Rheumatoid Arthritis Synovial Fibroblasts Research Articles

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.

Exploring the pharmacological mechanisms and key active ingredients of total flavonoids from Lamiophlomis rotata (Benth.) Kudo against rheumatoid arthritis based on multi-technology integrated network pharmacology

Ethnopharmacological relevanceLamiophlomis rotata (Benth.) Kudo (LR, Lamiaceae) is a traditional Tibetan medicinal material in China. Tibetan medicine classic and research report suggested that LR could be used to cure rheumatoid arthritis (RA). However, the anti-RA active ingredients and pharmacological mechanisms of LR have not been elucidated. Aim of the studyTo explore the mechanisms and key active ingredients of total flavonoids from LR (TFLR) against RA. Materials and methodsFirst, the mechanisms of TFLR against RA were investigated on collagen-induced arthritis (CIA) rat model by analyzing paw appearance, paw swelling, arthritis score, spleen index, thymus index, inflammatory cytokine (TNF-α, IL-1β, IL-6 and IL-17) levels in serum, histopathology of ankle joint and synovium from knee joint (hematoxylin-eosin, safranin O-fast green and DAB-TUNEL staining), and apoptosis-related protein (PI3K, Akt1, p-Akt, Bad, p-Bad, Bcl-xL and Bcl-2) levels in the synovium of ankle joints (Western blot). Then, the crucially active ingredients of TFLR against RA were explored by network pharmacology, ingredient analysis, in vitro metabolism and TNF-α-induced human RA synovial fibroblast MH7A proliferation assays. Network pharmacology was applied to predict the key active ingredients of TFLR against RA. The ingredient analysis and in vitro metabolism of TFLR were performed on HPLC, and MH7A proliferation assay were applied to evaluate the predicted results of network pharmacology. ResultsTFLR shown excellently anti-RA effect by reducing paw swelling, arthritis score, spleen index, thymus index and inflammatory cytokine (IL-1β, IL-6 and IL-17) levels, and improving the histopathological changes of ankle joint and synovium from knee joint in CIA rats. Results of Western blot indicated that TFLR reversed the changes of PI3K, p-Akt, p-Bad, Bcl-xL and Bcl-2 levels in the ankle joint synovium of CIA rats. Results of network pharmacology exhibited that luteolin was identified as the pivotal active ingredient of TFLR against RA. The ingredient analysis of TFLR indicated that the main ingredient in TFLR was luteoloside. The in vitro metabolism study of TFLR suggested that luteoloside could be converted to luteolin in artificial gastric juice and intestinal juice. Results of MH7A proliferation assay showed that there was no significant difference between TFLR and equal luteoloside on the viability of MH7A cells, indicating that luteoloside was the key active ingredient of TFLR against RA. Additionally, the luteolin (same mol as luteoloside) showed better inhibitory effect on the viability of MH7A cells than luteoloside. ConclusionTFLR showed anti-RA effect, and the mechanism was related to promoting synovial cell apoptosis mediated by PI3K/Akt/Bad pathway. Meanwhile, this work indicated that luteoloside was the key active ingredient of TFLR against RA. This work lays a foundation for providing TFLR product with clear mechanism and stable quality to treat RA.

Drivers of heterogeneity in synovial fibroblasts in rheumatoid arthritis

Inflammation of non-barrier immunologically quiescent tissues is associated with a massive influx of blood-borne innate and adaptive immune cells. Cues from the latter are likely to alter and expand activated states of the resident cells. However, local communications between immigrant and resident cell types in human inflammatory disease remain poorly understood. Here, we explored drivers of fibroblast-like synoviocyte (FLS) heterogeneity in inflamed joints of patients with rheumatoid arthritis using paired single-cell RNA and ATAC sequencing, multiplexed imaging and spatial transcriptomics along with in vitro modeling of cell-extrinsic factor signaling. These analyses suggest that local exposures to myeloid and T cell-derived cytokines, TNF, IFN-γ, IL-1β or lack thereof, drive four distinct FLS states some of which closely resemble fibroblast states in other disease-affected tissues including skin and colon. Our results highlight a role for concurrent, spatially distributed cytokine signaling within the inflamed synovium.

Differential effect of lactate on synovial fibroblast and macrophage effector functions.

The synovial membrane is the main site of inflammation in rheumatoid arthritis (RA). Here several subsets of fibroblasts and macrophages, with distinct effector functions, have been recently identified. The RA synovium is hypoxic and acidic, with increased levels of lactate as a result of inflammation. We investigated how lactate regulates fibroblast and macrophage movement, IL-6 secretion and metabolism via specific lactate transporters. Synovial tissues were taken from patients undergoing joint replacement surgery and fulfilling the 2010 ACR/EULAR RA criteria. Patients with no evidence of degenerative or inflammatory disease were used as control. Expression of the lactate transporters SLC16A1 and SLC16A3 on fibroblasts and macrophages was assessed by immunofluorescence staining and confocal microscopy. To test the effect of lactate in vitro we used RA synovial fibroblasts and monocyte-derived macrophages. Migration was assessed via scratch test assays or using trans-well inserts. Metabolic pathways were analysed by Seahorse analyser. IL-6 secretion was determined by ELISA. Bioinformatic analysis was performed on publicly available single cell and bulk RNA sequencing datasets. We show that: i) SLC16A1 and SLC16A3 which regulate lactate intake and export respectively, are both expressed in RA synovial tissue and are upregulated upon inflammation. SLC16A3 is more highly expressed by macrophages, while SLC16A1 was expressed by both cell types. ii) This expression is maintained in distinct synovial compartments at mRNA and protein level. iii) Lactate, at the concentration found in RA joints (10 mM), has opposite effects on the effector functions of these two cell types. In fibroblasts, lactate promotes cell migration, IL-6 production and increases glycolysis. In contrast macrophages respond to increases in lactate by reducing glycolysis, migration, and IL-6 secretion. In this study, we provide the first evidence of distinct functions of fibroblasts and macrophages in presence of high lactate levels, opening new insights in understanding the pathogenesis of RA and offering novel potential therapeutic targets.

CD5L aggravates rheumatoid arthritis progression via promoting synovial fibroblasts proliferation and activity.

Rheumatoid arthritis (RA) is a chronic inflammatory disease with progressive cartilage erosion and joint destruction. Synovial fibroblasts (SFs) play a crucial role in the pathogenesis of RA. This study aims to explore the function and mechanism of CD5L during RA progression. We examined the levels of CD5L in synovial tissues and SFs. The collagen-induced arthritis (CIA) rat models were used to investigate the effect of CD5L on RA progression. We also investigated the effects of exogenous CD5L on the behavior and activity of RA synovial fibroblasts (RASFs). Our results showed that CD5L expression was significantly upregulated in synovium of RA patients and CIA-rats. Histology and Micro-CT analysis showed that synovial inflammation and bone destruction were more severe in CD5L-treated CIA rats compared with control rats. Correspondingly, CD5L blockade alleviated bone damage and synovial inflammation in CIA-rats. The exogenous CD5L treatment promoted RASFs proliferation invasion and proinflammatory cytokine production. Knockdown of CD5L receptor by siRNA significantly reversed the effect of CD5L treatment on RASFs. Moreover, we observed that CD5L treatment potentiated PI3K/Akt signaling in the RASFs. The promoted effects of CD5L on IL-6 and IL-8 expression were significantly reversed by PI3K/Akt signaling inhibitor. In conclusion, CD5L promote RA disease progression via activating RASFs. CD5L blocking is a potential therapeutic approach for RA patients.

Microsomal prostaglandin E synthase-1 inhibition promotes shunting in arachidonic acid metabolism during inflammatory responses in vitro

Microsomal Prostaglandin E Synthase 1 (mPGES-1) is the key enzyme for the generation of the pro-inflammatory lipid mediator prostaglandin E2 (PGE2), which contributes to several pathological features of many diseases. Inhibition of mPGES-1 has been shown to be a safe and effective therapeutic strategy in various pre-clinical studies. In addition to reduced PGE2 formation, it is also suggested that the potential shunting into other protective and pro-resolving prostanoids may play an important role in resolution of inflammation. In the present study, we analysed the eicosanoid profiles in four in vitro inflammation models and compared the effects of mPGES-1 inhibition with those of cyclooxygenase-2 (Cox-2) inhibition. Our results showed a marked shift to the PGD2 pathway under mPGES-1 inhibition in A549 cells, RAW264.7 cells and mouse bone marrow-derived macrophages (BMDMs), whereas enhanced prostacyclin production was observed in rheumatoid arthritis synovial fibroblasts (RASFs) treated with an mPGES-1 inhibitor. As expected, Cox-2 inhibition completely suppressed all prostanoids. This study suggests that the therapeutic effects of mPGES-1 inhibition may be mediated by modulation of other prostanoids in addition to PGE2 reduction.

Serum Col3-4: A new type III and IV collagen biochemical marker of synovial tissue turnover in patients with rheumatoid arthritis.

The objective of this study was to develop a serum biochemical marker of the degradation of type III and IV collagens, as an index of synovium turnover, and evaluate its performance in patients with rheumatoid arthritis (RA). An enzyme-linked immunosorbent assay for serum synovial collagen fragments (Col3-4) was developed using an antibody recognizing a specific sequence from human type III collagen, which shares 70% homology with type IV collagen. Immunohistochemistry was performed to localize Col3-4 and the matrix metalloprotease MMP-9 which is upregulated in RA synovial fibroblasts in the synovial tissue from a RA patient. Serum Col3-4 was measured in patients with RA (n = 66, 73% women, mean age 62 years, median disease activity score 28 with erythrocyte sedimentation rate (DAS28-ESR) 2.6) and in sex and age matched healthy controls (n = 70, 76% women, mean age 59 years). Col3-4 immunoassay demonstrated adequate analytical performances and recognized a circulating neoepitope resulting from the cleavage of type III and IV collagens. In RA synovium tissue, Col3-4 fragments were localized in the lining layer where destructive fibroblasts are present and around blood vessels rich in type IV collagen. MMP-9 colocalized with Col3-4 staining and efficiently released Col3-4 fragments from type III and type IV collagen digestion. Serum Col3-4 was markedly increased in patients with RA (+240% vs controls, p < 0.0001) and correlated with DAS28-ESR (r = 0.53, p < 0.0001). Patients with RA and active disease (DAS28-ESR > 3.2, n = 20) had 896% (p < 0.0001) higher levels than subjects with low activity (n = 46). Serum Col3-4 is a specific and sensitive biochemical marker reflecting MMP- mediated type III and IV collagen degradation from synovial tissue. Serum Col3-4 levels are markedly increased in patients with RA, particularly in those with active disease, suggesting that it may be useful for the clinical investigation of RA.

The Role of TNF-α in Rheumatoid Arthritis

Rheumatoid arthritis (RA) induces articular damage through autoimmunity. Tumor necrosis factor-α (TNF-α) plays an important role in the pathogenesis of RA. TNF-α binds to its receptors to recruit certain molecules and forms two kinds of complexes (complex Ⅰ and Ⅱ) to regulate the pro-survival, apoptosis, and necrosis of cells. TNF-α upregulates RA by the methylation and acetylation of the RA synovial fibroblast gene and causes RA-derived joint damage through the induction of osteoclasts. Two TNF-α receptors mediate RA in opposite directions. TNF-α has been a target for the treatment of RA in which TNF-α inhibitors (TNFi) are administrated periodically for RA patients. Several mechanisms in the RA-related TNF-α signaling pathways remain imprecise including the regulation of regulatory T cells, osteoclast induction, and the potential interaction with the hypoxia-inducible factor. Further investigation into these questions may point out new methods for RA treatment to overcome the defects of current TNFi such as the anti-drug antibodies.

Circular RNA CircCDKN2B−AS_006 Promotes the Tumor-like Growth and Metastasis of Rheumatoid Arthritis Synovial Fibroblasts by Targeting the miR−1258/RUNX1 Axis

Rheumatoid arthritis (RA) is an autoimmune polyarthritis in which synovial fibroblasts (SFs) play a major role in cartilage and bone destruction through tumor-like proliferation, migration, and invasion. Circular RNAs (circRNAs) have emerged as vital regulators for tumor progression. However, the regulatory role, clinical significance, and underlying mechanisms of circRNAs in RASF tumor-like growth and metastasis remain largely unknown. Differentially expressed circRNAs in synovium samples from patients with RA and patients with joint trauma were identified via RNA sequencing. Subsequently, in vitro and in vivo experiments were performed to investigate the functional roles of circCDKN2B-AS_006 in RASF proliferation, migration, and invasion. CircCDKN2B-AS_006 was upregulated in synovium samples from patients with RA and promoted the tumor-like proliferation, migration, and invasion of RASFs. Mechanistically, circCDKN2B-AS_006 was shown to regulate the expression of runt-related transcription factor 1 (RUNX1) by sponging miR-1258, influencing the Wnt/β-catenin signaling pathway, and promoting the epithelial-to-mesenchymal transition (EMT) in RASFs. Moreover, in the collagen-induced arthritis (CIA) mouse model, intra-articular injection of lentivirus-shcircCDKN2B-AS_006 was capable of alleviating the severity of arthritis and inhibiting the aggressive behaviors of SFs. Furthermore, the correlation analysis results revealed that the circCDKN2B-AS_006/miR-1258/RUNX1 axis in the synovium was correlated with the clinical indicators of RA patients. CircCDKN2B-AS_006 promoted the proliferation, migration, and invasion of RASFs by modulating the miR-1258/RUNX1 axis.

Components of the sympathetic nervous system as targets to modulate inflammation – rheumatoid arthritis synovial fibroblasts as neuron-like cells?

BackgroundCatecholamines are major neurotransmitters of the sympathetic nervous system (SNS) and they are of pivotal importance in regulating numerous physiological and pathological processes. Rheumatoid arthritis (RA) is influenced by the activity of the SNS and its neurotransmitters norepinephrine (NE) and dopamine (DA) and early sympathectomy alleviates experimental arthritis in mice. In contrast, late sympathectomy aggravates RA, since this procedure eliminates anti-inflammatory, tyrosine hydroxylase (TH) positive cells that appear in the course of RA. While it has been shown that B cells can take up, degrade and synthesize catecholamines it is still unclear whether this also applies to synovial fibroblasts, a mesenchymal cell that is actively engaged in propagating inflammation and cartilage destruction in RA. Therefore, this study aims to present a detailed description of the catecholamine pathway and its influence on human RA synovial fibroblasts (RASFs).ResultsRASFs express all catecholamine-related targets including the synthesizing enzymes TH, DOPA decarboxylase, dopamine beta-hydroxylase, and phenylethanolamine N-methyltransferase. Furthermore, vesicular monoamine transporters 1/2 (VMAT1/2), dopamine transporter (DAT) and norepinephrine transporter (NET) were detected. RASFs are also able to degrade catecholamines as they express monoaminoxidase A and B (MAO-A/MAO-B) and catechol-O-methyltransferase (COMT). TNF upregulated VMAT2, MAO-B and NET levels in RASFs. DA, NE and epinephrine (EPI) were produced by RASFs and extracellular levels were augmented by either MAO, COMT, VMAT or DAT/NET inhibition but also by tumor necrosis factor (TNF) stimulation. While exogenous DA decreased interleukin-6 (IL-6) production and cell viability at the highest concentration (100 μM), NE above 1 μM increased IL-6 levels with a concomitant decrease in cell viability. MAO-A and MAO-B inhibition had differential effects on unstimulated and TNF treated RASFs. The MAO-A inhibitor clorgyline fostered IL-6 production in unstimulated but not TNF stimulated RASFs (10 nM-1 μM) while reducing IL-6 at 100 μM with a dose-dependent decrease in cell viability in both groups. The MAO-B inhibitor lazabemide hydrochloride did only modestly decrease cell viability at 100 μM while enhancing IL-6 production in unstimulated RASFs and decreasing IL-6 in TNF stimulated cells.ConclusionsRASFs possess a complete and functional catecholamine machinery whose function is altered under inflammatory conditions. Results from this study shed further light on the involvement of sympathetic neurotransmitters in RA pathology and might open therapeutic avenues to counteract inflammation with the MAO enzymes being key candidates.

Potential Anti-Rheumatoid Arthritis Activities and Mechanisms of Ganoderma lucidum Polysaccharides

Rheumatoid arthritis (RA) is a chronic and autoimmune disease characterized by inflammation, autoimmune dysfunction, and cartilage and bone destruction. In this review, we summarized the available reports on the protective effects of Ganoderma lucidum polysaccharides (GLP) on RA in terms of anti-inflammatory, immunomodulatory, anti-angiogenic and osteoprotective effects. Firstly, GLP inhibits RA synovial fibroblast (RASF) proliferation and migration, modulates pro- and anti-inflammatory cytokines and reduces synovial inflammation. Secondly, GLP regulates the proliferation and differentiation of antigen-presenting cells such as dendritic cells, inhibits phagocytosis by mononuclear macrophages and nature killer (NK) cells and regulates the ratio of M1, M2 and related inflammatory cytokines. In addition, GLP produced activities in balancing humoral and cellular immunity, such as regulating immunoglobulin production, modulating T and B lymphocyte proliferative responses and cytokine release, exhibiting immunomodulatory effects. Thirdly, GLP inhibits angiogenesis through the direct inhibition of vascular endothelial cell proliferation and induction of cell death and the indirect inhibition of vascular endothelial growth factor (VEGF) production in the cells. Finally, GLP can inhibit the production of matrix metalloproteinases and promote osteoblast formation, exerting protective effects on bone and articular cartilage. It is suggested that GLP may be a promising agent for the treatment of RA.

Anti-proliferation and anti-migration effects of Yishen Tongbi decoction in experimental rheumatoid arthritis by suppressing SLC3A2/integrin β3 signaling pathways

BackgroundYishen Tongbi (YSTB) decoction is a patented herbal formula that is used in China to treat rheumatoid arthritis (RA); however, the exact mechanism of its anti-synovial hyperplasia efficacy has not been fully elucidated. PurposeBased on our previous proteomics study, we aimed to reveal whether YSTB inhibits the proliferation and migration of RA-FLSs through the SLC3A2/integrin β3 pathway in vivo and in vitro. Study designThe study design consists of three parts, a comparison of the expression of SLC3A2 and integrin β3 in synovial tissues of RA and OA patients; an animal experiment to verify the pharmacodynamic effect of YSTB, and in vitro experiment to elucidate the specific mechanism of YSTB. MethodsThe expression of SLC3A2 and integrin β3 in the synovial tissues of patients with RA and osteoarthritis (OA) patients were detected by immunohistochemistry (IHC). In vitro, firstly, the proliferation and migration abilities of HFLS (human fibroblast-like synoviocytes) and HFLS-RA (human fibroblast-like synoviocytes-RA) cells were compared by EdU staining and wound healing assays, respectively, and the differences in the expression and localization of SLC3A2, integrin β3, p-FAK and p-Src between HFLS and HFLS-RA cells were detected by IF and WB. In vivo, DBA/1 mice were injected with bovine collagen II to construct a CIA mouse model. Paw swelling, body weight and the arthritis index (AI) were used as basic treatment evaluation indicators for YSTB. Micro-CT and histopathological analyses of the knee and ankle joints were also performed. In addition, the expression of SLC3A2, integrin β3, p-FAK and p-Src in the synovial tissue of mice was detected by IHC. Subsequently, CCK-8 was used to screen for suitable concentrations of YSTB for use in HFLS-RA cells. EdU staining and transwell migration assays were performed to evaluate the inhibitory effect of YSTB on cell proliferation and migration, and WB was conducted to assess whether YSTB inhibited HFLS-RA migration through downregulation of the SLC3A2/integrin β3 pathways. ResultsIHC showed that the expression of SLC3A2 and integrin β3 was higher in RA synovial tissues than in OA tissues. In vivo experiments showed that YSTB inhibited synovial hyperplasia, prevented bone destruction, and reduced the expression of SLC3A2, integrin β3, p-FAK and p-Src. In vitro experiments showed that YSTB inhibited HFLS-RA migration and proliferation by inhibiting the expression of SLC3A2/integrin β3 and downstream signaling molecules. ConclusionYSTB inhibits the proliferation and migration of synovial fibroblasts in RA by downregulating the SLC3A2/integrin β3 pathways.

Telotristat Etiprate alleviates rheumatoid arthritis by targeting LGALS3 and affecting MAPK signaling.

Rheumatoid arthritis (RA) is one of the most widespread chronic immune-mediated inflammatory diseases characterized by continuous erosion of bone and cartilage by synovial hyperplasia. Telotristat Etiprate is an inhibitor of tryptophan hydroxylase, a rate-limiting enzyme in the biosynthesis of serotonin. Telotristat Etiprate can be used in the treatment of carcinoid syndrome. The purpose of this study was to explore the effect of Telotristat Etiprate on RA and its mechanism. We investigated Telotristat Etiprate in collagen-induced arthritis (CIA) model mice and in rheumatoid arthritis synovial fibroblasts (RASFs). Results showed that Telotristat Etiprate had anti-inflammatory effects both in vitro and in vivo, can inhibit the invasion and migration of cells, inhibit the formation of pannus, and induce cell apoptosis. Transcriptome sequencing (RNA-seq) and mass spectrometry analysis showed that Galectins-3 (LGALS3) could be a newly identified target of Telotristat Etiprate, affecting the phosphorylation of the MAPK signaling pathway through UBE2L6, thereby improving RA.