorescence labeling was used to identify primary CFs. Cells were divided into normal control group, model group, 20% blank serum group, and 5%, 10%, and 20% LGZGD-medicated serum groups. Except for the normal control group, all other groups were stimulated with hydrogen peroxide(H_2O_2) after pretreatment with 20% blank serum or 5%, 10%, 20% LGZGD-medicated serum for 12 hours to establish a model of fibrosis in primary CFs. Scratch healing assay was used to observe cell migration ability. ELISA was used to detect the content of collagen type â (Col â ) and type â ¢(Col â ¢). Western blot was used to detect the protein expression of α-smooth muscle actin(α-SMA), Wnt1, glycogen synthase kinase 3ß(GSK-3ß), phosphorylated GSK-3ß(p-GSK-3ß), ß-catenin, and nuclear ß-catenin. RT-qPCR was used to detect the gene expression of ß-catenin and matrix metalloproteinase 9(MMP9), and immunofluorescence technique was used to detect the expression and localization of key proteins α-SMA and ß-catenin. CFs with Wnt1 overexpression were prepared and treated with H_2O_2. The following groups were set up: normal control group, model group, 20% LGZGD-medicated serum group, empty plasmid+20% LGZGD-medicated serum group, and Wnt1 overexpression+20% LGZGD-medicated serum group. ELISA was used to detect the content and ratio of Col â and Col â ¢. Western blot was used to detect the protein expression of α-SMA, Wnt1, GSK-3ß, p-GSK-3ß, ß-catenin, and nuclear ß-catenin. RT-qPCR was used to detect the gene expression of ß-catenin and MMP9. Immunofluorescence staining showed that CFs expressed Vimentin positively, appearing green, with blue nuclei and purity greater than 90%, which were identified as primary CFs. RESULTS:: showed that compared with the normal control group, CFs in the model group had enhanced healing rate, increased content of Col â and Col â ¢, increased ratio of Col â /Col â ¢, upregulated protein expression of α-SMA, Wnt1, p-GSK-3ß, ß-catenin, nuclear ß-catenin, decreased GSK-3ß expression, elevated mRNA expression of ß-catenin and MMP9, and enhanced fluorescence intensity and expression of ß-catenin and α-SMA. Compared with the model group, 5%, 10%, 20% LGZGD-medicated serum significantly inhibited cell migration ability, reduced the content of Col â and Col â ¢, decreased ratio of Col â /Col â ¢, downregulated protein expression of α-SMA, Wnt1, p-GSK-3ß, ß-catenin, nuclear ß-catenin, increased GSK-3ß expression, decreased mRNA expression of ß-catenin and MMP9, and reduced fluorescence intensity and expression of ß-catenin and α-SMA. Compared with the empty plasmid+20% LGZGD-medicated serum group, the effect of LGZGD-medicated serum was significantly reversed after overexpression of Wnt1. LGZGD can reduce excessive deposition of collagen fibers, inhibit excessive proliferation of fibroblasts, and improve the process of myocardial fibrosis. The improvement of myocardial fibrosis by LGZGD is related to the regulation of the Wnt/ß-catenin pathway, reduction of collagen deposition, and protection of myocardial cells.
Assuntos
Medicamentos de Ervas Chinesas , Fibrose , Miocárdio , Ratos Sprague-Dawley , Via de Sinalização Wnt , beta Catenina , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/administração & dosagem , Ratos , beta Catenina/metabolismo , beta Catenina/genética , Miocárdio/metabolismo , Miocárdio/patologia , Masculino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Células CultivadasRESUMO
High altitude environment is mainly characterized by low oxygen. Due to persistent hypoxia, nonhealing wounds are common in high-altitude areas. Moreover, Basic fibroblast growth factor (bFGF) is a versatile biologically active substance that has crucial impact on wound healing. Given the limited availability of atmospheric oxygen and reduced blood oxygen saturation in high-altitude area, and the challenge that arises from direct oxygen and bFGF delivery to wounds through the traumatized vascular structure, it necessitates an innovative solution for local and permeable delivery of oxygen and bFGF. In this study, we present a strategy that involves revamping traditional gel-based wound dressings through the incorporation of nanoparticles encapsulating oxygen and bFGF, engineered to facilitate the localized delivery of dissolved oxygen and bFGF to wound surfaces. The prospective evaluation of this delivery technique's therapeutic impacts on epithelial, endothelial and fibroblasts cells can be materialized. Further experiment corroborated these effects on a high-altitude wounds' murine model. Given its biocompatibility, efficacy, and utility, we posit that NOB-Gel exhibits remarkable translational potential for managing and hastening the healing process of an array of clinical wounds, more so for wounds inflicted at high altitudes.
Assuntos
Altitude , Bandagens , Fator 2 de Crescimento de Fibroblastos , Géis , Nanopartículas , Oxigênio , Cicatrização , Fator 2 de Crescimento de Fibroblastos/administração & dosagem , Animais , Cicatrização/efeitos dos fármacos , Oxigênio/administração & dosagem , Camundongos , Humanos , Masculino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismoRESUMO
This study investigated the proximate composition and inhibitory potential of hot water and ethanolic extracts of the pulp, peel and whole fruit of green banana (Musa sapientum) on α-amylase and α-glucosidase. Bioactive compounds were identified using GC-MS analysis. In addition, the cytotoxic effect on human gingival fibroblast (hGF) was evaluated using the sulphorhodamine B (SRB) assay. The results showed that the peel of green banana had the highest amount of ash (10.05%), fat (2.83%), protein (3.64%) and total dietary fibre (36.62%). The carbohydrate content of the whole fruit (81.79%) and pulp (81.50%) was higher than that of the peel (71.90%). The moisture content of the pulp (13.08%) was higher than that of the peel (11.58%) and whole fruit (11.30%). The ethanolic green banana peel extract showed a good inhibitory effect of α-amylase and α-glucosidase with the concentration necessary for 50% inhibition (IC50) of 0.512 and 0.100 mg·mL-1, respectively. The α-glucosidase inhibitory effect of the ethanolic green banana peel extract and the hot water green banana peel extract was not significantly different from that of acarbose (IC50 0.108 mg·mL-1). GC-MS analysis of the ethanolic green banana peel extract revealed fatty acids and fatty acid ester (9-octadecenamide (Z), octadecanamide and other compounds). The ethanolic peel extract exhibits a significant noncytotoxicity effect on hGF cells at concentrations ranging from 0.0001 to 1.0 mg·mL-1.
Assuntos
Inibidores de Glicosídeo Hidrolases , Musa , Extratos Vegetais , alfa-Amilases , alfa-Glucosidases , Musa/química , alfa-Amilases/antagonistas & inibidores , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Inibidores de Glicosídeo Hidrolases/farmacologia , alfa-Glucosidases/metabolismo , Humanos , Frutas/química , Pós , Cromatografia Gasosa-Espectrometria de Massas , Fibroblastos/efeitos dos fármacosRESUMO
OBJECTIVE: To develop a novel calcium silver zeolite (Ca-Ag-Zeo) and assess its biocompatibility, physiochemical properties and antimicrobial effects. METHODS: Ca-Ag-Zeo was synthesized using ion-exchange method with calcium chloride, silver nitrate and Zeolite X (Zeo). Silver zeolite X (Ag-Zeo) and Zeo were set as control. The chemical structure, morphology, crystal structure and elemental composition of Ca-Ag-Zeo was characterized by X-ray diffraction spectrum, scanning electron microscopy, transmission electron microscopy and energy dispersive spectroscopy, respectively. Its biocompatibility on the human gingival fibroblasts was assessed by cell counting kit-8 assay. Its physiochemical properties were determined by the released calcium and silver ion using Inductive Coupled Plasma Emission Spectrometry for up to 12 weeks. The antimicrobial properties on Streptococcus mutans, Lactobacillus acidophilus, Lactobacillus casei, and Candida albicans were assessed by minimum bactericidal concentration (MBC) or minimum fungicidal concentration (MFC) assay. RESULTS: Ca-Ag-Zeo with a hexagonal cage structure was synthesized. As for biocompatibility, the half-maximal inhibitory concentration (± SD in mg/mL) of Ca-Ag-Zeo, Ag-Zeo and Zeo in human gingival fibroblasts were 0.52 ± 0.05, 0.15 ± 0.01 and 3.35 ± 0.58, respectively (Zeo > Ca-Ag-Zeo > Ag-Zeo; p < 0.05). As for physiochemical properties, the accumulated ion release (± SD in mg) of Ca-Ag-Zeo, Ag-Zeo and Zeo were 0.011 ± 0.003, 0 and 0 for calcium ion, respectively (Ca-Ag-Zeo > Ag-Zeo, Zeo; p < 0.001), and 0.213 ± 0.032, 0.209 ± 0.019 and 0 for silver ion, respectively (Ca-Ag-Zeo, Ag-Zeo > Zeo; p < 0.001). As for anti-microbial ability, the MBC/MFC (mg/mL) of Ca-Ag-Zeo, Ag-Zeo and Zeo were 32, 16 and > 256 against Streptococcus mutans; 32, 16, > 256 against Lactobacillus acidophilus; 16, 16, and 256 against Lactobacillus casei; 0.25, 0.125; and 2, 1, > 256 against Candida albicans, respectively. CONCLUSION: A novel Ca-Ag-Zeo was developed. It presented better biocompatibility compared to Ag-Zeo. It released calcium and silver ions sustainably, and it could inhibit the growth of common cariogenic microorganisms.
Assuntos
Cálcio , Candida albicans , Cárie Dentária , Fibroblastos , Testes de Sensibilidade Microbiana , Prata , Streptococcus mutans , Zeolitas , Humanos , Zeolitas/farmacologia , Zeolitas/química , Streptococcus mutans/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Cárie Dentária/prevenção & controle , Cárie Dentária/microbiologia , Prata/farmacologia , Prata/química , Lactobacillus acidophilus/efeitos dos fármacos , Difração de Raios X , Gengiva/efeitos dos fármacos , Gengiva/citologia , Lacticaseibacillus casei/efeitos dos fármacos , Microscopia Eletrônica de Varredura , Materiais Biocompatíveis/farmacologia , Microscopia Eletrônica de Transmissão , Teste de Materiais , Nitrato de Prata/farmacologia , Antibacterianos/farmacologia , Anti-Infecciosos/farmacologiaRESUMO
A chemical investigation of an ethyl acetate-soluble layer in the culture broth of Perenniporia medulla-panis resulted in the isolation of eight novel sesquiterpenes conjugated Gly (1), l-Val (2), l-Ala (3), l-Tyr (4), l-Thr (5), l-Ile (6), l-Leu (7), and l-Phe (8). Elucidation of their structures was performed through comprehensive spectroscopic analysis. The absolute configuration of the sesquiterpene skeleton was ascertained using modified Mosher's methods. The configurations of the amino acid units in compounds 2-8 were identified through acid hydrolysis followed by LC-MS analysis employing Marfey's method. Compounds 1-3 and 5-8 showed significant regulating effect on MAP kinase activity (p-ERK and p-JNK) in human diploid fibroblast (HDF) cells.
Assuntos
Fibroblastos , Sesquiterpenos , Humanos , Sesquiterpenos/farmacologia , Sesquiterpenos/química , Sesquiterpenos/isolamento & purificação , Estrutura Molecular , Fibroblastos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
In response to pro-inflammatory cytokines such as interleukin (IL)-1ß, dental pulp fibroblasts produce various inflammatory mediators, including IL-6, IL-8, CC chemokine ligand 20 (CCL20), and CXC chemokine ligand 10 (CXCL10), leading to the progression of pulpitis. IL-17/IL-17A (IL-17A) is a pro-inflammatory cytokine secreted by T helper (Th) 17 cells following their recruitment to inflamed sites; however, the roles of IL-17A during pulpitis remain unclear. The purpose of this study was to investigate the effect of IL-17A on IL-6, IL-8, CCL20 and CXCL10 production by human dental pulp fibroblasts (HDPFs) in vitro. IL-17A at a concentration of 100 ng/ml induced the production of 10 times more IL-8 and 4 times more CXCL10, but not IL-6 and CCL20, compared to controls. Co-stimulation of HDPFs with IL-17A and IL-1ß synergistically enhanced the production of IL-6, CCL20, IL-8 and CXCL10. IL-1ß increased expression of IL-17 receptor/IL-17RA (IL-17R) on HDPFs. Moreover, the cell signal pathways of p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) were more potently activated by simultaneous stimulation with IL-17A and IL-1ß. These findings suggest that IL-17A participates in the progression of dental pulp inflammation through the enhanced production of inflammatory mediators in HDPFs.
Assuntos
Quimiocina CXCL10 , Polpa Dentária , Fibroblastos , Interleucina-17 , Interleucina-1beta , Interleucina-6 , Interleucina-8 , Humanos , Polpa Dentária/citologia , Polpa Dentária/metabolismo , Polpa Dentária/efeitos dos fármacos , Interleucina-17/farmacologia , Interleucina-17/metabolismo , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Interleucina-1beta/metabolismo , Quimiocina CXCL10/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Mediadores da Inflamação/metabolismo , Quimiocina CCL20/metabolismo , Pulpite/metabolismo , Células Cultivadas , NF-kappa B/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Receptores de Interleucina-17/metabolismoRESUMO
Oral submucous fibrosis (OSF) is a precancerous condition in the oral cavity, which is closely related to the myofibroblast conversion of buccal mucosal fibroblasts (BMFs) after chronic consumption of areca nut. Emerging evidence suggests pyroptosis, a form of programmed cell death that is mediated by inflammasome, is implicated in persistent myofibroblast activation and fibrosis. Besides, numerous studies have demonstrated the effects of non-coding RNAs on pyroptosis and myofibroblast activities. Herein, we aimed to target key long non-coding RNA PVT1 with natural compound, carvacrol, to alleviate pyroptosis and myofibroblast activation in OSF. We first identified PVT1 was downregulated in the carvacrol-treated fBMFs and then demonstrated that myofibroblast features and expression of pyroptosis makers were all reduced in response to carvacrol treatment. Subsequently, we analysed the expression of PVT1 and found that PVT1 was aberrantly upregulated in OSF specimens and positively correlated with several fibrosis markers. After revealing the suppressive effects of carvacrol on myofibroblast characterisitcs and pyroptosis were mediated by repression of PVT1, we then explored the potential mechanisms. Our data showed that PVT1 may serve as a sponge of microRNA(miR)-20a to mitigate the myofibroblast activation and pyroptosis. Altogether, these findings indicated that the anti-fibrosis effects of carvacrol merit consideration and may be due to the attenuation of pyroptosis and myofibroblast activation by targeting the PVT1/miR-20a axis.
Assuntos
Cimenos , MicroRNAs , Miofibroblastos , Fibrose Oral Submucosa , Piroptose , RNA Longo não Codificante , Fibrose Oral Submucosa/patologia , Fibrose Oral Submucosa/genética , Fibrose Oral Submucosa/metabolismo , Fibrose Oral Submucosa/tratamento farmacológico , Piroptose/efeitos dos fármacos , Piroptose/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Humanos , Cimenos/farmacologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Miofibroblastos/metabolismo , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/patologia , Progressão da Doença , Regulação para Baixo/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacosRESUMO
Long-term exposure to fine particulate matter (PM2.5) can lead to chronic lung injury, including inflammation, idiopathic pulmonary fibrosis, and cancer. Mesenchymal cells, such as fibroblasts, myeloid-derived suppressor cells (MDSCs), and interstitial macrophages (IMs), contribute to immune regulation in lung, yet their diversity and functions upon long-term exposure to particulate matter (PM) remain inadequately characterized. In this study, we conducted a 16-week real-ambient PM exposure experiment on C57BL/6â¯J male mice in Shijiazhuang, China. We used single-cell RNA sequencing to analyze the cellular and molecular changes in lung tissues. Notably, we revealed a significant increase in specific fibroblast (ATX+, Col5a1+Meg3+, universal fibroblasts) and monocyte-derived cell subpopulations (monocytic-MDSCs (M-MDSCs), Lyve1loMHC-â ¡hi IMs, Lyve1hiMHC-â ¡lo IMs) that exhibited pro-inflammatory and pro-fibrotic functions. These cell subpopulations engaged in immunosuppressive signaling pathways and interactions with various cytokines, shaping a pulmonary microenvironment similar to those associated with cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). This altered immune environment may promote the development of pulmonary fibrosis caused by PM exposure, underscoring the intricate roles of mesenchymal cells in chronic lung injury and highlighting the cancer-causing potential of PM2.5 exposure.
Assuntos
Fibroblastos , Lesão Pulmonar , Camundongos Endogâmicos C57BL , Monócitos , Material Particulado , Animais , Material Particulado/toxicidade , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Camundongos , Masculino , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/patologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Pulmão/patologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Células Supressoras Mieloides/efeitos dos fármacos , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismoRESUMO
BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant lung epithelial phenotypes, fibroblast activation, and increased extracellular matrix deposition. Transforming growth factor-beta (TGF-ß)1-induced Smad signaling and downregulation of peroxisomal genes are involved in the pathogenesis and can be inhibited by peroxisome proliferator-activated receptor (PPAR)-α activation. However, the three PPARs, that is PPAR-α, PPAR-ß/δ, and PPAR-γ, are known to interact in a complex crosstalk. METHODS: To mimic the pathogenesis of lung fibrosis, primary lung fibroblasts from control and IPF patients with comparable levels of all three PPARs were treated with TGF-ß1 for 24 h, followed by the addition of PPAR ligands either alone or in combination for another 24 h. Fibrosis markers (intra- and extracellular collagen levels, expression and activity of matrix metalloproteinases) and peroxisomal biogenesis and metabolism (gene expression of peroxisomal biogenesis and matrix proteins, protein levels of PEX13 and catalase, targeted and untargeted lipidomic profiles) were analyzed after TGF-ß1 treatment and the effects of the PPAR ligands were investigated. RESULTS: TGF-ß1 induced the expected phenotype; e.g. it increased the intra- and extracellular collagen levels and decreased peroxisomal biogenesis and metabolism. Agonists of different PPARs reversed TGF-ß1-induced fibrosis even when given 24 h after TGF-ß1. The effects included the reversals of (1) the increase in collagen production by repressing COL1A2 promoter activity (through PPAR-ß/δ activation); (2) the reduced activity of matrix metalloproteinases (through PPAR-ß/δ activation); (3) the decrease in peroxisomal biogenesis and lipid metabolism (through PPAR-γ activation); and (4) the decrease in catalase protein levels in control (through PPAR-γ activation) and IPF (through a combined activation of PPAR-ß/δ and PPAR-γ) fibroblasts. Further experiments to explore the role of catalase showed that an overexpression of catalase protein reduced collagen production. Additionally, the beneficial effect of PPAR-γ but not of PPAR-ß/δ activation on collagen synthesis depended on catalase activity and was thus redox-sensitive. CONCLUSION: Our data provide evidence that IPF patients may benefit from a combined activation of PPAR-ß/δ and PPAR-γ.