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1.
Braz. j. oral sci ; 20: e210053, jan.-dez. 2021. tab
Article in English | LILACS, BBO | ID: biblio-1253163

ABSTRACT

Aim: The literature has not yet reported investigations about the effect of laser photobiomodulation (LPBM) over the cytotoxicity of drugs for endodontic treatments. Thus, the aim of this study was to evaluate, in vitro, the effect of the association between LPBM and intracanal medications on fibroblasts viability in different exposure times. Methods: Calcium hydroxide (Ca(OH)2) and iodoform (IO) were used pure or associated to LPBM. Eluates of medications were prepared and placed in contact with the cells in three different periods: 24h, 48h and 72h. Laser irradiation (emitting radiation λ 660nm, power density of 10mW, energy density of 3 J/cm²) has been performed in two sessions within a six hour interval, for 12s per well. After each experimental time, the colorimetric assay (MTT) has been performed. Statistical analysis was applied for Mann-Whitney test with 5% α error admitted test. Results: At 24h, the use of LPBM did not increase cell viability while after 72h cell proliferation was stimulated in the group without medications. LPBM application did not increase cell viability in Ca(OH)2 group and IO at any tested time. Ca(OH)2 cytotoxicity at 24h was higher than iodoform, while at 72h not difference was observed. Therefore, after 72 hours was no statistical difference between the IO and Ca(OH)2 groups. Conclusion: LPBM was able to increase cell viability in 72h in the group without medication, although no improvement was observed in the other groups. Thus, LPBM was not able to reduce the cytotoxic effects of the materials on fibroblasts in vitro


Subject(s)
Low-Level Light Therapy , Endodontics , Fibroblasts
2.
Electron. j. biotechnol ; 52: 67-75, July. 2021. tab, graf, ilus
Article in English | LILACS | ID: biblio-1283594

ABSTRACT

BACKGROUND: Adipogenesis and fibrogenesis can be considered as a competitive process in muscle, which may affect the intramuscular fat deposition. The CCAAT/enhancer-binding protein beta (C/EBPb) plays an important role in adipogenesis, which is well-characterized in mice, but little known in bovine so far. RESULTS: In this study, real-time qPCR revealed that the level of C/EBPb was increased during the developmental stages of bovine and adipogenesis process of preadipocytes. Overexpression of C/EBPb promoted bovine fibroblast proliferation through mitotic clonal expansion (MCE), a necessary process for initiating adipogenesis, by significantly downregulating levels of p21 and p27 (p < 0.01). Also, the PPARc expression was inhibited during the MCE stage (p < 0.01). 31.28% of transfected fibroblasts adopted lipid-laden adipocyte morphology after 8 d. Real-time qPCR showed that C/EBPb activated the transcription of early stage adipogenesis markers C/EBPa and PPARc. Expression of ACCa, FASN, FABP4 and LPL was also significantly upregulated, while the expression of LEPR was weakened. CONCLUSIONS: It was concluded C/EBPb can convert bovine fibroblasts into adipocytes without hormone induction by initiating the MCE process and promoting adipogenic genes expression, which may provide new insights into the potential functions of C/EBPb in regulating intramuscular fat deposition in beef cattle.


Subject(s)
Cattle/metabolism , Adipocytes/metabolism , CCAAT-Enhancer-Binding Protein-beta/metabolism , Fibroblasts/metabolism , Adipose Tissue/metabolism , Clone Cells , Cell Proliferation , Adipogenesis , Real-Time Polymerase Chain Reaction , Mitosis , Muscles
3.
Rev. bras. oftalmol ; 80(1): 8-11, jan.-fev. 2021. graf
Article in Portuguese | LILACS | ID: biblio-1251324

ABSTRACT

RESUMO Objetivo: Avaliar a inibição da proliferação de fibroblastos in vitro das conjuntivas obtidas através de exérese de pterígios de pacientes utilizando mitomicina C (MMC) e ciclofosfamida (CF). Métodos: Os pterígios foram retirados de 7 pacientes e submetidos a cultivo celular. Após o cultivo, 3 fragmentos de dimensões iguais deste material foram colhidos de áreas adjacentes do pterígio removido de cada paciente. Eles foram randomicamente selecionados de tal forma que: um fragmento de cada paciente foi exposto: ao meio de cultura (grupo controle), a MMC e a CF por igual período de tempo nas concentrações de 0,4 mg/ml e 10 mg/ml respectivamente. Após este período realizou-se a contagem celular de fibroblastos destes 3 grupos. Cada grupo continha 7 fragmentos. Resultados: Com a utilização da MMC tivemos uma taxa de 95% da inibição da proliferação dos fibroblastos, enquanto com a CF 100%. Conclusões: Ambas as drogas apresentaram elevada taxa da inibição da proliferação de fibroblastos, porém a CF apresentou inibição maior que a MMC.


Abstract Objective: To evaluate the inhibition of fibroblast proliferation in vitro of conjunctiva obtained by excision of pterygium from patients using mitomycin (MMC) and cyclophosphamide (CF). Methods: Pterygiums were removed from 7 patients and subjected to cell culture. After cell cultivation, 3 fragments of equal dimensions of these tissues were collected from adjacent areas of each patient removed pterygium. They were randomly selected in such a way that one fragment of each patient was exposed to: the culture medium (group control), to MMC and to CF for an equal period of time at concentrations of 0,4 mg/dl and 10 mg/dl respectively. After this period, the fibroblast cell count of these groups were performed. Each group had seven fragments. Results: With the use of MMC we had a 95% rate of inhibition of fibroblast proliferation, while with CF 100%. Conclusion: Both drugs showed a high rate of inhibition of fibroblast proliferation, but CF showed greater inhibition than MMC.


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Wound Healing , Pterygium/surgery , Mitomycin/adverse effects , Cyclophosphamide/adverse effects , Cell Proliferation/physiology , Antimitotic Agents/adverse effects , Fibroblasts/physiology , In Vitro Techniques
4.
Arq. Inst. Biol ; 88: e00622019, 2021. graf
Article in English | ID: biblio-1146670

ABSTRACT

Aristolochia plants are notable from an ethnopharmacological viewpoint, but the relevance of these species for medicinal purposes has been debated because of their inherent toxicity. The convergence of these contrasting realities can be readily achieved using bioconversion methods, which have been shown to be useful tools for numerous applications, including the detoxification of biomass. In this context, methanolic extracts of leaves from Aristolochia triangularis and Aristolochia gibertii, as well as the feces of Battus polydamas larvae fed with leaves from these plants, were prepared, and their cytotoxic activities were evaluated on a human fibroblast cell line (GM07492). The leaf extracts were found to be cytotoxic, leading to reductions of 42.1 and 33.8% on cell viability, respectively, while the fecal extracts were considered inactive. In addition to evidencing the cytotoxicity of A. triangularis and A. gibertii, these findings demonstrated a potential bioconversion strategy for obtaining aristolochiaceous extracts with reduced toxicity using the larvae of a specialist phytophagous insect, thus renewing expectations in relation to the pharmacological importance of Aristolochia spp. The results were also ecologically relevant, as B. polydamas larvae were found to be able to detoxify compounds from host plants.(AU)


Subject(s)
Biodegradation, Environmental , Aristolochiaceae , Toxicity , Cell Line , Fibroblasts , Insecta , Larva
5.
J. venom. anim. toxins incl. trop. dis ; 27: e20200106, 2021. tab, graf, ilus
Article in English | ID: biblio-1154774

ABSTRACT

Avian pathogenic Escherichia coli (APEC) isolated from avian cellulitis lesions produces a toxin, named Escherichia coli vacuolating factor (ECVF), that causes cell vacuolization and induces inflammatory response in broiler chicken. Methods We investigated the intracellular activities of ECVF in avian fibroblasts using fluorescence staining, electron microscopy, MTT and LDH measurements. As ECVF act specifically in avian cells, we performed blotting assay followed by mass spectrometry to better understand its initial intracellular protein recognition. Results ECVF induced actin contraction, mitochondrial damage and membrane permeability alterations. Ultrastructural analysis showed intracellular alterations, as nuclear lobulation and the presence of degraded structures inside the vacuoles. Moreover, ECVF induced cell death in fibroblasts. ECVF-biotin associates to at least two proteins only in avian cell lysates: alpha-actinin 4 and vinculin, both involved in cytoskeleton structure. Conclusion These findings demonstrated that ECVF plays an important role in avian cellulitis, markedly in initial steps of infection. Taken together, the results place this toxin as a target for drug and/or vaccine development, instead of the use of large amounts antibiotics.(AU)


Subject(s)
Animals , Vacuoles , Actin Cytoskeleton , Chickens , Actins , Escherichia coli , Fibroblasts , Cellulitis
6.
Article in Chinese | WPRIM | ID: wpr-878992

ABSTRACT

The aim of this paper was to observe the effect of Xinfeng Capsules(XFC)-containing serum on the apoptosis and inflammation of fibroblast-like synoviocytes(FLS) in rheumatoid arthritis(RA) induced by tumor necrosis factor-α(TNF-α), so as to investigate the mechanism of XFC in the treatment of RA. RA-FLS immortalized cell line was established, and XFC drug-containing serum was prepared. CCK-8, ELISA, RT-qPCR, immunofluorescence and TUNEL were used to observe the effect of XFC-containing serum on RA-FLS apoptosis and inflammatory indexes. CCK-8 results showed that the optimal concentration and time of TNF-α on RA-FLS were 10 ng·mL~(-1) and 48 h, respectively; and the optimal concentration and time of XFC on RA-FLS were 6.48 mg·g~(-1) and 72 h, respectively. The results of ELISA showed that compared with RA-FLS group, the expressions of TNF-α, IL-1β, IL-6, IL-8 in TNF-α+RA-FLS group were significantly increased, while the expressions of IL-4 and IL-10 were significantly decreased(P<0.01); after intervention with XFC-containing serum, the expressions of TNF-α, IL-1β, IL-6, IL-8 were significantly decreased, whereas the expressions of IL-4 and IL-10 were significantly increased(P<0.01). The results of RT-qPCR showed that compared with RA-FLS group, the mRNA expressions of Fas, FasL, caspase-3, caspase-8, Bax, Bcl-X1 in TNF-α+RA-FLS group were significantly decreased, while the mRNA expression of Bcl-2 was significantly increased(P<0.001); after intervention with XFC-containing serum, the mRNA expressions of Fas, FasL, caspase-3, caspase-8, Bax, Bcl-X1 were significantly increased, whereas the mRNA expression of Bcl-2 was significantly decreased(P<0.01). The results of immunofluorescence showed that compared with RA-FLS group, the protein expressions of caspase-3 and Bax in TNF-α+RA-FLS group was significantly lower than those in RA-FLS group(P<0.05); after intervention with XFC-containing serum, the protein expressions of caspase-3 and Bax were significantly increased, whereas the protein expression of Bcl-2 was significantly decreased(P<0.05). TUNEL results showed that compared with RA-FLS group, the apoptosis of TNF-α+RA-FLS group was decreased(P<0.05); after intervention with XFC-containing serum, the apoptosis was significantly increased(P<0.05). One of the mechanisms of XFC in the treatment of RA is to promote the apoptosis of RA-FLS and inhibit its inflammatory reaction.


Subject(s)
Apoptosis , Arthritis, Rheumatoid/genetics , Capsules , Cells, Cultured , Drugs, Chinese Herbal , Fibroblasts , Humans , Inflammation , Synovial Membrane , Synoviocytes , Tumor Necrosis Factor-alpha/genetics
7.
Article in Chinese | WPRIM | ID: wpr-878988

ABSTRACT

The aim of this paper was to discuss the effect of swertiamarin, gentiopicrin and sweroside on rheumatoid arthritis fibroblast-like synoviocytes(RA-FLSs) and B-cell lymphoma-2(Bcl-2) and their mechanisms. ZINC database and RCSB PDB database were retrieved for 3 D chemical structures of swertiamarin, gentiopicrin and sweroside and 3 D target protein structures. AutoDock Mgltools 1.5.6, AutoDockVina 1.1.2 and pyMOL 2.2.0 were applied for molecular docking to analyze the relationship between Bcl-2(1 GJH) target protein and important ingredients. The cell apoptosis of RA-FLSs was tested by Annexin V-FITC. The Bcl-2 protein expression of RA-FLSs treated with different ingredients was tested by Western blot. The Bcl-2 mRNA expression of RA-FLSs treated with different ingredients was tested by RT-PCR. Swertiamarin, gentiopicrin and sweroside were docked well with Bcl-2(1 GJH). The binding energy of swertiamarin was-6.9 kcal·mol~(-1), the binding energy of gentiopicrin was-6.7 kcal·mol~(-1) and the binding energy of sweroside was-6.4 kcal·mol~(-1). Compared with the blank group, the Bcl-2 protein expression of each group were reduced, while that of the gentiopicrin group was the highest(P<0.01). Compared with the blank group, the Bcl-2 mRNA expression of each groups were reduced. Gentiopicrin can reduce the Bcl-2 protein expression and the Bcl-2 mRNA expression, so as to promote the RA-FLSs apoptosis.


Subject(s)
Apoptosis , Arthritis, Rheumatoid/genetics , Cell Proliferation , Cells, Cultured , Fibroblasts , Humans , Iridoid Glucosides , Molecular Docking Simulation , Proto-Oncogene Proteins c-bcl-2/genetics , Pyrones , Synoviocytes
8.
Article in English | WPRIM | ID: wpr-878449

ABSTRACT

OBJECTIVES@#The proliferation, migration capacity, and expression of activation-related proteins of NHGFs+Cal27-exo were determined by coculturing Cal27 exosome (Cal27-exo) with normal human gingival fibroblasts (NHGFs) to explore the effects of Cal27-exo on the activation and biological behavior of NHGFs.@*METHODS@#Cal27-exo was extracted using supercentrifugation, and exosomes were identified using Western blot, transmission electron microscopy (TEM), and particle size detection. Cal27-exo was cocultured with NHGFs to detect the uptake of Cal27-exo by NHGFs, and the proliferation and migration capacity of NHGFs+Cal27-exo were detected using CCK8 and wound healing tests, respectively. The expression levels of NHGF activation-related proteins, i.e., matrix metalloproteinase-9 (MMP-9), fibroblast-activating protein (FAP), alpha smooth muscle actin (αSMA), and transforming growth factor-β (TGF-β), were detected using real-time quantitative polymerase chain reaction (qRT-PCR).@*RESULTS@#Cal27-exo was extracted u-sing supercentrifugation, and Western blot showed the positive expression levels of Alix and CD63. TEM showed that Cal27-exo had a circular double-layer vesicle. The particle size was between 30 and 150 nm. Cal27-exo labeled with PKH67 entered NHGFs after the coculture method. The wound healing test showed that the migration capacity of NHGFs+Cal27-exo was stronger after the scratch compared with that of NHGFs. CCK8 results showed that the proliferation activity of NHGFs+Cal27-exo was enhanced. qRT-PCR results showed that the MMP-9 levels of NHGFs+Cal27-exo were upregulated, whereas the TGF-β and αSMA mRNA levels of NHGFs+Cal27-exo were downregulated (@*CONCLUSIONS@#The proliferation and migration ability of NHGFs+Cal27-exo are enhanced, and the mRNA expression of related proteins is changed. Cal27-exo can activate NHGFs, which suggests that Cal27-exo has potential significance in tumor invasion and metastasis.


Subject(s)
Cell Proliferation , Exosomes , Fibroblasts , Gingiva , Humans , Matrix Metalloproteinase 9
9.
Article in English | WPRIM | ID: wpr-878441

ABSTRACT

OBJECTIVES@#To study the effect and mechanism of low-level laser irradiation (LLLI) on lipopolysaccharide (LPS)-induced inflammatory injury of human periodontal ligament fibroblasts (hPDLFs).@*METHODS@#hPDLFs were inoculated into well plates and randomly divided into the normal group, LPS group, and LPS+LLLI group. The cells in the normal group were cultured in conventional medium. The hPDLFs in the LPS and LPS+LLLI groups were cultured in RPMI1640 medium containing 1 mg·L@*RESULTS@#Compared with the normal group, the LPS group showed increased apoptosis rate of hPDLFs and intracellular free Ca@*CONCLUSIONS@#LLLI has a protective effect on the inflammatory injury of hPDLFs induced by LPS, and the effect is most obvious when the irradiation intensity is 4 J·cm


Subject(s)
Cells, Cultured , Fibroblasts , Humans , Interleukin-1beta , Lasers , Lipopolysaccharides , Periodontal Ligament , Tumor Necrosis Factor-alpha
10.
Article in Chinese | WPRIM | ID: wpr-878424

ABSTRACT

OBJECTIVES@#This study was performed to clarify the effects of sitagliptin on @*METHODS@#Healthy gingival samples were collected from the donors. HGFs were isolated with enzymic digestion method and identified. The effects of LPS and sitagliptin on cell viability were detected by cell-counting kit-8 (CCK8). The mRNA levels of inflammatory cytokines, namely, interleukin (IL)-6, IL-8, C-C motif ligand 2 (CCL2), and superoxide dismutase 2 (SOD2), were evaluated by quantity real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immune sorbent assay (ELISA) was used to measure the secretion protein levels of IL-6, IL-8, and CCL2. Western blot analysis was used to further investigate the activation of nuclear factor (NF)-κB signaling pathway. The effect of NF-κB pathway inhibitor BAY11-7082 on LPS-induced HGF inflammatory cytokines at the gene level was verified by qRT-PCR.@*RESULTS@#Low concentrations of sitagliptin (0.1, 0.25, and 0.5 µmol·L@*CONCLUSIONS@#Sitagliptin could significantly inhibit LPS-induced HGF inflammatory response by blocking the NF-κB signaling pathway activation.


Subject(s)
Fibroblasts , Gingiva/metabolism , Humans , Lipopolysaccharides , NF-kappa B/metabolism , Signal Transduction , Sitagliptin Phosphate
11.
Chinese Medical Journal ; (24): 261-274, 2021.
Article in English | WPRIM | ID: wpr-878043

ABSTRACT

There have been recent extensive studies and rapid advancement on the pathogenesis underlying idiopathic pulmonary fibrosis (IPF), and intricate pathogenesis of IPF has been suggested. The purpose of this study was to clarify the logical relationship between these mechanisms. An extensive search was undertaken of the PubMed using the following keywords: "etiology," "pathogenesis," "alveolar epithelial cell (AEC)," "fibroblast," "lymphocyte," "macrophage," "epigenomics," "histone," acetylation," "methylation," "endoplasmic reticulum stress," "mitochondrial dysfunction," "telomerase," "proteases," "plasminogen," "epithelial-mesenchymal transition," "oxidative stress," "inflammation," "apoptosis," and "idiopathic pulmonary fibrosis." This search covered relevant research articles published up to April 30, 2020. Original articles, reviews, and other articles were searched and reviewed for content; 240 highly relevant studies were obtained after screening. IPF is likely the result of complex interactions between environmental, genetic, and epigenetic factors: environmental exposures affect epigenetic marks; epigenetic processes translate environmental exposures into the regulation of chromatin; epigenetic processes shape gene expression profiles; in turn, an individual's genetic background determines epigenetic marks; finally, these genetic and epigenetic factors act in concert to dysregulate gene expression in IPF lung tissue. The pathogenesis of IPF involves various imbalances including endoplasmic reticulum, telomere length homeostasis, mitochondrial dysfunction, oxidant/antioxidant imbalance, Th1/Th2 imbalance, M1-M2 polarization of macrophages, protease/antiprotease imbalance, and plasminogen activation/inhibition imbalance. These affect each other, promote each other, and ultimately promote AEC/fibroblast apoptosis imbalance directly or indirectly. Excessive AEC apoptosis and impaired apoptosis of fibroblasts contribute to fibrosis. IPF is likely the result of complex interactions between environmental, genetic, and epigenetic factors. The pathogenesis of IPF involves various imbalances centered on AEC/fibroblast apoptosis imbalance.


Subject(s)
Alveolar Epithelial Cells , Apoptosis , Endoplasmic Reticulum Stress , Fibroblasts , Humans , Idiopathic Pulmonary Fibrosis/genetics
12.
Article in Chinese | WPRIM | ID: wpr-880831

ABSTRACT

OBJECTIVE@#To investigate the role of NOV/CCN3 in regulating the proliferation of mesenchymal stem cells (MSCs) and its regulatory mechanism and assess the value of CCN3 as a proliferative factor in bone tissue engineering.@*METHODS@#Mouse embryonic fibroblasts (MEFs) were used as the MSC model, in which CCN3 expression was up-regulated and downregulated by transfection with the recombinant adenovirus vectors Ad-CCN3 and Ad-siCCN3, respectively. Flow cytometry was used to analyze the changes in cell cycle and apoptosis of the transfected cells. Western blotting was used to detect the expression levels of the proliferation indicators (PCNA, cyclin E, and cyclin B1) and the apoptosis indicators (Bax and Bcl-2) to assess the effect of modulation of CCN3 expression on MEF proliferation and apoptosis. CCN3 protein secretion by the cells was detected using ELISA. RT-qPCR and Western blotting were employed to analyze the changes in the expressions of Notch1, ligand DLL1, the downstream key proteins or genes (Hey1, P300, H3K9) and MAPK pathway-related proteins ERK1+2 and p-ERK1+2.@*RESULTS@#Flow cytometry showed that compared with the control cells, MEFs transfected with Ad-CCN3 exhibited significantly increased cell proliferation index (@*CONCLUSIONS@#CCN3 over-expression promotes the proliferation and inhibits apoptosis of MEFs possibly by inhibiting the classical Notch signaling pathway and activating the MAPK pathway


Subject(s)
Animals , Apoptosis , Cell Cycle , Cell Proliferation , Fibroblasts , Mice , Nephroblastoma Overexpressed Protein
13.
Article in English | WPRIM | ID: wpr-880634

ABSTRACT

OBJECTIVES@#Silence of SET domain containing lysine methyltransferase 7 (SET7) alleviates myocardial tissue injury caused by ischemia-reperfusion. But the effects of SET7 on angiotensin II (Ang II)-induced myocardial fibroblast proliferation and the collagen synthesis are not clear. The purpose of this study was to explore the effect of SET7 on the proliferation and collagen synthesis of myocardial fibroblasts and its mechanisms.@*METHODS@#Myocardial fibroblasts were isolated and identified by immunofluorescence. Myocardial fibroblasts were randomly divided into 4 groups: a control group (cells were normally cultured), an Ang II group (cells were treated with 100 nmol/L Ang II for 24 h), a siCtrl group (cells were transfected with siRNA control and were then treated with 100 nmol/L Ang II for 24 h), and a siSET7 group (cells were transfected with siRNA SET7 and were then treated with 100 nmol/L Ang II for 24 h). Cell counting kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assay were used to evaluate cell proliferation. Real-time PCR was used to detect the mRNA levels of SET7, collagen I, collagen III, and α-smooth muscle actin (α-SMA). Western blotting was used to detect the protein expression of SET7, collagen I, collagen III, α-SMA, sonic hedgehog (Shh), ptched1 (Ptch1), and glioma-associated oncogene homolog 1 (Gli1).@*RESULTS@#Fluorescence microscopy showed positive vimentin staining, and myocardial fibroblasts were in good condition. As compared to the control group, the mRNA and protein levels of SET7 in the Ang II group were significantly upregulated; cell proliferation rate and EdU fluorescence intensity in the Ang II group were significantly increased; the mRNA and protein levels of collagen I, collagen III, and α-SMA were significantly upregulated (all @*CONCLUSIONS@#Silence of SET7 gene inhibits Ang II-induced proliferation and collagen synthesis of myocardial fibroblasts. Shh signaling pathway may be involved in this process.


Subject(s)
Angiotensin II/pharmacology , Cell Proliferation , Cells, Cultured , Collagen/genetics , Fibroblasts , Hedgehog Proteins
14.
Article in English | WPRIM | ID: wpr-879959

ABSTRACT

: To assess the () recombinant gingivalis gingipain R2 (rRgpB)-induced Ca mobilization in human gingival fibroblast (HGF) mediated by protease-activated receptor (PAR) and its downstream signal transduction pathways. : Flow cytometry was used to detect the expression of PAR in HGF. The proliferation of HGF was measured by CCK-8. The dynamic changes of intracellular Ca concentration in HGF induced by rRgpB and the blocking effect of PAR-1 antagonist were observed by laser confocal microscopy. Western blot was performed to determine the phosphorylation levels of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) 1/2, p38 mitogen-activated protein kinase (p38 MAPK) and p65 in HGF. : PAR-1 and PAR-3 were expressed in HGF, and the rRgpB could promote the proliferation of HGF. rRgpB caused a transient increase in [Ca], which could be completely suppressed by vorapaxar, a PAR-1 antagonist. The phosphorylation levels of JNK, ERK1/2 and p65 were significantly up-regulated after the induction of rRgpB for and (all <0.05), which was completely inhibited by vorapaxar. However, the phosphorylation level of p38 MAPK had no significant change after rRgpB stimulation. : rRgpB causes an increase in [Ca] in HGF mediated by PAR-1. JNK, ERK1/2 and nuclear factor-κB may be involved in intracellular signal transduction after PAR-1 activation.


Subject(s)
Fibroblasts , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , MAP Kinase Signaling System , Phosphorylation , Signal Transduction , p38 Mitogen-Activated Protein Kinases/metabolism
15.
Braz. j. med. biol. res ; 54(4): e10692, 2021. tab, graf
Article in English | LILACS | ID: biblio-1153536

ABSTRACT

Fibrosis caused by the increase in extracellular matrix in cardiac fibroblasts plays an important role in the occurrence and development of atrial fibrillation (AF). The aim of this study was to investigate the role of hsa-miR-4443 in AF, human cardiac fibroblast (HCFB) proliferation, and extracellular matrix remodeling. TaqMan Stem-loop miRNA assay was used to measure hsa-miR-4443 expression in patients with persistent AF (n=123) and healthy controls (n=100). Patients with AF were confirmed to have atrial fibrosis by late gadolinium enhancement. At the cellular level, after hsa-miR-4443 mimic and inhibitor were transfected with HCFBs, proliferation, apoptosis, migration, and invasion were analyzed. Lastly, hsa-miR-4443-targeted gene and transforming growth factor (TGF)-β1/α-SMA/collagen pathway were evaluated by dual-luciferase reporter assay and western blot, respectively. In patients with AF, hsa-miR-4443 decreased significantly and collagen metabolism level increased significantly. Logistic regression analysis showed that low hsa-miR-4443 level was a risk factor of AF (P<0.001). The receiver operating characteristic curve revealed that hsa-miR-4443 was useful for predicting AF (area under the curve: 0.828, sensitivity: 0.71, specificity: 0.78, P<0.001). In HCFBs, hsa-miR-4443 targeted thrombospondin-1 (THBS1) and downregulated TGF-β1/α-SMA/collagen pathway. The inhibition of hsa-miR-4443 expression promoted HCFB proliferation, migration, invasion, myofibroblast differentiation, and collagen production. The significant reduction of hsa-miR-4443 can be used as a biomarker for AF. hsa-miR-4443 protected AF by targeting THBS1 and regulated TGF-β1/α-SMA/collagen pathway to inhibit HCFB proliferation and collagen synthesis.


Subject(s)
Humans , Atrial Fibrillation , MicroRNAs/genetics , Fibrosis , Collagen , Contrast Media , Thrombospondin 1/genetics , Cell Proliferation , Transforming Growth Factor beta1 , Fibroblasts , Gadolinium
16.
Mem. Inst. Oswaldo Cruz ; 116: e200592, 2021. tab, graf
Article in English | LILACS | ID: biblio-1154883

ABSTRACT

BACKGROUND Paracoccidioidomycosis (PCM) is a systemic mycosis with high prevalence in Latin America that is caused by thermodimorphic fungal species of the Paracoccidioides genus. OBJECTIVES In this study, we used quantitative polymerase chain reaction (qPCR) to investigate the expression of genes related to the virulence of Paracoccidioides brasiliensis (Pb18) and P. lutzii (Pb01) strains in their mycelial (M) and yeast (Y) forms after contact with alveolar macrophages (AMJ2-C11 cell line) and fibroblasts (MRC-5 cell line). METHODS The selected genes were those coding for 43 kDa glycoprotein (gp43), enolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 14-3-3 protein (30 kDa), phospholipase, and aspartyl protease. FINDINGS In the Pb18 M form, the aspartyl protease gene showed the highest expression among all genes tested, both before and after infection of host cells. In the Pb18 Y form after macrophage infection, the 14-3-3 gene showed the highest expression among all genes tested, followed by the phospholipase and gp43 genes, and their expression was 50-fold, 10-fold, and 6-fold higher, respectively, than that in the M form. After fibroblast infection with the Pb18 Y form, the 14-3-3 gene showed the highest expression, followed by the phospholipase and aspartyl protease genes, and their expression was 25-fold, 10-fold, and 10-fold higher, respectively, than that in the M form. Enolase and aspartyl protease genes were expressed upon infection of both cell lines. After macrophage infection with the Pb01 Y form, the 14-3-3 gene showed the highest expression, followed by the phospholipase and aspartyl protease genes, and their expression was 18-fold, 12.5-fold, and 6-fold higher, respectively, than that in the M form. MAIN CONCLUSIONS In conclusion, the data show that the expression of the genes analysed may be upregulated upon fungus-host interaction. Therefore, these genes may be involved in the pathogenesis of paracoccidioidomycosis.


Subject(s)
Humans , Paracoccidioides/genetics , Paracoccidioidomycosis/genetics , Virulence Factors/genetics , Fibroblasts , Macrophages , Paracoccidioides/pathogenicity , Gene Expression , Latin America
17.
Braz. dent. sci ; 24(1): 1-9, 2021. tab, ilus
Article in English | LILACS, BBO | ID: biblio-1145441

ABSTRACT

Objective: Unlike traditional composite resins, bulk-fill composite resins could be polymerized as thicker layers. This study aims to contribute to the field by investigating the cytotoxic effects of various bulk-fill composite resins on L929 mouse fibroblast cells in vitro. Material and Methods: In our study, six bulk fill and one conventional composite resin were used. Composite resin samples (8×4 mm) were prepared in a sterile cabinet by using a glass mod and polymerizing with a led light device (DTE LUX E, Germany). Composite samples (n:3) of which surface area was calculated according to ISO 10993-12: 2012 standards (3 cm2/ml), were kept in media for 24 h and 72 h in 37 oC incubator, their extracts were filtered in 1:1 and 1:2 proportion and were added on L929 mouse fibroblast cells. Cell viability was examined by the MTT assay and cell death by the LDH test. Cell viability results were evaluated using one-way analysis of variance (ANOVA) test (p<0.05). Results: When the 1:1 extracts from 4 mm thick bulk-fill composite samples were applied on L929 mouse fibroblast cells, cell viability rates showed significant differences compared to the control group at the end of 24 h and 72 h (except for Estelite Bulk Fill Flow). Although the extracts of the tested composite samples at 1:1 and 1:2 ratio at the end of 72 hours caused a decrease in L929 mouse fibroblast cell viability, the cell viability rate of only PRG-containing bulk fill composite and conventional composite remained below the cell viability ratio (70%) specified in ISO standards. Bulk fill composites did not produce toxic effects (except Beautifil Bulk Restorative) according to the LDH test. Conclusions: Despite decreasing in general the cell viability, bulk-fill composite resins used in 4 mm thick layers provided cell viability rates over the acceptability level, except PRG-containing bulk fill composite (Beautifil Bulk Restorative), which was cytotoxic to L929 mouse fibroblasts. (AU)


Objetivo: Ao contrário das resinas compostas tradicionais, as resinas compostas bulk-fill podem ser polimerizadas como camadas mais espessas. Este estudo visa investigar in vitro os efeitos citotóxicos de várias resinas compostas bulk-fill em células de fibroblastos de camundongo L929.Material e Métodos: Em nosso estudo, seis resinas tipo bulk fill e uma resina composta convencional foram usadas. Amostras de resina composta (8 × 4 mm) foram preparadas em gabinete estéril usando um molde de vidro e polimerizado com um dispositivo de luz LED (DTE LUX E, Alemanha). Amostras compostas (n=3) cuja área de superfície foi calculada de acordo com os padrões ISO 10993-12:2012 (3cm2/ml), foram mantidas em meio e incubadas por 24 h e 72 h a 37 ºC, seus extratos foram filtrados na Proporção de 1:1 e 1:2 e foram acondicionados em cultura de células de fibroblastos de camundongo L929. A viabilidade celular foi examinada pelo ensaio MTT e a morte celular pelo teste LDH. Os resultados de viabilidade celular foram avaliados usando o teste de análise de variância (ANOVA) um fator (p <0,05). Resultados: Quando os extratos foram plaqueados na proporção 1:1 de amostras de compósito bulk-fill de 4 mm de espessura com as células de fibroblastos de camundongo L929, as taxas de viabilidade celular mostraram diferenças significativas em comparação com o grupo controle no final de 24 h e 72 h (exceto para Estelite Bulk Fluxo de enchimento). Embora os extratos das amostras compostas testadas na proporção de 1:1 e 1:2 ao final de 72 horas tenham causado uma diminuição na viabilidade das células de fibroblastos de camundongo L929, a taxa de viabilidade celular apenas do compósito de preenchimento total contendo PRG e o compósito convencional permaneceram abaixo a taxa de viabilidade celular (70%) especificada nas normas ISO. Os compósitos de preenchimento a granel não produziram efeitos tóxicos (exceto Beautifil Bulk Restorative) de acordo com o teste de LDH. Conclusão: Apesar de diminuir em geral a viabilidade celular, as resinas compostas bulk-fill usadas em camadas de 4 mm de espessura forneceram taxas de viabilidade celular acima do nível aceitável, exceto o compósito bulk fill contendo PRG (Beautifil Bulk Restorative), que foi citotóxico para fibroblastos de camundongos L929 (AU)


Subject(s)
Animals , Mice , Composite Resins , Toxicity , Fibroblasts
18.
Braz. arch. biol. technol ; 64: e21200093, 2021. tab, graf
Article in English | LILACS | ID: biblio-1153294

ABSTRACT

HIGHLIGHTS Sodium arsenite can cause neoplastic transformation in cells. Curcumin reduced cell viability and increased LDH activity in transformed Balb/c 3T3 cells. Curcumin caused DNA damage in transformed Balb/c 3T3 cells. Curcumin may play a protective role in sodium arsenite-induced toxicity.


Abstract Arsenic is a toxic substance that spreads widely around the environment and accumulates as metalloid in the earth's crust. Arsenic and its derivatives are found in drinking water, nutrients, soil, and air. Exposure to arsenic is associated with lung, blood, skin cancer and various lesions. Curcumin is a polyphenolic compound derived from Curcuma longa (turmeric) rhizome and is one of the main curcuminoids. Curcumin is known to be antioxidant, antibacterial, anti-inflammatory, analgesic effects. This study aimed to investigate the potential of sodium arsenite to transform embryonic fibroblast cells and to evaluate the cytotoxic and genotoxic effects of curcumin in neoplastic transformed cells. Neoplastic cells transformation was induced by sodium arsenite in Balb/c 3T3 cells at the end of 32 days. After transformation assay, the transformed cells were treated with various concentration of curcumin to evaluate cell viability, lactate dehydrogenase activity and DNA damage for 24h. The results revealed that curcumin decreased cell viability and increased the activity of lactate dehydrogenase enzyme in neoplastic transformed Balb/c 3T3 cells. In conclusion, the results demonstrated that curcumin has an anticancer effect on neoplastic transformed Balb/c 3T3 cells by causing DNA damage.


Subject(s)
Animals , Mice , Arsenic/toxicity , DNA Damage , Cell Transformation, Neoplastic , Curcumin/pharmacology , Fibroblasts/drug effects , BALB 3T3 Cells , Fibroblasts/pathology
19.
Clin. biomed. res ; 41(1): 57-64, 2021. tab, graf
Article in English | LILACS | ID: biblio-1255192

ABSTRACT

Introduction: Several studies in the literature have evaluated the role of oxidative stress and adjuvant therapies for X-linked adrenoleukodystrophy (X-ALD). Here, we investigated whether n-acetyl-L-cysteine (NAC) and rosuvastatin (RSV) could influence the generation of reactive species, redox status and nitrative stress in fibroblasts from asymptomatic patients with X-ALD. Methods: Skin biopsy samples were cultured and treated for 2 hours (37 °C) with NAC and RSV. Results: X-ALD fibroblasts generated high levels of reactive oxygen species. These levels were significantly lower in fibroblasts treated with NAC and RSV relative to untreated samples. The X-ALD fibroblasts from asymptomatic patients also had higher catalase activity, and only NAC was able to increase enzyme activity in the samples. Conclusions: Our results indicated that NAC and RSV were able to improve oxidative stress parameters in fibroblasts from asymptomatic patients with X-ALD, showing that adjuvant antioxidant therapy may be a promising treatment strategy for asymptomatic patients with this disease. (AU)


Subject(s)
Humans , Male , Female , Acetylcysteine , Oxidative Stress , Adrenoleukodystrophy/therapy , Rosuvastatin Calcium , Fibroblasts
20.
Acta odontol. Colomb. (En linea) ; 11(2): 25-38, 2021. ilus, ilus, ilus, ilus
Article in Spanish | LILACS, COLNAL | ID: biblio-1281693

ABSTRACT

Objetivo: identificar, describir y diferenciar las características fenotípicas de los fibroblastos gingivales (FGs) en pacientes con hiperplasia gingival idiopática (HGI) e individuos periodontalmente sanos. Métodos: los FGs fueron aislados a partir de tejido gingival de individuos periodontalmente sanos (n=2) y pacientes con HGI (n=2). Los FGs se cultivaron en el medio DMEM (Dulbecco's Modified of Eagle Medium) a 37°C con 5% de CO2. La identificación y localización de la actina, vimentina y mitocondrias en FGs fue realizada y evaluada microscópicamente mediante inmunofluorescencia con anticuerpos monoclonales. La capacidad de migración de los FGs en los pacientes con HGI e individuos sanos también fue estudiada. Resultados: todos los FGs fueron mononucleares, fusiformes y con prolongaciones citoplasmáticas visibles. La faloidina permitió identificar una densa red de actina en los FGs de pacientes con HGI, contrariamente a los FGs de individuos periodontalmente sanos. La vimentina y mitocondrias fueron identificadas en los FGs de individuos sanos y pacientes con HGI sin ninguna alteración en su expresión y localización. La migración de la monocapa de los FGs indicó una actividad de migración celular importante en los FGs de los pacientes con HGI, en relación a los FGs de los individuos periodontalmente sanos. Conclusión: los FGs de pacientes con HGI conservan características fenotípicas celulares similares a los FGs de individuos periodontalmente sanos. Sin embargo, los FGs de pacientes con HGI simulan tener una mayor capacidad migratoria que amerita ser explorada en futuros trabajos de investigación.


Objective: To identify and to describe the phenotypic characteristics of gingival fibroblasts from patients with idiopathic gingival hyperplasia (IGH) and periodontally healthy individuals. Methods: Gingival fibroblasts (GFs) were isolated from gingival tissue from periodontally healthy individuals (n=2) and patients with IGH (n=2). The GFs were grown in DMEM (Dulbecco's Modified of Eagle Medium) at 37°C with 5% CO2. The identification and location of actin, vimentin and mitochondria in GFs were performed and evaluated microscopically by immunofluorescence with monoclonal antibodies. The migration capacity of GFs from IGH and healthy individuals was also studied. Results: All the GFs were mononuclear, fusiform and with visible cytoplasmic extensions. The phalloidin allowed to identify a dense actin network in the GFs of patients with IGH, contrary to the GFs of periodontally healthy individuals. Vimentin and mitochondria were identified in the GFs of healthy individuals and patients with IGH without any alteration in their expression and location. Monolayer migration of GFs indicates significant cell migration activity in the GFs of patients with IGH in relation to the GFs of periodontally healthy individuals. Conclusion: GFs from patients with IGH retain cellular phenotypic characteristic similar to GFs from periodontally healthy individuals. However, the GFs of patients with IGH simulate having a greater migratory capacity that deserves to be explored in future research works.


Subject(s)
Humans , Fibroblasts/physiology , Gingival Hyperplasia , Patients , Cell Movement , Fluorescent Antibody Technique, Indirect , Healthy Volunteers
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