Rheumatoid Arthritis Fibroblast-like Synoviocytes Research Articles

Anti-rheumatic effect of KMU-11361 in various rheumatoid arthritis-related cells targeting multiple protein kinase

Abstract Protein kinases important for regulating protein phosphorylation, which is a promising therapeutic target for inflammatory disease, such as rheumatoid arthritis (RA). In this study, we synthesized a novel multi-protein kinase inhibitor, KMU-11361, a derivative of indolin-2-one and investigated the mechanisms of anti-inflammatory and anti-rheumatic effects using THP-1 cells, RAW264.7 cells, Jurkat T cells, and human RA fibroblast-like synoviocytes (RA-FLS). Our results revealed that KMU-11361 inhibited LPS-induced upregulation of inducible iNOS, and COX-2, and suppressed LPS-induced phosphorylation of TAK1, JNK, inhibitor of NF-κB kinase α/β (IKKα/β), IκBα, and NF-κB p65 as well as nuclear translocation of NF-κB p65 in THP-1 cells. Furthermore, KMU-11361 attenuated proinflammatory cytokines such as IL-1β, TNF-α, and IL-6, and, remarkably, inhibited LPS-induced activation of the NLRP3 inflammasome in THP-1 cells. Additionally, in activated Jurkat T cells, KMU-11361 inhibited the TAK1-IKKα-mediated NF-κB pathway as well as the phosphorylation of MAPKs. Moreover, KMU-11361 inhibited anti-CD3/CD28 antibodies-induced upregulation of proinflammatory cytokine IL-2. Moreover, it suppressed LPS-induced upregulation of proinflammatory cytokines, p-TAK1, p-IKKα/β, and p-NF-κB p65 in RA-FLS. Surprisingly, KMU-11361 also inhibited the receptor activator of NF-κB-induced osteoclast differentiation and up-regulation of nuclear factor of activated T-cells, NFATc-1 and c-Fos in RAW264.7 cells. Taken together, our results suggest that KMU-11361 has anti-inflammatory and anti-rheumatic effects in various RA-related cells and could be developed as a potent anti-rheumatic drug.

HIF-1α dependent RhoA as a novel therapeutic target to regulate rheumatoid arthritis fibroblast-like synoviocytes migration in vitro and in vivo.

The purpose of this work is to investigate how the Rho family of GTPases A (RhoA) mediates the pathogenesis of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). The expression of RhoA in the synovial tissues of RA and Healthy people (Control) was detected using immunohistochemistry methods. The expression of RhoA and hypoxia-inducible factor-1α (HIF-1α) is inhibited by small interfering RNAs (siRNAs). The inhibition effect on RA-FLS migration was further investigated. The protein expression level of HIF-1α, RhoA, focal adhesion kinase (FAK), and myosin light chain (MLC) was also analysed using western blotting (WB). DBA1 mice were immunised with the mixture of bovine type II collagen and Freund's adjuvant to establish collagen induced arthritis (CIA) mouse model. Lip-siRhoA is administered through joint injection every two days. Micro-computed tomography (micro-CT) was used to detect mouse ankle joint destruction and evaluate the bone loss of the periarticular side. Destruction of the ankle articular cartilage was tested by histology. Expressions of P-RhoA, P-FAK and P-MLC in the ankle joint was detected by immunohistochemistry assay. The expression level of RhoA in the synovial tissues of RA patients was significantly higher than that in control group. Hypoxia was able to up-regulate the expression of RhoA. Whereas, HIF-1α siRNA (siHIF-1α) could down-regulate the expression of RhoA. Additionally, both of siHIF-1α and RhoA siRNA (siRhoA) delivered by liposome (Lip-siHIF-1α and Lip-siRhoA) were found to suppress FAK and MLC phosphorylation in vitro. In CIA mouse model, Lip-siRhoA was demonstrated to ameliorate the destruction of ankle joint and reduce the severity of ankle joint cartilage damage by micro-CT and histological staining, respectively. Therefore, inhibition of FLS cell migration can protect articular bone from destruction. Furthermore, the expression of P-RhoA, P-FAK and P-MLC was evaluated and found to be down-regulated by Lip-siRhoA in vivo. The results demonstrated that under hypoxic environment, HIF-1α dependent RhoA pathway played an important role on cytoskeleton remodelling and RA-FLS migration. Through down-regulating RhoA expression, it could effectively treat RA in vitro and in vivo. Our study provides new evidence for the potential clinical application of RhoA as a candidate for the treatment of RA.

Akuammiline alkaloid derivatives: divergent synthesis and effect on the proliferation of rheumatoid arthritis fibroblast-like synoviocytes.

During the past decades, rheumatoid arthritis had become a serious problem, torturing millions of patients because of unclear pathogenesis and no ideal therapies. Natural products remain an important source of medicines to treat various major diseases such as rheumatoid arthritis (RA) given their excellent biocompatibility and structural diversity. Herein, we have developed a versatile synthetic method for constructing various skeletons of akuammiline alkaloid analogs based on our previous research on the total synthesis of the related indole alkaloids. We have also evaluated the effect of these analogs on the proliferation of RA fibroblast-like synoviocytes (FLSs) in vitro and analyzed the corresponding structure-activity relationship (SAR). Among these analogs, compounds 9 and 17c have demonstrated a promising inhibitory effect on the proliferation of RA-FLSs, with IC50 values of 3.22 ± 0.29 μM and 3.21 ± 0.31μM, respectively. Our findings provide a solid foundation for future pharmacological studies on akuammiline alkaloid derivatives and inspiration for the development of anti-RA small molecule drugs derived from natural products.

LKB1 Regulates Inflammation of Fibroblast-like Synoviocytes from Patients with Rheumatoid Arthritis via AMPK-Dependent SLC7A11-NOX4-ROS Signaling.

Fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) patients have increased reactive oxygen species (ROS) levels and an impaired redox balance compared with FLS from control patients. Liver kinase B1 (LKB1) plays a key role in ROS scavenging and cellular metabolism in various cancers. Here, we aimed to determine the specific mechanism of LKB1 in RA pathogenesis. FLS were obtained from RA patients (n = 10). siRNA-induced LKB1 deficiency in RA FLS increased ROS levels via NADPH oxidase 4 (NOX4) upregulation. RA FLS migration and expression of inflammatory factors, including interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF), were enhanced by LKB1 deficiency. LKB1-deficient RA FLS showed increased sensitivity to oxidative stress damage caused by hydrogen peroxidase exposure. siRNA-induced solute carrier family 7 member 11 (SLC7A11) deficiency in RA FLS enhanced NOX4 and ROS expression and increased cell migration. When LKB1-deficient RA FLS were stimulated with an AMP-activated protein kinase (AMPK) activator, the LKB1-inhibition-induced cell migration significantly decreased through the restoration of SLC7A11/NOX4 expression. LKB1 regulates the AMPK-mediated SLC7A11-NOX4-ROS pathway to control cell migration and inflammation. Our data indicate that LKB1 is a key regulator of redox homeostasis in RA FLS.

E076 To investigate the mechanisms of action of novel compounds ex vivo in cells obtained from the inflammatory synovium of patients with rheumatoid arthritis

Abstract Background/Aims To investigate the ex vivo effect of JAK1/2 inhibitor baricitinib on the expression of pro-inflammatory mediators in normal and RA diseased fibroblasts stimulated with oncostatin M (OSM) as well as in cells derived from RA inflamed synovial membrane cells (RA SMC). Methods RA SMC and fibroblast like synoviocytes (RA FLS) were isolated from synovial membranes of patients undergoing surgery using enzymatic digestion and cell propagation. Normal fibroblast-like synoviocytes (FLS) and normal human skin fibroblasts (HSF) were purchased from commercial sources. All fibroblasts were cultured in media supplemented foetal bovine serum and antibiotics in incubators using established laboratory procedures, and serum-starved prior to stimulation with OSM with or without baricitinib. RA SMC were cultured in the presence of baricitinib without OSM as they spontaneously release pro-inflammatory cytokines. Levels of mRNA and proteins of the various inflammatory cytokines (IL-6, IL-8, MCP-1, CCL5 and IP-10) were determined by qPCR, ELISA and MSD. Western blot was used to measure levels of phospho-JAK specific components. Results qPCR analysis found that baricitinib inhibited OSM induced IP-10 and MCP-1 mRNA expression but not RANTES mRNA expression in normal human skin fibroblasts and RA FLS. In normal FLS however, baricitinib was found to inhibit RANTES and MCP-1 mRNA expression, but not IP-10 mRNA expression. Expression levels of IL-8 were too low for accurate conclusions to be drawn in all cell types apart from the RA SMCs, and in this case baricitinib did not inhibit IL-8 expression. ELISA results showed that baricitinib inhibited OSM induced MCP-1 expression in all cell types investigated. OSM induced IL-6 expression was inhibited in RA FLS but no other cell type. Conclusion Baricitinib inhibits OSM stimulated pro-inflammatory cytokine production through JAK1, 2. It has a varying inhibitory effect on the production of different cytokines: it is shown to particularly inhibit MCP-1. Baricitinib was found to have no effect on the production of spontaneously released pro-inflammatory cytokines in RA mixed cell populations. Disclosure M. Turner: None.

5-aminolevulinic acid-loaded dissolving microneedle array for photodynamic therapy of rheumatoid arthritis on rats

Photodynamic therapy (PDT) is a noninvasive technique that can be used to treat rheumatoid arthritis (RA) by irradiating photosensitizers with specific wavelengths of light to generate reactive oxygen species (ROS), thus leading to targeted cell necrosis. However, efficient delivery of photosensitizers with low side effects is a key issue. We developed a 5-aminolevulinic acid-loaded dissolving microneedle array (5-ALA@DMNA) that can locally and efficiently deliver photosensitizers for RA treatment by PDT. 5-ALA@DMNA was fabricated through a two-step molding process, which was characterized. The effects of 5-ALA-mediated PDT on RA fibroblast-like synoviocytes (RA-FLs) were investigated via in vitro experiments. Adjuvant arthritis rat models were established to evaluate the therapeutic effect of 5-ALA@DMNA-mediated PDT on RA. The results showed that 5-ALA@DMNA could penetrate the skin barrier and efficiently deliver photosensitizers. 5-ALA-mediated PDT can significantly inhibit the migration ability and selectively induce apoptosis of RA-FLs. Moreover, 5-ALA-mediated PDT had a significant therapeutic effect on rats with adjuvant arthritis, which may be related to the upregulation of interleukin (IL)− 4 and IL-10 and downregulation of TNF-α, IL-6, and IL-17. Thus, 5-ALA@DMNA-mediated PDT may be a potential therapy for RA.

Evaluation of 18F-FAPI-04 Imaging in Assessing the Therapeutic Response of Rheumatoid Arthritis.

Fibroblast activating protein (FAP) is highly expressed in the synovial tissues of rheumatoid arthritis (RA) patients. The aim of this study was to determine the feasibility of PET imaging with an Al[18F] F-NOTA-labeled FAP inhibitor 04(18F-FAPI-04) for the evaluation of arthritic progression and therapeutic response in experimental arthritis. Fibroblast-like synoviocytes (FLSs) were obtained from patients with RA or osteoarthritis (OA), and the relationship between 18F-FAPI-04 uptake and the inflammatory activity of RA FLSs was investigated. Collagen-induce arthritis (CIA) mice models were established and treated with methotrexate (MTX) or etanercept (ETC). Then, PET imaging was performed 24h following 18F-FAPI-04 injection. The imaging results were compared by assessing macroscopic arthritis scores and histological staining. 18F-FAPI-04 uptake was obvious in RA FLSs that characterizing FAP activation. The higher the uptake of 18F-FAPI-04, the more severity of the inflammatory phenotype in RA FLS. Furthermore, the uptake of 18F-FAPI-04 in inflamed joints could be found even before the deformity of the parental joints could be observed by histological examination. Both MTX and ETC were effective in inhibiting the progression of arthritis in CIA mice was confirmed by macroscopic, histological, and radiographic pathology scores. Importantly, 18F-FAPI-04 uptake declined accordingly in CIA models following MTX and ETC treatment. These findings suggest that PET imaging of 18F-FAPI-04 can be used to monitor treatment response in RA, and is more sensitive in disease speculation than macroscopic arthritis scoring.

IL-37 alleviates TNF-α-induced pyroptosis of rheumatoid arthritis fibroblast-like synoviocytes by inhibiting the NF-κB/GSDMD signaling pathway

ObjectivePyroptosis is crucial to rheumatoid arthritis (RA) by inducing and aggravating inflammation. TNF-α is abundant in fibroblast-like synoviocytes of RA (RA-FLSs) and plays a key role in pyroptosis by inducing nuclear factor (NF)-κB activation. Additionally, interleukin (IL)-37 is involved in autoimmune diseases as an anti-inflammatory cytokine and innate and acquired immune response inhibitor. However, the effect of IL-37 on pyroptosis in RA-FLSs remains unclear. Therefore, this study investigated the effects and mechanism of IL-37 on RA-FLS pyroptosis induced by TNF-α. MethodsIn this study, the serum cytokines in patients with RA and healthy controls were detected using ELISA. The RA-FLSs were then cultured with TNF-α, with or without various IL-37 concentrations, to test the cytokine levels in the cell supernatant. 5-Ethynyl-2ʹ-Deoxyuridine (EdU) assay assessed the effects of IL-37 on RA FLS proliferation. RA-FLS apoptosis was assessed using flow cytometry and mitochondrial membrane potential (MMP) measurement. In addition, transmission electron microscopy (TEM) was used to examine cell pyroptosis. We selected the optimal concentration for the following experiments and detected the signal pathway of IL-37 on pyroptosis of RA FLSs by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blotting. Finally, we validated the therapeutic effects of IL-37 on CIA rat model in vivo. ResultsIL-37 inhibited inflammation in vitro and in vivo and reduced pyroptosis-related protein expression in RA FLSs. Furthermore, we determined that nuclear factor κB (NF-κB) signaling is required for GSDMD-mediated pyroptosis in RA FLSs. ConclusionIL-37 alleviates TNF-α-induced pyroptosis of RA FLSs by inhibiting NF-κB/GSDMD signaling. Additionally, our data revealed a novel mechanism for IL-37 in RA FLSs, suggesting a new potential therapy for IL-37 to treat RA.

Retraction notice to "MLL1 promotes migration and invasion of fibroblast-like synoviocytes in rheumatoid arthritis by activating the TRIF/NF-κB signaling pathway via H3K4me3 enrichment in the TLR4 promoter region" [Int. Immunopharmacol. 82 (2020) 106220

Retraction notice to "MLL1 promotes migration and invasion of fibroblast-like synoviocytes in rheumatoid arthritis by activating the TRIF/NF-κB signaling pathway via H3K4me3 enrichment in the TLR4 promoter region" [Int. Immunopharmacol. 82 (2020) 106220

Effect of Siegesbeckiae Herba on immune-inflammation of rheumatoid arthritis: Data mining and network pharmacology

Rheumatoid arthritis (RA) is an autoimmune disease, resulting in an overall poor prognosis. Siegesbeckiae Herba is a natural botanical herb that possesses a variety of biological activities, particularly its anti-rheumatism property. This study collected the clinical data of 7657 RA patients who received treatment with traditional Chinese medicine prescriptions before December 2021 in the Department of Rheumatology, The First Affiliated Hospital of Anhui University of Traditional Chinese Medicine. The levels of immune-inflammatory indicators including Hs-CRP, ESR, RF, C3, C4, IgA, IgG, and IgM were observed before and after the treatment. Association rules between Siegesbeckiae Herba and immune-inflammatory indicators were analyzed by Apriori algorithm. Protein-protein interaction network diagram, core gene and drug-active ingredient-target diagram were drawn by Cytoscape3.7.2 software. GO and KEGG analysis was performed by DAVID database. Autodock software was used to conduct molecular docking and in vitro experiments were performed to verify the action mechanism of Siegesbeckiae Herba on RA. Data mining results showed that the use of Siegesbeckiae Herba was strongly associated with the improvement of immune-inflammatory markers of RA. The results of network pharmacology and molecular docking showed that the main target proteins including Bax, CASP3, CASP8, CASP9, COX2, TNF, and IL-17 of Siegesbeckiae Herba were closely related to apoptosis and inflammatory signaling pathways in the treatment of RA. In vitro cell experiments results showed that Kirenol, the main active ingredient of Siegesbeckiae Herba, up-regulated the expressions of pro-apoptotic proteins Bax, caspase3, caspase8, and caspase9 in RA-fibroblast-like synoviocytes (RA-FLSs) and down-regulated the expressions of COX2, TNF, and IL-17. Siegesbeckiae Herba promoted the apoptosis of RA-FLSs and alleviated immune-inflammatory responses in RA.

Identification and preliminary validation of synovial tissue-specific genes and their-mediated biological mechanisms in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease. It is well known that the formation of positive feedback between synovial hyperplasia and inflammatory infiltration is intimately associated with the occurrence and development of RA. However, the exact mechanisms still remain unknown, making the early diagnosis and therapy of RA difficult. This study was designed to identify prospective diagnostic and therapeutic biomarkers, as well as their-mediated biological mechanisms in RA. Three microarray datasets (GSE36700, GSE77298 and GSE153015) and two RNA-sequencing datasets (GSE89408 and GSE112656) of synovial tissues, as well as three other microarray datasets (GSE101193, GSE134087 and GSE94519) of peripheral blood were downloaded for integrated analysis. The differently expressed genes (DEGs) were identified by "limma" package of R software. Then, weight gene co-expression analysis and gene set enrichment analysis were performed to investigate synovial tissue-specific genes and their-mediated biological mechanisms in RA. The expression of candidate genes and their diagnostic value for RA were verified by quantitative real-time PCR and receiver operating characteristic (ROC) curve, respectively. Relevant biological mechanisms were explored through cell proliferation and colony formation assay. The suggestive anti-RA compounds were discovered by CMap analysis. We identified a total of 266 DEGs, which were mainly enriched in cellular proliferation and migration, infection and inflammatory immune signaling pathways. Bioinformatics analysis and molecular validation revealed 5 synovial tissue-specific genes, which exhibited excellent diagnostic value for RA. The infiltration level of immune cells in RA synovial tissue was significantly higher than that in control individuals. Moreover, preliminary molecular experiments suggested that these characteristic genes may be responsible for the high proliferation potential of RA fibroblast-like synoviocytes (FLSs). Finally, 8 small molecular compounds with anti-RA potential were obtained. We have proposed 5 potential diagnostic and therapeutic biomarkers (CDK1, TTK, HMMR, DLGAP5, and SKA3) in synovial tissues that may contribute to the pathogenesis of RA. These findings may shed light on the early diagnosis and therapy of RA.

Exosomes derived from synovial fibroblasts from patients with rheumatoid arthritis promote macrophage migration that can be suppressed by miR-124-3p

ObjectivesExosomes are potent vehicles for intercellular communication. Rheumatoid arthritis (RA) is a chronic systemic disease of unknown etiology. Local administration of miR-124 precursor to rats with adjuvant-induced arthritis suppresses systemic arthritis and bone destruction. Thus, exosomes may be involved in this disease. We aimed to determine the role of exosomes in the pathology of RA. MethodsFibroblast-like synoviocytes (FLS) were collected from patients with RA and osteoarthritis (OA). miR-124-3p mimic was transfected into the RA FLS (RA miR-124 FLS). Exosomes were collected from the culture medium by ultracentrifugation. Macrophages were produced from THP-1 cells. MicroRNAs in the exosomes were analyzed using real-time PCR. Proteomics analysis was performed using nanoscale liquid chromatography-tandem mass spectrometry. Macrophage migration was evaluated using a Transwell migration assay. SiRNA was used to knockdown proteins of interest. ResultsMicroRNAs in the RA FLS, RA miR-124 FLS, and OA FLS exosomes were similar. Proteomics analysis revealed that pentraxin 3 (PTX3) levels were higher in RA FLS exosomes than in RA miR-124 FLS and OA FLS exosomes, and proteasome 20S subunit beta 5 (PSMB5) levels were lower in RA FLS exosomes than in RA miR-124 FLS and OA FLS exosomes. The RA FLS exosomes promoted and the RA miR-124 FLS exosomes suppressed macrophage migration. PTX3-silenced RA FLS exosomes suppressed and PSMB5-silenced OA FLS exosomes promoted macrophage migration. ConclusionsRA FLS exosomes promote macrophage migration via PTX3 and PSMB5, and miR-124-3p suppresses this migration.

Circ_0000479 promotes proliferation, invasion, migration and inflammation and inhibits apoptosis of rheumatoid arthritis fibroblast-like synoviocytes via miR-766/FKBP5 axis

Circular RNAs have been demonstrated to play a critical role in the progression of autoimmune diseases. This study aimed to investigate the function of circ_0000479 in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs). Circ_0000479 was found to be upregulated in RA-FLSs. Flow cytometry analysis, cell counting Kit-8, transwell, wound-healing and enzyme-linked immunosorbent assays were conducted to evaluate RA-FLS apoptosis, proliferation, invasion, migration and inflammation. The results confirmed that circ_0000479 knockdown suppressed pathogenic properties of RA-FLSs. Through bioinformatics analysis and screening, we obtained 18 miRNAs that can bind to circ_0000479, of which miR-766 was most significantly up-regulated after circ_0000479 knockdown. MiR-766 was confirmed to be down-regulated in RA-FLSs and the combination between circ_0000479 and miR-766 was verified by dual-luciferase reporter assays. Moreover, the inhibitory effect of circ_0000479 knockdown in RA-FLS progression was attenuated by miR-766 inhibitor. By intersecting the target genes of miR-766 with the up-regulated genes in RA, we obtained 8 genes, of which FKBP5 was most significantly down-regulated after miR-766 overexpression. The results of dual-luciferase reporter assays also verified that FKBP5 was the target gene of miR-766. In addition, FKBP5 overexpression abated the inhibition of RA-FLS progression caused by circ_0000479 silencing. In summary, circ_0000479 binds to miR-766 to promote RA progression via FKBP5.

LncRNA OIP5-AS1/miR-410-3p/Wnt7b axis promotes the proliferation of rheumatoid arthritis fibroblast-like synoviocytes via regulating the Wnt/β-catenin pathway

LncRNA OIP5-AS1 has a common gene imbalance in various cancers and tumours, which plays an important role in regulating its biological function. However, there are few studies on lncRNA OIP5-AS1 in rheumatoid arthritis (RA). The purpose of the present study was to investigate the role of lncRNA OIP5-AS1 in the pathogenesis of RA. In the present study, we established an adjuvant arthritis (AA) rat model to obtain primary fibroblast-like synoviocytes (FLSs);The subcellular localisation of lncRNA OIP5-AS1 was detected by fluorescence in situ hybridisation (FISH) assay; Cell proliferation of FLSs was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay;IL-1β, IL-6 and TNF-α concentrations were measured by enzyme-linked immunosorbent assay (ELISA);Quantitative real-time PCR (qRT-PCR), Western blots(WB) and immunofluorescence were used to detect the expression of lncRNA OIP5-AS1/miR-410-3p/wnt7b signal axis and Wnt/β-catenin signal pathway related indicators in FLSs. FISH assay confirmed the presence of lncRNA OIP5-AS1 in the cytoplasm, suggesting that it acts as a competing endogenous RNA (ceRNA). qRT-PCR showed that the expression of lncRNA OIP5-AS1 was upregulated in FLSs, while the expression of miR-410-3p was downregulated in FLSs. We also found that lncRNA OIP5-AS1 knockdown inhibited the proliferation and inflammation of FLSs. Moreover, the expression of Wnt7b, the downstream target gene of miR-410-3p, and the activation of the Wnt/β-catenin signalling pathway were also inhibited by lncRNA OIP5-AS1 knockdown. These results suggested that lncRNA OIP5-AS1 promotes the activation of the Wnt/β-catenin signalling pathway by regulating the miR-410-3p/Wnt7b signalling axis, thereby participating in the occurrence and development of RA.

Role and mechanism of fibroblast-activated protein-α expression on the surface of fibroblast-like synoviocytes in rheumatoid arthritis.

Fibroblast-activated protein-α (FAP) is a type II integrated serine protease expressed by activated fibroblasts during fibrosis or inflammation. Fibroblast-like synoviocytes (FLSs) in rheumatoid arthritis (RA) synovial sites abundantly and stably overexpress FAP and play important roles in regulating the cellular immune, inflammatory, invasion, migration, proliferation, and angiogenesis responses in the synovial region. Overexpression of FAP is regulated by the initial inflammatory microenvironment of the disease and epigenetic signaling, which promotes RA development by regulating FLSs or affecting the signaling cross-linking FLSs with other cells at the local synovium and inflammatory stimulation. At present, several treatment options targeting FAP are in the process of development. This review discusses the basic features of FAP expressed on the surface of FLSs and its role in RA pathophysiology and advances in targeted therapies.

PINK1/Parkin-Mediated Mitochondrial Autophagy Participates in H2O2-Induced Abnormal Proliferation of Fibroblast-Like Synoviocytes in Rheumatoid Arthritis.

To explore the role of PINK1/Parkin-mediated mitochondrial autophagy in H2O2-induced abnormal proliferation of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). Firstly, we isolated fibroblast like synoviocytes (RA-FLS) from RA patients. H2O2-induced oxidative stress, and NAC (a ROS inhibitor) or FCCP (a mitochondrial autophagy activator) treatment inhibited ROS level or activate mitochondrial autophagy in RA-FLS. MitoSOX Red, JC-1 kit, DCFH-DA kit and CCK8 kit were used to evaluate mitochondrial redox status, mitochondrial membrane potential, intracellular ROS level and cell activity, respectively. Western blot was used to detect the protein expression. The rat model of Freund's complete adjuvant arthritis (AA) was established and treated with NAC and FCCP, respectively. The pathological changes of synovium and the percentage of apoptotic cells in synovium were detected by H&E and TUNEL staining, respectively. We have successfully isolated synovial cells from RA patients. Using 5μM H2O2 to stimulate RA-FLS could induce mitochondrial abnormalities of RA-FLS and inhibit RA-FLS autophagy. FCCP could reverse the effect of H2O2 on RA-FLS cell proliferation and apoptosis. NAC could reverse the effect of H2O2 on PINK1/Parkin. Overexpression of PINK1 or Parkin reversed the effect of H2O2 on RA-FLS mitochondrial autophagy, proliferation and apoptosis. The in vivo experiment results showed that both NAC and FCCP could prevent the pathogenesis of RA, reduce RA-FLS cell viability and increase RA-FLS cell apoptosis. The PINK1/Parkin-mediated mitochondrial autophagy participates in H2O2-induced abnormal proliferation of RA-FLS, and targeting of PINK1/Parkin-mediated mitochondrial autophagy may be the key mechanism in the treatment of RA.

Tanshinone IIA relieves arthritis by inhibiting autophagy of fibroblast-like synoviocytes via matrix metalloproteinase9/receptor for advanced glycation end product/toll-like receptor 9 signal axis in mice with collagen-induced arthritis.

In this study, we probed into the related mechanism underlying the role of Tanshinone IIA (TIIA) in RA fibroblast-like synoviocytes (RA-FLSs). We constructed a mouse model of RA using the collagen-induced arthritis (CIA) method. Gain- or loss-of-function approaches were used to manipulate matrix metalloproteinase9 (MMP9), receptor for advanced glycation end product (RAGE), and toll-like receptor 9 (TLR9) in both CIA mice and RA-FLSs following treatment with TIIA to study the in vivo and in vitro effect of TIIA through analysis of cell viability, and measurement of autophagy and inflammatory proteins as well as severity of RA. In vitro and in vivo animal experiments results showed that TIIA could inhibit the proliferation of RA-FLSs and affect autophagy, thereby improving the symptoms of RA in mice. Mechanically, TIIA could inhibit the expression of MMP9 in RA-FLSs, thereby inhibiting the shedding of RAGE and thus inhibiting the activation of TLR9. Finally, animal experiments confirmed that TIIA affected autophagy by regulating the MMP9/RAGE/TLR9 axis, and finally improve the symptoms of RA in mice. Conclusively, TIIA may inhibit expression of MMP9 to suppress the combination of RAGE and TLR9, thereby inhibiting RA-FLS proliferation and affecting autophagy, eventually improve the RA.

Identification of anti-citrullinated osteopontin antibodies and increased inflammatory response by enhancement of osteopontin binding to fibroblast-like synoviocytes in rheumatoid arthritis

BackgroundAnti-citrullinated protein/peptide antibodies (ACPAs) are present in patients at onset and have important pathogenic roles during the course of rheumatoid arthritis (RA). The characteristics of several molecules recognized by ACPA have been studied in RA, but the positivity rate of autoantibodies against each antigen is not high, and the pathogenic mechanism of each antibody is not fully understood. We investigated the role of anti-citrullinated osteopontin (anti-cit-OPN) antibodies in RA pathogenesis.MethodsEnzyme-linked immunosorbent assays on RA patients’ sera were used to detect autoantibodies against OPN. Fibroblast-like synoviocytes (FLS) isolated from RA patients were used to test the binding activity and inflammatory response of OPN mediated by anti-cit-OPN antibodies, and their effect was tested using an inflammatory arthritis mouse model immunized with cit-OPN. Anti-cit-OPN antibody positivity and clinical characteristics were investigated in the patients as well.ResultsUsing sera from 224 RA patients, anti-cit-OPN antibodies were positive in approximately 44% of RA patients, while approximately 78% of patients were positive for the cyclic citrullinated peptide (CCP2) assay. IgG from patients with anti-cit-OPN antibody increased the binding activity of OPN to FLSs, which further increased matrix metalloproteinase and interleukin-6 production in TNF-stimulated FLSs. Mice immunized with cit-OPN antibodies experienced severe arthritis. Anti-cit-OPN antibodies in RA patients decreased the drug survival rate of tumor necrosis factor (TNF) inhibitors, while it did not decrease that of CTLA4-Ig.ConclusionsAnti-cit-OPN antibodies were detected in patients with RA. IgG from patients with anti-cit-OPN antibodies aggravated RA, and anti-cit-OPN antibody was a marker of reduced the survival rate of TNF inhibitors in RA patients.

MiR-326 regulates cell proliferation and apoptosis in fibroblast-like synoviocytes in rheumatoid arthritis.

The dysregulation of microRNAs plays a critical role in the development of rheumatoid arthritis (RA). This study aims to explore the functional significance of miR-326 in RA. TheRT-qPCR results showed that miR-326 was downregulated in synovial tissues of RA patients and RA fibroblast-like synoviocytes (RA-FLS). We found that miR-326 couldtarget and reduce the expression of inhibitor of DNA binding 1 (Id1). MTT assay and flow cytometry were conducted to explore the biological function of miR-326.Our datarevealed that the upregulation of miR-326suppressed cell proliferation and induced apoptosis in RA-FLS. Incollagen-induced arthritis mice,intraarticular injection of lentivirus carryingmiR-326overexpression vectors could reduce the arthritis score and attenuate synovial inflammation and cartilage destruction. We also found thatlong non-coding RNA-Ewing sarcoma-associated transcript 1 (lncRNA-EWSAT1) was significantly increased in RAsynovial tissues and RA-FLS. TheRNA immunoprecipitation and RNApull-down assay indicated thatlncRNA-EWSAT1 directly bound and negatively regulated the expression of miR-326.Knockdown of lncRNA-EWSAT1 could upregulatemiR-326 expression and attenuate its proliferation inhibition and apoptosis induction effect in RA-FLS. In conclusion,the lncRNA-EWSAT1/miR-326 axis might provide a novel therapeutic target in the treatment of RA.

Berberine inhibits RA-FLS cell proliferation and adhesion by regulating RAS/MAPK/FOXO/HIF-1 signal pathway in the treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a common chronic immune disease. Berberine, as its main active ingredient, was also contained in a variety of medicinal plants such as Berberaceae, Buttercup, and Rutaceae, which are widely used in digestive system diseases in traditional Chinese medicine with anti-inflammatory and antibacterial effects. The aims of this article were to explore the therapeutic effect and mechanism of berberine on rheumatoid arthritis. Cell Counting Kit-8 was used to evaluate the effect of berberine on the proliferation of RA fibroblast-like synoviocyte (RA-FLS) cells. The effect of berberine on matrix metalloproteinase (MMP)-1, MMP-3, receptor activator of nuclear factor kappa-Β ligand (RANKL), tumour necrosis factor alpha (TNF-α), and other factors was determined by enzyme-linked immunoassay (ELISA) kit. Transcriptome technology was used to screen related pathways and the potential targets after berberine treatment, which were verified by reverse transcription-polymerase chain reaction (RT-qPCR) and Western blot (WB) technology. Berberine inhibited proliferation and adhesion of RA-FLS cells, and significantly reduced the expression of MMP-1, MMP-3, RANKL, and TNF-α. Transcriptional results suggested that berberine intervention mainly regulated forkhead box O (FOXO) signal pathway, prolactin signal pathway, neurotrophic factor signal pathway, and hypoxia-inducible factor 1 (HIF-1) signal pathway. The effect of berberine on RA was related to the regulation of RAS/mitogen-activated protein kinase/FOXO/HIF-1 signal pathway in RA-FLS cells.Cite this article: Bone Joint Res2023;12(2):91-102.