Fibroblast-like Synovial Cells Research Articles

Cold Air Plasma Inhibiting Tumor-Like Biological Behavior of Rheumatoid Arthritis Fibroblast-Like Synovial Cells via G2/M Cell Cycle Arrest

Cold Air Plasma Inhibiting Tumor-Like Biological Behavior of Rheumatoid Arthritis Fibroblast-Like Synovial Cells via G2/M Cell Cycle Arrest

The Hippo-YAP Signaling Pathway in Osteoarthritis and Rheumatoid Arthritis.

Arthritis is the most prevalent joint disease and is characterized by articular cartilage degradation, synovial inflammation, and changes in periarticular and subchondral bone. Recent studies have reported that Yes-associated protein (YAP) and the transcriptional coactivator with PDZ-binding motif (TAZ) have significant effects on the proliferation, migration, and survival of chondrocytes and fibroblast-like synovial cells (FLSs). YAP/TAZ signaling pathway, as well as the related Hippo-YAP signaling pathway, are responsible for the condition of cells and articular cartilage in joints. They are tightly regulated to maintain metabolism in chondrocytes and FLSs because abnormal expression may result in cartilage damage. However, the roles and mechanisms of the Hippo-YAP pathway in arthritis remain largely unknown. This review summarizes the roles and key functions of YAP/TAZ and the Hippo-YAP signaling pathway in FLSs and chondrocytes for the induction of proliferation, migration, survival, and differentiation in rheumatoid arthritis (RA) and osteoarthritis (OA) research. We also discuss the therapeutic strategies involving YAP/TAZ and the related Hippo-YAP signaling pathway involved in OA.

Wilforine inhibits rheumatoid arthritis pathology through the Wnt11/β-catenin signaling pathway axis

BackgroundWilforine (WFR) is a monomeric compound of the anti-RA plant Tripterygium wilfordii Hook. f. (TwHF). Whether WFR has anti-RA effect, its molecular mechanism has not been elucidated.Aim of the studyOur study aims to clarify how WFR inhibits fibroblast-like synovial cells (FLS) activation and improves RA through Wnt11 action on the Wnt11/β-catenin signaling pathway.MethodsThe therapeutic effect of WFR on collagen-induced arthritis (CIA) rats was evaluated using methods such as rat arthritis score. The inhibitory effects and signaling pathways of WFR on the proliferation and inflammatory response of CIA FLS and RA FLS were studied using ELISA, CCK-8, RT-qPCR, Western blot, and immunofluorescence methods.ResultsWFR could effectively alleviate the arthritis symptoms of CIA rats; reduce the levels of IL-6, IL-1β, and TNF-α in the peripheral blood of CIA rats; and inhibit the expression of MMP3 and fibronectin. The data showed that WFR has a significant inhibitory effect on FLS proliferation. Furthermore, WFR inhibited the activation of Wnt/β-catenin signaling pathway and decreased the expression of Wnt11, β-catenin, CCND1, GSK-3β, and c-Myc, while the effects of WFR were reversed after overexpression of Wnt11.ConclusionsWFR improves RA by inhibiting the Wnt11/β-catenin signaling pathway, and Wnt11 is the direct target of WFR. This study provides a new molecular mechanism for WFR to improve RA and contributes to the clinical promotion of WFR.

Network Pharmacology and Molecular Docking Explore the Mechanism of Mubiezi-Yinyanghuo Herb Pair in the Treatment of Rheumatoid Arthritis.

Our previous studies have shown that the Mubiezi-Yinyanghuo (MBZ-YYH) herb pair inhibits rheumatoid arthritis (RA) cell proliferation and glycolysis, promising results with an obscure mechanism of action. Therefore, it is necessary to explore the main components of MBZ-YYH and unravel the potential mechanism in RA based on network pharmacology and molecular docking methods. Components and targets of MBZ-YYH were retrieved from the TCMSP. Relevant targets of RA were searched in GeneCards, therapeutic target database (TTD), and DisGeNET databases; the common targets of the MBZ-YYH compounds and RA were obtained by comparison; and a component-target interaction network was established by Cytoscape 3.9.1. Gene ontology (GO) analysis and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway enrichment analysis were performed through the David database. Molecular docking was performed by PyMoL2.3.0 and AutoDock Vina1.1.2 software. 7 active ingredients and 58 putatively identified target genes were screened from MBZ, and 16 effective components of YYH and 230 potential targets were identified. There were 29 mutual targets between the two herbs and RA. Through the PPI network, 9 hub targets which contain JUN, CASP3, PPARG, PTGS2, GSK3B, CASP8, HMOX1, ICAM1, and HK2 were screened out. GO term enrichment analysis indicated that positive regulation of the apoptotic process, response to drugs, and response to hypoxia were significantly enriched. Based on KEGG analysis, it was mainly associated with the IL-17 signaling pathway, the TNF signaling pathway, and the p53 signaling pathway. The docking analysis revealed that the effective components showed strong binding activity with the receptors. The effects of the MBZ-YYH herb pair on RA were coordinated by the interaction of diverse components, which may be through the IL-17 signaling pathway and the TNF signaling pathway, which target GSK3B, HK2, caspase 3, and caspase 8, inhibiting the proliferation and glycolysis of rheumatoid arthritis fibroblast-like synovial cells (RA-FLS) and tending towards an increasing efficacy and decreasing toxicity effect on RA.

Mechanism of Rhodiola rosea-Euonymus alatus drug pair against rheumatoid arthritis: Network pharmacology and experimental validation.

The present study aimed to explore the potential components and mechanisms of Rhodiola rosea-Euonymus alatus drug pair (TY) that ameliorate rheumatoid arthritis (RA). The main active components, core targets, and important pathways of TY against RA were predicted by network pharmacology analysis. The binding activity between the main active components and the core targets was verified by the molecular docking technique. Collagen-induced arthritis (CIA) rat model and tumor necrosis factor (TNF)-α-induced fibroblast-like synovial cells in human RA (HFLS-RA) model were established, respectively. The core targets were verified by cell counting kit-8 assay, hematoxylin eosin, enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and Western blot analysis, and the therapeutic effect of TY was evaluated. A total of 18 possible components and 34 core targets were obtained by network pharmacology, among which inflammatory response, phosphatidylinositide 3-kinases (PI3K)-AKT and MAPK pathways were involved in the therapeutic effect of TY on RA. The results of molecular docking showed that kaempferol and quercetin had high binding affinity to interleukin (IL)-1β, IL-6, matrix metalloproteinase (MMP)9, and TNF-α. In vivo and in vitro experiments showed that TY dose-dependently inhibited the proliferation of HFLS-RA cells induced by TNF-α, and significantly reduced the paw swelling and arthritis scores in CIA rats. At the same time, TY inhibited the production of inflammatory factors in CIA rat serum and TNF-α-induced HFLS-RA cells. It also decreased the expression of PI3K, phospho-protein kinase B, MMP1, MMP3, MMP9, and increased the protein and mRNA levels of tissue inhibitors of MMPs (TIMP)1 in synovial tissue. TY can inhibit the PI3K/AKT signaling pathway and regulate the balance between MMPs and TIMP, thus playing a therapeutic role in RA.

2-hydroxy-4-methoxy benzaldehyde from Hemidesmus indicus root extract suppresses toll-like receptor2-mediated migration and invasive mechanisms in rheumatoid arthritis

IntroductionElevated expression of Toll-like receptor2 (TLR2) in synovial fibroblasts, endothelial and other infiltrating cells in rheumatoid arthritis promotes their migration and invasion. Our aim was to isolate a TLR2 inhibitor from Hemidesmus indicus root extract and test its effectiveness in regulating migration and invasion of fibroblast-like synoviocytes, endothelial cells and ex vivo synovial explant outgrowth. MethodFractionation of TLR2 inhibitor by a bioassay, followed by testing the inhibitor in the cultures of isolated fibroblast-like synoviocytes, endothelial cells and ex vivo synovial explant outgrowth in the presence of a TLR2 agonist, Pam3CSK4 was undertaken. ResultsTLR2 inhibition using 2-hydroxy-4-methoxy benzaldehyde significantly lowered Pam3CSK4-induced cell migration, invasion, MMP-1, MMP-3, Pro-MMP-2 and Pro-MMP-9 expression in fibroblast-like synovial cells isolated from the discarded biopsy specimens of rheumatoid arthritis patients as well as in ex vivo synovial explant culture. TLR2 inhibition lowered active Rac1 protein and β1 integrin mRNA levels in synovial fibroblasts and suppressed filopodia and lamellipodia formation in response to Pam3CSK4. In endothelial cells, 2-hydroxy-4-methoxy benzaldehyde inhibited Pam3CSK4-stimulated vascular endothelial growth factor (VEGF) and ICAM-1 expression and thereby lowered tube formation and endothelial migration. TLR2 inhibition using 2-hydroxy-4-methoxy benzaldehyde also attenuated the Pam3CSK4-evoked outgrowth formation in ex vivo synovial explants. 2-hydroxy-4-methoxy benzaldehyde targeted TLR2-MyD88 interactions and prevented dimerization. Conclusions2-hydroxy-4-methoxy benzaldehyde is the active constituent in the roots of H indicus that appears to act through TLR2 inhibition, followed by modulation of the agonist-induced cell migration, invasion and angiogenesis.

Hederagenin inhibits proliferation, angiogenesis and inflammation of fibroblast-like synovial cells in rheumatoid arthritis

Purpose: To determine the effect of Hederagenin (Hed) on rheumatoid arthritis (RA) in a cell model, and to elucidate the mechanism of action of Hed.
 Methods: MTT, EDU, and Immunoblot assays were used to determine the effects of Hed on the viability of fibroblast-like synovial cells, while the effects of Hed on inflammation were examined by enzymelinked immunosorbent assay (ELISA) and immunoblot assay. The influence of Hed on cell motility angiogenesis was evaluated by Transwell and tube formation assays, while immunoblot analysis was used to determine the mechanism of action of Hed.
 Results: Hed inhibited the viability of RA-FLS cells and suppressed the inflammation of RA-FLS cells (p < 0.05). Furthermore, Hed suppressed the migration and angiogenesis of RA-FLSl cells, as well as regulated MAPK pathway (p < 0.05).
 Conclusion: Hed inhibits the proliferation, angiogenesis and inflammation of fibroblast-like synovial cells in RA by regulating MAPK pathway. Therefore, Hed is a drug for the treatment of RA, However, in vivo studies to validate these findings are recommended

Angelica sinensis polysaccharide inhibits inflammation of collagen-induced arthritis rat fibroblast-like synoviocytes by inhibiting JAK2/STAT3 and MAPK signaling

A. sinensis polysaccharide (ASP), one of the effective components of A. sinensis, has been used to treat inflammatory diseases in China. However, its effect on rheumatoid arthritis (RA) is unknown. The purpose of this study was to explore the anti-inflammatory effects and mechanisms of ASP on RA using a rat model of collagen-induced arthritis (CIA). For evaluation of the therapeutic effects of ASP in vitro, the CIA model was used. Our study showed that ASP (100 and 200 μg/mL) dose-dependently inhibited tumor necrosis factor (TNF)-α-induced (10 ng/mL) proliferation, migration, and invasion of fibroblast-like synovial (FLS) cells, promoted apoptosis, and arrested the cell cycle in G0/G1 phase (P < 0.05). In vivo, ASP increased body weight in CIA rats while decreasing paw swelling, arthritis score, and synovial tissue proliferation (P < 0.05). ASP also reduced the expression of pro-inflammatory cytokines (interleukin (IL) −6, IL-1β, inducible nitric oxide synthetase (iNOS), matrix metalloproteinase (MMP)-1 and MMP-3) (P < 0.05). ASP inhibited the phosphorylation of JAK2/STAT3 and MAPK signaling pathway components (P < 0.05) induced by TNF-α in CIA-FLS cells. Using JAK and p38 inhibitors, we found that JAK2/STAT3 might be an upstream pathway of MAPK. In conclusion, our study investigated the therapeutic effects of ASP in RA in vitro and in vivo. Our research suggests that ASP can inhibit the invasiveness and secretion of inflammatory cytokines of FLS cells in CIA rats via JAK2/STAT3 and MAPK signaling. Elucidation of the underlying mechanism will provide a theoretical basis for clinical application of ASP.

Chinese herbal formula, modified Xianfang Huoming Yin, alleviates the inflammatory proliferation of rat synoviocytes induced by IL-1β through regulating the migration and differentiation of T lymphocytes.

Xianfang Huoming Yin (XFH) is a traditional Chinese herbal formula, which has the effect of clearing heat and detoxifying toxins, dispersing swellings, activating blood circulation, and relieving pain. It is usually applied to treat various autoimmune diseases, including Rheumatoid arthritis (RA). The migration of T lymphocytes plays an indispensable role in the pathogenesis of RA. Our previous studies demonstrated that modified Xianfang Huoming Yin (XFHM) could modulate the differentiation of T, B, and NK cells, and contribute to the restoration of immunologic balance. It also could downregulate the production of pro-inflammatory cytokines by regulating the activation of NF-κ B and JAK/STAT signaling pathways in the collagen-induced arthritis mouse model. In this study, we want to investigate whether XFHM has therapeutic effects on the inflammatory proliferation of rat fibroblast-like synovial cells (FLSs) by interfering with the migration of T lymphocytes in vitro experiments. High performance liquid chromatography-electrospray ionization/mass spectrometer system was used to identify the constituents of the XFHM formula. A co-culture system of rat fibroblast-like synovial cells (RSC-364cells) and peripheral blood lymphocytes stimulated by interleukin-1 beta (IL-1β) was used as the cell model. IL-1β inhibitor (IL-1βRA) was used as a positive control medicine, and two concentrations (100μg/mL and 250μg/mL) of freeze-dried XFHM powder were used as intervention measure. The lymphocyte migration levels were analyzed by the Real-time xCELLigence analysis system after 24h and 48h of treatment. The percentage of CD3+CD4+ T cells and CD3+CD8+ T cells, and the apoptosis rate of FLSs were detected by flow cytometry. The morphology of RSC-364cells was observed by hematoxylin-eosin staining. The protein expression of key factors for T cell differentiation and NF-κ B signaling pathway-related proteins in RSC-364cells were examined by western-blot analysis. The migration-related cytokines levels of P-selectin, VCAM-1, and ICAM-1 in the supernatant were measured by enzyme-linked immunosorbent assay. Twenty-one different components in XFHM were identified. The migration CI index of T cells was significantly decreased in treatment with XFHM. XFHM also could significantly downregulate the levels r of CD3+CD4+T cells and CD3+CD8+T cells that migrated to the FLSs layer. Further study found that XFHM suppresses the production of P-selectin, VCAM-1, and ICAM-1. Meanwhile, it downregulated the protein levels of T-bet, ROR γ t, IKKα/β, TRAF2, and NF-κ B p50, upregulated the expression of GATA-3 and alleviated synovial cells inflammation proliferation, contributing to the FLSs apoptosis. XFHM could attenuate the inflammation of synovium by inhibiting T lymphocyte cell migration, regulating differentiation of T cells through modulating the activation of the NF-κ B signaling pathway.

Microplastics aggravates rheumatoid arthritis by affecting the proliferation/migration/inflammation of fibroblast-like synovial cells by regulating mitochondrial homeostasis

Rheumatoid arthritis (RA) is an autoimmune disease involving multiple joints. RA is a systemic disease characterized by chronic synovial inflammation and destruction of articular cartilage and bone. As a new pollutant, microplastics can enter the body through the respiratory and digestive tract and cause health damage. However, to date, the impact of microplastics on RA has not been revealed. Therefore, in the current research, we explored the impact of microplastics on RA. First, FLS (fibroblast-like synoviocytes) from RA was isolated and identified. FLS has been used as a cell model in vivo to study the potential impact of microplastics on FLS. Therefore, a series of biochemical experiments have been carried out, such as indirect immunofluorescence, western blotting and flow cytometry. First, we found that microplastics promote the proliferation of RA-FLSs through the MTT assay and the detection of cell proliferation markers and the cell cycle analysis through flow cytometry. On this basis, further research showed that microplastics also promoted the invasion and migration ability of RA-FLSs through Transwell experiments. In addition, microplastics also promote the secretion of inflammatory factors in RA-FLSs. In in vivo studies, the effect of microplastics on RA cartilage damage was evaluated. The results showed that RA cartilage damage was aggravated by microplastics, as determined by Alcian blue, toluidine blue and safranin O-fast green staining. Current research shows that microplastics, as a new pollutant, can promote sustained damage in RA.

Mechanism underlying the action of Duanteng-Yimu Tang in regulating Treg/Th17 imbalance and anti-rheumatoid arthritis

BackgroundRheumatoid arthritis (RA) is a chronic immune disease characterised by synovitis and cartilage destruction. Currently, many patients experience poor remission after new antirheumatic drug treatments. Duanteng-Yimu Tang (DTYMT), a traditional Chinese medicine, is effective in the treatment of RA. In this research, we designed to investigate the anti-RA effects of DTYMT and explore its potential mechanisms. MethodsNetwork pharmacology was adopted to explore the main pathways of DTYMT in patients with RA. Collagen-induced arthritis models of male DBA/1 mice were established, and their histopathological changes were observed by hematoxylin-eosin staining and micro-CT. qRT-PCR was performed to detect the expression of Foxp3 and RORγt in the serum and synovial tissue and IL-17, IL-1β, TNF-α, and IL-10 mRNA in vivo. The proliferation and invasion of synovial cells were analyzed using Cell Counting Kit-8 and transwell assays, respectively. The ratio of T helper 17 (Th17) to regulatory T (Treg) cells was analyzed by flow cytometry. ResultsNetwork pharmacology analysis revealed that Th17 cell differentiation may be the key pathway of DTYMT in RA. DTYMT ameliorated joint damage, inhibited RORγt expression, and increased Foxp3 expression in CIA mice. DTYMT significantly decreased IL-1β, IL-17, and TNF-α mRNA levels, and increased IL-10 mRNA levels in IL-6-induced cells. Additionally, DTYMT inhibited Th17 cell differentiation and promoted Treg cell production, thus improving the Treg/Th17 imbalance. DTYMT also inhibited the proliferation, migration, and invasion of RA fibroblast-like synovial cells. ConclusionsThese results indicate that DTYMT could regulate the Treg/Th17 cell balance, which is a possible mechanism of DTYMT in treating RA.

Berberine inhibits autophagy and promotes apoptosis of fibroblast-like synovial cells from rheumatoid arthritis patients through the ROS/mTOR signaling pathway

To evaluate the regulatory effect of berberine on autophagy and apoptosis balance of fibroblast-like synoviocytes (FLSs) from patients with in rheumatoid arthritis (RA) and explore the mechanism. The inhibitory effect of 10, 20, 30, 40, 50, 60, 70, and 80 μmol/L berberine on RA-FLS proliferation was assessed using CCK-8 method. Annexin V/PI and JC-1 immunofluorescence staining was used to analyze the effect of berberine (30 μmol/L) on apoptosis of 25 ng/mL TNF-α- induced RA-FLSs, and Western blotting was performed to detect the changes in the expression levels of autophagy- and apoptosis-related proteins. The cells were further treated with the autophagy inducer RAPA and the autophagy inhibitor chloroquine to observe the changes in autophagic flow by laser confocal detection of mCherry-EGFP-LC3B. RA-FLSs were treated with the reactive oxygen species (ROS) mimic H2O2 or the ROS inhibitor NAC, and the effects of berberine on ROS, mTOR and p-mTOR levels were observed. The results of CCK-8 assay showed that berberine significantly inhibited the proliferation of RA-FLSs in a time- and concentration-dependent manner. Flow cytometry and JC-1 staining showed that berberine (30 μmol/L) significantly increased apoptosis rate (P < 0.01) and reduced the mitochondrial membrane potential of RA-FLSs (P < 0.05). Berberine treatment obviously decreased the ratios of Bcl-2/Bax (P < 0.05) and LC3B-II/I (P < 0.01) and increased the expression of p62 protein in the cells (P < 0.05). Detection of mCherry-EGFP-LC3B autophagy flow revealed obvious autophagy flow block in berberine-treated RA-FLSs. Berberine significantly reduced the level of ROS in TNF-α-induced RA-FLSs and upregulated the expression level of autophagy-related protein p-mTOR (P < 0.01); this effect was regulated by ROS level, and the combined use of RAPA significantly reduced the pro-apoptotic effect of berberine in RA-FLSs (P < 0.01). Berberine can inhibit autophagy and promote apoptosis of RA-FLSs by regulating the ROS-mTOR pathway.

Qinteng Tongbi Decoction Medicated Serum Exerts Regulates the Proliferation, Migration, and Apoptosis of Synovial Fibroblasts in Adjuvant-Induced Arthritis Rats Model via Phosphatidylinositol 3-Kinase/Protein Kinase B Signaling Pathway

Background Qinteng Tongbi decoction (QTTBD) was an empirical prescription that could effectively prevent and treat rheumatoid arthritis (RA), but there was no report of pharmacological studies on this prescription. The purpose of this paper was to report the effects of QTTBD on the proliferation, migration, and apoptosis of synovial fibroblasts in adjuvant arthritis model rats, and to reveal its anti-RA regulatory mechanism. Methods To divide the fibroblast-like synovial (FLS) cells of experimental rats into 6 groups (blank control group, model control group, positive drug group, QTTBD high, medium, and low dose groups) and cultured with serum-containing drugs. And using Cell Counting Kit-8 to detect the proliferation rate of FLS cells, flow cytometry to detect the apoptosis of FLS cells, the enzyme-linked immunosorbent assay method to detect the levels of interleukin (IL)-6, IL-1β, and tumor necrosis factor-α (TNF-α), Western Blot to detect phosphatidylinositol 3-kinase (PI3K), AKT1, p-AKT1, Bax and Bcl-2 gene and protein expression. Results Experimental results showed that QTTBD-containing serum could effectively inhibit the proliferation of FLS cells ( p &lt;0 .05), induce the apoptosis of FLS cells, reduce the expression levels of inflammatory factors such as IL-6, IL-1β, and TNF-α ( p &lt;0 .05), reduce expression of PI3K, AKT1, p-AKT1, and Bax ( p &lt;0 .05), while the Bcl-2 expression increased ( p &lt;0 .05). Conclusion QTTBD could effectively regulate the proliferation, migration, and apoptosis of FLS cells in adjuvant-induced arthritis (AIA) rats, and its mechanism might be related to regulating the level of inflammatory factors and intervening in the PI3K/protein kinase B (AKT) signaling pathway.

Identification and experimental validation of ferroptosis-related gene SLC2A3 is involved in rheumatoid arthritis

BackgroundFerroptosis, an iron-dependent manner of lipid peroxidative cell death, has recently been reported to be strongly associated with rheumatoid arthritis (RA). Targeted ferroptosis may be a potential treatment for RA. MethodsWe combined bioinformatics analysis and machine learning algorithm to screen the characteristic gene of RA. Moreover, we used gene set enrichment analysis (GSEA) to investigate the biological function of feature gene and CIBERSORT algorithm to analyze the correlation between selected hub gene and immune cells. The CellMiner database was used to predict potential drugs for RA. Finally, it was further verified by in vitro cell experiment. ResultsSLC2A3 was identified as an important potential biomarker based on bioinformatics methods and machine learning algorithms. SLC2A3 encodes the predominantly neuronal glucose transporter 3 (GLUT3). GSEA showed that SLC2A3 high-expression group was correlated with metabolic pathways. Immune cell infiltration analysis showed that SLC2A3 was positively correlated with activated mast cell expression. RSL3 is an activator of ferroptosis that binds to and inactivates GPX4, mediating ferroptosis regulated by GPX4. In our experiment, we treated synovial fibroblast-like cells of RA (RA-FLS) with RSL3 (Ferroptosis inducers) and found that RSL3 can downregulate SLC2A3 expression and induce ferroptosis in RA-FLS. ConclusionsOur study identifies and validates ferroptosis-related gene SLC2A3 as a potential biomarker for the diagnosis and treatment of RA. It was also found that RSL3 can induce ferroptosis in RA-FLS via lead to the downregulation of SLC2A3.

4-Iodo-6-phenylpyrimidine (4-IPP) suppresses fibroblast-like synoviocyte- mediated inflammation and joint destruction associated with rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic immune-mediated inflammatory disease that significantly impacts patients' quality of life. Fibroblast-like synovial cells (FLSs) within the synovial intima exhibit "tumor-like" properties such as increased proliferation, migration, and invasion. Activation of FLSs and secretion of pro-inflammation factors result in pannus formation and cartilage destruction. As an inhibitor of the cytokine, macrophage migration inhibitory factor (MIF), 4-Iodo-6-phenylpyrimidine (4-IPP) has been shown to reduce cell proliferation, migration, invasion, and the secretion of pro-inflammatory mediators in a variety of diseases. However, the usefulness of 4-IPP for RA treatment has not been assessed and was the purpose of this study. In vitro, 4-IPP was demonstrated to inhibit proliferation, migration, and invasion of RA FLSs, as well as the expression of pro-inflammatory cytokines. 4-IPP was also shown to inhibit MIF-induced phosphorylation of ERK, JNK, and p38, as well as reduce expression of COX2 and PGE2. In order to efficiently deliver 4-IPP to anatomical RA sites, we developed lactic-co-glycolic acid (PLGA) nanospheres, which not only protected 4-IPP from degradation but also controlled the release of 4-IPP. 4-IPP/PLGA nanospheres had potent anti-inflammatory activity and a high degree of biosafety. Results showed that local 4-IPP concentration was increased by nanosphere delivery, effectively reducing the inflammatory microenvironment as well as synovial inflammation, joint swelling, and cartilage destruction in a collagen-induced rheumatoid arthritis (CIA) rat model. Therefore, 4-IPP nanospheres are a sustained-release delivery system that may be an effective therapeutic strategy for RA treatment.

DHA Induces Cell Death through the Production of ROS and the Upregulation of CHOP in Fibroblast-like Synovial Cells from Human Rheumatoid Arthritis Patients.

Rheumatoid arthritis (RA) is an inflammatory disease marked by a massive proliferation of synovial cells in the joints. In this study, we investigated the pro-apoptotic effects of docosahexaenoic acid (DHA) in human fibroblast-like synovial cells from RA patients (RA-FLS). An in vitro study using MH7A cells showed that DHA treatment induced caspase-8-dependent apoptosis in a dose-dependent manner and reduced the TNF-α-mediated induction of MMP-9 and IL-1β. DHA also induced the phosphorylation of eIF2α, the expression of the ER stress markers ATF4 and C/EBP homologous protein (CHOP), and death receptor 5 (DR5). The knockdown of CHOP or DR5 increased cell viability and reduced apoptosis in DHA-treated cells. Furthermore, the knockdown of CHOP reduced DHA-mediated DR5 expression, while the overexpression of CHOP increased DR5 expression. We also found that DHA treatment induced the accumulation of reactive oxygen species (ROS), and pretreatment with the anti-oxidant Tiron effectively abrogated not only the expression of CHOP and DR5, but also DHA-induced apoptosis. Under this condition, cell viability was increased, while PARP-1 cleavage and caspase-8 activation were reduced. All the findings were reproduced in human primary synovial cells obtained from RA patients. These results suggest that the DHA-mediated induction of ROS and CHOP induced apoptosis through the upregulation of DR5 in RA-FLSs, and that CHOP could be used as a therapy for RA.

Orientin inhibits the progression of fibroblast-like synovial cells in rheumatoid arthritis by regulating MAPK-signaling pathway.

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. 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. 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. 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.

Sensory Neuron-Specific Deletion of Tropomyosin Receptor Kinase A (TrkA) in Mice Abolishes Osteoarthritis (OA) Pain via NGF/TrkA Intervention of Peripheral Sensitization.

Tropomyosin receptor kinase A (TrkA/NTRK1) is a high-affinity receptor for nerve growth factor (NGF), a potent pain mediator. NGF/TrkA signaling elevates synovial sensory neuronal distributions in the joints and causes osteoarthritis (OA) pain. We investigated the mechanisms of pain transmission as to whether peripheral sensory neurons are linked to the cellular plasticity in the dorsal root ganglia (DRG) and are critical for OA hyperalgesia. Sensory neuron-specific deletion of TrkA was achieved by tamoxifen injection in 4-week-old TrkAfl/fl;NaV1.8CreERT2 (Ntrk1 fl/fl;Scn10aCreERT2) mice. OA was induced by partial medial meniscectomy (PMM) in 12-week-old mice, and OA-pain-related behavior was analyzed for 12 weeks followed by comprehensive histopathological examinations. OA-associated joint pain was markedly improved without cartilage protection in sensory-neuron-specific conditional TrkA knock-out (cKO) mice. Alleviated hyperalgesia was associated with suppression of the NGF/TrkA pathway and reduced angiogenesis in fibroblast-like synovial cells. Elevated pain transmitters in the DRG of OA-induced mice were significantly diminished in sensory-neuron-specific TrkA cKO and global TrkA cKO mice. Spinal glial activity and brain-derived neurotropic factor (BDNF) were significantly increased in OA-induced mice but were substantially eliminated by sensory-neuron-specific deletion. Our results suggest that augmentation of NGF/TrkA signaling in the joint synovium and the peripheral sensory neurons facilitate pro-nociception and centralized pain sensitization.

Study on the Therapeutic Effect of Lenalidomide on Hemophilic Arthropathy

To explore the effect of lenalidomide on human fibroblast-like synovial cells (HFLS) and the therapeutic efficacy on hemophilic arthropathy in hemophilia A mice model. In vitro, to remodel the inflammatory environment of synovial tissue after hemorrhage, ferric citrate and recombinant TNF-α were added into the cell culture medium of HFLS. Cell Counting Kit-8 (CCK-8), Enzyme-linked immunosorbent assay (ELISA), Quantitative Real-time PCR (RT-qPCR) and flow cytometry were employed for detection of the effects of lenalidomide on the proliferation ability, pro-inflammatory cytokines release and apoptosis of HFLS cells. In vivo, hemophilia arthropathy was remodeled in hemophilia A mice by induction of hemarthrosis. A series of doses of lenalidomide (0.1, 0.3 and 1.0 g/kg) was administrated intra-articularly. Tissues of knee joints were collected on the 14th day after administration, and the protective effect of lenalidomide on arthritis in hemophilia A mice were evaluated by RT-qPCR and histological grading. In vitro, compared with the untreated control group, lenalidomide could significantly inhibit the proliferation of HFLS cells (P<0.05), and the effect was the most significant when the concentration was 0.01 μmol/L (P<0.001). Compared with the control group, lenalidomide could significantly inhibit the expression levels of TNF-α, IL-1β, IL-6 and IFN-γ in HFLS cells (P<0.05). The flow cytometry results showed that lenalidomide could enhance the apoptotis of HFLS cells (P<0.05). The results of RT-qPCR showed that lenalidomide could significantly reduce the mRNA expression levels of TNF-α, IL-1β, IL-6,MCP-1 and VEGF in the joint tissues (P<0.05). Histological results showed that compared with the injured group, lenalidomide could significantly reduce the pathological sequela after hemarthrosis induction, e.g. synovial thickening and neo-angiogenesis in the synovium. The protection displayed a dose-response pattern roughly. In vitro, lenalidomide can inhibit the proliferation of HFLS cells, promote their apoptosis, and inhibit the expression of pro-inflammatory cytokines. In vivo, lenalidomide can significantly decrease the expression of pro-inflammatory cytokines in the joints of mice, and prevent the development of inflammation and neo-angiogenesis. The results provide a theoretical and experimental basis for the clinical application of lenalidomide in the treatment of hemophilic arthropathy.

Triptolide improves chondrocyte proliferation and secretion via down-regulation of miR-221 in synovial cell exosomes

BackgroundRheumatoid 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. PurposeThis study aimed to explore the molecular mechanisms underlying triptolide-mediated improvements in RA and identify the miRNA pathway responsible for these effects. MethodsWe 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. ResultsOur 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. ConclusionOur 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.