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1.
Methods Mol Biol ; 2582: 369-390, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36370364

RESUMO

The matricellular protein Wnt-induced secreted protein 1 (WISP1) is the fourth member of the CCN family of proteins, which has been shown to affect tissues of the musculoskeletal system. In the context of the musculoskeletal disorder osteoarthritis, our lab studied the function of CCN4/WISP1 in joint tissues, including synovium and cartilage, using both gain- and loss-of-function approaches. In mice, this was done by genetic engineering and recombination to generate mice deficient in CCN4/WISP1 protein. Various experimental models of osteoarthritis with different characteristics were induced in these mice. Moreover, CCN4/WISP1 levels in joints were experimentally increased by adenoviral transfections. Osteoarthritis pathology was determined using histology, and the effect of different CCN4/WISP1 levels on gene expression was evaluated in individual tissues. Effects of high levels of CCN4/WISP1 on chondrocytes were studied with an in vitro chondrocyte pellet model. In this chapter, we describe the procedures to conduct these experiments.


Assuntos
Proteínas de Sinalização Intercelular CCN , Osteoartrite , Camundongos , Animais , Proteínas de Sinalização Intercelular CCN/genética , Proteínas de Sinalização Intercelular CCN/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Condrócitos/metabolismo , Membrana Sinovial/metabolismo , Osteoartrite/genética , Osteoartrite/metabolismo
2.
Allergol Immunopathol (Madr) ; 50(6): 154-162, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36335459

RESUMO

BACKGROUND: Natural compounds are found to play an essential role in diverse inflammatory diseases, including rheumatoid arthritis (RA). Orientin, a flavonoid compound, is closely related to diverse pathological processes. Nevertheless, the role of orientin in RA is still unknown. METHODS: The cell viability was tested through cell counting kit 8 (CCK-8) assay, and the number of cell colonies was calculated via colony formation assay. In addition, flow cytometry assay was employed to detect apoptosis rate in human RA fibroblast-like synoviocytes (RA-FLS). Besides, Transwell assay was introduced to determine cell migratory and invasive abilities. Moreover, the level of cytokines (IL-8, IL-1ß, and IL-6) was estimated with quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent serologic assay. Furthermore, western blotting analysis was used to test the protein levels of cleaved-caspase-3, Bax, BCL-2, matrix metalloproteinase (MMP)-2, MMP-9, phosphorylated c-Jun N-terminal kinase, p-P38, and phospho-extracellular signal-related kinase. RESULTS: Orientin inhibited cell viability, migration as well as invasion in a concentration- dependent manner in human RA-FLS. Additionally, treatment of orientin facilitated apoptosis and decreased the secretion of cytokines induced by tumor necrosis factor alpha (TNF-α) in human RA-FLS. Moreover, orientin inactivated mitogen-activated protein kinase (MAPK)-related signaling pathway, notably in human RA-FLS. CONCLUSION: These findings confirmed that orientin inhibited human RA-FLS development and decreased TNF-α-induced inflammatory factors, at least partly, by modulating MAPK-signaling pathway, which implied that orientin might be an effective agent for treating RA.


Assuntos
Artrite Reumatoide , Sinoviócitos , Humanos , Sinoviócitos/metabolismo , Sinoviócitos/patologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/farmacologia , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Fator de Necrose Tumoral alfa/metabolismo , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/metabolismo , Fibroblastos , Flavonoides/farmacologia , Transdução de Sinais , Citocinas/metabolismo , Células Cultivadas , Proliferação de Células
3.
Arthritis Res Ther ; 24(1): 247, 2022 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-36324152

RESUMO

BACKGROUND: The cytoskeletal protein, PSTPIP2, is associated with inflammation and is predominantly expressed in macrophages. Previous data have shown that PSTPIP2 inhibits articular bone damage in arthritic rats. The aim of this study is to explore the molecular mechanism of PSTPIP2's resistance to bone erosion. METHODS: In the current study, peripheral blood and surgically excised synovial tissue from RA patients, DBA/1 mice, Pstpip2CreR26-ZsGreen reporter mice, and Esr2fl/fl/Adgre-Cre tool mice were used for in vivo studies. Adeno-associated viral vector was used to overexpress PSPTIP2 protein in vivo. RESULTS: We found that The level of PSTPIP2 in synovial macrophages is negatively correlated with RA disease activity, which is mediated by synovial macrophages polarization. PSTPIP2hi synovial macrophages form a tight immunological barrier in the lining layer. Notably, the ability of PSTPIP2 to regulate synovial macrophages polarization is dependent on ERß. Additionally, PSTPIP2 regulates the dynamics of synovial macrophages via ERß. CONCLUSIONS: Together, this study reveals that PSTPIP2 regulates synovial macrophages polarization and dynamics via ERß to form an immunological barrier (F4/80+PSTPIP2hi cell-enriched zone) for the joints. Thus, local modulation of PSTPIP2 expression in the joint microenvironment may be a potential strategy for controlling bone erosion in rheumatoid arthritis. PSTPIP2 regulates synovial macrophages polarization and dynamics via ERß to form F4/80+PSTPIP2hi cellular barrier in joint microenvironment.


Assuntos
Artrite Reumatoide , Receptor beta de Estrogênio , Animais , Camundongos , Ratos , Receptor beta de Estrogênio/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos DBA , Membrana Sinovial/metabolismo
4.
Int J Mol Sci ; 23(21)2022 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-36361990

RESUMO

The morphology of fibroblast-like synoviocytes (FLS) issued from the synovial fluid (SF) of patients suffering from osteoarthritis (OA), rheumatoid arthritis (RA), or from healthy subjects (H), as well as the ultrastructure and mechanical properties of the FLS-secreted extracellular vesicles (EV), were analyzed by confocal microscopy, transmission electron microscopy, atomic force microscopy, and tribological tests. EV released under healthy conditions were constituted of several lipid bilayers surrounding a viscous inner core. This "gel-in" vesicular structure ensured high mechanical resistance of single vesicles and good tribological properties of the lubricant. RA, and to a lesser extent OA, synovial vesicles had altered morphology, corresponding to a "gel-out" situation with vesicles surrounded by a viscous gel, poor mechanical resistance, and poor lubricating qualities. When subjected to inflammatory conditions, healthy cells developed phenotypes similar to that of RA samples, which reinforces the importance of inflammatory processes in the loss of lubricating properties of SF.


Assuntos
Artrite Reumatoide , Vesículas Extracelulares , Osteoartrite , Sinoviócitos , Humanos , Sinoviócitos/fisiologia , Membrana Sinovial , Células Cultivadas , Fibroblastos
5.
Int J Mol Sci ; 23(21)2022 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-36362408

RESUMO

The pathophysiology of early-stage hip osteoarthritis (EOA) is not fully understood. Although a previous study in an age-unmatched cohort reported that the number of macrophages was increased in knee EOA compared to late OA (LOA), it remained unclear whether increased macrophages in EOA accurately reflect EOA pathology. We investigated the differences in CD14 expression levels between EOA and LOA using age-unmatched and -matched cohorts. Synovial tissues were obtained from 34 EOA (Tönnis grades 0 and 1) and 80 LOA (Tönnis grades 2 and 3) patients. To correct for differences in demographics between patients with LOA and EOA, we also created propensity score-matched cohorts (16 EOA and 16 LOA). CD14 expression and its association with pain was estimated in LOA and EOA before and after propensity matching. We performed flow cytometry on tissues from the 16 patients, with 8 from each group, to assess for CD14+ subsets in the cells. The CD14 expression in EOA was higher than that in LOA both before and after propensity matching. The proportion of CD14high subsets in EOA was higher than that in LOA. The CD14 expression was associated with pain in EOA before matching. However, no difference was observed between the pain and CD14 expression after matching in EOA. The increased CD14 expression and the proportion of CD14high subsets may be important features associated with hip EOA pathology. To accurately compare early and late OA, the analysis of a propensity score-matched cohort is necessary.


Assuntos
Osteoartrite do Quadril , Humanos , Osteoartrite do Quadril/genética , Membrana Sinovial , Articulação do Joelho , Dor , RNA Mensageiro/genética
6.
Turk J Med Sci ; 52(4): 1355-1361, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-36326383

RESUMO

BACKGROUND: Epidermal growth factor receptor (EGFR) family members and their associated ligands may be related to bone and joint destruction in rheumatoid arthritis. Matrix metalloproteinases are responsible for joint and bone tissue degradation. This study is intended to investigate the effect of epidermal growth factor receptor inhibition by lapatinib on the synthesis of matrix metalloproteinases in in vitro. METHODS: Synovial fibroblast cell culture was obtained from a patient with rheumatoid arthritis who underwent knee arthroplasty. Interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) were added to the cell culture to stimulate synovial fibroblast cells and create an inflammatory character. Understimulated and nonstimulated conditions, lapatinib was applied to the culture in four different concentrations of 25, 50, 100, and 200 µmol. Then, matrix metalloproteinase -1, -3, and, -13 levels were assessed. RESULTS: When stimulated with IL-1ß and TNF-α, the synthesis of matrix metalloproteinases from synovial fibroblast was increased significantly. When lapatinib is added to the stimulated synovial fibroblasts, matrix metalloproteinases synthesis is significantly suppressed. DISCUSSION: Inhibition of the EGFR pathway with lapatinib suppresses matrix metalloproteinases synthesis. Our results suggest EGFR pathway inhibition may be a promising option to prevent joint destruction in the treatment of rheumatoid arthritis.


Assuntos
Artrite Reumatoide , Membrana Sinovial , Humanos , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Fator de Necrose Tumoral alfa , Lapatinib/farmacologia , Lapatinib/metabolismo , Metaloproteinases da Matriz/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Artrite Reumatoide/patologia , Receptores ErbB/metabolismo , Células Cultivadas
7.
Zhong Nan Da Xue Xue Bao Yi Xue Ban ; 47(9): 1208-1216, 2022.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-36411704

RESUMO

OBJECTIVES: Rheumatoid arthritis is a common autoimmune disease, and microRNAs (miRNAs) are involved in its pathogenesis. This study aims to examine the differentially expressed miRNAs in collagen-induced arthritis (CIA) rats, to analyze the biological functions and the related pathways of the miRNA target genes. METHODS: The total RNA in the synovium of experimental animals was extracted. The miRNA gene profile was obtained by miRNA microarray. Then the differentially expressed miRNAs were screened and the relevant target mRNAs were predicted. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed on the significantly differentially expressed miRNAs. RESULTS: There were 69 differentially expressed miRNAs including rno-miR-6215 and rno-miR-709 in CIA rats, of which 22 (31.9%) were up-regulated and 47 (68.1%) were down-regulated. GO and KEGG enrichment analysis showed that the up-regulated miRNA target genes were mainly enriched in cellular metabolism, and they were involved in MAPK and Wnt signaling pathways. The down-regulated miRNA target genes were mainly enriched in nervous system development, and they were involved in axon guidance signaling pathway. CONCLUSIONS: There are differentially expressed miRNAs in the CIA rat model, which may be involved in metabolism biological functions and signal pathways such as MAPK and Wnt.


Assuntos
Artrite Experimental , MicroRNAs , Ratos , Animais , Artrite Experimental/genética , MicroRNAs/genética , Membrana Sinovial , RNA Mensageiro , Via de Sinalização Wnt
8.
Photobiomodul Photomed Laser Surg ; 40(11): 751-762, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36395086

RESUMO

Background: Inflammatory cytokine secretion from fibroblast-like synoviocytes (FLS) plays a vital role in the pathological process of rheumatoid arthritis (RA). Photobiomodulation (PBM) has been widely used in the treatment of RA. However, the mechanism of PBM in RA has not been clarified. Objective: In this study, we investigated the underlying mechanism of 630 nm light-emitting diode (LED) irradiation on anti-inflammation using mRNA sequencing analysis. Methods and results: Reverse transcription (RT)-quantitative polymerase chain reaction (RT-qPCR) results showed that 630 nm LED irradiation significantly inhibited interleukin (IL)-1ß, IL-6, and IL-8 mRNA expression in rheumatoid arthritis fibroblast synovial cells (RA-FLS) and MH7A cells. A total of 1730 differentially expressed genes (DEGs) were identified between tumor necrosis factor α (TNF-α)+LED and TNF-α-treated RA-FLS and 1219 DEGs in MH7A cells by mRNA sequencing analysis. A total of 646 intersecting DEGs from the 2 cell models were used for gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses. Protein-protein interaction (PPI) network of DEGs was used, and 502 nodes and 1452 edges were found. A total of 14 clusters were generated in MCODE, and the top 3 clusters were selected as hub modules. PPI network showed that most of the nodes were DEGs of the heat shock protein (HSP) family. RT-qPCR verified that 630 nm LED irradiation significantly increased HSP70 mRNA expression in FLS. Conclusions: Taken together, our results revealed the correlation between HSP70 and the inhibition of inflammation caused by 630 nm LED irradiation. These findings suggested that HSP may be a novel target of 630 nm LED irradiation to alleviate inflammation in the treatment of RA.


Assuntos
Artrite Reumatoide , Sinoviócitos , Humanos , Sinoviócitos/química , Sinoviócitos/metabolismo , Sinoviócitos/patologia , Membrana Sinovial/química , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Fator de Necrose Tumoral alfa/metabolismo , Proteínas de Choque Térmico , Células Cultivadas , Fibroblastos/metabolismo , Artrite Reumatoide/radioterapia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Inflamação , RNA Mensageiro/genética , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Análise de Sequência de RNA
9.
Allergol. immunopatol ; 50(6): 154-162, 01 nov. 2022. ilus, tab, graf
Artigo em Inglês | IBECS | ID: ibc-211517

RESUMO

Background Natural compounds are found to play an essential role in diverse inflammatory diseases, including rheumatoid arthritis (RA). Orientin, a flavonoid compound, is closely related to diverse pathological processes. Nevertheless, the role of orientin in RA is still unknown. Methods The cell viability was tested through cell counting kit 8 (CCK-8) assay, and the number of cell colonies was calculated via colony formation assay. In addition, flow cytometry assay was employed to detect apoptosis rate in human RA fibroblast-like synoviocytes (RA-FLS). Besides, Transwell assay was introduced to determine cell migratory and invasive abilities. Moreover, the level of cytokines (IL-8, IL-1β, and IL-6) was estimated with quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent serologic assay. Furthermore, western blotting analysis was used to test the protein levels of cleaved-caspase-3, Bax, BCL-2, matrix metalloproteinase (MMP)-2, MMP-9, phosphorylated c-Jun N-terminal kinase, p-P38, and phospho-extracellular signal-related kinase. Results Orientin inhibited cell viability, migration as well as invasion in a concentration- dependent manner in human RA-FLS. Additionally, treatment of orientin facilitated apoptosis and decreased the secretion of cytokines induced by tumor necrosis factor alpha (TNF-α) in human RA-FLS. Moreover, orientin inactivated mitogen-activated protein kinase (MAPK)-related signaling pathway, notably in human RA-FLS. Conclusion These findings confirmed that orientin inhibited human RA-FLS development and decreased TNF-α-induced inflammatory factors, at least partly, by modulating MAPK-signaling pathway, which implied that orientin might be an effective agent for treating RA (AU)


Assuntos
Humanos , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/metabolismo , Sinoviócitos/metabolismo , Sinoviócitos/patologia , Proliferação de Células , Células Cultivadas , Citocinas , Fibroblastos , Flavonoides/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/farmacologia , Transdução de Sinais , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Fator de Necrose Tumoral alfa/metabolismo
10.
RMD Open ; 8(2)2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36270740

RESUMO

OBJECTIVES: Programmed cell death protein 1 (PD-1)-expressing T cells are implicated in the pathogenesis of autoimmune inflammatory diseases such as rheumatoid arthritis. A subset of CXCR5- T cells, termed T peripheral helper (Tph) cells, which drive B cell differentiation, have been identified in ectopic lymphoid structures in established rheumatoid arthritis synovial tissue. Here, we aimed to characterise these in treatment-naïve, early rheumatoid arthritis to determine whether these cells accumulate prior to fully established disease. METHODS: Fresh dissociated tissue and peripheral blood mononuclear cell (PBMC) suspensions were stained with Zombie UV, followed by anti-CD45RO, PD-1, CD3, ICOS, CD8, CD4, CD20, CXCR5, TIGIT and CD38 antibodies prior to analysis. For histology, rheumatoid arthritis synovial sections were prepared for Opal multispectral immunofluorescence with anti-CD45RO, CD20, PD-1 and CXCR5 antibodies. Images were acquired on the Perkin Elmer Vectra V.3.0 imaging system and analysed using InForm Advanced Image Analysis software. RESULTS: Flow cytometry revealed T cell infiltration in the rheumatoid arthritis synovium with differential expression of PD-1, CD45RO, ICOS, TIGIT and CD38. We observed a higher frequency of PD1hiCXCR5- Tph in rheumatoid arthritis synovial tissue and PBMCs versus controls, and no significant difference in T follicular helper cell frequency. Microscopy identified a 10-fold increase of Tph cells in early rheumatoid arthritis synovial follicular and diffuse regions, and identified Tph adjacent to germinal centre B cells. CONCLUSIONS: These data demonstrate that PD-1hi Tph cells are present in early rheumatoid arthritis, but not osteoarthritis synovium, and therefore may provide a target for treatment of patients with early rheumatoid arthritis.


Assuntos
Artrite Reumatoide , Osteoartrite , Humanos , Receptor de Morte Celular Programada 1/metabolismo , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Auxiliares-Indutores/patologia , Membrana Sinovial/metabolismo , Receptores CXCR5/metabolismo , Osteoartrite/patologia
11.
Arthritis Res Ther ; 24(1): 234, 2022 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-36253807

RESUMO

BACKGROUND: Abnormal proliferation of fibroblast-like synoviocytes (FLSs) in the synovial lining layer is the primary cause of synovial hyperplasia and joint destruction in rheumatoid arthritis (RA). Currently, the relationship between metabolic abnormalities and FLS proliferation is a new focus of investigation. However, little is known regarding the relationship between amino acid metabolism and RA. METHODS: The concentrations of amino acids and cytokines in the synovial fluid of RA (n = 9) and osteoarthritis (OA, n = 9) were detected by LC-MS/MS and CBA assay, respectively. The mRNA and protein expression of cationic amino acid transporter-1 (CAT-1) were determined in FLSs isolated from RA and OA patients by real-time PCR and western blotting. MTT assay, cell cycle, apoptosis, invasion, and cytokine secretion were determined in FLSs knocked down of CAT-1 using siRNA or treated with D-arginine under normoxic and hypoxic culture conditions. A mouse collagen-induced arthritis (CIA) model was applied to test the therapeutic potential of blocking the uptake of L-arginine in vivo. RESULTS: L-rginine was upregulated in the synovial fluid of RA patients and was positively correlated with the elevation of the cytokines IL-1ß, IL-6, and IL-8. Further examination demonstrated that CAT-1 was the primary transporter for L-arginine and was overexpressed on RA FLSs compared to OA FLSs. Moreover, knockdown of CAT-1 using siRNA or inhibition of L-arginine uptake using D-arginine significantly suppressed L-arginine metabolism, cell proliferation, migration, and cytokine secretion in RA FLSs under normoxic and hypoxic culture conditions in vitro but increased cell apoptosis in a dose-dependent manner. Meanwhile, in vivo assays revealed that an L-arginine-free diet or blocking the uptake of L-arginine using D-arginine suppressed arthritis progression in CIA mice. CONCLUSION: CAT-1 is upregulated and promotes FLS proliferation by taking up L-arginine, thereby promoting RA progression.


Assuntos
Arginina , Artrite Experimental , Artrite Reumatoide , Transportador 1 de Aminoácidos Catiônicos , Sinoviócitos , Animais , Camundongos , Aminoácidos/metabolismo , Artrite Experimental/metabolismo , Artrite Reumatoide/tratamento farmacológico , Transportador 1 de Aminoácidos Catiônicos/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Cromatografia Líquida , Citocinas/metabolismo , Fibroblastos/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Camundongos Endogâmicos CBA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/farmacologia , RNA Interferente Pequeno/uso terapêutico , Membrana Sinovial/metabolismo , Sinoviócitos/metabolismo , Espectrometria de Massas em Tandem
12.
Nat Commun ; 13(1): 6221, 2022 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-36266270

RESUMO

Rheumatoid arthritis (RA) is an immune-mediated disease affecting diarthrodial joints that remains an unmet medical need despite improved therapy. This limitation likely reflects the diversity of pathogenic pathways in RA, with individual patients demonstrating variable responses to targeted therapies. Better understanding of RA pathogenesis would be aided by a more complete characterization of the disease. To tackle this challenge, we develop and apply a systems biology approach to identify important transcription factors (TFs) in individual RA fibroblast-like synoviocyte (FLS) cell lines by integrating transcriptomic and epigenomic information. Based on the relative importance of the identified TFs, we stratify the RA FLS cell lines into two subtypes with distinct phenotypes and predicted active pathways. We biologically validate these predictions for the top subtype-specific TF RARα and demonstrate differential regulation of TGFß signaling in the two subtypes. This study characterizes clusters of RA cell lines with distinctive TF biology by integrating transcriptomic and epigenomic data, which could pave the way towards a greater understanding of disease heterogeneity.


Assuntos
Artrite Reumatoide , Sinoviócitos , Humanos , Sinoviócitos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Biologia de Sistemas , Fator de Transferência/metabolismo , Artrite Reumatoide/metabolismo , Fibroblastos/metabolismo , Proliferação de Células/genética , Linhagem Celular , Fator de Crescimento Transformador beta/metabolismo , Células Cultivadas , Membrana Sinovial/metabolismo
13.
Int J Mol Sci ; 23(19)2022 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-36232539

RESUMO

Obesity is a risk factor for knee osteoarthritis (KOA). Neuromedin U (NMU) and NMU receptors (NMUR1 and NMUR2) are associated with obesity-related disorders and found in mast cells (MCs), which are elevated in osteoarthritis. However, NMU/NMUR expression was not examined in the synovial membrane (SM) or synovial MCs of obese osteoarthritis patients. We compared expression of NMU, NMUR1, NMUR2, and the mast cell (MC) marker, CPA3, in the SM of KOA patients categorized as normal weight (NW; BMI < 25 kg/m2, n = 79), overweight (OW; BMI ≥ 25 and <30 kg/m2, n = 87), and obese (OB; ≥30 kg/m2, n = 40). To study NMU/NMUR expression in MCs, we compared the MC-rich fraction (MC-RF), CD88(+) MC-RF, and CD88(-) MC-RF, extracted using magnetic isolation, with the MC-poor fraction (MC-PF). While NMU and NMUR2 expression were comparable, NMUR1 was significantly elevated in OW and OB compared to NW. Moreover, CPA3 levels were significantly greater in OB than NW. NMUR1 and CPA3 expression were significantly higher in both the CD88(+) and CD88(-) MC-RF than MC-PF. Therefore, NMUR1 expression was elevated in the SM of OB KOA patients, and its expression was found in MCs. Further investigation to analyze the NMU/NMUR1 pathway in MC may provide a link between obesity and KOA pathology.


Assuntos
Mastócitos , Osteoartrite , Humanos , Obesidade/complicações , Receptores de Neurotransmissores , Membrana Sinovial
14.
Int J Mol Sci ; 23(19)2022 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-36233118

RESUMO

Osteoarthritis (OA) is one of the most common joint pathologies and a major cause of disability among the population of developed countries. It manifests as a gradual degeneration of the cartilage and subchondral part of the bone, leading to joint damage. Recent studies indicate that not only the cells that make up the articular cartilage but also the synoviocytes, which build the membrane surrounding the joint, contribute to the development of OA. Therefore, the aim of the study was to determine the response to inflammatory factors of osteoarthritic synoviocytes and to identify proteins secreted by them that may influence the progression of OA. This study demonstrated that fibroblast-like synoviocytes of OA patients (FLS-OA) respond more strongly to pro-inflammatory stimulation than cells obtained from control patients (FLS). These changes were observed at the transcriptome level and subsequently confirmed by protein analysis. FLS-OA stimulated by pro-inflammatory factors [such as lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNFα) were shown to secrete significantly more chemokines (CXCL6, CXCL10, and CXCL16) and growth factors [angiopoietin-like protein 1 (ANGPTL1), fibroblast growth factor 5 (FGF5), and insulin-like growth factor 2 (IGF2)] than control cells. Moreover, the translation of proteolytic enzymes [matrix metalloprotease 3 (MMP3), cathepsin K (CTSK), and cathepsin S (CTSS)] by FLS-OA is increased under inflammatory conditions. Our data indicate that the FLS of OA patients are functionally altered, resulting in an enhanced response to the presence of pro-inflammatory factors in the environment, manifested by the increased production of the previously mentioned proteins, which may promote further disease progression.


Assuntos
Osteoartrite , Somatomedinas , Sinoviócitos , Catepsina K/metabolismo , Células Cultivadas , Fator 5 de Crescimento de Fibroblastos/metabolismo , Fibroblastos/metabolismo , Humanos , Inflamação/patologia , Lipopolissacarídeos/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Osteoartrite/metabolismo , Somatomedinas/metabolismo , Membrana Sinovial/patologia , Sinoviócitos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
15.
Front Immunol ; 13: 997621, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36275686

RESUMO

Rheumatoid arthritis (RA) is a common autoimmune disease characterized by chronic inflammation. Immune dysfunction is an essential mechanism in the pathogenesis of RA and directly linked to synovial inflammation and cartilage/bone destruction. Intermediate conductance Ca2+-activated K+ channel (KCa3.1) is considered a significant regulator of proliferation, differentiation, and migration of immune cells by mediating Ca2+ signal transduction. Earlier studies have demonstrated abnormal activation of KCa3.1 in the peripheral blood and articular synovium of RA patients. Moreover, knockout of KCa3.1 reduced the severity of synovial inflammation and cartilage damage to a significant extent in a mouse collagen antibody-induced arthritis (CAIA) model. Accumulating evidence implicates KCa3.1 as a potential therapeutic target for RA. Here, we provide an overview of the KCa3.1 channel and its pharmacological properties, discuss the significance of KCa3.1 in immune cells and feasibility as a drug target for modulating the immune balance, and highlight its emerging role in pathological progression of RA.


Assuntos
Artrite Experimental , Artrite Reumatoide , Camundongos , Animais , Membrana Sinovial , Artrite Experimental/patologia , Inflamação , Modelos Animais de Doenças , Colágeno
17.
Sci Rep ; 12(1): 16619, 2022 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-36198727

RESUMO

The possibility that mesenchymal stem cells (MSCs) can adhere to partial defects or degenerative areas in cartilage remains to be established. The purposes of the present study were to verify the adhesion of synovial MSCs to degenerated cartilage, the time course of that adhesion, and the morphological changes that MSCs might undergo during the adhesion process. The surface of pig cartilage was abraded, and a human synovial MSC suspension was placed on the abraded surface. The proportion/number of MSCs that adhered to the cartilage was quantified by counting non-adhered MSCs, measuring the fluorescence intensity of DiI-labeled MSCs, and scanning electron microscopy (SEM) observations. The presence of microspikes or pseudopodia on the MSCs that adhered to the cartilage was also evaluated. SEM confirmed the adhesion of synovial MSCs to degenerated cartilage. The three independent quantification methods confirmed increases in the proportion/number of adhered MSCs within 10 s of placement and over time up to 24 h. The MSCs that adhered at 10 s had a high proportion of microspikes, whereas those that adhered after 1 h had that of pseudopodia. MSCs showed time-dependent morphological changes and increased adhesion to degenerated cartilage after placement of the human synovial MSC suspension.


Assuntos
Cartilagem Articular , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Animais , Cartilagem , Diferenciação Celular , Humanos , Transplante de Células-Tronco Mesenquimais/métodos , Suínos , Membrana Sinovial
18.
Sci Rep ; 12(1): 17367, 2022 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-36253398

RESUMO

Synovium is critical for maintaining joint homeostasis and may contribute to mechanobiological responses during joint movement. We investigated mechanobiological responses of whole synovium from patients with late-stage knee osteoarthritis (OA). Synovium samples were collected during total knee arthroplasty and assigned to histopathology or cyclic 10% tensile strain loading, including (1) static (control); (2) low-frequency (0.3 Hz); and iii) high-frequency (1.0 Hz) for 30-min. After 6-h incubation, tissues were bisected for RNA isolation and immunostaining (3-nitrotyrosine; 3-NT). RNA sequencing was analyzed for differentially expressed genes and pathway enrichment. Cytokines and lactate were measured in conditioned media. Compared to controls, low-frequency strain induced enrichment of pathways related to interferon response, Fc-receptor signaling, and cell metabolism. High-frequency strain induced enrichment of pathways related to NOD-like receptor signaling, high metabolic demand, and redox signaling/stress. Metabolic and redox cell stress was confirmed by increased release of lactate into conditioned media and increased 3-NT formation in the synovial lining. Late-stage OA synovial tissue responses to tensile strain include frequency-dependent increases in inflammatory signaling, metabolism, and redox biology. Based on these findings, we speculate that some synovial mechanobiological responses to strain may be beneficial, but OA likely disturbs synovial homeostasis leading to aberrant responses to mechanical stimuli, which requires further validation.


Assuntos
Osteoartrite do Joelho , Meios de Cultivo Condicionados/metabolismo , Citocinas/metabolismo , Humanos , Interferons/metabolismo , Lactatos/metabolismo , Proteínas NLR/metabolismo , Osteoartrite do Joelho/patologia , RNA/metabolismo , Membrana Sinovial/metabolismo
19.
Life Sci ; 309: 121031, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36206833

RESUMO

Choline kinase (ChoK) has been well documented as a major enzyme involved in the anomalous cellular lipid metabolic profile of chronic inflammatory disorders. However, new research has now been unveiled that helps us to better understand how changes in lipid metabolism influence the transformational phenotype, drug resistance, and antiapoptotic characteristics of invasive cells, leading to rheumatoid arthritis (RA) disease progression. It is still unknown how ChoK modulates the lipid metabolic aberrations that may promote altered cell phenotype and functionality in RA. Herein, we review the current understanding of ChoK's role in altered metabolism in diverse cell types involved in RA progression, and for the first time, we take a step forward to complete the puzzle and summarise striking facts that link choline metabolism to its transformed phenotype, in order to postulate ChoK as a robust therapeutic target in RA. This review forms a foundation on which ChoK can be tackled as a potential biomarker, opening doors for RA diagnosis and prognosis. It frameworks several ChoK inhibitors that rewire the lipid metabolic profile in the inflammatory disease landscape and envisages its being translated to clinics.


Assuntos
Artrite Reumatoide , Colina Quinase , Humanos , Colina Quinase/genética , Colina Quinase/metabolismo , Artrite Reumatoide/tratamento farmacológico , Colina/metabolismo , Apoptose , Lipídeos , Membrana Sinovial/metabolismo
20.
Phytomedicine ; 107: 154479, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36194972

RESUMO

BACKGROUND: Rheumatoid arthritis (RA), the most common type of inflammatory arthritis, can cause bone damage and disability. Triptolide, a prominent treatment for RA, has satisfactory anti-inflammatory effects. However, the mechanism of action of triptolide in RA remains unknown. PURPOSE: This study aimed to explore the molecular mechanisms underlying triptolide-mediated improvements in RA and identify the miRNA pathway responsible for these effects. METHODS: We identified various dysregulated miRNAs associated with RA by mining previously described microarray data and verified and screened these candidates using RT-qPCR. Hematoxylin-eosin staining was then applied to identify pathological changes in the affected joints, and cell counting kit-8 analysis and flow cytometry were employed to examine cell proliferation and apoptosis, respectively. Extracted exosomes were verified using transmission electron microscopy. RESULTS: Our results revealed that the legs of rats with collagen-induced arthritis presented with obvious swelling and bone damage, a high degree of inflammatory cell infiltration into the synovium, and structural changes to the cartilage. Data mining identified 39 dysregulated miRNAs in these tissues, and RT-qPCR further refined these observations to highlight miR-221 as a potential RA biomarker. Subsequent evaluations revealed that fibroblast-like synovial (FLS) cells secrete Exs carrying dysregulated miR-221 in vitro. These Exs mediate miR-221 levels, inflammation, and TLR4/MyD88 signaling via their fusion with chondrocytes, leading to changes in chondrocyte growth and metabolic factor levels. Additionally, the addition of triptolide impaired miR-221 expression, cell proliferation, inflammatory factors, and the protein levels of TLR4/MyD88 in RA-FLS and promoted the apoptosis of FLS. The therapeutic effect of triptolide on miR-221 Exs was reversed by miR-221 inhibitor in both normal and RA FLS. CONCLUSION: Our research shows that effective treatment with triptolide is mediated by its regulation of growth and secretory functions of chondrocytes via the inhibition of miR-221 secretion by FLS, providing a new target and natural medicinal candidate for future RA treatments.


Assuntos
Artrite Reumatoide , Exossomos , MicroRNAs , Animais , Anti-Inflamatórios/farmacologia , Apoptose , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Proliferação de Células , Células Cultivadas , Condrócitos/metabolismo , Diterpenos , Regulação para Baixo , Amarelo de Eosina-(YS)/metabolismo , Amarelo de Eosina-(YS)/farmacologia , Compostos de Epóxi , Exossomos/metabolismo , Exossomos/patologia , Fibroblastos/metabolismo , Hematoxilina/metabolismo , Hematoxilina/farmacologia , MicroRNAs/genética , MicroRNAs/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Fenantrenos , Ratos , Membrana Sinovial/patologia , Receptor 4 Toll-Like/metabolismo
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