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IL-33 is a member of the IL-1 family of cytokines whose role remains controversial in rheumatoid arthritis (RA). The present study was performed to evaluate the correlation of IL-33 with other cytokines and chemokines in serum and the synovia, and to explore the nature of the association. The concentration of IL-33 in samples from 96 patients with RA was analyzed. The response of fibroblast-like synoviocytes (FLSs) to treatment with different concentrations of IL-33 was assessed in vitro. IL-33 was indicated to exhibit an association with multiple cytokines and chemokines in synovial fluid with an inverted-U-shaped trend, including IL-6, IL-1β, IL-8, MIG and IP-10, but not in the serum. Furthermore, in vitro experiments confirmed that IL-33 also exerted a U-type dose-dependent regulatory effect on FLS function. In addition, the data-points do not exactly follow the U-shaped curve fit in most cases, therefore, the applicability of this mathematical model in clinical practice is limited. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA)-related fibrosis is a possible cause of temporomandibular joint (TMJ) stiffness. However, the molecular mechanisms underlying the fibrogenic activity in fibroblast-like synoviocytes (FLSs) remain to be clarified. The present study examined the effects of receptor tyrosine kinase (RTK) ligands, such as fibroblast growth factor (FGF)-1 and epidermal growth factor (EGF), on myofibroblastic differentiation of the FLS cell line FLS1, which is derived from the mouse TMJ. The present study revealed that both FGF-1 and EGF dose-dependently suppressed the expression of the myofibroblast (MF) markers, including α-smooth muscle actin (α-SMA) and type I collagen, in FLS1 cells. Additionally, both FGF-1 and EGF activated extracellular signal-regulated kinase (ERK) in FLS1 cells. In addition, the mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor U0126 abrogated the FGF-1- and EGF-mediated suppression of MF marker expression. On the other hand, inflammatory cytokines, such as interleukin-1β and tumor necrosis factor-α, also suppressed the expression of MF markers in FLS1 cells. Importantly, U0126 abrogated the inflammatory cytokine-mediated suppression of MF marker expression. Interestingly, RTK ligands and inflammatory cytokines additively suppressed the expression of type I collagen. These results suggested that RTK ligands and inflammatory cytokines cooperatively inhibited the fibrogenic activity in FLSs derived from the TMJ in a MEK/ERK-dependent manner. The present findings partially clarify the molecular mechanisms underlying the development of OA-related fibrosis in the TMJ and may aid in identifying therapeutic targets for this condition. Additionally, FGF-1 and EGF could be therapeutically utilized to prevent OA-related fibrosis around the inflammatory TMJ. [ABSTRACT FROM AUTHOR]

Transforming growth factor β-activated kinase-1 (TAK1), a member of the mitogen-activated protein kinase family, plays a key role in the pathogenesis and progression of rheumatoid arthritis (RA). Estrogen has been previously reported to delay arthritis progression. However, the exact association between TAK1 and estrogen remains elusive. The present study demonstrated that TAK1 was upregulated in synoviocytes of patients with RA compared with patients with osteoarthritis and healthy controls. In addition, TAK1 was also expressed in cultured fibroblast-like synoviocytes (FLS), and its levels decreased significantly in 17β-estradiol (E2)-treated cells in a dose-dependent manner. Furthermore, administration of E2 significantly decreased TAK1 expression and attenuated the development of collagen-induced arthritis (CIA). Taken together, the findings of the present study suggested that E2 mediates a decrease of TAK1 in both FLS and CIA, which subsequently results in a suppression of the pathological process of CIA. Therefore, estrogen may serve as a potential therapeutic agent for the treatment of RA by targeting TAK1. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is an autoimmune disease that causes chronic inflammation in synovial tissues. Hyperplasia of synovial tissue leads to the formation of pannus, which invades joint cartilage and bone resulting in joint destruction. Tumor necrosis factor-like ligand 1A (TL1A), a member of the tumor necrosis factor superfamily (TNFSF15), contributes to the pathogenesis of autoimmune diseases, including RA. In the present study, a cDNA microarray was used to search for genes whose expression in rheumatoid fibroblast-like synoviocytes (RA-FLS) were regulated by TL1A. Four individual lines of primary cultured RA-FLS were incubated either with recombinant human TL1A protein or phosphate-buffered saline, as an unstimulated control, for 12 h. Gene expression was then detected through the microarray assay. The results revealed the expression profiles of genes in RA-FLS regulated by TL1A. The present study also demonstrated the functions of those genes whose expression in RA-FLS was regulated by TL1A. Among the genes in this profile, the present study focused on the following genes: Spectrin repeat-containing nuclear envelope 1, Fc receptor-like 2, PYD (pyrin domain)-containing 1, cell division cycle 45 homolog, signal transducer and activator of transcription 5B, and interferon regulatory factor 4. These genes may affect the pathogenesis of RA, including proliferation, regulation of B cells and T cells, inflammation, and cytokine processing. The present study revealed for the first time, to the best of our knowledge, the expression profile of genes in RA-FLS regulated by TL1A. The data indicate that TL1A may regulate the gene expression of various key molecules in RA-FLS, thus affecting the pathogenesis of RA. Further investigations of the genes detected in the current profiles may provide a deeper understanding of the pathogenesis and a novel target for the treatment of RA. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is a chronic autoimmune disease that significantly affects patient quality of life. Galangin is an extract with multiple health benefits, including anti-oxidative, anti-proliferative, immunoprotective and cardioprotective effects. However, to the best of the authors' knowledge, no detailed studies have investigated its regulatory effects on the nuclear factor (NF)-κB/NLR family pyrin domain containing 3 (NLRP3) signaling pathway. The present study aimed to investigate the protective mechanism of galangin in RA fibroblast-like synoviocytes with regards to the NF-κB/NLRP3 signaling pathway. Human RA fibroblast-like synovium cells (RAFSCs) were treated with lipopolysaccharide (LPS) to induce inflammation. The levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-18, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, prostaglandin E2 (PGE2), and nitric oxide (NO) were measured by enzyme-linked immunosorbent assay or western blotting in the absence or presence of different concentrations of galangin. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were additionally evaluated. Furthermore, factors involved in the NF-κB/NLRP3 pathway, including NLRP3, apoptosis-associated speck-like protein containing A, IL-1β, pro-caspase-1, caspase-1, phosphorylated (p)-NF-κB inhibitor α and p-NF-κB, were assessed by western blotting. The results revealed that LPS significantly stimulated IL-1β, TNF-α, IL-18, PGE2, NO, iNOS, COX-2 and NF-κB/NLRP3 factor expression, compared with the control. SOD activity was reduced. Pre-treatment with galangin significantly attenuated the effects of LPS, and galangin was demonstrated to have effective anti-oxidative properties. In conclusion, galangin protected RAFSCs through downregulation of the NF-κB/NLRP3 signaling pathway. These findings suggested that galangin may provide a novel direction for the development of RA therapies in the future. [ABSTRACT FROM AUTHOR]

Homeobox D10 (HOXD10) belongs to the human homeobox (HOX) gene family, and the homologous protein encoded by HOX primarily controls cell differentiation and morphogenesis during embryonic development. The current study aimed to explore the roles and mechanisms of HOXD10 in the migration of human fibroblast-like synoviocytes in rheumatoid arthritis (RAFLS). Cell counting kit-8, cell migration and wound healing assays were performed to examine the cell viability and migration, respectively. Western blot and reverse transcription-quantitative polymerase chain reaction assays were used to evaluate the association between mRNA and protein expression levels. The results revealed HOXD10 expression was upregulated in tissues from patients with RA. HOXD10 silencing inhibited the viability of RAFLS. In addition, HOXD10 silencing suppressed the migration of RAFLS through modulating the expression of cadherin-11, N-cadherin, E-cadherin, vimentin, zonula occludens-1, integrinß1 and paxillin. In conclusion, HOXD10 silencing downregulates the p38/c-Jun N-terminal kinase signaling pathway, which in turn may suppress the migration of RAFLS. [ABSTRACT FROM AUTHOR]

Pyroptosis is triggered by inflammasomes after its activation by various inflammatory stimulations, including lipopolysaccharide (LPS) and improper pH. This may result in programmed death of the affected cell. It is well known that NLRP1 and NLRP3 inflammasomes mediate the production of various cytokines in inflammatory disorders; however, it is still unknown whether NLRP1 and NLRP3 inflammasomes can influence the LPS‑induced pyroptosis in the progression of knee osteoarthritis (KOA). In the present study, the correlation between the NLRP inflammasomes and fibroblast‑like synoviocytes (FLSs) pyroptosis was investigated in vivo and in vitro. Human synovial samples were collected from KOA patients and the expression of NLRP1 and NLRP3 inflammasomes was analyzed. Human FLS were isolated in vitro and stimulated with LPS. To determine whether NLRP1 and NLRP3 inflammasomes are involved in FLS pyroptosis, NLRP1 and NLRP3 small interfering RNAs (siRNAs) were used. The results showed that the expression of NLRPs and inflammasome‑related proteins were upregulated and FLS stimulated with LPS+ATP resulted in cell pyroptosis. However, LPS+ATP‑induced pyroptosis was attenuated by NLRP1 and NLRP3 siRNAs. The results of the present study indicate that LPS‑induced FLS pyroptosis may be mediated by either NLRP1 or NLRP3 inflammsomes. Overall, based on the data obtained from patients and in vitro cells, the present finsings showed that NLRP1 and NLRP3 inflammasomes are highly involved in the FLS inflammation and pyroptosis. Furthermore, inhibition of NLRP1 and NLRP3 led to a remarkable reduction of pyroptosis‑related cytokines. Thus, NLRP1 and NLRP3 inflammasomes may be important in the pathogenesis of OA and may represent a novel therapeutic target. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is a common degenerative joint disease; however, its underlying pathogenesis remains to be elucidated. Previous studies have demonstrated that the transforming growth factor-β (TGF-β) signaling pathway has a role in the initiation and development of OA. Additionally, latent TGF-β-binding protein-1 (LTBP-1) modulates the activity of the TGF-β-mothers against decapentaplegic (Smad) signaling pathway in numerous diseases, including malignant glioma. The present study demonstrated that expression of LTBP-1 is increased in OA synovial tissues compared with normal synovial tissues. The effect of TGF-β was identified to be mediated by phosphorylated(p)-(Smad)2/3, which may activate activin-like kinase (ALK)5 receptor, and by p-Smad1/5/8, which may induce ALK1, thereby stimulating expression of matrix metalloproteinase-(MMP)-13 in OA fibroblast-like synoviocytes (FLS). Compared with normal FLS, OA FLS demonstrated an increased p-Smad1/5/8:p-Smad2 ratio, which led to elevated MMP-13 expression and aggravation of OA. Furthermore, knockdown of the LTBP-1 gene by siRNA transfection in OA FLS reduced p-Smad1/5/8 expression without affecting TGF-β mRNA levels, although p-Smad2 expression increased. It was also demonstrated that OA FLS exhibited increased proliferation compared with normal FLS in vitro. Furthermore, siRNA-mediated downregulation of LTBP-1 reduced proliferation of OA FLS. In conclusion, the present study demonstrated that an alteration in the p-Smad1/5/8:p-Smad2 ratio as well as association between p-Smad1/5/8 and MMP-13 expression in human OA FLS, may contribute to the development of OA. The results of the present study suggested that LTBP-1 is a modulator of the TGF-β signaling pathway in human OA FLS, which may aid in elucidating the mechanism underlying the pathology of OA. [ABSTRACT FROM AUTHOR]

The aim of the present study was to investigate the effects of daphnetin combined with B cell lymphoma 2 (Bcl2)-targeted small interfering (si)RNA (si-Bcl2) on antiapoptotic genes in fibroblast-like synoviocytes (FLS) in rats with collagen II-induced arthritis (CIA). The roles of si-Bcl2 and daphnetin were determined by measuring the expression levels of Bcl2. Protein and mRNA expression levels of Bcl2 in FLS were determined by flow cytometry and reverse transcription-quantitative polymerase chain reaction. Apoptosis of FLS was also determined by flow cytometry. It was revealed that treatment with si-Bcl2 or daphnetin alone resulted in downregulation of Bcl2 mRNA and protein expression. In addition, the mRNA expression levels of the signal transducer and activator of transcription 3 (STAT3), which transcriptionally regulates the activity of mitochondria, were reduced. The combination of si-Bcl2 and daphnetin exhibited an enhanced effect on rheumatoid arthritis FLS (RAFLS), in which the apoptotic rate was significantly higher than either treatment alone. The results suggested that si-Bcl combined with daphnetin may have an enhanced effect in promoting apoptosis of RAFLS derived from CIA rats, and a possible underlying molecular mechanism may function through the downregulation of Bcl2 expression and STAT3, which is located upstream of Bcl2 in the mitochondrial apoptotic pathway. The results of the present study are expected to provide theoretical and experimental basis for the treatment of RA and the medicinal development of daphnetin combined siRNA. [ABSTRACT FROM AUTHOR]

Increasing evidence indicates the important role of inflammation in the pathogenesis and progression of osteoarthritis (OA). Dual specificity phosphatase 1 (DUSP1), a negative regulator of the mitogen-activated protein kinase (MAPK) signaling pathway, has anti-inflammatory properties. In the present study, the expression of DUSP1 was investigated in human OA fibroblast-like synoviocytes (FLSs), human normal FLSs and OA FLSs pretreated with dexamethasone at the mRNA and protein levels. Then, the activation of MAPK pathway proteins and the expression of matrix metalloproteinase-13 (MMP-13) and cyclooxygenase-2 (COX-2) were measured by western blot analysis in the three groups of cells. Dexamethasone induced the expression of DUSP1 and inhibited the activation of the MAPK pathway and reduced the expression of MMP-13 and COX-2 in OA FLSs. However, the role of DUSP1 remained unclear. To clarify this, the effects of overexpression of DUSP1 in OA FLSs were determined using a DUSP1-overexpressing lentivirus. The results demonstrated that overexpression of DUSP1 in OA FLSs inhibited the activation of the MAPK pathway and expression of OA-associated mediators. The findings of the present study indicate that DUSP1 has a protective role in OA FLSs and may be a potential target in the treatment of OA. [ABSTRACT FROM AUTHOR]

The aim of the current study was to investigate the role and mechanism of sirtuin 1 (Sirt1) in the regulation of synovial cell invasion and joint destruction in rheumatoid arthritis (RA). The Sirt1 protein and mRNA levels in fibroblast‑like synoviocytes (FLS) isolated from RA synovial tissues were compared with normal tissues by western blot and reverse transcription‑polymerase chain reaction. RA FLS were then treated with the Sirt1 agonist resveratrol (1, 3 and 10 μg/ml) for 48 h, and their invasiveness and expression of matrix metalloproteinase (MMP) 1 and MMP13 protein and mRNA were measured. Furthermore, a collagen‑induced arthritis (CIA) rat model was established and the rats were divided into a model group, and low‑ and high‑dose resveratrol (2.5 and 10 mg/kg/day) groups to receive an intraperitoneal injection of resveratrol for 42 consecutive days. The joint morphology, arthritis index (AI), and MMP1 and MMP13 expression in synovial tissues was monitored. The Sirt1 protein and mRNA levels in RA FLS were significantly lower compared with normal FLS (P<0.01). The resveratrol treatment significantly inhibited the invasive ability of RA FLS (P<0.01) and reduced MMP1 and MMP13 expression (P<0.01). The AI in low‑ and high‑dose groups was significantly lower compared with the model group from day 28 (P<0.01). Resveratrol also reduced the swelling and damage and decreased MMP1 and MMP13 expression levels in CIA rats (P<0.01). The resveratrol‑induced upregulation of Sirt1 in RA FLS may significantly inhibit the invasion of these cells and reduce the degree of joint damage, which may be mediated through the inhibition of MMP1 and MMP13 expression. The present results suggested a regulatory role for Sirt1 in RA pathogenesis, and demonstrated the beneficial effects of resveratrol, which may have potential as an alternative therapeutic strategy for the treatment of patients with RA. [ABSTRACT FROM AUTHOR]

The objective of the present study was to investigate the effects of Tim-3 silencing on cell viability and lipopolysaccharide (LPS)-induced inflammatory reactions in fibroblast-like synoviocytes (FLS). T-cell immunoglobulin mucin domain molecule (Tim)-3 expression in FLS obtained from patients with rheumatoid arthritis (RA) and normal controls were detected by western blot analysis and reverse transcription-polymerase chain reaction (RT-PCR). Small interfering (si)RNA was transfected using Lipofectamine® 2000 to decrease Tim-3 expression. Following transfection, FLS were stimulated by LPS. An MTT assay, RT-PCR and western blot analysis were performed to measure cell viability, Toll-like receptor 4 (TLR4) signaling pathway-related protein expression and inflammatory cytokine release, respectively. The results of the present study indicated that Tim-3 expression was increased in FLS from patients with RA compared with FLS from healthy controls. Transfection of Tim-3 siRNA significantly decreased Tim-3 expression in FLS from patients with RA. Notably, Tim-3 silencing decreased FLS cell viability. Following stimulation with LPS, cell viability and the expression of TLR4, myeloid differentiation protein gene 88 (MyD88) and nuclear factor-κB (NF-κB) p65 were enhanced in FLS. By contrast, Tim-3 silencing attenuated LPS-induced cell proliferation and the expression of TLR4, MyD88 and NF-κB p65. In addition, LPS significantly increased levels of cytokines in the supernatant, including tumor necrosis factor-a, interferon-? and interleukin-6 (P<0.01). By contrast, Tim-3 silencing significantly decreased LPS-induced cytokine release (P<0.01). However, Tim-3 silencing did not affect TLR4, MyD88 and NF-κB p65 expression and the release of cytokines in cells that did not undergo treatment with LPS. Therefore, the results of the present study indicate that Tim-3 silencing decreases the viability of FLS in RA and attenuates the LPS-induced inflammatory reaction. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is an inflammatory and autoimmune disease that affects ∼1% of the world's population. Although the precise mechanism of RA has yet to be elucidated, accumulating evidence suggests that fibroblast-like synoviocytes (FLSs) serve critical roles in the initiation and progression of RA. However, the underlying molecular mechanisms of FLS proliferation have yet to be elucidated. Long noncoding-interleukin-7 receptor (lnc-IL7R) has been recently identified, which is activated by lipopolysaccharide (LPS) stimulation and diminishes the LPS-induced inflammatory response. In the present study, gain- and loss-of-function assays were performed in order to investigate the role of lnc-IL7R in FLS. It is demonstrated, to the best of the authors' knowledge for the first time, that lnc-IL7R promotes cell proliferation, cell cycle progression and inhibits apoptosis in FLS. Further investigation identified that lnc-IL7 interacts with enhancer of zeste homolog 2 (EZH2) and is required for polycomb repressive complex 2 (PRC2)-mediated suppression, including cyclin-dependent kinase inhibitor 1A and cyclin-dependent kinase inhibitor 2A. Lnc-IL7R may be a promising therapeutic target for the treatment of RA. [ABSTRACT FROM AUTHOR]

Long non-coding RNAs (lncRNAs) assume pivotal roles in various autoimmune diseases including ankylosing spondylitis (AS). Inflammation affects the progression of multiple diseases. The current research aimed at dissecting out the possible role and mechanism of lncRNAs NONHSAT227927.1 in the pathogenesis of AS. In clinical trials, 50 patients with AS and 30 healthy persons were enrolled, followed by the extraction of peripheral blood mononuclear cells (PBMCs). NONHSAT227927.1 expression was detected using reverse transcription quantitative PCR. Enzyme-linked immunosorbent assay was used to determine the levels of inflammatory cytokines. Human fibroblast-like synoviocytes (FLSs) were induced by PBMC supernatant, after which the activity of FLSs was measured by Cell Counting Kit-8 and the signaling pathway were detected by western blotting. Cell migratory capacity was assessed by Transwell migration assay. NONHSAT227927.1 expression was obviously enhanced in the PBMCs of AS patients. NONHSAT227927.1 expression was positively correlated with immunoglobin A (IgA), erythrocyte sedimentation rate (ESR), complement C3 (C3), visual analog scale, IL-17, and IL-23 Levels but was negatively correlated with IL-10 level. The results of association rules showed that the increase of NONHSAT227927.1 expression was strongly associated with the elevation of IgA, C3, ESR, self-rating anxiety scale and IL-17 levels. Overexpression of NONHSAT227927.1 remarkably augmented the proliferation and migration of AS-PBMCs stimulated by AS-FLSs, facilitated the levels of IL-17 and IL-23, and reduced the IL-10 level. By contrast, NONHSAT227927.1 knockdown decreased cell proliferation and migration and cell viability as well as the levels of IL-17 and IL-23, but increased the level of IL-10. overexpression of NONHSAT227927.1 promoted the phosphorylation of JAK2 and STAT3 proteins, while knockout of NONHSAT227927.1 decreased their phosphorylation. Conclusively, lncRNA NONHSAT227927.1 was overexpressed in AS, which activated the JAK2/STAT3 signaling pathway to upregulate inflammatory factors. [ABSTRACT FROM AUTHOR]

The present study aimed to investigate the effect of co-culture of fibroblast-like synoviocytes (FLS) with human umbilical cord-mesenchymal stem cells (UC-MSCs) on rheumatoid arthritis (RA) and to understand the mechanisms that mediate the induced changes. FLS and UC-MSCs were isolated and cultured individually, FLS were then cultured with or without UC-MSCs. The phenotype of UC-MSCs was analyzed prior to co-culture. The UC-MSCs were successfully isolated and expanded, and exhibited a fibroblast-like morphology. Enzyme-linked immunosorbent assay (ELISA) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were performed to determine the expression levels of interleukin (IL)-1β, IL-6, and chemokine (C-C motif) ligand (CCL)-2. The cell apoptosis rate was determined by flow cytometry. Furthermore, the RNAs of aggrecan and collagen type II were isolated and assessed in a chondrogenesis assay following co-culture for 7, 14, 21 and 28 days. Protein expression levels of apoptosis-related proteins, including B-cell lymphoma (Bcl-2), Bcl-2-associated X protein, p53 and phospho (p)-AKT, and growth differentiation factor-5 were analyzed by western blotting. ELISA and qRT-PCR demonstrated that compared with FLS cultured alone, co-culture with UC-MSCs significantly downregulates the expression levels of IL-1β, IL-6 and CCL-2. Additionally, the percentage of apoptotic cells was significantly increased in the co-cultured cells (P<0.05), and the relative RNAs levels of aggrecan and collagen type II were increased compared with FLS alone. Furthermore, the expression levels of Bcl-2 (P<0.05) and p-AKT (P<0.05) were significantly decreased, whereas, p53 (P=0.001), Bax (P<0.01) and GDF-5 (P<0.01) were increased by co-culture of FLS with UC-MSCs compared with FLS alone. In conclusion, co-culture of FLS with UC-MSCs may be important and clinically useful for the treatment of RA by inhibiting the expression of pro-inflammatory mediators, inducing apoptosis and promoting chondrogenesis. [ABSTRACT FROM AUTHOR]

Chlorogenic acid (CGA) is the primary constituent of Caulis Lonicerae, a Chinese herb used for the treatment of rheumatoid arthritis (RA). The present study aimed to investigate whether CGA was able to inhibit the proliferation of the fibroblast-like synoviocyte cell line (RSC-364), stimulated by interleukin (IL)-6, through inducing apoptosis. Following incubation with IL-6 or IL-6 and CGA, the cellular proliferation of RSC-364 cells was detected by MTT assay. The ratio of apoptosed cells were detected by flow cytometry. Western blot analysis was performed to observe protein expression levels of key molecules involved in the Janus-activated kinase/signal transducer and activator of transcription 3 (JAK/STAT) signaling pathway [phosphorylated (p)-STAT3, JAK1 and gp130] and the nuclear factor κB (NF-κB) signaling pathway [phosphorylated (p)-inhibitor of κB kinase subunit a/β and NF-κB p50). It was revealed that CGA was able to inhibit the inflammatory proliferation of RSC-364 cells mediated by IL-6 through inducing apoptosis. CGA was also able to suppress the expression levels of key molecules in the JAK/STAT and NF-κB signaling pathways, and inhibit the activation of these signaling pathways in the inflammatory response through IL-6-mediated signaling, thereby resulting in the inhibition of the inflammatory proliferation of synoviocytes. The present results indicated that CGA may have potential as a novel therapeutic agent for inhibiting inflammatory hyperplasia of the synovium through inducing synoviocyte apoptosis in patients with RA. [ABSTRACT FROM AUTHOR]

Decoy receptor 3 (DcR3) competitively binds to three ligands, Fas ligand, lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells and tumor necrosis factor-like ligand 1A (TL1A), to prevent their effects. Recent studies have suggested that DcR3 directly affects cells as a ligand. Using a microarray assay, our group newly identified interleukin (IL)-12B, which encodes the p40 subunit common to IL-12 and IL-23, as one of the genes for which expression in fibroblast-like synoviocytes from patients with rheumatoid arthritis (RA-FLS) is induced by DcR3. The present study demonstrated that IL-12B mRNA expression was upregulated by DcR3-Fc in RA-FLS in a dose-dependent manner, but not in OA-FLS. IL-12B p40 protein in RA-FLS was increased when stimulated with DcR3-Fc. Pre-treatment with anti-TL1A antibody suppressed the upregulation of IL-12B mRNA in RA-FLS stimulated with DcR3-Fc. DcR3 mRNA expression in RA-FLS was induced by IL-23, but not by IL-12. These results indicated that DcR3 may increase IL-12 or IL-23 by inducing IL-12B p40 expression via membrane-bound TL1A on RA-FLS and that IL-23 reciprocally induces DcR3 expression in RA-FLS. DcR3 and IL-23 may interact in a feedback loop that aggravates local inflammation in patients with RA. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is a common systemic autoimmune disorder of unknown etiology, which threatens public health. The regulatory role of tripartite motif-containing 22 (TRIM22) has been reported in multiple types of cancers and disease, but not in RA. The aim of the present study was therefore to elucidate the potential roles and underlying mechanisms of TRIM22 in fibroblast-like synoviocytes (FLSs) in RA. The Gene Expression Omnibus database was used to examine TRIM22 mRNA expression levels in synovial tissue samples of patients with RA and healthy controls. TRIM22 and forkhead box C1 (FOXC1) mRNA and protein expression levels in normal FLSs and RA-FLSs were assessed using reverse transcription-quantitative PCR (RT-qPCR) and western blotting, respectively. The Cell Counting Kit-8 assay was used to assess cell proliferation. Cell apoptosis was analyzed using flow cytometry. The migratory and invasive abilities of RA-FLSs were assessed using Transwell assays. Western blotting was used to analyze the protein expression levels of apoptosis-related factors, MMP2, MMP9 and NF-κB signaling pathway-related proteins. Inflammatory factors levels were assessed via ELISA and RT-qPCR. Furthermore, the JASPAR database, chromatin immunoprecipitation and the dual-luciferase reporter assays were used to determine the interaction between FOXC1 and the TRIM22 promoter. The results of the present study demonstrated that TRIM22 expression levels were significantly elevated in the synovial tissue samples of patients with RA and RA-FLSs. Moreover, FOXC1 was also significantly overexpressed in RA-FLSs. TRIM22 knockdown significantly reduced cell proliferation, migration, invasion and the inflammatory response, whereas cell apoptosis was significantly increased. Furthermore, the results demonstrated that FOXC1 may have positively mediated TRIM22 expression via binding to the TRIM22 promoter. Moreover, FOXC1 overexpression significantly reversed the outcome of TRIM22 knockdown on the proliferation, apoptosis, migration, invasion and inflammation of RA-FLSs. FOXC1 overexpression also significantly reversed the inactivation of the NF-κB signaling pathway caused by TRIM22 knockdown. In summary, the present study demonstrated that TRIM22 was potentially activated via FOXC1, which contributed to the progression of RA via the NF-κB signaling pathway. [ABSTRACT FROM AUTHOR]

The aim of the present study was to assess the effects of pro-protein convertase subtilisin/kexin type 6 (PCSK6), a proteinase implicated in the proteolytic activity of various precursor proteins and involved in the regulation of protein maturation, in fibroblast-like synoviocytes (FLS) of a rat model of collagen-induced arthritis (CIA). Cultured FLS from CIA models were subjected to small interfering RNA mediated PCSK6 knockdown, followed by assessment of the proliferation, invasive and migratory capacity, the secretion of inflammation factors and the cell cycle. Expression of genes associated with proliferation, invasion, migration and inflammation was detected by reverse transcription polymerase chain reaction. The results showed that PCSK6 knockdown significantly decreased the cell proliferation, invasion and migration of FLS from rats with CIA. ELISA showed an obvious decrease of tumor necrosis factor α and interleukin 1β secretion, and flow cytometric analysis revealed G0/G1 arrest of FLS following PCSK6 knockdown. Furthermore, a decrease in the mRNA levels of inflammation-associated chemokine CXCL9, angiogenesis-associated genes MMP-2, MMP-9 and NOSTRIN, hypoxia-associated gene HIF-1α, adhesion-associated gene MPZL2, proliferation-associated gene IGF-2 and citrullination-associated gene PADI4 was detected after PCSK6 knockdown. The results of the present study indicated that inhibition of PCSK6 may have a protective role against synovitis in rheumatoid arthritis. [ABSTRACT FROM AUTHOR]

Fibroblast-like synoviocytes (FLSs) have a pivotal role in the destruction of joints in rheumatoid arthritis (RA). Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a critical mediator in the inflammatory pathway and of the activity of osteoclasts. The aim of the present study was to investigate whether TRAF6 is involved in the progression of RA in mouse collagen-induced arthritis (CIA) and human RA FLSs in vitro. In vivo mouse models were transfected with TRAF6 small interfering (si)RNA (siTRAF6) and TRAF6 inhibition was achieved in FLSs using an anti-TRAF6 monoclonal antibody in vitro in order to assess the effects of TRAF6 inhibition on the migration and invasion of FLSs. Inhibition of TRAF6 using mouse specific siTRAF6 reduced the severity of arthritis and joint inflammation. Serum anti-collagen II antibodies, matrix metalloproteinase (MMP)-1, MMP-3 and MMP-9 were also inhibited in CIA mice by siTRAF6. The levels of MMPs produced by IL-1β-stimulated human RA-FLSs were reduced by anti-TRAF6 monoclonal antibody. TRAF6 blockade significantly suppressed the IL-1β-stimulated migration and invasion of human RA-FLSs. These results support a role for TRAF6 in the pathogenesis of RA, and suggest that the TRAF6 blockade may be a potential strategy in the management of RA. [ABSTRACT FROM AUTHOR]

Piperlonguminine (PL), a key compound from the Piper longum fruit, is known to exhibit anti-tumor and anti-inflammatory activities. However, little is known about its effects on collagen-induced arthritis (CIA). Fibroblast-like synoviocytes (FLS) have a pivotal role in the development of rheumatoid arthritis (RA). Myeloid-derived suppressor cells (MDSCs) are able to suppress T cell responses and have important roles in the regulation of autoimmune arthritis. The current study investigated whether PL alters the progression of RA. It was determined that PL reduces the arthritis score and histopathologic lesions in a mouse model of CIA. PL also reduces the expression levels of serum anti-collagen II antibodies (anti-CII), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-23 and IL-17 in CIA mice. In draining lymph nodes (DLNs), MDSCs were significantly expanded, however, the number of Th17 cells was markedly decreased by PL treatment. Additionally, PL reduced secretion of IL-1β, IL-23 and IL-17 by TNF-α-stimulated human RA FLS. PL significantly inhibited the migration and invasion of TNF-α-stimulated human RA FLS. These results indicate that PL may be a candidate therapeutic agent for the treatment of RA, via the expansion of MDSCs and the inhibition of the Th17 response and activation of FLS. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is a chronic autoimmune disease. Enhanced G protein coupled receptor kinase 2 (GRK2) translocation and prostaglandin E4 receptor (EP4) desensitization play a critical role in fibroblast-like synoviocytes (FLS) dysfunction. Paeoniflorin-6'O-benzene sulfonate (CP-25) exerts a protective effect in arthritis in the RA animal models. To demonstrate the role of Gβγ in EP4 desensitization and the mechanisms of CP-25 that protects FLS in RA, RA-FLS and adjuvant-induced arthritis (AA-FLS) were isolated from synovium of RA patients and AA rats. RA-FLS, AA-FLS and MH7A were treated with CP-25, Gβγ agonist and antagonist. The cell membrane expression of EP4, GRK2, and Gβγ were detected using western blot analysis. Co-immunoprecipitation (Co-IP) and immunofluorescence were adopted to detect the interactions of GRK2-Gβγ, GRK2-EP4, and EP4-Gβγ. Cell Counting Kit-8 and Transwell assay were used to analyze the proliferation and migration of the FLS. An increased membrane expression of GRK2 and Gβγ, enhanced GRK2-Gβγ interaction and decreased EP4 membrane expression in the RA synovial tissue were identified. In vitro, prostaglandin E2 (PGE2) enhanced the proliferation and migration of FLS. CP-25 exhibited an inhibition effect similar to Gβγ inhibitor, which downregulated GRK2-EP4 interaction, blocked the translocation of GRK2, and reversed EP4 desensitization, leading to the suppression of the proliferation and migration induced by PGE2. These results elucidated that an enhanced GRK2-Gβγ interaction was involved in the EP4 desensitization and dysfunction. CP-25 regulated EP4-GRK2-Gβγ signaling and re-sensitized EP4 by inhibiting GRK2-Gβγ interaction. The regulation of EP4-Gβγ-GRK2 signaling may be a novel potential therapeutic target in RA. [ABSTRACT FROM AUTHOR]

Interleukin-1β (IL-1β) has a significant role in osteoarthritis (OA). The purinergic receptor, P2X4, has previously been implicated in IL-1β secretion. The NLRP1 inflammasome mediates the production of IL-1β in inflammatory disorders. However, it is unknown whether P2X4 modulates NLRP1-mediated IL-1β release. In the present study, the correlation between the P2X4 receptor and NLRP1 was investigated in OA fibroblast-like synoviocytes (OAFLS). The expression of P2X4 and NLRP1 was detected in the OAFLS. The OAFLS were stimulated with P2X4 and the levels of IL-1β and matrix metalloproteinases (MMPs) were measured. To determine whether P2X4 is involved in NLRP1-triggered IL-1β production, NLRP1 small interfering RNA (siRNA) was used. In the OAFLS, a markedly higher expression of P2X4 and NLRP1 was revealed compared with that in the normal FLS. OAFLS stimulated by P2X4 resulted in concentration-dependent increases in the production of IL-1β, MMP-3 and MMP-9. Furthermore, P2X4-mediated IL-1β production was attenuated by the NLRP1 siRNA. The results of the present study indicate that P2X4 induced IL-1β, MMP-3 and MMP-9 production in the OAFLS. IL-1β induced by P2X4 is mediated via NLRP1. P2X4/NLRP1 may be important in the pathogenesis of OA and may represent a novel therapeutic target. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is a chronic inflammatory disease that mainly targets the synovial membrane, thus causing stiffness, deformity and dysfunction of joints. To date, no effective anti-inflammatory treatments are available for RA. Piceatannol (PIC) is a natural derivative of resveratrol, which has been reported to attenuate the inflammatory response. To evaluate the effect of PIC on RA and to determine the underlying molecular target of PIC, both in vitro and in vivo experiments were performed in the present study. A CIA rat model was established to evaluate the therapeutic effects of PIC. TNF-α, IL-1β and IL-6 levels in blood were measured by ELISA. Western blotting, immunofluorescence analysis and reverse transcription-quantitative PCR (RT-qPCR) were used to analyze the expression levels of protein and mRNA. In vitro, RA-fibroblast-like synoviocytes (FLSs) were pretreated with PIC and subsequently stimulated with TNF-α. The results revealed that PIC significantly upregulated the expression levels of proapoptotic proteins such as Bax and cleaved caspase-3. PIC also significantly reduced the production of proinflammatory cytokines, including PGE2, IL-6 and IL-1β, and significantly downregulated the expression of cyclooxygenase-2 at both the mRNA and protein expression levels. Furthermore, PIC downregulated the expression of MMP-3 and MMP-13, which have been found to be highly expressed in the synovium of patients with RA. Mechanistically, PIC was capable of significantly downregulating the expression levels of proteins involved in the NF-κB and MAPK signaling pathways. The results of the in vivo experiments using a rat collagen-induced arthritis model demonstrated that PIC decreased the arthritis score and exerted beneficial effects in cartilage and significantly reduced the expression of MMP-13. In conclusion, the findings of the present study revealed that PIC could suppress the inflammatory response, promote apoptosis, and exert a significant regulatory effect on the NF-κB and MAPK signaling pathways in RA-FLSs. Therefore, PIC may represent a potential drug for the future treatment of RA. [ABSTRACT FROM AUTHOR]

Fibroblast-like synoviocytes (FLSs) have functions in the pathogenesis of rheumatoid arthritis (RA) through the onset of synovitis, the growth of pannus and the destruction of cartilage and bone. The significant increase in the proliferation, migration and invasion of FLSs induces the onset and advancement of RA. To date, the exact function of corepressor element-1 silencing transcription factor (CoREST) in RA remains unclear, but its expression has been determined in RA synovial tissues. In this study, the effects of CoREST were investigated in a TNF-α-induced FLS activation model. Following the silencing of CoREST expression with small interfering (si)RNA, the viability and migration of FLSs were evaluated. Furthermore, the possible molecular mechanisms were explored by detecting the expression of key factors, including matrix metalloproteinases (MMPs), lysine-specific histone demethylase 1 (LSD1) and associated cytokines, via reverse transcription-quantitative PCR and western blotting. CoREST expression increased not only in the RA synovial tissues, but also in the TNF-α-induced FLS activation model. Following the silencing of CoREST in the FLSs treated with TNF-α, cell viability was inhibited, and the migratory capacity of FLSs was suppressed, which was accompanied by the reduced expression of MMP-3 and MMP-9. The expression of LSD1 was also downregulated. There was a notable decrease in the synthesis of interferon-γ and interleukin (IL)-17, while IL-10 expression was increased. The knockdown of CoREST inhibited the viability and migration of FLSs stimulated with TNF-α. Thus, the suppression of CoREST may have crucial roles in the occurrence and development of RA. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is an autoimmune disease that causes chronic inflammation in synovial tissues. Hyperplasia of synovial tissues leads to the formation of pannus that invades the joint cartilage and bone, resulting in joint destruction. Fas ligand (FasL), which is a member of the tumor necrosis factor superfamily, contributes to the pathogenesis of autoimmune diseases, including RA. The current study attempted to identify genes whose expressions in rheumatoid fibroblast-like synoviocytes (RA-FLS) were regulated by FasL, using cDNA microarray. A total of four individual lines of primary cultured RA-FLS were incubated either with recombinant human FasL protein or PBS as an unstimulated control for 12 h. Gene expression was detected using a microarray assay. The results revealed the expression profiles of genes in RA-FLS regulated by Fas and investigated the functions of the genes that were regulated. Among the genes in this profile, the mRNA expression changes of the following genes were indicated to be of note using RT-qPCR: Dual specificity phosphatase 6, epiregulin, interleukin 11, angiopoietin-like 7, protein inhibitor of activated STAT 2 and growth differentiation factor 5. These genes may affect the pathogenesis of RA by affecting apoptosis, proliferation, cytokine production, cytokine-induced inflammation, intracellular signaling, angiogenesis, bone destruction and chondrogenesis. To the best of our knowledge, the current study is the first study to reveal the expression profile of genes in RA-FLS regulated by FasL. The data demonstrated that FasL may regulate the expression of a number of key molecules in RA-FLS, thus affecting RA pathogenesis. Further studies of the genes detected may improve the understanding of RA pathogenesis and provide novel treatment targets for RA. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by joint disorders. Long non-coding RNA zinc finger antisense 1 (ZFAS1) is aberrantly expressed in numerous human diseases, including RA. The present study aimed to investigate the functions and underlying mechanisms of ZFAS1 in RA. Reverse transcription-quantitative PCR was performed to determine the expression levels of ZFAS1, microRNA (miR)-3926 and follistatin-like protein 1 (FSTL1). MTT assay, flow cytometric analysis and Transwell assay were performed to examine the proliferation, apoptosis, migration and invasion of fibroblast-like synoviocytes (FLSs), respectively. Western blotting was employed to measure the protein expression levels of cleaved caspase-3, interleukin (IL)-6, IL-1β, tumor necrosis factor-α and FSTL1. Dual-luciferase reporter assay was performed to verify the interaction between miR-3926 and ZFAS1 or FSTL1. The results demonstrated that ZFAS1 and FSTL1 were upregulated, and miR-3926 was downregulated in RA synovial tissues and RA-FLSs. ZFAS1 knockdown suppressed cell proliferation, migration, invasion and inflammatory cytokine production, and induced apoptosis in RA-FLSs. ZFAS1 acted as a sponge for miR-3926, and ZFAS1 overexpression abolished the impact of miR-3926 on the development of RA-FLSs. FSTL1 was a direct target of miR-3926, and the effect of FSTL1 knockdown on the progression of RA-FLSs was rescued by miR-3926 inhibition. Furthermore, ZFAS1 regulated FSTL1 expression levels via sponging miR-3926 in RA-FLSs. In conclusion, ZFAS1 knockdown inhibited RA-FLS proliferation, migration, invasion and inflammatory cytokine production, and induced apoptosis in RA via the miR-3926/FSTL1 axis. [ABSTRACT FROM AUTHOR]

Fibroblast-like synoviocytes (FLS) in the synovial lining play a key role in the pathological process of rheumatoid arthritis (RA), which produce pro-inflammatory mediators to perpetuate inflammation and proteases to contribute to cartilage destruction. Ginkgolide J (GJ) is a subclass of ginkgolides (GGs) that exhibits anti-inflammatory activity. In the present study, the protective effect of GJ on lipopolysaccharide (LPS)-treated human synovial cells SW982 and its related mechanisms were investigated using various methods, including ELISA, Griess assay, western blotting, immunofluorescence analysis and p38 kinase activity assay. The results revealed that GJ pretreatment significantly attenuated LPS-induced excess production of pro-inflammatory mediators in SW982 cells via suppression of tumor necrosis factor-α/interleukin (IL)-1β/IL-18/NF-κB/NLR family pyrin domain containing 3, prostaglandin E2/cyclooxygenase-2 and inducible nitric oxide synthase/nitric oxide signaling. Mechanistic studies revealed that p38 activation contributed to the LPS-induced inflammatory response, and GJ pretreatment dose-dependently attenuated p38 activation, indicating that the suppressive effect of GJ was achieved by targeting p38 signaling. These findings may contribute to the prevention and treatment of RA. [ABSTRACT FROM AUTHOR]