Rheumatoid Arthritis Synovial Fibroblasts Research Articles

ARID5B is a negative modulator of IL-6 production in rheumatoid arthritis synovial fibroblasts.

Recent single-cell RNA-sequencing analysis of rheumatoid arthritis (RA) synovial tissues revealed the heterogeneity of RA synovial fibroblasts (SFs) with distinct functions such as high IL-6 production. The molecular mechanisms responsible for high IL-6 production will become a promising drug target of RASFs to treat RA. In this study, we performed siRNA screening of 65 transcription factors (TFs) differentially expressed among RASF subsets to identify TFs involved in IL-6 production. The siRNA screening identified 7 TFs including ARID5B, a RA risk gene, that affected IL-6 production. Both long and short isoforms of ARID5B were expressed and negatively regulated by TNF-α in RASFs. The siRNA knockdown and lentiviral overexpression of long and short isoforms of ARID5B revealed that the long isoform suppressed IL-6 production stimulated with TNF-α. eQTL analysis using 58 SFs demonstrated that RA risk allele, rs10821944, in intron 4 of the ARID5B gene had a trend of eQTL effects to the expression of long isoform of ARID5B in SFs treated with TNF-α. ARID5B was found to be a negative modulator of IL-6 production in RASFs. The RA risk allele of ARID5B intron may cause high IL-6 production, suggesting that ARID5B will become a promising drug target to treat RA.

Regulation of cytokine and chemokine expression by histone lysine methyltransferase MLL1 in rheumatoid arthritis synovial fibroblasts

Histone lysine methylation is thought to play a role in the pathogenesis of rheumatoid arthritis (RA). We previously reported aberrant expression of the gene encoding mixed-lineage leukemia 1 (MLL1), which catalyzes methylation of histone H3 lysine 4 (H3K4), in RA synovial fibroblasts (SFs). The aim of this study was to elucidate the involvement of MLL1 in the activated phenotype of RASFs. SFs were isolated from synovial tissues obtained from patients with RA or osteoarthritis (OA) during total knee joint replacement. MLL1 mRNA and protein levels were determined after stimulation with tumor necrosis factor α (TNFα). We also examined changes in trimethylation of H3K4 (H3K4me3) levels in the promoters of RA-associated genes (matrix-degrading enzymes, cytokines, and chemokines) and the mRNA levels upon small interfering RNA-mediated depletion of MLL1 in RASFs. We then determined the levels of H3K4me3 and mRNAs following treatment with the WD repeat domain 5 (WDR5)/MLL1 inhibitor MM-102. H3K4me3 levels in the gene promoters were also compared between RASFs and OASFs. After TNFα stimulation, MLL1 mRNA and protein levels were higher in RASFs than OASFs. Silencing of MLL1 significantly reduced H3K4me3 levels in the promoters of several cytokine (interleukin-6 [IL-6], IL-15) and chemokine (C–C motif chemokine ligand 2 [CCL2], CCL5, C-X-C motif chemokine ligand 9 [CXCL9], CXCL10, CXCL11, and C-X3-C motif chemokine ligand 1 [CX3CL1]) genes in RASFs. Correspondingly, the mRNA levels of these genes were significantly decreased. MM-102 significantly reduced the promoter H3K4me3 and mRNA levels of the CCL5, CXCL9, CXCL10, and CXCL11 genes in RASFs. In addition, H3K4me3 levels in the promoters of the IL-6, IL-15, CCL2, CCL5, CXCL9, CXCL10, CXCL11, and CX3CL1 genes were significantly higher in RASFs than OASFs. Our findings suggest that MLL1 regulates the expression of particular cytokines and chemokines in RASFs and is associated with the pathogenesis of RA. These results could lead to new therapies for RA.

Endogenous Melanin and Hydrogen-Based Specific Activated Theranostics Nanoagents: A Novel Multi-Treatment Paradigm for Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disorder characterized by excessive proliferation of rheumatoid arthritis synovial fibroblasts (RASFs) and accumulation of inflammatory cytokines. Exploring the suppression of RASFs and modulation of the RA microenvironment is considered a comprehensive strategy for RA. In this work, specifically activated nanoagents (MAHI NGs) based on the hypoxic and weakly acidic RA microenvironment are developed to achieve a second near-infrared fluorescence (NIR-II FL)/photoacoustic (PA) dual-model imaging-guided multi-treatment. Due to optimal size, the MAHI NGs passively accumulate in the diseased joint region and undergo rapid responsive degradation, precisely releasing functionalized components: endogenous melanin-nanoparticles (MNPs), hydrogen gas (H2), and indocyanine green (ICG). The released MNPs play a crucial role in ablating RASFs within the RA microenvironment through photothermal therapy (PTT) guided by accurate PA imaging. However, the regional hyperthermia generated by PTT may exacerbate reactive oxygen species (ROS) production and inflammatory response following cell lysis. Remarkably, under the acidic microenvironment, the controlled release of H2 exhibits precise synergistic antioxidant and anti-inflammatory effects with MNPs. Moreover, the ICG, the second near-infrared dye currently approved for clinical use, possesses excellent NIR-II FL imaging properties that facilitate the diagnosis of deep tissue diseases and provide the right time-point for PTT.

Targeting TNF-α-induced expression of TTR and RAGE in rheumatoid arthritis: Apigenin's mediated therapeutic approach

BackgroundRheumatoid arthritis (RA) is a chronic inflammatory disease induced by TNF-α, which increases fibroblast-like synoviocytes inflammation, resulting in cartilage destruction. The current work sought to comprehend the pathophysiological importance of TNF-α stimulation on differential protein expression and their regulation by apigenin using in-vitro and in-vivo models of RA. MethodsThe human RA synovial fibroblast cells were stimulated with or without TNF-α (10 ng/ml) and treated with 40 μM apigenin. In-silico, in-vitro and in-vivo studies were performed to confirm the pathophysiological significance of apigenin on pro-inflammatory cytokines and on differential expression of TTR and RAGE proteins. ResultsTNF-α induced inflammatory response in synoviocytes revealed higher levels of IL-6, IL-1β, and TNF-α cytokines and upregulated differential expression of TTR and RAGE. In-silico results demonstrated that apigenin has a binding affinity towards TNF-α, indicating its potential effect in the inflammatory process. Both in-vitro and in-vivo results obtained by Western Blot analysis suggested that apigenin reduced the level of p65 (p = 0.005), TTR (p = 0.002), and RAGE (p = 0.020). ConclusionThe findings of this study suggested that TNF-α promotes the differential expression of pro-inflammatory cytokines, TTR, and RAGE via NF-kB pathways activation. Anti-inflammatory effect of apigenin impedes TNF-α mediated dysregulation or expression associated with RA pathogenesis.

Synovial fibroblast gene expression is associated with sensory nerve growth and pain in rheumatoid arthritis.

It has been presumed that rheumatoid arthritis (RA) joint pain is related to inflammation in the synovium; however, recent studies reveal that pain scores in patients do not correlate with synovial inflammation. We developed a machine-learning approach (graph-based gene expression module identification or GbGMI) to identify an 815-gene expression module associated with pain in synovial biopsy samples from patients with established RA who had limited synovial inflammation at arthroplasty. We then validated this finding in an independent cohort of synovial biopsy samples from patients who had early untreated RA with little inflammation. Single-cell RNA sequencing analyses indicated that most of these 815 genes were most robustly expressed by lining layer synovial fibroblasts. Receptor-ligand interaction analysis predicted cross-talk between human lining layer fibroblasts and human dorsal root ganglion neurons expressing calcitonin gene-related peptide (CGRP+). Both RA synovial fibroblast culture supernatant and netrin-4, which is abundantly expressed by lining fibroblasts and was within the GbGMI-identified pain-associated gene module, increased the branching of pain-sensitive murine CGRP+ dorsal root ganglion neurons in vitro. Imaging of solvent-cleared synovial tissue with little inflammation from humans with RA revealed CGRP+ pain-sensing neurons encasing blood vessels growing into synovial hypertrophic papilla. Together, these findings support a model whereby synovial lining fibroblasts express genes associated with pain that enhance the growth of pain-sensing neurons into regions of synovial hypertrophy in RA.

New biflavonoids isolated from Xylia kerrii leaves extract with selective cytotoxicity against MH7A human rheumatoid arthritis synovial fibroblast cells.

Three new biflavonoids (1-3) and two known flavonoids (4, 5) were isolated from Xylia kerrii collected in Thailand. Compounds 1-5 showed selective cytotoxicity against the rheumatoid fibroblast-like synovial MH7A cell line, and these compounds showed weak cytotoxicity against the human lung synovial fibroblast WI-38 VA13 sub 2 RA cell line. Notably, compound 1 was highly selective toward MH7A cells with an IC50 value of 6.9μM, whereas the IC50 value for WI-38 VA13 sub 2 RA cells was > 100μM. The western blotting analysis of MH7A cells treated with compound 1 showed increased CDKN2A /p16INK4A and caspase-8 levels.

Biomimetic nanoparticles for effective Celastrol delivery to targeted treatment of rheumatoid arthritis through the ROS-NF-κB inflammasome axis

Previous study has indicated that Celastrol (Cel) has various physiological and pharmacological effects, including antibacterial, antioxidant, pro-apoptotic, anticancer and anti-rheumatoid arthritis (RA) effects. However, low water solubility, low oral bioavailability, narrow treatment window, and high incidence of systemic adverse reactions still limit the further clinical application of Cel. Here, aiming at effectively overcome those shortcomings of Cel to boost its beneficial effects for treating RA, we developed the leukosome (LEUKO) coated biomimetic nanoparticles (NPs) for the targeted delivery of Cel to arthritis injury area in RA. LEUKO were synthesized using membrane proteins purified from activated J774 macrophage. LEUKO and Cel-loaded LEUKO (Cel@LEUKO) were characterized using dynamic light scattering and transmission electron microscopy. Our results demonstrated that Cel@LEUKO can inhibit the inflammatory response of lipopolysaccharide (LPS) induced mouse monocyte macrophage leukemia cells (RAW264.7 cells) and human rheumatoid arthritis synovial fibroblasts (MH7A) cells through the inhibition of reactive oxygen species (ROS)-NF-κB pathway. In addition, research has shown that LEUKO effectively targets and transports Cel to the inflammatory site of RA, increased drug concentration in affected areas, reduced systemic toxicity of Cel, and reduced clinical symptoms, inflammatory infiltration, bone erosion, and serum inflammatory factors in collagen-induced arthritis (CIA) rats.

FGF23 facilitates IL-1β synthesis in rheumatoid arthritis through activating PI3K, Akt, and NF-κB pathways.

Rheumatoid arthritis (RA) is a well-known autoimmune disorder related with joint pain, joint swelling, cartilage and bone degradation as well as deformity. Fibroblast growth factor 23 (FGF23) is an endocrine factor of the FGF family primarily produced by osteocytes and osteoblasts, involves an essential effect in pathogenesis of RA. IL-1β is a vital proinflammatory factor in the development of RA. However, the role of FGF23 on IL-1β synthesis in RA has not been fully explored. Our analysis of database revealed higher levels of FGF23 and IL-1β in RA samples compared with healthy controls. High-throughput screening demonstrated that IL-1β is a potential candidate factor after FGF23 treatment in RA synovial fibroblasts (RASFs). FGF23 concentration dependently promotes IL-1β synthesis in RASFs. FGF23 enhances IL-1β expression by activating the PI3K, Akt, and NF-κB pathways. Our findings support the notion that FGF23 is a promising target in the remedy of RA.

NGF facilitates ICAM-1-dependent monocyte adhesion and M1 macrophage polarization in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disorder in which monocytes adhering to synovial tissue differentiate into the pro-inflammatory M1 macrophage phenotype. Nerve growth factors (NGF) referred to as neurotrophins have been associated with inflammatory events; however, researchers have yet to elucidate the role of NGF in RA. Our examination of clinical tissue samples and analysis of data sourced from the Gene Expression Omnibus dataset unveiled elevated expression levels of M1 macrophage markers in human RA synovial tissue samples compared to normal tissue, with no such distinction observed for M2 markers. Furthermore, immunofluorescence data depicted increased expression levels of NGF and M1 macrophages in RA mice in contrast to normal mice. It appears that NGF stimulation facilitates macrophage polarization from the M0 to the M1 phenotype. It also appears that NGF promotes ICAM-1 production in human RA synovial fibroblasts, which enhances monocyte adhesion through the TrkA, MEK/ERK, and AP-1 signaling cascades. Our findings indicate NGF/TrkA axis as a novel target for the treatment of RA.

The long non-coding RNA HOTAIR contributes to joint-specific gene expression in rheumatoid arthritis

Although patients with rheumatoid arthritis (RA) typically exhibit symmetrical joint involvement, some patients develop alternative disease patterns in response to treatment, suggesting that different molecular mechanism may underlie disease progression depending on joint location. Here, we identify joint-specific changes in RA synovium and synovial fibroblasts (SF) between knee and hand joints. We show that the long non-coding RNA HOTAIR, which is only expressed in knee SF, regulates more than 50% of this site-specific gene expression in SF. HOTAIR is downregulated after stimulation with pro-inflammatory cytokines and is expressed at lower levels in knee samples from patients with RA, compared with osteoarthritis. Knockdown of HOTAIR in knee SF increases PI-Akt signalling and IL-6 production, but reduces Wnt signalling. Silencing HOTAIR inhibits the migratory function of SF, decreases SF-mediated osteoclastogenesis, and increases the recruitment of B cells by SF. We propose that HOTAIR is an important epigenetic factor in joint-specific gene expression in RA.

Efficient delivery of the lncRNA LEF1-AS1 through the antibody LAIR-1 (CD305)-modified Zn-Adenine targets articular inflammation to enhance the treatment of rheumatoid arthritis

BackgroundsRheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by synovial hyperplasia. Maintaining a balance between the proliferation and apoptosis of rheumatoid arthritis synovial fibroblasts (RASFs) is crucial for preventing the erosion of bone and cartilage and, ultimately, mitigating the progression of RA. We found that the lncRNA LEF1-AS1 was expressed at low levels in the RASFs and inhibited their abnormal proliferation by targeting PIK3R2 protein and regulating the PI3K/AKT signal pathway through its interaction with miR-30-5p. In this study, we fabricated a nano-drug delivery system for LEF1-AS1 using Zn-Adenine nanoparticles (NPs) as a novel therapeutic strategy against RA.MethodsThe expression levels of LEF1-AS1, miR-30-5p, PIK3R2, p-PI3K, and p-AKT were detected in the primary RASFs and a human fibroblast-like synovial cell line (HFLS). Zn-Adenine nanoparticles (NPs) were functionalized with anti-CD305 antibody to construct (Zn-Adenine)@Ab. These NPs were then loaded with LEF1-AS1 to form (Zn-Adenine)@Ab@lncRNA LEF1-AS1. Finally, the (Zn-Adenine)@Ab@lncRNA LEF1-AS1 NPs were locally injected into a rat model with collagen-induced arthritis (CIA). The arthritic injuries in each group were evaluated by HE staining and other methods.ResultsLEF1-AS1 was expressed at low levels in the primary RASFs. High expression levels of LEF1-AS1 were detected in the HFLS cells, which corresponded to a significant downregulation of miR-30-5p. In addition, the expression level of PIK3R2 was significantly increased, and that of p-PI3K and p-AKT were significantly downregulated in these cells. The (Zn-Adenine)@Ab@lncRNA LEF1-AS1 NPs significantly inhibited the proliferation of RASFs and decreased the production of inflammatory cytokines (IL-1β, IL-6, TNF-α). Intra-articular injection (IAI) of (Zn-Adenine)@Ab@lncRNA LEF1-AS1 NPs significantly alleviated cartilage destruction and joint injury in the CIA-modeled rats.ConclusionsLEF1-AS1 interacts with miR-30-5p to inhibit the abnormal proliferation of RASFs by regulating the PI3K/AKT signal pathway. The (Zn-Adenine)@Ab NPs achieved targeted delivery of the loaded LEF1-AS1 into the RASFs, which improved the cellular internalization rate and therapeutic effects. Thus, LEF1-AS1 is a potential target for the treatment of RA.

BRD3 Regulates the Inflammatory and Stress Response in Rheumatoid Arthritis Synovial Fibroblasts.

Individual functions of members of the bromodomain (BRD) and extra-terminal (BET) protein family underlying the anti-inflammatory effects of BET inhibitors in rheumatoid arthritis (RA) are incompletely understood. Here, we aimed to analyze the regulatory functions of BRD3, an understudied member of the BET protein family, in RA synovial fibroblasts (FLS). BRD3 was silenced in FLS prior to stimulation with TNF. Alternatively, FLS were treated with I-BET. Transcriptomes were analyzed by RNA sequencing (RNAseq), followed by pathway enrichment analysis. We confirmed results for selective target genes by real-time PCR, ELISA, and Western blotting. BRD3 regulates the expression of several cytokines and chemokines in FLS, and positively correlates with inflammatory scores in the RA synovium. In addition, RNAseq pointed to a profound role of BRD3 in regulating FLS proliferation, metabolic adaption, and response to stress, including oxidative stress, and autophagy. BRD3 acts as an upstream regulatory factor that integrates the response to inflammatory stimuli and stress conditions in FLS and executes many functions of BET proteins that have previously been identified using pan-BET inhibitors.

A multifunctional supramolecular hydrogel that rapidly binds TNF-α for efficient reduction of synovial inflammation and cartilage destruction in rheumatoid arthritis

TNF-α is a key inflammatory cytokine in the progression of rheumatoid arthritis (RA), which is the most common inflammatory joint disorder and is characterized by persistent synovitis and cartilage destruction. However, systematic use of TNF-α inhibitors may cause undesirable side effects and fail to repair damaged cartilage. Here we design a self-assembling Nap-DFDFDEGPIRRSDS (termed FP) peptide that can form injectable supramolecular hydrogels and inhibit TNF-α within 15 min. Following integration of metformin-loaded hollow copper sulfide (CuS/MET, termed CM) nanoparticles (NPs) into these FPCM hydrogels, invasion by RA synovial fibroblasts and polarization of pro-inflammatory M1 type macrophages is reduced by inhibiting TNF-α and releasing MET. In addition, CuS NPs are chondroinductive, allowing the hydrogels to provide a suitable microenvironment for cartilage regeneration. More importantly, intraarticular injection of FPCM hydrogels significantly alleviates synovial inflammation and promotes cartilage repair in an adjuvant-induced arthritis mouse model. The therapeutic mechanism was shown to involve inhibition of the PI3K/AKT signaling pathway. Our approach synergistically inhibits inflammation and promotes tissue repair, thus holding promise for treatment of many kinds of inflammatory arthritis where TNF-α is overexpressed.

Circ_0000396 suppresses the proliferation and inflammation of rheumatoid arthritis synovial fibroblasts by targeting miR-574-5p/RSPO1 axis

BackgroundCircular RNAs (circRNAs) are important regulators on the onset and progression of rheumatoid arthritis (RA). Our purpose is to explore the role and underpin mechanism of circ_0000396 in RA progression.MethodsRA patients (n = 39) and healthy volunteers (n = 33) were recruited from the Affiliated Hospital of Shaanxi University of Chinese Medicine for the present work. Circ_0000396, microRNA-574-5p (miR-574-5p) and R-spondin 1 (RSPO1) RNA levels were analyzed by reverse transcription-quantitative polymerase chain reaction. Cell proliferation was analyzed by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay, and 5-ethynyl-2′-deoxyuridine (EDU) assay. Cell apoptosis was assessed by flow cytometry. Protein expression levels of proliferating cell nuclear antigen (PCNA), Cyclin D1, Cyclin E1, BCL2-associated × protein (Bax), B-cell lymphoma-2 (Bcl2), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and RSPO1 were detected by western blot assay. Enzyme-linked immunosorbent assay (ELISA) was conducted to analyze the secretion of pro-inflammatory cytokines including IL-1β and TNF-α. The interaction between miR-574-5p and circ_0000396 or RSPO1 was confirmed by dual-luciferase reporter assay and RNA-pull down assay.ResultsCirc_0000396 expression was notably down-regulated in RA patients compared with healthy controls. Circ_0000396 overexpression suppressed the proliferation and inflammatory response and triggered the apoptosis of RA synovial fibroblasts (RASFs), accompanied by decreases in PCNA, Cyclin D1, Cyclin E1, Bcl2, IL-1β and TNF-α protein expression and an increase in Bax protein expression. Circ_0000396 acted as a molecular sponge for miR-574-5p, and circ_0000396 overexpression-mediated protective effects on RASFs dysfunction were largely reversed by the introduction of miR-574-5p mimics. miR-574-5p interacted with the 3’ untranslated region (3’UTR) of RSPO1, and miR-574-5p negatively regulated RSPO1 expression in RASFs. Circ_0000396 could up-regulate the expression of RSPO1 by sponging miR-574-5p in RASFs. RSPO1 interference largely overturned circ_0000396 overexpression-mediated effects in RASFs.ConclusionCirc_0000396 restrained the proliferation and inflammation and induced the apoptosis of RASFs by mediating miR-574-5p/RSPO1 axis, which provided novel potential targets for RA treatment.

Arecoline hydrobromide suppresses PI3K/AKT pathway in rheumatoid arthritis synovial fibroblasts and relieves collagen-induced arthritis in mice

ObjectiveThis study investigated the effectiveness of arecoline hydrobromide (AH) on the functions of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs) and collagen-induced arthritis (CIA) mice. MethodsImmunofluorescence was used to identify RA-FLSs. Cell Counting Kit-8 (CCK-8) was used to determine the viability of RA-FLSs and the half maximal inhibitory concentration (IC50) of AH. The 5-ethynyl-2′-deoxyuridine (EdU) assay was used to detect DNA replication in RA-FLSs. Cell cycle and apoptosis were examined by flow cytometry. Migration and invasion, as well as wound healing assays, were employed to determine cell migration and invasion ability. Proteins and mRNA expression levels were investigated using Western blot, quantitative real-time PCR (RT-qPCR), and immunofluorescence. The CIA mice model was used to assess the effect of AH in vivo. RNA-sequencing (RNA-seq) was used to find the potential signaling pathways of AH against RA, and Western blot was used to verify the key signaling pathway of AH on RA-FLSs. Network pharmacology and molecular docking were used to predict drug targets. ResultsAH inhibited the proliferation and DNA replication of RA-FLSs, promoted cell cycle arrest by reducing the levels of cyclin-dependent kinase 1 (CDK1), cyclin A2, and cyclin B1, promoted apoptosis by suppressing B-cell lymphoma-2 (Bcl-2) expression, and suppressed migration and invasion by inhibiting vimentin expression in RA-FLSs. AH was also effective in relieving arthritis in vivo. RNA sequencing analyses suggested that AH inhibited the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway in RA-FLSs, which was also confirmed in Western blot analysis. Furthermore, network pharmacology and molecular docking suggested that F2, MAPK14, SRC, AKT1, and CTSK might be the direct targets of AH. ConclusionAH can modulate the pathological process of RA-FLSs by blocking the PI3K/AKT pathway and relieve CIA in mice, making it a potential new small molecule candidate.

CXCL1 enhances COX-II expression in rheumatoid arthritis synovial fibroblasts by CXCR2, PLC, PKC, and NF-κB signal pathway

Rheumatoid arthritis (RA) is the most common autoimmune disease, affecting the joints of the hands and feet. Several chemokines and their receptors are crucial in RA pathogenesis through immune cell recruitment. C-X-C Motif Chemokine Ligand 1 (CXCL1), a chemokine for the recruitment of various immune cells, can be upregulated in patients with RA. However, the discussion on the role of CXCL1 in RA pathogenesis is insufficient. Here, we found that CXCL1 promoted cyclooxygenase-2 (COX-II) expression in a dose- and time-dependent manner in rheumatoid arthritis synovial fibroblasts (RASFs). CXCL1 overexpression in RASFs led to a significant increase in COX-II expression, while the transfection of RASFs with the shRNA plasmid resulted in a noticeable decrease in COX-II expression. Next, we delineated the molecular mechanism underlying CXCL1-promoted COX-II expression and noted that CXC chemokine receptor 2 (CXCR2), phospholipase C (PLC), and protein kinase C (PKC) signal transduction were responsible for COX-II expression after CXCL1 incubation for RASFs. Finally, we confirmed the transcriptional activation of nuclear factor κB (NF-κB) in RASFs after incubation with CXCL1. In conclusion, the current study provided a novel insight into the role of CXCL1 in RA pathogenesis.

Regulatory effect of zinc finger protein A20 on rheumatoid arthritis through NLRP3/Caspase-1 signaling axis mediating pyroptosis of HFLS- RA cells.

As a chronic inflammatory autoimmune disease, rheumatoid arthritis (RA) causes significant destruction to joints and cartilage. So far, from RA patients, the synovial cells and subsynovial tissues reflected the positive expression of IL-18, IL-1β, Caspase-1 and NLRP3, with the synovial tissues of those patients also expressing the zinc finger protein A20 at a significantly lower level compared with osteoarthritis (OA) ones. Thus, the inhibition of the NLRP3/caspase-1 signaling pathway can effectively down-regulate the expression of IL-1β, but when NLRP3 inflammasomes are activated, they can also shear GSDMD and induce pyroptosis. These suggest that the Gasdermin family of proteins, downstream of the NLRP3 inflammasome, could be involved in pyroptosis. Previous studies have shown that A20 contributes largely as an anti-inflammatory factor in many inflammatory diseases, but it remains unclear whether zinc finger protein A20, as an inhibitor of NLRP3 inflammasomes, can play a protective role against RA by inhibiting NLRP3 inflammasome-mediated pyroptosis. Therefore, this study aimed to verify the effects of zinc finger protein A20 on NLRP3/ Caspase-1-mediated pyroptosis in rheumatoid arthritis synovial fibroblasts (HFLS-RA) cells through cell experiments and clinical bidirectional verification, aim to understand the regulatory mechanism of A20 on RA. The results of clinical trials showed that NLRP3, Caspase-1, IL-1β and IL-18 were positively scattered in RA synovial cells and subsynovial tissue. The expression level of the zinc finger protein A20 in RA synovial tissues was significantly lower than that in OA synovial tissue and was negative, while zinc finger protein A20 was strongly positive in OA synovial tissue. In addition, HFLS-RA cells with siRNA-interfering zinc finger protein A20 were constructed at the cellular level, with the results also confirming that zinc finger protein A20 can play a protective role against RA by inhibiting NLRP3 inflammasome-mediated pyroptosis. In conclusion, this study is of great significance for understanding the role of the NLRP3-caspase-1-IL-1β/ pyroptosis signaling pathway in the occurrence and development of RA. It is expected that the results will provide a theoretical basis for the immune regulation of innate immunity in the occurrence and development of RA, while providing a new therapeutic target for the clinical treatment of RA.

Knockdown of BUB1 inhibits tumor necrosis factor-α-induced proliferation and migration of rheumatoid arthritis synovial fibroblasts by regulating PI3K/Akt pathway.

Rheumatoid arthritis (RA) is a common disease with joint cartilage destruction. BUB1 Mitotic Checkpoint Serine/Threonine Kinase (BUB1) is abnormally expressed in synovial tissues of RA patients, but its effect on RA remains unclear. In this study, we explored the role of BUB1 in RA. An RA cell model was constructed by treating MH7A cells with tumor necrosis factor-α (TNF-α). The levels of BUB1, GAPDH, phosphorylated phosphatidylinositol 3 kinase (p-PI3K)/PI3K, and phosphorylated serine/threonine kinase (p-Akt)/Akt in MH7A cells were examined by Western blot. The MH7A cell proliferation was examined by colony formation assay. Wound healing assay and transwell assay were carried out to detect MH7A cell migration and invasion. The mRNA levels of proinflammatory cytokines were assessed by quantitative reverse transcription polymerase chain reaction. The results showed that knockdown BUB1 inhibited TNF-α-induced MH7A cell proliferation, migration, and invasion. Silencing BUB1 repressed the PI3K/Akt pathway in TNF-α-induced MH7A cells. We also found that the TNF-α-induced MH7A cell proliferation, migration, and invasion were repressed by si-BUB1 transfection, whereas these effects were attenuated by 740Y-P (an activator of the PI3K pathway) co-treatment. Knockdown of BUB1 reduced the expression of the proinflammatory cytokines. Knockdown BUB1 repressed TNF-α-induced MH7A cell proliferation, migration and invasion through the PI3K/Akt pathway.

MRNA vaccine against fibroblast activation protein ameliorates murine models of inflammatory arthritis.

Synovial fibroblasts in patients with rheumatoid arthritis (RA) contribute substantially to the perpetuation of synovitis and invasion to cartilage and bone, and are potential therapeutic targets. Fibroblast activation protein (FAP) is highly expressed by RA synovial fibroblasts and the expression is relatively specific. We tested whether FAP can serve as a molecular target to modulate synovial fibroblasts for therapy in experimental arthritis. mRNA encoding consensus FAP (cFAP) was encapsulated in lipid nanoparticles (LNP) and was injected intramuscularly as vaccine prior to induction of collagen-induced arthritis (CIA) and collagen antibody induced arthritis (CAIA) in mice. Development of CIA and CAIA was assessed clinically and by histology. cFAP mRNA-LNP vaccine provoked immune response to cFAP and mouse FAP (mFAP); prevented onset of CIA in 40% of mice and significantly reduced the severity of arthritis. In CAIA, cFAP mRNA-LNP did not prevent onset of arthritis but significantly reduced the severity of arthritis. cFAP mRNA-LNP vaccine was able to provoke immune response to mFAP and suppress inflammatory arthritis.

Nesfatin-1 stimulates BMP5 expression and osteoclastogenesis in rheumatoid arthritis

Rheumatoid arthritis (RA) is a common autoimmune disease marked by immune cell activation and chronic inflammation in the synovium accompanied by osteoclast activation and local joint destruction. Increased levels of the adipokine nesfatin-1 in RA synovium are associated with proinflammatory cytokines. Our analysis of datasets from the Gene Expression Omnibus (GEO) database and synovial tissue samples from RA patients revealed that these had higher levels of nesfatin-1 and osteoclast markers compared with normal synovium. These findings were the same in tissue samples from mice with collagen-induced arthritis (CIA) and normal healthy controls. RNA sequencing analysis revealed that nesfatin-1 increased levels of bone morphogenetic protein-5 (BMP5) expression via JAK/STAT signaling in RA synovial fibroblasts. Finally, we found that nesfatin-1 short hairpin RNA reduced BMP5 and osteoclast formation in CIA mice. These findings provide new insights into the pathogenesis of RA.