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OBJECTIVE: Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovial hyperplasia and progressive bone destruction. The tumor-like growth of fibroblast-like synoviocytes (FLSs) plays a crucial role in the pathogenesis of RA. The N6 methyladenine (m6A) mRNA methylation modification, regulated by methyltransferases (METTL3) and demethylation enzymes, is a novel epigenetic regulator in the development of RA. However, there is limited research on m6A methylation modifications in RA synovitis and a lack of mechanistic studies on their impact on the function of RA-FLSs. METHODS: This study utilized clinical synovial tissue specimens and FLSs as research subjects. The m6A methylation level and the expression of methyltransferases and demethylation enzymes were detected. RNA interference and gene overexpression methods were employed to investigate the mechanism of METTL3 in RA-FLSs. The study also examined the proliferation, apoptosis, migration, invasion, and cytokine levels of RA-FLSs, as well as the expression of METTL3 in RA animal models. RESULTS: In this study, we found that m6A methylation levels were elevated in synovial tissues and FLSs of RA patients. Immunohistochemical staining showed that METTL3 and METTL14 levels were up-regulated in synovial tissues of RA, the mRNA levels of METTL3, METTL14, WTAP, FTO, and ALKBH5 were significantly higher in synovial tissues and FLSs of RA patients. Overexpression of METTL3 could promote the proliferation, migration, and secretion of IL-6, RANKL of RA-FLSs; inhibition of METTL3 expression could inhibit the abnormal proliferation, migration, invasion, and secretion of IL-6, RANKL, at the same time promoted the apoptosis and secretion of OPG, thus inhibited RA-FLSs tumor-like growth. In CIA mice, the use of MTX and STM2457 reduced METTL3 expression, synovial hyperplasia and bone destruction. CONCLUSION: Abnormal modification of m6A methylation exists in synovial tissues and FLSs of RA patients, and inhibition of METTL3 can reduce synovitis and bone destruction. Our findings suggest that m6A methylation might control FLS-mediated tumor-like phenotype, and be a novel target for RA treatment.

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Brucine is a weak alkaline indole alkaloid with wide pharmacological activities and has been identified to protect against rheumatoid arthritis (RA) process. Circular RNAs (circRNAs) are also reported to be involved in the pathogenesis of RA. Here, we aimed to probe the role and mechanism of Brucine and circ_0139658 in RA progression. The fibroblast-like synoviocytes of RA (RA-FLSs) were isolated for functional analysis. Cell proliferation, apoptosis, invasion, migration, as well as inflammatory response were evaluated by CCK-8 assay, EdU assay, flow cytometry, transwell assay, and ELISA analysis, respectively. qRT-PCR and western blotting analyses were utilized to measure the levels of genes and proteins. The binding between miR-653-5p and circ_0139658 or Yin Yang 1 (YY1), was verified using dual-luciferase reporter and RNA pull-down assays. Brucine suppressed the proliferation, migration, and invasion of RA-FLSs, and alleviated inflammation by reducing the release of pro-inflammatory factors and macrophage M1 polarization. RA-FLSs showed increased circ_0139658 and YY1 levels and decreased miR-653-5p levels. Circ_0139658 is directly bound to miR-653-5p to regulate YY1 expression. Brucine treatment suppressed circ_0139658 and YY1 expression but increased YY1 expression in RA-FLSs. Functionally, circ_0139658 overexpression reversed the suppressing effects of Brucine on RA-FLS dysfunction and inflammation. Moreover, circ_0139658 silencing alleviated the dysfunction and inflammation in RA-FLSs, which were reverted by YY1 overexpression. Brucine suppressed the proliferation, migration, invasion, and inflammation in RA-FLSs by decreasing YY1 via circ_0139658/miR-653-5p axis.

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OBJECTIVES: Rheumatoid arthritis (RA) is a chronic inflammatory joint disease, which might trigger cartilage, bone damage, and disability. Recent studies have suggested that Tetramethylpyrazine (TMP), an alkaloid monomer isolated from the rhizome of the traditional herbal medicine Ligusticum wallichii Franch, exerts a broad spectrum of pharmacological properties, containing anti-inflammatory. This study aimed to analyze the role and underlying mechanism of TMP in RA. METHODS: Under Hypoxia condition, RA-Fibroblast-like synoviocyte (FLS) were treated with TMP at different doses. Cell viability, proliferation, cell cycle progression, and migration were detected using Cell Counting Kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay, flow cytometry assay, wound healing assay, and transwell assay. Cyclin D1, Proliferating cell nuclear antigen (PCNA), Matrix metalloproteinase-2 (MMP2), MMP9, and hypoxia-inducible factor-1α (HIF-1α) protein levels were measured using western blot assay. Interleukin-6 (IL-6) and IL-8 were evaluated using ELISA. Circular RNA (circRNA) hsa_circ_0005178 (circCDC42BPB), CDC42BPB, and HIF-1α expression were determined using real-time quantitative polymerase chain reaction (RT-qPCR). Binding between HIF-1α and CDC42BPB promoter was predicted by JASPAR and verified using dual-luciferase reporter and Chromatin immunoprecipitation (ChIP) assays. RESULTS: TMP might hinder FLS proliferation, cycle progression, migration, and inflammatory response under hypoxic conditions. CircCDC42BPB expression was increased in RA patients and RA-FLSs treated with hypoxia, while its level was obviously reduced in RA-FLSs treated with hypoxia and TMP. TMP might abolish hypoxia-induced circCDC42BPB expression. Upregulation of circCDC42BPB might partially overturn the repression of TMP on hypoxia-caused RA-FLS damage. TMP might regulate circCDC42BPB level via HIF-1α in RA-FLSs under hypoxic conditions. CONCLUSION: TMP might block RA-FLS injury partly via regulating the HIF-1α- circCDC42BPB pathway, providing a promising therapeutic target for RA.

HIGHLIGHTS: • TMP suppressed hypoxia-induced RA-FLS growth and inflammatory response.• TMP might repress circCDC42BPB expression in RA-FLSs under hypoxic conditions.• TMP might inhibit HIF-1α-induced circCDC42BPB transcription under hypoxic conditions.

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OBJECTIVE: This 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. METHODS: Immunofluorescence 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. RESULTS: AH 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. CONCLUSION: AH 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.

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

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BACKGROUND: Circular RNAs (circRNAs) have emerged as vital regulators in the development of rheumatoid arthritis (RA). In this study, we aimed to explore the functions and mechanisms of circ_0001947 in RA. METHODS: The expression of circ_0001947, microRNA-671-5p (miR-671-5p) and signal transducer and activator of transcription 3 (STAT3) was determined by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. Cell Counting Kit-8 (CCK-8) assay, 5'-ethynyl-2'-deoxyuridine (EdU) assay, flow cytometry analysis, transwell assay and wound-healing assay were performed to assess cell proliferation, apoptosis, invasion and migration. The concentrations of inflammatory factors were examined with enzyme-linked immunosorbent assay (ELISA) kits. Dual-luciferase reporter assay was used to analyze the relationships of circ_0001947, miR-671-5p and STAT3. RESULTS: Circ_0001947 was upregulated in RA patients and RA-FLSs. Knockdown of circ_0001947 repressed cell proliferation, invasion, migration and inflammatory response and facilitated apoptosis in RA-FLSs. Circ_0001947 served as the sponge for miR-671-5p and the inhibitory effect of circ_0001947 in RA-FLS progression was reversed by miR-671-5p inhibition. STAT3 was the target gene of miR-671-5p. MiR-671-5p overexpression restrained RA-FLS growth, invasion, migration and inflammation and promoted apoptosis, but STAT3 upregulation reversed the impacts. CONCLUSION: Circ_0001947 contributed to the progression of RA-FLSs by elevating STAT3 through adsorbing miR-671-5p.

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Activated fibroblast-like synoviocytes (FLSs) play a crucial role in the pathogenesis and progression of rheumatoid arthritis (RA). It is urgent to develop new drugs that can effectively inhibit the abnormal activation of RA-FLS. In our study, the RA-FLS cell line, MH7A, and mice with collagen-induced arthritis (CIA) were used to evaluate the effect of paclitaxel (PTX). Based on the results, PTX inhibited the migration of RA-FLS in a dose-dependent manner and significantly reduced the spontaneous expression of IL-6, IL-8, and RANKL mRNA and TNF-α-induced transcription of the IL-1 ß, IL-8, MMP-8, and MMP-9 genes. However, PTX had no significant effect on apoptosis in RA-FLS. Mechanistic studies revealed that PTX significantly inhibited the TNF-α-induced phosphorylation of ERK1/2 and JNK in the mitogen-activated protein kinase (MAPK) pathway and suppressed the TNF-α-induced activation of AKT, p70S6K, 4EBP1, and HIF-1α in the AKT/mTOR pathway. Moreover, PTX alleviated synovitis and bone destruction in CIA mice. In conclusion, PTX inhibits the migration and inflammatory mediator production of RA-FLS by targeting the MAPK and AKT/mTOR signaling pathways, which provides an experimental basis for the potential application in the treatment of RA.

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BACKGROUND: Isopsoralen (IPRN), one of the active ingredients of Psoralea corylifolia Linn, has anti-inflammatory properties. We attempted to investigate the inhibitory effects of IPRN on rheumatoid arthritis (RA) and characterize its potential mechanism. METHODS: RA fibroblast-like synoviocytes (FLSs) and mice with collagen-induced arthritis (CIA) were used as in vitro and in vivo models to analyze the antiarthritic effect of IPRN. Histological analysis of the inflamed joints from mice with CIA was performed using microcomputed tomography (micro-CT) and hematoxylin-eosin (HE) staining. RNA sequencing (RNA-Seq), network pharmacology analysis, molecular docking, drug affinity responsive target stability (DARTS) assay, and cellular thermal shift assay (CETSA) were performed to evaluate the targets of IPRN. RESULTS: IPRN ameliorated the inflammatory phenotype of RA FLSs by inhibiting their cytokine production, migration, invasion, and proangiogenic ability. IPRN also significantly reduced the severity of CIA in mice by decreasing paw thickness, arthritis score, bone damage, and serum inflammatory cytokine levels. A mechanistic study demonstrated that macrophage migration inhibitory factor (MIF), a key protein in the inflammatory process, was the specific target by which IPRN exerted its anti-inflammatory effects in RA FLSs. CONCLUSION: Our study demonstrates the antiarthritic effect of IPRN, which suggests the therapeutic potential of IPRN in RA.

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Rheumatoid arthritis is a chronic systemic disease characterised by an unknown aetiology of inflammatory synovitis. A large number of studies have shown that synoviocytes show tumour-like dysplasia in the pathological process of RA, and the changes in the expression of related cytokines are closely related to the pathogenesis of RA. In this thesis, a series of novel 3-(4-aminophenyl) coumarins containing different substituents were synthesised to find new coumarin anti-inflammatory drugs for the treatment of rheumatoid arthritis. The results of preliminary activity screening showed that compound 5e had the strongest inhibitory activity on the proliferation of fibroid synovial cells, and it also had inhibitory effect on RA-related cytokines IL-1, IL-6, and TNF-α. The preliminary mechanism study showed that compound 5e could inhibit the activation of NF-κB and MAPKs signal pathway. The anti-inflammatory activity of compound 5e in vivo was further determined in the rat joint inflammation model.

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ETHNOPHARMACOLOGICAL RELEVANCE: Ranunculus japonicus Thunb. (short for R. japonicus) is a topically applied herb with the activities of removing jaundice, nebula and edema, preventing malaria, stopping asthma, promoting diuresis and relieving pain. It was firstly recorded in Zhouhou Beiji Fang and has been used for the treatment of malaria, ulcers, carbuncle, jaundice, migraine, stomachache, toothache and arthritis for over 1800 years. AIM OF THE STUDY: This study aimed to uncover the potentially effective components of R. japonicus and the pharmacological mechanisms against rheumatoid arthritis (RA) by combing LC-MS and network pharmacology. MATERIALS AND METHODS: Firstly, the chemical constituents of R. japonicus were qualitatively identified by UPLC-ESI-LTQ-Orbitrap MS. Then we performed target prediction by PharmMapper, protein-protein interaction (PPI) analysis via String, GO and KEGG pathway enrichment analysis by DAVID and constructed the compound-target-pathway network using Cytoscape. Thirdly, crucial compounds in the network were quantitatively analyzed to achieve quality control of R. japonicus. Finally, the pharmacological activities of R. japonicus and two potentially bioactive ingredients were validated in RA-FLSs (Rheumatoid Arthritis Fibroblast-like Synoviocytes) in vitro. RESULTS: Overall fifty-four ingredients of R. japonicus were identified and forty-five components were firstly discovered in R. japonicus. Among them, twenty-seven validated compounds were predicted to act on twenty-five RA-related targets and they might exhibit therapeutic effects against RA via positive regulation of cell migration, etc. Nine potentially bioactive components of R. japonicus which played important roles in the compound-target-pathway network were simultaneously quantified by an optimized UPLC-ESI-Triple Quad method. In vitro, compared to control group, R. japonicus extract, berberine and yangonin significantly inhibited the migration capacity of RA-FLSs after 24 h treatment. CONCLUSION: This study clarified that R. japonicus and the bioactive ingredients berberine and yangonin might exert therapeutic actions for RA via suppressing the aggressive phenotypes of RA-FLSs through combined LC-MS technology and network pharmacology tools for the first time. The present research provided deeper understanding into the chemical profiling, pharmacological activities and quality control of R. japonicus and offered reference for further scientific research and clinical use of R. japonicus in treating RA.

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Flares of joint inflammation and resistance to currently available biologic therapeutics in rheumatoid arthritis (RA) patients could reflect activation of innate immune mechanisms. Herein, we show that a TLR7 GU-rich endogenous ligand, miR-Let7b, potentiates synovitis by amplifying RA monocyte and fibroblast (FLS) trafficking. miR-Let7b ligation to TLR7 in macrophages (MΦs) and FLSs expanded the synovial inflammatory response. Moreover, secretion of M1 monokines triggered by miR-Let7b enhanced Th1/Th17 cell differentiation. We showed that IRAK4 inhibitor (i) therapy attenuated RA disease activity by blocking TLR7-induced M1 MΦ or FLS activation, as well as monokine-modulated Th1/Th17 cell polarization. IRAK4i therapy also disrupted RA osteoclastogenesis, which was amplified by miR-Let7b ligation to joint myeloid TLR7. Hence, the effectiveness of IRAK4i was compared with that of a TNF inhibitor (i) or anti-IL-6R treatment in collagen-induced arthritis (CIA) and miR-Let7b-mediated arthritis. We found that TNF or IL-6R blocking therapies mitigated CIA by reducing the infiltration of joint F480+iNOS+ MΦs, the expression of certain monokines, and Th1 cell differentiation. Unexpectedly, these biologic therapies were unable to alleviate miR-Let7b-induced arthritis. The superior efficacy of IRAK4i over anti-TNF or anti-IL-6R therapy in miR-Let7b-induced arthritis or CIA was due to the ability of IRAK4i therapy to restrain the migration of joint F480+iNOS+ MΦs, vimentin+ fibroblasts, and CD3+ T cells, in addition to negating the expression of a wide range of monokines, including IL-12, MIP2, and IRF5 and Th1/Th17 lymphokines. In conclusion, IRAK4i therapy may provide a promising strategy for RA therapy by disconnecting critical links between inflammatory joint cells.

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Rheumatoid arthritis (RA) is a chronic and progressive autoimmune disease in which activated RA fibroblast-1ike synoviocytes (RA-FLSs) are one of the main factors responsible for inducing morbidity. Previous reports have shown that RA-FLSs have proliferative features similar to cancer cells, in addition to causing cartilage erosion that eventually causes joint damage. Thus, new therapeutic strategies and drugs that can effectively contain the abnormal hyperplasia of RA-FLSs and restrain RA development are necessary for the treatment of RA. Tanshinone IIA (Tan IIA), one of the main phytochemicals isolated from Salvia miltiorrhiza Bunge, is capable of promoting RA-FLS apoptosis and inhibiting arthritis in an AIA mouse model. In addition, RA patients treated at our clinic with Tan IIA showed significant improvements in their clinical symptoms. However, the details of the molecular mechanism by which Tan IIA effects RA are unknown. To clarify this mechanism, we evaluated the antiproliferative and inhibitory effects of proinflammatory factor production caused by Tan IIA to RA-FLSs. We demonstrated that Tan IIA can restrict the proliferation, migration, and invasion of RA-FLSs in a time- and dose-dependent manner. Moreover, Tan IIA effectively suppressed the increase in mRNA expression of some matrix metalloproteinases and proinflammatory factors induced by TNF-α in RA-FLSs, resulting in inflammatory reactivity inhibition and blocking the destruction of the knee joint. Through the integration of network pharmacology analyses with the experimental data obtained, it is revealed that the effects of Tan IIA on RA can be attributed to its influence on different signaling pathways, including MAPK, AKT/mTOR, HIF-1, and NF-kB. Taken together, these data suggest that the compound Tan IIA has great therapeutic potential for RA treatment.

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In rheumatoid arthritis(RA) pathogenesis, activated RA fibroblast-like synoviocytes (RA-FLSs) exhibit similar proliferative features as tumor cells and subsequent erosion to cartilage will eventually lead to joint destruction. Therefore, it is imperative to search for compounds, which can effectively inhibit the abnormal activation of RA-FLSs, and retard RA progression.3'3-Diindolylmethane (DIM), the major product of the acid-catalyzed oligomerization of indole-3-carbinol from cruciferous vegetables, has been reported to be functionally relevant to inhibition of migration, invasion and carcinogenesis in some solid tumors. In this study, we explored the anti-proliferation, anti-metastasis and anti-inflammation effects of DIM on RA-FLSs as well as the underlying molecular mechanisms. To do this, primary RA-FLSs were isolated from RA patients and an animal model. Cell proliferation, migration and invasion were measured using CCK-8, scratch, and Transwell assays, respectively. The effects of DIM on Matrix metalloproteinases (MMPs) and some inflammatory factors mRNA and key molecules such as some inflammatory factors and those involved in aberrantly-activated signaling pathway in response to tumor necrosis factor α(TNF-α), a typical characteristic mediator in RA-FLS, were quantitatively measured by real-time PCR and western blotting. Moreover, the effect of DIM on adjuvant induced arthritis(AIA) models was evaluated with C57BL/6 mice in vivo. The results showed that DIM inhibited proliferation, migration and invasion of RA-FLS in vitro. Meanwhile, DIM dramatically suppressed TNF-α-induced increases in the mRNA levels of MMP-2, MMP-3, MMP-8, and MMP-9; as well as the proinflammatory factors IL-6, IL-8, and IL-1ß. Mechanistic studies revealed that DIM is able to suppress phosphorylated activation not only of p38, JNK in MAPK pathway but of AKT, mTOR and downstream molecules in the AKT/mTOR pathway. Moreover, DIM treatment decreased expression levels of proinflammatory cytokines in the serum and alleviated arthritis severity in the knee joints of AIA mice. Taken together, our findings demonstrate that DIM could inhibit proliferation, migration and invasion of RA-FLSs and reduce proinflammatory factors induced by TNF-α in vitro by blocking MAPK and AKT/mTOR pathway and prevent inflammation and knee joint destruction in vivo, which suggests that DIM might have therapeutic potential for RA.

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AIMS: Hypoxia is implicated in the pathogenesis of rheumatoid arthritis (RA), contributing to the tumor-like phenotypes of RA fibroblast-like synoviocytes (RA-FLSs). Andrographolide is the main bioactive component of Andrographis paniculata, an herbal medicine that shows therapeutic benefits in RA patients. Here, we explored the effects of andrographolide on hypoxia-induced migration and invasion of RA-FLSs. MATERIALS AND METHODS: RA-FLSs were exposed to hypoxia in the presence or absence of andrographolide and cell migration and invasion were tested by Transwell assays. The expression of hypoxia-inducible factor-1 alpha (HIF-1α), matrix metalloproteinase (MMP)-1, MMP-3 and MMP-9 was measured by semi-quantitative reverse transcription polymerase chain reaction and Western blot analysis. HIF-1α DNA binding activity was assessed by electrophoretic mobility shift assay. The effects of overexpression of exogenous HIF-1α on the action of andrographolide in RA-FLSs were investigated. KEY FINDINGS: Andrographolide inhibited FLS migration and invasion under hypoxic conditions in a dose-dependent manner. The upregulation of MMP-1, MMP-3 and MMP-9 in response to hypoxia was significantly (P<0.05) attenuated by andrographolide. Moreover, the expression and DNA binding activity of HIF-1α were dose-dependently decreased in andrographolide-treated cells under hypoxic conditions. Overexpression of HIF-1α almost completely reversed the suppressive effects of andrographolide on the migration, invasion and MMP expression of hypoxic RA-FLSs. SIGNIFICANCE: These results indicate the ability of andrographolide to attenuate hypoxia-induced invasiveness of RA-FLSs via inhibition of HIF-1α signaling, and warrant further exploration of andrographolide for the treatment of RA.

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