lncRNA GAS5 suppresses rheumatoid arthritis by inhibiting miR-361-5p and increasing PDK4.

Rheumatoid arthritis (RA) is an inflammatory disease that causes hyperplasia of synovial tissue and cartilage destruction. This research was to investigate the effects of lncRNA GAS5/miR-361-5p/PDK4 on rheumatoid arthritis. By qRT-PCR, GAS5 and PDK4 were found to be overexpressed in synovial tissue, fibroblast-like synoviocytes of RA patients and LPS-induced chondrocytes, while the miR-361-5p expression was significantly reduced. GAS5 overexpression resulted in a decrease in the proliferation and Bcl-2 protein expression, and an increase in the Bax protein level. On the contrary, miR-361-5p sponged by GAS5 could accelerate chondrocyte proliferation, inhibit apoptosis. PDK4 targeted by miR-361-5p could inhibit RA, and partially eliminated the effect of miR-361-5p on RA. Our study suggested that GAS5 suppressed RA by competitively adsorbing miR-361-5p to modulate PDK4 expression.

Carvacrol suppresses inflammatory responses in rheumatoid arthritis fibroblast-like synoviocytes.

BACKGROUND: Fibroblast-like synoviocytes (FLSs) play an essential role in the chronic inflammatory process of rheumatoid arthritis (RA). Carvacrol is a natural monoterpenic phenol that retains significant anti-inflammatory activity. However, the effect of carvacrol on inflammatory response in RA-FLSs has not yet been reported. The present study aimed to investigate the role of carvacrol in lipopolysaccharides (LPS)-induced inflammatory response in human RA-FLSs. METHODS: Cell viability and proliferation were measured by MTT and Cell Counting Kit-8 assays, respectively. The migration was detected by transwell assay. The production of inflammatory cytokines and matrix metalloproteinases (MMPs) were analyzed by enzyme-linked immunosorbent assay. The expressions of toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), NF-κB, p38, p-p38, ERK1/2, p-ERK1/2, c-Jun N-terminal kinase (JNK), and p-JNK were detected by Western blot analysis. RESULTS: Carvacrol-inhibited LPS-induced cell proliferation and migration of RA-FLSs. The production of inflammatory cytokines, including tumor necrosis factor alpha, interleukin (IL)- 6, and IL-8, was reduced by carvacrol in LPS-induced RA-FLSs. Meanwhile, the induction of MMPs, including MMP-1, MMP-3, and MMP-13, caused by LPS stimulation was inhibited by carvacrol in RA-FLSs. Furthermore, carvacrol prevented LPS-induced activation of the TLR4/MyD88/NF-κB, p38, and ERK1/2 pathways in RA-FLSs. CONCLUSIONS: Carvacrol-mitigated LPS-induced cell proliferation, migration, and inflammation in RA-FLSs. The TLR4/MyD88/NF-κB, p38 and ERK1/2 pathways might be involved in the protective effect of carvacrol.

Circular RNA circ_0130438 suppresses TNF-α-induced proliferation, migration, invasion and inflammation in human fibroblast-like MH7A synoviocytes by regulating miR-130a-3p/KLF9 axis.

BACKGROUND: Circular RNAs (circRNAs) can play a critical role in rheumatoid arthritis (RA) pathogenesis by involving gene regulation by competing for shared microRNAs (miRNAs), a family of small noncoding RNAs. MiR-130a-3p is a disease-related miRNA and Kruppel-like factor 9 (KLF9) is a zinc finger transcription factor, which are involved in RA pathogenesis. Here, we identified the action of circRNA circ_0130438 in regulating fibroblast-like synoviocytes (FLSs) stimulated by tumor necrosis factor α (TNF-α). METHODS: The direct relationship between miR-130a-3p and circRNA circ_0130438 or KLF9 was predicted by bioinformatics analysis and examined by a dual-luciferase reporter or RNA immunoprecipitation (RIP) assay. CircRNA circ_0130438, miR-130a-3p and KLF9 factor expression levels were gauged by a quantitative real-time PCR (qRT-PCR) or a western blot method. Cell proliferation ability was analyzed by a 5-Ethynyl-2'-Deoxyuridine (EdU) staining assay. The transwell assay was used to evaluate cell migration and invasion capacities. The production levels of interleukin-1ß (IL)-1ß, IL-6 and IL-8 were assessed by enzyme-linked immunosorbent assay (ELISA). RESULTS: The level of circRNA circ_0130438 was reduced in RA tissues (P = 0.0001) and FLSs isolated from RA tissues (P = 0.0001) compared with corresponding normal controls. Exposure of human fibroblast-like MH7A synoviocytes to TNF-α suppressed circRNA circ_0130438 expression (P < 0.0001). In contrast, the elevated expression of circRNA circ_0130438 suppressed the TNF-α-induced proliferation (P = 0.0047) and migration (P = 0.0023) of MH7A cells, as well as their pro-inflammatory cytokines (IL-1ß, IL-6 and IL-8) production (P < 0.0001, P < 0.0001 and P < 0.0001). The circRNA circ_0130438 contained a miR-130a-3p binding site. Furthermore, the increase of miR-130-3p in TNF-α-stimulated MH7A cells reversed the effects of circRNA circ_0130438 elevation on cell proliferation (P = 0.0006), migration (P = 0.0406) and pro-inflammatory cytokines (IL-1ß, IL-6 and IL-8) production (P = 0.0036, P < 0.0001 and P = 0.0004), indicating that miR-130a-3p was a functional mediator of circRNA circ_0130438 regulation. We also documented that KLF9 was a direct target and downstream effector of miR-130a-3p. Importantly, circRNA circ_0130438 enhanced KLF9 expression (P < 0.0001) in TNF-α-stimulated MH7A cells by functioning as a competing endogenous RNA (ceRNA) for miR-130a-3p (P = 0.0004). CONCLUSION: Our findings demonstrate that the elevated expression of circRNA circ_0130438 suppresses TNF-α-induced migration, proliferation and pro-inflammatory cytokines (IL-1ß, IL-6 and IL-8) production of human MH7A cells by enhancing KLF9 expression by operating as a ceRNA for miR-130a-3p.

Inhibition of lncRNA NEAT1 induces dysfunction of fibroblast-like synoviocytes in rheumatoid arthritis via miRNA-338-3p-mediated regulation of glutamine metabolism.

BACKGROUND: Rheumatoid arthritis (RA) is a systemic chronic autoimmune disease; cellular glutamine metabolism in fibroblast-like synoviocytes (FLSs) of RA was known to be essential for RA pathogenesis and progression. NEAT1, a long non-coding RNA, functions as an oncogene in diverse cancers. The exact roles and molecular mechanisms of NEAT1 in fibroblast-like synoviocytes (FLSs) of RA patients are unknown. METHODS: Expression of NEAT1 and miR-338-3p was measured by qRT-PCR. lncRNA-miRNA and miRNA-mRNA interactions were predicted from starBase and validated by RNA pull-down and luciferase assay. The glutamine metabolism of FLSs was evaluated by glutamine uptake and glutaminase activity. Cell death in FLSs in response to H2O2 was assessed by MTT and Annexin V assays. RESULTS: NEAT1 was significantly upregulated, and miR-338-3p was significantly downregulated in FLSs from RA patients compared to normal FLSs. Silencing of NEAT1 and overexpression of miR-338-3p suppressed glutamine metabolism in FLSs-RA and promoted H2O2-induced apoptosis. Bioinformatics analysis showed that NEAT1 sponges miR-338-3p to form competing endogenous RNA (ceRNAs), which was verified by RNA pull-down assay and luciferase assay FLSs-RA had an increased rate of glutamine metabolism compared to normal FLSs increased compared to normal FLSs. The results confirmed that GLS (Glutaminase), a key enzyme in glutamine metabolism, is a direct target of miR-338-3p in FLSs-RA. miR-338-3p inhibition of glutamine metabolism was verified by rescue experiments verified. Finally, restoration of miR-338-3p in FLSs-RA expressing NEAT1 overcomes NEAT1-promoted glutamine metabolism and resistance to apoptosis. CONCLUSIONS: This study reveals the essential role and molecular targets of NEAT1-regulated glutamine metabolism and FLSs-RA dysfunction in fibroblast-like synoviocytes of RA and indicates that blocking the molecular pathway via non-coding RNAs may be beneficial for RA patients.

Long Non-Coding RNA NR-133666 Promotes the Proliferation and Migration of Fibroblast-Like Synoviocytes Through Regulating the miR-133c/MAPK1 Axis.

Long non-coding RNA (lncRNA) is involved in the regulation of rheumatoid arthritis (RA) and many other diseases. In this study, a new lncRNA, NR-133666, was identified to be highly expressed in the adjuvant-induced arthritis rat model using the Agilent lncRNA microarray assay. qRT-PCR verified that NR-133666 was upregulated in fibroblast-like synoviocyte of a collagen-induced arthritis (CIA) rat model. Fluorescence in situ hybridization analysis showed that NR-133666 is mainly expressed in the cytoplasm of collagen-induced arthritis FLS. MTT assay and EdU staining results showed that the proliferation of CIA FLS was inhibited after NR-133666 was knocked down, and the wound healing assay showed that the migration of CIA FLS was also suppressed. Dual luciferase detection was used to confirm the relationship among NR-133666, miR-133c and MAPK1. MAPK1 is the target gene of miR-133c, where NR-133666 acts as a sponge of miR-133c to reduce the inhibitory effect of miR-133c on MAPK1. Overexpression of NR-133666 and MAPK1 can promote the proliferation and migration of CIA FLS, and overexpression of miR-133c can reverse this phenomenon. Western blot indicated that it may be related to the ERK/MAPK signaling pathway. Collectively, we identified that lncRNA NR-133666 acted as a miR-133c sponge that can promote the proliferation and migration of CIA FLS through regulating the miR-133c/MAPK1 axis.

cGAS/STING signaling in the regulation of rheumatoid synovial aggression.

Background: Fibroblast-like synoviocytes (FLSs) play a critical role in promoting synovial aggression and joint destruction in rheumatoid arthritis (RA). Cyclic GMP-AMP synthase (cGAS)/stimulator of interferon gene (STING) signaling plays an important role in controlling a series of cellular biological processes. However, it is still unclear whether cGAS/STING signaling regulates rheumatoid synovial aggression. Methods: Cell migration and invasion were detected using a Transwell chamber. Gene expression was measured using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and protein expression was detected by western blotting. Reactive oxygen species (ROS) levels were measured by 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) probe. F-actin staining and immunofluorescence assays were used to investigate lamellipodia formation and nuclear translocation, respectively. A severe combined immunodeficiency (SCID) mouse model was established to observe the migration and invasion of RA FLSs in vivo. Results: Our results showed that cytosolic double-stranded DNA (dsDNA)-induced cGAS/STING activation promoted the in vitro migration and invasion of RA FLSs. Moreover, RA FLSs treated with cGAS or STING short hairpin RNA (shRNA) exhibited reduced invasion into cartilage in the SCID model. Mechanistically, we determined that cGAS/STING activation leads to increased mitochondrial ROS levels, and thereby increases phosphorylation of mammalian sterile 20-like kinase 1 (MST1), a core component of the Hippo pathway, subsequently promoting activation of forkhead box1 (FOXO1). MST1 and FOXO1 knockdown also diminished the migration and invasion of RA FLSs. Conclusions: Our findings suggest that cGAS/STING signaling has an important role in regulating rheumatoid synovial aggression and that targeting cGAS/STING may represent a novel potential therapy for RA.

Mucin 1 aggravates synovitis and joint damage of rheumatoid arthritis by regulating inflammation and aggression of fibroblast-like synoviocytes.

AIMS: To explore the synovial expression of mucin 1 (MUC1) and its role in rheumatoid arthritis (RA), as well as the possible downstream mechanisms. METHODS: Patients with qualified synovium samples were recruited from a RA cohort. Synovium from patients diagnosed as non-inflammatory orthopaedic arthropathies was obtained as control. The expression and localization of MUC1 in synovium and fibroblast-like synoviocytes were assessed by immunohistochemistry and immunofluorescence. Small interfering RNA and MUC1 inhibitor GO-203 were adopted for inhibition of MUC1. Lysophosphatidic acid (LPA) was used as an activator of Rho-associated pathway. Expression of inflammatory cytokines, cell migration, and invasion were evaluated using quantitative real-time polymerase chain reaction (PCR) and Transwell chamber assay. RESULTS: A total of 63 RA patients and ten controls were included. Expression of MUC1 was observed in both the synovial lining and sublining layer. The percentage of MUC1+ cells in the lining layer of synovium was significantly higher in RA than that in control, and positively correlated to joint destruction scores of RA. Meanwhile, MUC1+ cells in the sublining layer were positively correlated to the Krenn subscore of inflammatory infiltration. Knockdown of MUC1, rather than GO-203 treatment, ameliorated the expression of proinflammatory cytokines, cell migration, and invasion of rheumatoid synoviocytes. Knockdown of MUC1 decreased expression of RhoA, Cdc42, and Rac1. Treatment with LPA compromised the inhibition of migration and invasion, but not inflammation, of synoviocytes by MUC1 knockdown. CONCLUSION: Upregulated MUC1 promotes the aggression of rheumatoid synoviocytes via Rho guanosine triphosphatases (GTPases), thereby facilitating synovitis and joint destruction during the pathological process of RA.Cite this article: Bone Joint Res 2022;11(9):639-651.

Destructive Roles of Fibroblast-like Synoviocytes in Chronic Inflammation and Joint Damage in Rheumatoid Arthritis.

Fibroblast-like synoviocytes (FLSs) are important non-immune cells located mostly in the inner layer of the synovium. Indeed, these cells are specialized mesenchymal cells, implicated in collagen homeostasis of the articular joint and provide extracellular matrix (ECM) materials for cartilage and contribute to joint destruction via multiple mechanisms. RA FLS interactions with immune and non-immune cells lead to the development and organization of tertiary structures such as ectopic lymphoid-like structures (ELSs), tertiary lymphoid organs (TLOs), and secretion of proinflammatory cytokines. The interaction of RA FLS cells with immune and non-immune cells leads to stimulation and activation of effector immune cells. Pathological role of RA FLS cells has been reported for many years, while molecular and cellular mechanisms are not completely understood yet. In this review, we tried to summarize the latest findings about the role of FLS cells in ELS formation, joint destruction, interactions with immune and non-immune cells, as well as potential therapeutic options in rheumatoid arthritis (RA) treatment. Our study revealed data about interactions between RA FLS and immune/non-immune cells as well as the role of RA FLS cells in joint damage, ELS formation, and neoangiogenesis, which provide useful information for developing new approaches for RA treatment.

PLD1 knockdown reduces metastasis and inflammation of fibroblast-like synoviocytes in rheumatoid arthritis by modulating NF-κB and Wnt/ß-catenin pathways.

Considered as an autoimmune disease, rheumatoid arthritis (RA) is an chronic inflammatory disorder that causes inflammation of the joints. This study is performed with the aim to clarify the expression of phospholipase D1 (PLD1) in RA and its specific regulation role of RA as well as the underlying mechanisms. In this study, synovial tissue samples were collected from RA patients, and RA-fibroblast-like synoviocytes (FLSs) were subsequently isolated. The expression levels of PLD1 and pathway-related proteins were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blotting or immunohistochemistry (IHC). Upon shPLD1 treatment, cell viability, proliferation, migration, invasion, and the level of inflammation-related factors were measured by Cell Counting Kit-8 (CCK-8), Edu, wound healing, Transwell and enzyme-linked immunosorbent assay (ELISA). Furthermore, C-reactive protein (CRP), rheumatoid factor (RF), arthritis score and synovial tissue lesions were assessed by collecting the blood or tissues from collagen induced arthritis (CIA) model rats. Our results showed that PLD1 level was increased in RA synovial tissues. Cell viability, proliferation, migration, invasion, and the level of inflammatory factors were reduced upon PLD1 knockdown in RA-FLSs. Moreover, p-IκBα/IκBα, ß-catenin, p-IKKß/IKKß and TCF-4 were inhibited under PLD1 knockdown treatment. PLD1 knockdown alleviated the collagen-induced addition of arthritis score, CRP and RF, as well as the filling of inflammatory cells and proliferation of synovium in CIA model rat. To sum up, knockdown of PLD1 could reduce RA-FLSs metastasis as well as inflammatory response by modulating the activity of NF-κB and Wnt/ß-catenin pathways.

Ginkgolide B attenuates collagen-induced rheumatoid arthritis and regulates fibroblast-like synoviocytes-mediated apoptosis and inflammation.

BACKGROUND: Rheumatoid arthritis (RA) is a systemic disease characterized by chronic synovial infiltration and proliferation, cartilage destruction, and joint injury. Ginkgolide B (GB) is an extract of the leaves of Ginkgo biloba, and pharmacological studies have shown that it has anti-inflammatory and anti-apoptotic activities. The purpose of this study was to investigate the anti-RA properties of GB. METHODS: In vivo, we established a collagen II-induced arthritis (CIA) mouse model. Mice were divided into five groups (n=10): sham, CIA, GB (10 µM), GB (20 µM), and GB (40 µM). We measured arthritis score, synovial histopathological change, and peripheral blood cytokine levels. In vitro, we used lipopolysaccharide (LPS)-induced-fibroblast-like synoviocytes (RA-FLSs) as the study subject. Cell viability, apoptosis, and inflammatory cytokines levels were detected by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, flow cytometry, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), respectively. Finally, the protein expression of wingless-type family member 5A (Wnt5a), c-Jun N-terminal kinase (JNK), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 were detected by Western blot. RESULTS: Arthritis scores, synovial hyperplasia, and cartilage and bone destruction were significantly ameliorated by GB. Additionally, GB decreased the serum levels of interleukin (IL)-1ß, IL-6, monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor alpha (TNF-α), matrix metalloproteinase (MMP)-3 and MMP-13, and increased IL-10. In vitro, we found that GB remarkably inhibited RA-FLSs viability at 24 or 48 h in a concentration-dependent manner. The apoptotic ratio was reduced by GB, and it increased the expression of cleaved-Caspase-3 and Bax while decreasing Bcl-2 expression in RA-FLSs. Furthermore, GB attenuated the progression of inflammation by mediating inflammatory cytokine release and MMPs gene expression. Meanwhile, GB inactivated the expression levels of Wnt5a, phosphorylated (p)-JNK, and p-P65 in the synovial tissues and RA-FLSs. CONCLUSIONS: This study was the first to demonstrate that the anti-RA effect of GB is related to reducing articular cartilage and bone destruction, inducing RA-FLSs apoptosis, and regulating inflammatory cytokine release and the Wnt5a/JNK/NF-κB axis. All the findings highlight that GB might provide a novel treatment approach for RA.

LncRNA NEAT1 Targets Fibroblast-Like Synoviocytes in Rheumatoid Arthritis via the miR-410-3p/YY1 Axis.

LncRNA NEAT1 functions as an oncogene in multiple human cancers. However, its expression and role in fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA) remain unclear. Thus, we investigated the expression of NEAT1 in synovial tissues and FLSs in RA, to determine its role in the development of RA. Quantitative real-time polymerase chain reaction was used to measure the expression of NEAT1. FLS proliferation was evaluated using cell proliferation assays. Flow cytometry was used to analyze cell cycle progression and apoptosis in FLSs. Binding between NEAT1 and miR-410-3p was demonstrated by dual-luciferase assays. We found that NEAT1 was upregulated in synovial tissues and FLSs in RA. Upregulation of NEAT1 promoted cell proliferation, induced S-to G2/M phase transition, and suppressed apoptosis in RA FLSs. NEAT1 directly bound to and negatively modulated miR-410-3p expression, while positively regulating YinYang 1(YY1; a miR-410-3p target). Inhibiting miR-410-3p and upregulating YY1 partially restored the inhibitory role in cell viability induced by the depletion of NEAT1 in RA FLSs. Besides pro-proliferative and anti-apoptotic roles, upregulation of NEAT1 promoted migration, invasion, and inflammatory cytokines secretion in RA FLSs. Taken together, our results suggest that the NEAT1 may serve as a novel diagnostic and therapeutic target for patients with RA.

Protocatechuic acid inhibits proliferation, migration and inflammatory response in rheumatoid arthritis fibroblast-like synoviocytes.

Rheumatoid arthritis (RA) is a chronic joint inflammatory disease that is closely associated with dysregulation of fibroblast-like synoviocytes (FLSs). Protocatechuic acid (PCA), a phenolic compound of anthocyanins, has been proven to possess anti-inflammatory activity. However, the role of PCA in RA has not been investigated. In the present study, we aimed to explore the effects of PCA on the RA-FLSs. The results showed that PCA suppressed the proliferation, invasion, and migration of RA-FLSs in a dose-dependent manner. PCA treatment also inhibited the expressions of matrix metalloproteinase (MMP)-3 and MMP-13, as well as the secretion of inflammatory cytokines including TNF-α, IL-1ß, IL-6 in RA-FLSs. Moreover, cell apoptosis of RA-FLSs was significantly induced by PCA treatment. PCA was found to repress the activation of NF-κB signalling, which was evidenced by the decreased expression of p-p65 and increased expression of IκBα. Furthermore, PCA significantly decreased the phosphorylation levels of Akt and mTOR in RA-FLSs. In conclusion, the results indicated that PCA exhibited an inhibitory effect on RA-FLSs via inhibiting the NF-κB and Akt/mTOR signalling pathways. These findings supported the concept that PCA might be a therapeutic agent for RA treatment.

The P2X7 receptor (P2X7R)-specific antagonist A804598 inhibits inflammatory reaction in human fibroblast-like synoviocytes.

Activation of the P2X7 receptor (P2X7R) has been found to increase expression of tumor necrosis factor-α (TNF-α) in the joints and synovial lining of patients with rheumatoid arthritis (RA). Increased expression of TNF-α promotes joint destruction through deterioration of type II collagen by matrix metalloproteinases (MMPs), expression of proinflammatory cytokines, oxidative stress, and activation of cellular signaling pathways. In the present study, we exposed fibroblast-like synoviocytes (FLSs) to TNF-α in the presence and absence of the P2X7R antagonist A804598. We then employed real time PCR and western blot analysis to analyze the mRNA and protein expression levels of P2X7R in both control and RA-FLSs. We confirmed that P2X7R is expressed on FLSs and is upregulated in RA-FLSs and FLSs exposed to TNF-α. Importantly, we also demonstrate the ability of P2X7R antagonism using A804598 to suppress oxidative stress, expression of interleukin (IL)-1ß, IL-6, MMP-1, MMP-3, MMP-13 as well as activation of the Janus family of tyrosine kinase/signal transducer and activator of transcription (JAK1/STAT3) proinflammatory signaling pathway. These findings implicate a novel role of antagonism of P2X7R as a target for the treatment and prevention of RA.

Dihydroartemisinin derivative DC32 inhibits inflammatory response in osteoarthritic synovium through regulating Nrf2/NF-κB pathway.

Synovitis is an aseptic inflammation that leads to joint effusion, pain and swelling. As one of the main drivers of pathogenesis in osteoarthritis (OA), the presence of synovitis contributes to pain, incidence and progression of OA. In our previous study, DC32 [(9α,12α-dihydroartemisinyl) bis(2'-chlorocinnmate)], a dihydroartemisinin derivative, was found to have an antirheumatic ability via immunosuppression, but the effect of DC32 on synovitis has not been fully illuminated. In this study, we chose to evaluate the effect and mechanism of DC32 on attenuating synovial inflammation. Fibroblast-like synoviocytes (FLSs) of papain-induced OA rats were isolated and cultured. And DC32 significantly inhibited the invasion and migration of cultured OA-FLSs, as well as the transcription of IL-6, IL-1ß, CXCL12 and CX3CL1 in cultured OA-FLSs measured by qPCR. DC32 remarkably inhibited the activation of ERK and NF-κB pathway, increased the expression of Nrf2 and HO-1 in cultured OA-FLSs detected by western blot. DC32 inhibited the degradation and phosphorylation of IκBα which further prevented the phosphorylation of NF-κB p65 and the effect of DC32 could be relieved by siRNA for Nrf2. In papain-induced OA mice, DC32 significantly alleviated papain-induced mechanical allodynia, knee joint swelling and infiltration of inflammatory cell in synovium. DC32 upregulated the mRNA expression of Type II collagen and aggrecan, and downregulated the mRNA expression of MMP2, MMP3, MMP13 and ADAMTS-5 in the knee joints of papain-induced OA mice measured by qPCR. The level of TNF-α in the serum and secretion of TNF-α in the knee joints were also reduced by DC32 in papain-induced OA mice. In conclusion, DC32 inhibited the inflammatory response in osteoarthritic synovium through regulating Nrf2/NF-κB pathway and attenuated OA. In this way, DC32 may be a potential agent in the treatment of OA.

Regulation of Immune Responses and Chronic Inflammation by Fibroblast-Like Synoviocytes.

Synovial tissue is a membranous non-immune organ lining joint cavities where it supports local immune responses, and functions directly and indirectly in joint destruction due to chronic inflammatory diseases such as rheumatoid arthritis (RA). Fibroblast-like synoviocytes (FLS), the dominant non-immune cells of synovial tissues, mainly contribute to joint destruction via multiple mechanisms. In RA, FLS respond to endogenous ligands of pattern recognition receptors (PRRs) and inflammatory cytokines as non-immune cells. In addition, FLS aid in the activation of immune responses by interacting with immune cells and by supporting ectopic lymphoid-like structure (ELS) formation in synovial tissues. Moreover, FLS directly cause the pathogenicity of RA i.e., joint deformities. Here, we describe new findings and review the mechanisms underlying the regulation of immune reactions by non-immune FLS and their roles in inflammatory diseases such as RA.

The protective effects of lixisenatide against inflammatory response in human rheumatoid arthritis fibroblast-like synoviocytes.

Rheumatoid arthritis (RA) is a major debilitating systemic disease characterized by chronic inflammation of the synovium and joint destruction. Despite major advancements in our understanding of RA in recent decades, it remains a disease of unknown etiology. To our knowledge, this is the first study exploring the effects of agonism of the glucagon-like peptide-1 (GLP-1) receptor using lixisenatide, a licensed drug used for the treatment of type II diabetes, on the pathological characteristics of RA in human fibroblast-like synoviocytes. Our findings indicate that lixisenatide inhibited the inflammatory response through downregulation of proinflammatory cytokines, such as tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8); inhibition of matrix metalloproteinases (MMPs); and blockade of cellular signaling pathways, including the c-Jun N-terminal kinase (JNK), activator protein 1 (AP-1), and nuclear factor κ B (NF-κB) pathways. Furthermore, lixisenatide improved oxidative stress, rescued mitochondrial membrane potential (ΔΨm), and prevented cell death in fibroblast-like synoviocytes. These findings suggest that agonism of the GLP-1 receptor using lixisenatide may serve as a novel therapeutic option for the treatment and prevention of RA.

Dihydroartemisinin derivative DC32 attenuates collagen-induced arthritis in mice by restoring the Treg/Th17 balance and inhibiting synovitis through down-regulation of IL-6.

Imbalance of Treg/Th17 and chronic synovitis characterized by the recruitment and infiltration of inflammatory cells are the typical features of rheumatoid arthritis (RA). IL-6 promotes the differentiation and function of Th17 cells, which contributes to the imbalance of Treg/Th17 and aggravates lymphocytic infiltration in joints. DC32, a dihydroartemisinin derivative, was found to have anti-inflammatory and immunosuppressive activities in previous study. The aim of this study is to evaluate the effects and mechanisms of DC32 in immunodeficiency and inflammatory infiltration of RA. In vivo, the antirheumatic effect of DC32 was evaluated in a collagen-induced arthritis (CIA) mouse model in DBA/1 mice. The percentages of Treg and Th17 and transcription of IL-6 in the spleen were assayed. In vitro, a coculture system of ConA-activated lymphocytes and fibroblast-like synoviocytes (FLSs) from rat with adjuvant arthritis (AA) was established. The effects and mechanisms of DC32 on synovitis were investigated. It was shown that DC32 inhibited footpad swelling and lymphocytic infiltration in mice with CIA and significantly restored the Treg/Th17 balance by reducing the transcription of IL-6 in splenocytes. DC32 significantly inhibited the lymphocyte-induced invasion and migration of FLSs by decreasing the secretion of MMPs (MMP-2, MMP-3) in vitro. DC32 also reduced the transcription of chemokines (CXCL12, CX3CL1) and IL-6 in FLSs, as well as IL-6 levels in the supernatant. These results demonstrated that DC32 may attenuate RA by restoring Treg/Th17 balance and inhibiting lymphocytic infiltration through downregulation of the expression and transcription of IL-6. This study supports the potential of DC32 to down-regulate IL-6 for the treatment of RA and other related autoimmune diseases.

TNF-α，IL-1β induce mechano growth factor expression in human fibroblast-like synoviocytes through the PKA pathway / 医用生物力学

Objective To investigate the effects of inflammatory factors TNF-α, IL-1β, IL-6 on expression of mechano growth factor (MGF). Methods In the experimental group, TNF-α and IL-6 at concentration of 25, 50, 100 ng/mL, or IL-1β at concentration of 2.5, 5.0, 10 ng/mL were applied to fibroblast-like synoviocytes (FLSs) for 12 hours. The inhibitor groups were pretreated with PKA pathway inhibitor KT5720 at concentration of 1.0 mmol for 1 hour. The control group remained under the same culture condition as the experimental group, but without any growth factor. Real-time PCR was used to measure the gene expression of MGF. Results Treated with TNF-α at concentration of 25 ng/mL and IL-1β at concentration of 10 ng/mL, the MGF expression in FLSs was significantly increased (P＜0.05). IL-6 had no effect on MGF expression. A specific inhibitor of cAMP-dependent protein kinase, at concentration of 1.0 mmol significantly decreased the activation of MGF synthesis by TNF-α and IL-1β in FLSs (P＜0.05). Conclusions TNF-α at concentration of 25 ng/mL and IL-1β at concentration of 10 ng/mL significantly induce the MGF expression in FLSs, which activate MGF synthesis via the PKA pathway. This study is of significance in improving the application of MGF used in tissue repair area to make up the insufficient stress stimulation.