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Atherosclerosis preferentially occurs in areas with low shear stress (LSS) and oscillatory flow. LSS has been demonstrated to correlate with the development of atherosclerosis. The sphingosine 1-phosphate receptor 1 (S1PR1), involving intravascular blood flow sensing, regulates vascular development and vascular barrier function. However, whether LSS affects atherosclerosis via regulating S1PR1 remains incompletely clear. In this study, immunostaining results of F-actin, ß-catenin, and VE-cadherin indicated that LSS impaired endothelial barrier function in human umbilical vein endothelial cells (HUVECs). Western blot analysis showed that LSS resulted in blockage of autophagic flux in HUVECs. In addition, autophagy agonist Rapamycin (Rapa) antagonized LSS-induced endothelial barrier dysfunction, whereas autophagic flux inhibitor Bafilomycin A1 (BafA1) exacerbated it, indicating that LSS promoted endothelial barrier dysfunction by triggering autophagic flux blockage. Notably, gene expression analysis revealed that LSS downregulated S1PR1 expression, which was antagonized by Rapa. Selective S1PR1 antagonist W146 impaired endothelial barrier function of HUVECs under high shear stress (HSS) conditions. Moreover, our data showed that expression of GAPARAPL2, a member of autophagy-related gene 8 (Atg8) proteins, was decreased in HUVECs under LSS conditions. Autophagic flux blockage induced by GAPARAPL2 knockdown inhibited S1PR1, aggravated endothelial barrier dysfunction of HUVECs in vitro, and promoted aortic atherosclerosis in ApoE-/- mice in vivo. Our study demonstrates that autophagic flux blockage induced by LSS downregulates S1PR1 expression and impairs endothelial barrier function. GABARAPL2 inhibition is involved in LSS-induced autophagic flux blockage, which impairs endothelial barrier function via downregulation of S1PR1.
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Aterosclerose , Autofagia , Células Endoteliais da Veia Umbilical Humana , Receptores de Esfingosina-1-Fosfato , Estresse Mecânico , Animais , Autofagia/efeitos dos fármacos , Aterosclerose/metabolismo , Aterosclerose/patologia , Aterosclerose/genética , Humanos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Receptores de Esfingosina-1-Fosfato/genética , Camundongos , Camundongos Endogâmicos C57BL , Masculino , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Endotélio Vascular/efeitos dos fármacosRESUMO
The effects of eight oral anti-coronavirus drugs (lopinavir, ritonavir, chloroquine, darunavir, ribavirin, arbidol, favipiravir, oseltamivir) on the metabolism of four specific glycosides (polydatin, geniposide, quercitrin, glycyrrhizin) and on the activities of three major glycosidases (ß-glucosidase, α-rhamnosidase, ß-glucuronidase) from gut microflora were explored in vitro and determined by LC-MS/MS. The metabolism of polydatin, geniposide, quercitrin and glycyrrhizin was significantly inhibited by one or several anti-coronavirus drugs of 100 µM around 1 h and 4 h (P<0.05), among which darunavir could strongly reduce the production of genipin (70.6% reduction), quercitin (80.6% reduction) and glycyrrhetinic acid (37.9% reduction), which may cause a high risk of herb-drug interactions (HDI). Additionally, chloroquine reduced the production of genipin and quercitin by more than 75% (P<0.05), whereas arbidol had no significant influence on the metabolism of polydatin, quercitrin and glycyrrhizin (P>0.05) so that its risk may be lower. The inhibition of darunavir on ß-glucosidase was relatively strong (IC50 = 193±23 µM), and the inhibition became weaker on ß-glucuronidase and α-rhamnosidase (IC50>500 µM). The consistency between gut microflora and glycosidase system indicated that the inhibition of darunavir on the activity of ß-glucosidase and ß-glucuronidase may be the main reason for affecting the metabolism of geniposide, glycyrrhizin and polydatin in gut microflora. However, for the inhibition of darunavir and chloroquine on the metabolism of quercetrin, there was no correlation between gut microflora and α-rhamnosidase system. Assessing the risk of HDI mediated by glycosidases in gut microflora may be conducive to the safety and efficacy of combining traditional herbal and Western medicine for the treatment of patients with Covid-19.
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Antivirais/efeitos adversos , Tratamento Farmacológico da COVID-19 , Microbioma Gastrointestinal , Glicosídeo Hidrolases/metabolismo , Glicosídeos/metabolismo , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Cloroquina/farmacologia , Darunavir/farmacologia , Humanos , Segurança do Paciente , Preparações de Plantas/efeitos adversos , Espectrometria de Massas em TandemRESUMO
P53 is a famous cancer suppressor and plays key roles in metabolism. Intervertebral disc (IVD) is the largest avascular cartilaginous structure in humans and its degeneration is a common cause of spine diseases initiated from damaged nucleus pulposus (NP) cells. The potential cause of disc degeneration has been attributed to aging, genetic factors, mechanical factors and nutrition. In this study, we found that p53 decreased and leaked to the cytoplasm in NP cells as the glucose level decreases, in contrast to cancer cells in which p53 increases and concentrates to the nuclei. Comparing with in p53 knockdown NP cells, relative high p53 expression in normal control NP cells inhibited autophagy and the pentose phosphate pathway. Furthermore, the expression of Sox 9 and type II collagen were higher in p53 normal control than p53 knockdown NP cells. Based on these results, we believe that relative high p53 facilitates NP cell viability and integrity.
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Condrócitos/efeitos dos fármacos , Glucose/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Núcleo Pulposo/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Núcleo Pulposo/citologia , Núcleo Pulposo/metabolismo , Via de Pentose Fosfato/efeitos dos fármacos , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismoRESUMO
Degeneration of the intervertebral disc (IVD) is a major chronic medical condition associated with back pain. To better understand the pathogenesis of IVD degeneration, we performed comparative and quantitative proteomic analyses of normal and degenerated human annulus fibrosus (AF) cells and identified proteins that are differentially expressed between them. Annulus fibrosus cells were isolated and cultured from patients with lumbar disc herniation (the experimental group, degenerated AF cells) and scoliosis patients who underwent orthopaedic surgery (the control group, normal AF cells). Comparative proteomic analyses of normal and degenerated cultured AF cells were carried out using 2-D electrophoresis, mass spectrometric analyses, and database searching. Quantitative analyses of silver-stained 2-D electrophoresis gels of normal and degenerated cultured AF cells identified 10 protein spots that showed the most altered differential expression levels between the two groups. Among these, three proteins were decreased, including heat shock cognate 71-kDa protein, glucose-6-phosphate 1-dehydrogenase, and protocadherin-23, whereas seven proteins were increased, including guanine nucleotide-binding protein G(i) subunit α-2, superoxide dismutase, transmembrane protein 51, adenosine receptor A3, 26S protease regulatory subunit 8, lipid phosphate phosphatase-related protein, and fatty acyl-crotonic acid reductase 1. These differentially expressed proteins might be involved in the pathophysiological process of IVD degeneration and have potential values as biomarkers of the degeneration of IVD.
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Degeneração do Disco Intervertebral/metabolismo , Disco Intervertebral/citologia , Disco Intervertebral/patologia , Proteômica , Adolescente , Idoso , Humanos , Disco Intervertebral/metabolismoRESUMO
Osteolytic bone diseases are commonly presented with enhanced osteoclast formation and bone resorption. Sesquiterpene lactone natural compounds have been found to possess anti-inflammatory and immune-modulation effects. Here, we identified three germacrane sesquiterpenes using computer-based virtual screening for the structural similarity with sesquiterpene lactone, parthenolide. We showed that natural germacrane sesquiterpene compounds A, B, and C inhibit osteoclast formation and bone resorption in a dose-dependent manner, with relative potency compound A > compound C > compound B based on their equimolar concentrations. Mechanistic studies by Luciferase reporter gene assay and Western blot analysis showed that germacrane sesquiterpene compound A inhibits RANKL-induced activation of NF-κB and IκBα degradation. This study reveals that natural germacrane sesquiterpene compounds are inhibitors for osteoclast formation and bone resorption, and provides evidence that naturally-occurring compounds might be beneficial as alternative medicine for the prevention and treatment of osteolysis.
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Produtos Biológicos/farmacologia , Reabsorção Óssea/metabolismo , Proteínas I-kappa B/metabolismo , NF-kappa B/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Ligante RANK/metabolismo , Sesquiterpenos de Germacrano/farmacologia , Animais , Ativação Enzimática/efeitos dos fármacos , Macrófagos , Camundongos , Inibidor de NF-kappaB alfa , Proteólise/efeitos dos fármacosRESUMO
Extracellular-signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family and regulates a wide variety of cellular processes such as proliferation, differentiation, necrosis, apoptosis and degeneration. However, the expression of ERK5 and its role in degenerated human nucleus pulposus (NP) is hitherto unknown. In this study, we observed the differential expression of ERK5 in normal and degenerated human nucleus pulposus tissues by using immunohistochemical staining and Western blot. Treatment of NP cells with Pro-inflammatory cytokine, TNF-α decreased ERK5 gene expression as well as NP marker gene expression; including the type II collagen and aggrecan. Suppression of ERK5 gene expression in NP cells by ERK5 siRNA resulted in decreased gene expression of type II collagen and aggrecan. Furthermore, inhibition of ERK5 activation by BIX02188 (5µM) decreased the gene expression of type II collagen and aggrecan in NP cells. Our results document the expression of ERK5 in degenerated nucleus pulposus tissues, and suggest a potential involvement of ERK5 in human degenerated nucleus pulposus.
Assuntos
Degeneração do Disco Intervertebral/fisiopatologia , Proteína Quinase 7 Ativada por Mitógeno/biossíntese , Adolescente , Agrecanas/biossíntese , Compostos de Anilina/farmacologia , Células Cultivadas , Colágeno Tipo II/biossíntese , Humanos , Indóis/farmacologia , Disco Intervertebral/citologia , Disco Intervertebral/efeitos dos fármacos , Disco Intervertebral/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/antagonistas & inibidores , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
OBJECTIVE: To explore the mechanism of Shangke Jiegu Tablet (SJT)in repairing the mandibular defect. METHODS: Totally 72 healthy male New Zealand rabbits were randomly divided into the normal control group (n = 24), the model group (n = 24), and the SJT group (n = 24). Then the mandibular defect model was established. Animals in the normal control group and the model group were fed with normal forage, while those in the SJT group were fed with SJT forage. On the day 7, 14, 28, and 56 after model establishment, 6 rabbits were killed in each group. The bone was collected from the mandibular defect. The gene expressions of osteoprotegerin (OPG) and osteoprotegerin ligand (OPGL) were detected by means of RT-PCR. The positive dyeing strength and area of the bone tissue were detected by means of immunohistochemical technique. RESULTS: Compared with the normal control group, the degree of OPGmRNA expression was remarkably up-regulated on day 7 after model establishment (P < 0.05) and the degree of OPGLmRNA expression was remarkably up-regulated on day 14 after model establishment (P < 0.05) in the model group. Compared with the model group, the degree of OPGmRNA expression was remarkably up-regulated (P < 0.05), and the positive dyeing strength and area of bone tissue were stronger and broader on day 14, 28, and 56 after model establishment in the SJT group. The degree of OPGLmRNA expression was remarkably down-regulated (P < 0.05), and the positive dyeing strength and area of bone tissue were weaker and smaller on day 14 after model establishment in the SJT group. The ratio of OPGmRNA/OPGLmRNA was remarkably up-regulated on day 14, 28, and 56 after model establishment (P < 0.05). CONCLUSION: The effect mechanism of promoting mandibular defect repairing by SJT may be correlated to regulating the expressions of OPGmRNA and OPGLmRNA.
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Medicamentos de Ervas Chinesas/farmacologia , Traumatismos Mandibulares/metabolismo , Osteoprotegerina/metabolismo , Ligante RANK/metabolismo , Cicatrização/efeitos dos fármacos , Animais , Expressão Gênica/efeitos dos fármacos , Ligantes , Masculino , Traumatismos Mandibulares/genética , CoelhosRESUMO
[This corrects the article DOI: 10.3389/fcell.2022.910626.].
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Background: Single-cell RNA sequencing (scRNA-seq) enables specific analysis of cell populations at single-cell resolution; however, there is still a lack of single-cell-level studies to characterize the dynamic and complex interactions between osteoporotic vertebral compression fractures (OVCFs) and Kümmell's disease (KD) in the osteoimmune microenvironment. In this study, we used scRNA-seq analysis to investigate the osteoimmune microenvironment and cellular composition in OVCFs and KD. Methods: ScRNA-seq was used to perform analysis of fractured vertebral bone tissues from one OVCF and one KD patients, and a total of 8,741 single cells were captured for single-cell transcriptomic analysis. The cellularity of human vertebral bone tissue was further analyzed using uniform manifold approximation and projection. Pseudo-time analysis and gene enrichment analysis revealed the biological function of cell fate and its counterparts. CellphoneDB was used to identify the interactions between bone cells and immune cells in the osteoimmune microenvironment of human vertebral bone tissue and their potential functions. Results: A cellular profile of the osteoimmune microenvironment of human vertebral bone tissue was established, including mesenchymal stem cells (MSCs), pericytes, myofibroblasts, fibroblasts, chondrocytes, endothelial cells (ECs), granulocytes, monocytes, T cells, B cells, plasma cells, mast cells, and early erythrocytes. MSCs play an immunoregulatory function and mediate osteogenic differentiation and cell proliferation. The differentiation trajectory of osteoclasts in human vertebral bone tissue was also revealed. In addition, ECs actively participate in inflammatory infiltration and coupling with bone cells. T and B cells actively participate in regulating bone homeostasis. Finally, by identifying the interaction of ligand-receptor pairs, we found that immune cells and osteoclasts have bidirectional regulatory characteristics, have the effects of regulating bone resorption by osteoclasts and promoting bone formation, and are essential for bone homeostasis. It is also highlighted that CD8-TEM cells and osteoclasts might crosstalk via CD160-TNFRSF14 ligand-receptor interaction. Conclusion: Our analysis reveals a differential landscape of molecular pathways, population composition, and cell-cell interactions during OVCF development into KD. OVCFs exhibit a higher osteogenic differentiation capacity, owing to abundant immune cells. Conversely, KD results in greater bone resorption than bone formation due to depletion of MSCs and a relatively suppressed immune system, and this immune imbalance eventually leads to vertebral avascular necrosis. The site of action between immune cells and osteoclasts is expected to be a new therapeutic target, and these results may accelerate mechanistic and functional studies of osteoimmune cell types and specific gene action in vertebral avascular necrosis and pathological bone loss diseases, paving the way for drug discovery.
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The nucleus pulposus (NP) cells are chondrocyte-like cells that are required for the resistance of compressive loads through the synthesis of collagen fibrils and proteoglycan aggrecans, and the generation of a hydrostatic swelling pressure, and thus play an important role in the intervertebral disc. Here, we report the production and characterization of an immortalized human NP cell line from normal NP cells using stable transfection of recombinant human telomerase reverse transcriptase (hTERT) gene. The hTERT-transfected NP cells exhibited morphological characteristics typical of native cells. When compared with the first generation of normal NP cells, the hTERT-transfected NP cells grew faster and had an increased level of IGF-1 and TGF-ß gene expression. They were successfully passaged over 20 generations without significant change in the levels of type II collagen and proteoglycan aggrecan expression. In addition, they showed resistance to serum starvation-induced apoptosis, G1 cell cycle arrest, and gene expression of p53, CCNE1, Fas, and Caspase 3. Moreover, histology revealed that no tumorigenicity of NP cells over expressing hTERT was observed after they were implanted in nude mice. Taken together, an immortalized human NP cell line was established, which has an extended lifespan, retains phenotypic features similar to primary parent NP cells, and should provide a suitable model for studying the biology of NP cells.
Assuntos
Apoptose , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Disco Intervertebral/citologia , Telomerase/genética , Animais , Caspase 3/biossíntese , Técnicas de Cultura de Células , Proliferação de Células , Condrócitos , Colágeno/biossíntese , Ciclina E/biossíntese , Humanos , Pressão Hidrostática , Fator de Crescimento Insulin-Like I/biossíntese , Disco Intervertebral/metabolismo , Disco Intervertebral/fisiologia , Camundongos , Camundongos Nus , Proteínas Oncogênicas/biossíntese , Proteoglicanas/biossíntese , Telomerase/biossíntese , Transfecção , Fator de Crescimento Transformador beta/biossíntese , Proteína Supressora de Tumor p53/biossíntese , Receptor fas/biossínteseRESUMO
OBJECTIVE: To explore the effect and mechanism of long noncoding RNA ERVK13-1 on osteosarcoma (OS) cell development by regulation of miR-873-5p/KLF5 axis. METHODS: The expression of ERVK13-1 in the collected tissue was detected by RT-qPCR, and then the relationship between ERVK13-1 expression and clinical characteristics of OS patients was analyzed. After OS cell lines were transfected with miR-873-5p inhibitor, si-ERVK13-1, si-KLF5 or their negative controls, the expression of ERVK13-1, miR-873-5p, and KLF5 in OS cell lines were measured, followed by determination of their effects on cell proliferation, migration, and invasion abilities. Moreover, the binding relationships of ERVK13-1 and miR-873-5p, as well as miR-873-5p and KLF5, were verified by the dual-luciferase reporter gene assay. RESULTS: Highly expressed ERVK13-1 was found in OS tissues, which was closely related to tumor size, tumor node metastasis, and distant metastasis. The overall survival of OS patients with high expression of ERVK13-1 was poorer than those with low expression of ERVK13-1. Elevated ERVK13-1 and KLF5 but suppressed miR-873-5p was observed in the OS cell lines U2OS and MG63. Transfection with miR-873-5p inhibitor enhanced the malignant potentials of OS cells, and transfection with si-ERVK13-1 or si-KLF5 reduced these abilities of OS cells. ERVK13-1 bound to miR-873-5p and KLF5 was a target gene of miR-873-5p. CONCLUSION: The ERVK13-1/miR-873-5p/KLF5 axis confers vital effect on the occurrence and progression of OS, thus providing possible guidance for the clinical treatment of OS.
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Neoplasias Ósseas , MicroRNAs , Osteossarcoma , RNA Longo não Codificante , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Regulação Neoplásica da Expressão Gênica , Movimento Celular/genética , Osteossarcoma/genética , Osteossarcoma/patologia , Proliferação de Células/genética , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismoRESUMO
This study aimed to analyze the application of a responsive nano-drug-loading system in injury model of articular chondrocyte in rabbits, as well as its effect on expression of matrix metalloprotein 13 (MMP13). The nanoprecipitation method was adopted to prepare camptothecin (CPT)-loaded poly ethylene glycol (PEG)-Poly caprolactone (PCL) and PEG-PCL nanoparticles without CPT. Afterward, the above mentioned nano-drug-loaded system was used to treat an in vitro scratch model of articular chondrocytes. According to different treatment plans, they were divided into groups: G0 (administered CPT-PEG-PCL nanomedicine), G1 (administered PEG-PCL drug), G2 (saline control), and G3 (healthy control). Results showed that the drug-loading capacity and efficiency of CPT-PEG-PCL was higher than that of PEG-PCL. The levels of type II collagen and hyaluronic acid in G0 was higher than that in G1 and G2. The levels of type II collagen and hyaluronic acid in G0 were not obviously different from those in G3. The level of MMP13 in G0 was lower than that in G1 and G2 and the level of tissue inhibitor of metalloproteinases 1 (TIMP1) in G0 was higher than that in G1 and G2. The proliferation activity of cells in G0 was higher than that in G1 and G2, but there was no obvious difference when compared with G3. In conclusion, CPT-PEG-PCL has stronger long-term circulation capacity and drug-loading efficiency. It can effectively up-regulate the levels of type II collagen, hyaluronic acid, and TIMP1, as well as reduce the synthesis and secretion of MMP13 and promote the repair of articular cartilage damage.
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Metaloproteínas , Nanopartículas , Animais , Condrócitos , Colágeno Tipo II , Ácido Hialurônico , Metaloproteinase 13 da Matriz , Nanopartículas/uso terapêutico , Poliésteres , Polietilenoglicóis , CoelhosRESUMO
[This corrects the article DOI: 10.3389/fcell.2022.910626.].
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Background: The nucleus pulposus is a constituent structure of the human intervertebral disc, and its degeneration can cause intervertebral disc degeneration (IDD). However, the cellular and molecular mechanisms involved remain elusive. Methods: Through bioinformatics analysis, the single-cell transcriptome sequencing expression profiles of human normal nucleus pulposus (NNP) cells and human degenerative nucleus pulposus (DNP) cells were compared to clarify the transcriptome differential expression profiles of human NNP and DNP. The single-cell sequencing results of the two samples were analyzed using bioinformatics methods to compare the differences in histiocytosis between human NNP and DNP, map the histiocytes of NNP and DNP, perform cell differentiation trajectories for the cell populations of interest and predict cell function, and explore their heterogeneity by pathway analysis and Gene Ontology analysis. Results: Nine cell types were identified, which were chondrocyte 1, chondrocyte 2, chondrocyte 3, chondrocyte 4, chondrocyte 5, endothelial, macrophage, neutrophil, and T cells. Analysis of the proportion of chondrocytes in different tissues revealed that chondrocyte 1 accounted for a higher proportion of NNP cells and highly expressed COL2A1 compared with DNP cells; chondrocyte 2, chondrocyte 3, chondrocyte 4, and chondrocyte 5 accounted for a higher proportion of DNP cells compared with NNP cells. Among them, chondrocyte 2 was an inhibitory calcified chondrocyte with high expression of MGP, chondrocytes 3 were fibrochondrocytes with high expression of COL1A1, chondrocytes 4 were chondrocytes that highly express pain inflammatory genes such as PTGES, and chondrocytes 5 were calcified chondrocytes with high expression of FN1 (chondrocytes 4 and chondrocytes 5 were found for the first time in a study of single-cell transcriptome sequencing of disc tissue). Cell trajectory analysis revealed that chondrocyte 1 was at the beginning of the trajectory and chondrocyte 3 was at the end of the trajectory, while chondrocyte 5 appeared first in the trajectory relative to chondrocyte 2 and chondrocyte 4. Conclusion: After functional identification of the specifically expressed genes in five chondrocytes, it was found that chondrocyte 1 was a chondrocyte with high expression of COL2A1, COL9A2, COL11A2, and CHRDL2 in a high proportion of NNP cells, and chondrocyte 3 was a fibrochondrocyte with high expression of COL1A1, COL6A3, COL1A2, COL3A1, AQP1, and COL15A1 in an increased proportion during nucleus pulposus cell degeneration. Through cell trajectory analysis, it was found that chondrocytes 5 specifically expressing FN1, SESN2, and GDF15 may be the key cells leading to degeneration of nucleus pulposus cells. Chondrocytes 2 expressing MGP, MT1G, and GPX3 may play a role in reversing calcification and degeneration, and chondrocytes 4 expressing PTGES, TREM1, and TIMP1 may play a role in disc degeneration pain and inflammation.
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Aims: To explore the role of the Sphingosine 1-Phosphate (S1P)/Receptor2 (S1PR2) pathway in thrombin-induced hyperpermeability (TIP) and to test whether bivalirudin can reverse TIP via the S1P-S1PRs pathway. Methods and Results: Using western blot, we demonstrated that Human umbilical vein endothelial cells (HUVECs) that were cultured with 2 U/ml thrombin showed significantly increased S1PR2 expression while S1PR1and three kept unchanged. Such increment was attenuated by JTE-013 pretreatment and by presence of bivalirudin. Exposure of 2 U/ml of thrombin brought a higher level of S1P both intracellularly and extracellularly within the HUVECs by using ELISA detecting. Thrombin induced S1P and S1PR2 increment was restored by usage of PF543 and bivalirudin. Bivalirudin alone did not influenced the level of S1P and S1PR1,2, and S1PR3 compare to control group. As a surrogate of cytoskeleton morphology, phalloidin staining and immunofluorescence imaging were used. Blurry cell edges and intercellular vacuoles or spaces were observed along thrombin-exposed HUVECs. Presence of JTE-013 and bivalirudin attenuated such thrombin-induced permeability morphological change and presence of heparin failed to show the protective effect. Transwell chamber assay and probe assay were used to measure and compare endothelial permeability in vitro. An increased TIP was observed in HUVECs cultured with thrombin, and coculture with bivalirudin, but not heparin, alleviated this increase. JTE-013 treatment yielded to similar TIP alleviating effect. In vivo, an Evans blue assay was used to test subcutaneous and organ microvascular permeability after the treatment of saline only, thrombin + saline, thrombin + bivalirudin, thrombin + heparin or thrombin + JTE-013. Increased subcutaneous and organ tissue permeability after thrombin treatment was observed in thrombin + saline and thrombin + heparin groups while treatment of bivalirudin and JTE-013 absent this effect. Conclusion: S1P/S1PR2 mediates TIP by impairing vascular endothelial barrier function. Unlike heparin, bivalirudin effectively blocked TIP by inhibiting the thrombin-induced S1P increment and S1PR2 expression, suggesting the novel endothelial protective effect of bivalirudin under pathological procoagulant circumstance.
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The preparation of collagen sponges was studied in order to develop tissue engineering scaffolds. Collagen solutions with varying concentrations were obtained by condensing the initial collagen with polyethylene glycol (PEG) at 4 degrees C for different periods of time, and then were freeze-dried to make collagen scaffolds. The porous characteristics of the prepared scaffolds were characterized by use of different methods, including laser scanning confocal microscopy (LSCM), scanning electron microscopy (SEM) and tensile tests. All collagen sponges were shown to have similar interconnected porous structures but were found to have different pore size, porosity, water capacity and the mechanical property, depending on the concentration of collagen solutions. These findings indicate that the way of controlling the concentration of collagen solutions with PEG permits the freeze-drying fabrication of collagen sponges with varying porous features suitable for different tissue engineering purposes.
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Colágeno/química , Polietilenoglicóis/química , Engenharia Tecidual , Alicerces Teciduais , Colágeno/ultraestrutura , Liofilização , PorosidadeRESUMO
Enhancer of zeste homolog 2 (EZH2) is an important member of the epigenetic regulatory factor polycomb group proteins (PcG) and is abnormally expressed in a wide variety of tumors, including osteosarcoma. Scientists consider EZH2 as an attractive target for the treatment of osteosarcoma and have found many potential EZH inhibitors, such as GlaxoSmithKline 343 (GSK343). It has been reported that GSK343 can be used as an inhibitor in different types of cancer. This study demonstrated that GSK343 not only induced apoptosis by increasing cleaved Casp-3 and poly ADP-ribose polymerase (PARP) expression, but also induced autophagic cell death by inhibiting p62 expression. Apoptosis and autophagic cell death induced by GSK343 were confirmed by the high expression of cleaved caspase-3, LC3-II and transmission electron microscopy. GSK343 inhibited the expression of EZH2 and c-Myc. Additionally, GSK343 inhibited the expression of FUSE binding protein 1 (FBP1), which was identified by its regulatory effects on c-Myc expression. Since c-Myc is a common target of EZH2 and FBP1, and GSK343 inhibited the expression of these proliferation-promoting proteins, a mutual regulatory mechanism between EZH2 and FBP1 was proposed. The knockdown of EZH2 suppressed the expression of FBP1; similarly, the knockdown of FBP1 suppressed the expression of EZH2. These results suggest the mutual regulatory association between EZH2 and FBP1. The knockdown of either EZH2 or FBP1 accelerated the sensitivity of osteosarcoma cells to GSK343. Based on these results, this study clarified that GSK343, an EZH2 inhibitor, may have potential for use in the treatment of osteosarcoma. The underlying mechanisms of the effects of GSK343 are partly mediated by its inhibitory activity against c-Myc and its regulators (EZH2 and FBP1).
Assuntos
Apoptose , Neoplasias Ósseas/patologia , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Indazóis/farmacologia , Osteossarcoma/patologia , Piridonas/farmacologia , Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Proliferação de Células , Inibidores Enzimáticos/farmacologia , Frutose-Bifosfatase/antagonistas & inibidores , Humanos , Osteossarcoma/tratamento farmacológico , Osteossarcoma/metabolismo , Células Tumorais CultivadasRESUMO
Ovarian cancer is the seventh most common cancer and the second most common cause of cancer-associated mortality among gynecological malignancies worldwide. The combination of antimitotic agents, such as taxanes, and the DNA-damaging agents, such as platinum compounds, is the standard treatment for ovarian cancer. However, due to chemoresistance, development of novel therapeutic strategies for the treatment of ovarian cancer remains critical. Amentoflavone (AMF) is a biflavonoid derived from the extracts of Selaginella tamariscina, which has been used as a Chinese herb for thousands of years. A previous study demonstrated that AMF inhibits angiogenesis of endothelial cells and induces apoptosis in hypertrophic scar fibroblasts. In order to check the influence of AMF on cell proliferation, the effects of AMF on cell cycle and DNA damage were measured by cell viability, flow cytometry, immunofluorescence and western blotting assays in SKOV3 cells, an ovarian cell line. In the present study, treatment with AMF inhibited ovarian cell proliferation, increased P21 expression, decreased CDK1/2 expression, interrupted the balance of microtubule dynamics and arrested cells at the G2 phase. Furthermore, treatment with AMF increased the expression levels of phospho-Histone H2AX (γ-H2AX; a variant of histone 2A, that belongs to the histone 2A family member X) and the DNA repair protein RAD51 homolog 1 (Rad51), indicating the occurrence of DNA damage since γ-H2AX and Rad51 are both key markers of DNA damage. Consistent with previous findings, the results of the present study suggest that AMF is a potential therapeutic agent for the treatment of ovarian cancer. In addition, the effects of AMF on cell cycle arrest and DNA damage induction may be the molecular mechanisms by which AMF might exert its potential therapeutic benefits in ovarian cancer.
RESUMO
AIMS: Forkhead box C1 (FoxC1) is essential for maintaining the hair follicle stem cell niche. The role of FoxC1 in maintaining mesenchymal stem cell (MSC) niches after myocardial infarction (MI) has not been directly determined to date. In this study, we determined to explore the possible roles and mechanisms of FoxC1 on MSC survival and function in the ischemic niche. METHODS AND RESULTS: MI model was established in this study, and expression level of FoxC1 was overexpressed or knocked down through efficient delivery of FoxC1 transfection or siFoxC1. Fifteen days later, the animals were allocated randomly to receive phosphate-buffered saline (PBS) injection or MSC transplantation. We identified FoxC1 as a key regulator of maintaining the vascular niche in the infarcted hearts (IHs) by driving proangiogenic and anti-inflammatory cytokines while repressing inflammatory and fibrotic factor expression. This vascular niche improved MSC survival and capacity in the IHs. Importantly, FoxC1 interacted with MSCs and was required for vessel specification and differentiation of engrafted MSCs in the ischemic niches, promoting myocardial repair. Inhibiting FoxC1 abolished these effects. CONCLUSION: These results definitively implicate FoxC1 signaling in maintaining ischemic vascular niche, which may be helpful in myocardial repair induced by MSC therapy.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Infarto do Miocárdio/patologia , Miocárdio/patologia , Cicatrização , Animais , Diferenciação Celular , Sobrevivência Celular , Microambiente Celular , Fibrose , Fatores de Transcrição Forkhead/genética , Inflamação/patologia , Macrófagos/patologia , Isquemia Miocárdica/patologia , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Neovascularização Fisiológica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Endogâmicos Lew , Regulação para CimaRESUMO
BACKGROUND: Bone-tendon junction (BTJ) is a unique structure connecting tendon and bone through a fibrocartilage zone. Owing to its unique structure, the regeneration of BTJ remains a challenge. Here, we study the fibrochondrogenic differentiation of human tendon-derived stem/progenitor cells (TSPCs) both in vitro and in vivo. METHODS: TSPCs were isolated from human patellar tendon tissues and investigated for their multidifferentiation potential. TSPCs were cultured in chondrogenic medium with transforming growth factor beta 3 (TGF-ß3) and BMP-2 in vitro âand examined for the expression of fibrochondrogenic marker genes by quantitative real-time reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and immunofluorescence. TSPCs pretreated were also seeded in collage II sponge and then transplanted in immunocompromised nude mice to examine if the fibrochondrogenic characteristics were conserved in vivo. RESULTS: We found that TSPCs were differentiated towards fibrochondrogenic lineage, accompanied by the expression of collagen I, collagen II, SRY-box transcription factor 9 (Sox 9), and tenascin C. Furthermore, after TSPCs were seeded in collagen II sponge and transplanted in immunocompromised nude mice, they expressed fibrochondrogenic genes, including proteoglycan, collagen I, and collagen II. CONCLUSION: Taken together, this study showed that TSPCs are capable of differentiating towards fibrocartilage-like cells, and the fibrochondrogenic characteristics were conserved even in vivo, and thus might have the potential application for fibrocartilage regeneration in BTJ repair. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: TSPCs are able to differentiate into fibrocartilage-like cells and thus might well be one potential cell source for fibrocartilage regeneration in a damaged BTJ repair.