Human Fibroblast-like Synoviocytes Research Articles

Dehydroevodiamine suppresses inflammatory responses in adjuvant-induced arthritis rats and human fibroblast-like synoviocytes

ABSTRACT Dehydroevodiamine (DHE) is an effective natural active substance extracted from Euodiae Fructus, which is a widely used herbal drug in traditional Chinese medicine. The focus of this study was to test the possibility of using DHE in the treatment of rheumatoid arthritis (RA) diseases. A rat model of adjuvant-induced arthritis (AIA) was generated using Complete Freund’s Adjuvant (CFA). Body weight changes, arthritis scores, ankle pathology, tumor necrosis factor-alpha (TNF-α), interleukin-1β(IL-1β), interleukin-6 (IL-6), and interleukin-17 (IL-17) secretion, as well as matrix metalloproteinase (MMP) expression in joint tissue, were measured as indicators of viability of DHE medicated AIA rats. Human fibroblast-like synoviocytes (MH7A cells) were connected to check these impacts. The results confirmed that DHE administration had an excellent therapeutic impact on the AIA rat model, substantially relieving joint swelling, inhibiting synovial pannus hyperplasia, and decreasing joint scores. In addition, the serum enzyme-linked immunosorbent assay (ELISA) showed that DHE treatment reduced the expression of pro-inflammatory factors in AIA rats. The immunohistochemical results showed that DHE treatment could reduce the synthesis of MMPs such as matrix metalloproteinase-1(MMP-1) and matrix metalloproteinase-3 (MMP-3) in the ankle tissue of AIA rats. In vitro, DHE inhibited cell proliferation, mRNA transcription, protein synthesis of proinflammatory factors such as IL-1βand IL-6, and matrix metalloproteinases such as MMP-1 and MMP-3. Furthermore, DHE inhibited the phosphorylation levels of p38, JNK, and ERK proteins in TNF-α-treated MH7A cells.This work assessed the effect of DHE in AIA rats and revealed its mechanism in vitro.

LncRNA cardiac autophagy inhibitory factor is downregulated in rheumatoid arthritis and suppresses the apoptosis of fibroblast-like synoviocytes by promoting the maturation of miRNA-20a.

In this study, we aimed to investigate the effects of LncRNA cardiac autophagy inhibitory factor (CAIF) and miR-20a on the apoptosis of synovial cells in rheumatoid arthritis (RA) and the regulatory mechanism. Between May 2018 and March 2020, a total of 62 RA patients (24 males, 38 females; mean age: 55.2±4.9 years; range, 42 to 68 years) and 62 controls (24 males, 38 females; mean age: 55.3±4.8 years; range, 41 to 68 years) were included in this study. Plasma samples were collected from all participants. The expression levels of CAIF, mature miR-20a, and miR-20a precursor in these plasma samples were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Correlations were analyzed using linear regression analysis. Overexpression of CAIF was achieved in human fibroblast-like synoviocytes (HFLSs) and the expression levels of mature miR-20a and miR-20a precursor were determined using RT-qPCR. Cell apoptosis was analyzed by cell apoptosis assay. The CAIF was downregulated in RA and positively correlated with the expression of mature miR-20a. In HFLSs, LPS treatment resulted in downregulation of both CAIF and miR-20a in a dose-dependent manner. In HFLSs, overexpression of CAIF did not affect the expression of miR-20a precursor, but upregulated the expression of mature miR-20a. Cell apoptosis analysis showed that overexpression of CAIF and miR-20a inhibited the apoptosis of HFLSs induced by LPS. The combination of overexpression of CAIF and miR-20a showed a stronger effect. The CAIF may suppress the apoptosis of HFLSs in RA by promoting the maturation of miR-20a.

14-3-3η Promotes Invadosome Formation via the FOXO3-Snail Axis in Rheumatoid Arthritis Fibroblast-like Synoviocytes.

Erosive destruction of joint structures is a critical event in the progression of rheumatoid arthritis (RA), in which fibroblast-like synoviocytes (FLS) are the primary effectors. We previously reported that the ability of RA FLS to degrade extracellular matrix (ECM) components depends on the formation of actin-rich membrane protrusions, called invadosomes, through processes that remain elusive. 14-3-3η belongs to a family of scaffolding proteins involved in a wide range of cellular functions, and its expression is closely related to joint damage and disease activity in RA patients. In this study, we sought to assess the role of 14-3-3η in joint damage by examining its contribution to the invadosome formation phenotype of FLS. Using human primary FLS, we show that 14-3-3η expression is closely associated with their ability to form invadosomes. Furthermore, knockdown of 14-3-3η using shRNAs decreases the level of invadosome formation in RA FLS, whereas addition of the recombinant protein to FLS from healthy individuals promotes their formation. Mechanistic studies suggest that 14-3-3η regulates invadosome formation by increasing Snail expression, a mechanism that involves nuclear exclusion of the transcription repressor FOXO3. Our results implicate the 14-3-3η–FOXO3–Snail axis in promoting the aggressive ECM-degrading phenotype of RA FLS, and suggest a role for this scaffolding protein in cartilage degradation.

Methotrexate-loaded folic acid of solid-phase synthesis conjugated gold nanoparticles targeted treatment for rheumatoid arthritis

PurposeMethotrexate (MTX) is a first-line drug for rheumatoid arthritis (RA). Targeting of MTX to inflamed joints is essential to the prevention of potential toxicity and improving therapeutic effects. Gold nanoparticles (GNPs) are characterized by controllable particle sizes and good biocompatibilities, therefore, they are promising drug delivery systems. We aimed at developing a GNPs drug delivery system incorporating MTX and folic acid (FA) with strong efficacies against RA. MethodsMTX-Cys-FA was synthesized through solid-phase organic synthesis. Then, it was coupled with sulfhydryl groups in GNPs, thereby successfully preparing a GNPs/MTX-Cys-FA nanoconjugate with targeting properties. Physical and chemical techniques were used to characterize it. Moreover, we conducted its stability, release, pharmacokinetics, biodistribution and cell cytotoxicity, cell uptake, cell migration, as well as its therapeutic effect on CIA rats. The histopathology was conducted to investigate anti-RA effects of GNPs/MTX-Cys-FA nanoconjugates. ResultsThe GNPs/MTX-Cys-FA nanoconjugate exhibited a spherical appearance, had a particle size of 103.06 nm, a zeta potential of -33.68 mV, drug loading capacity of 11.04 %, and an encapsulation efficiency of 73.61%. Cytotoxicity experiments revealed that GNPs had good biocompatibilities while GNPs/MTX-Cys-FA exhibited excellent drug-delivery abilities. Cell uptake and migration experiment showed that nanoconjugates containing FA by LPS activated mouse mononuclear macrophages (RAW264.7) was significantly increased, and they exerted significant inhibitory effects on human fibroblast-like synoviocytes (HFLS) of RA (p<0.01). In addition, the nanoconjugate prolonged blood circulation time of MTX in collagen-induced arthritis (CIA) rats (p<0.01), enhanced MTX accumulation in inflamed joints (p<0.01), enhanced their therapeutic effects (p<0.01), and reduced toxicity to major organs (p<0.01). ConclusionGNPs/MTX-Cys-FA nanoconjugates provide effective approaches for RA targeted therapeutic strategies.

Ellagic Acid Alleviates Rheumatoid Arthritis in Rats through Inhibiting MTA1/HDAC1-Mediated Nur77 Deacetylation.

Ellagic acid (EA) was reported to play protective roles in rheumatoid arthritis (RA). It was found that the level of metastasis-associated gene 1 (MTA1)/histone deacetylase 1 (HDAC1) protein complex was downregulated by polyphenols in several human disorders. Notably, inhibition of MTA1 or HDAC1 has anti-inflammatory effects on RA. Therefore, our study is aimed at investigating whether EA prevents RA progression through regulating the MTA1/HDAC1 complex. Herein, the human fibroblast-like synoviocyte (FLS) cell line MH7A was treated with TNF-α to induce an inflammation model in vitro and then incubated with different concentrations of EA. Western blot analysis showed that EA reduced MTA1 expression in a dose-dependent manner in MH7A cells. Then, TNF-α-treated MH7A cells were incubated with EA alone or together with MTA1 overexpression plasmid (pcDNA-MTA1), and we found that EA inhibited proliferation, inflammation cytokine levels, and oxidative stress marker protein levels and promoted apoptosis in MH7A cells, while MTA1 overexpression abolished these effects. Moreover, coimmunoprecipitation assay verified the interaction between MTA1 and HDAC1. EA downregulated the MTA1/HDAC1 complex in MH7A cells. MTA1 knockdown inhibited proliferation, inflammation, and oxidative stress and promoted apoptosis in MH7A cells, while HDAC1 overexpression reversed these effects. Moreover, chromatin immunoprecipitation assay indicated that EA inhibited HDAC1-mediated Nur77 deacetylation. Rescue experiments demonstrated that Nur77 knockdown reversed the effects of EA on MH7A cell biological behaviors. Additionally, EA treatment attenuated arthritis index, paw swelling, synovial hyperplasia, and inflammation in collagen-induced arthritis (CIA) rats. In conclusion, EA inhibited proliferation, inflammation, and oxidative stress and promoted apoptosis in MH7A cells and alleviated the severity of RA in CIA rats though downregulating MTA1/HDAC1 complex and promoting HDAC1 deacetylation-mediated Nur77 expression.

MicroRNA-4423-3p inhibits proliferation of fibroblast-like synoviocytes by targeting matrix metalloproteinase 13 in rheumatoid arthritis

ABSTRACT Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that is increasing in incidence worldwide. RA is regulated by a variety of microRNAs (miRNAs/miR). Moreover, analysis of public data has revealed that miR-4423-3p is significantly downregulated in peripheral blood mononuclear cells of RA patients. This study investigated the role of miR-4423-3p in RA. The levels of miR-4423-3p and matrix metalloproteinase 13 (MMP13) in RA patients and the regulatory relationship between miR-4423-3p and MMP13 were analyzed using public data. A dual-luciferase reporter assay was performed to verify that miR-4423-3p targets MMP13 in human fibroblast-like synoviocyte (HFLS) RA cells (HFLS-RA). Following the overexpression of miR-4423-3p, miR-4423-3p inhibitor, and MMP13 in HFLS-RA, viability, proliferation, cell cycle, apoptosis, and invasion/migration assays were used to detect the effects of miR-4423-3p targeting MMP13 on cell biological processes. The results revealed that miR-4423-3p was downregulated in peripheral blood mononuclear cells of RA patients and MMP13 was upregulated in synovial tissue of RA patients. miR-4423-3p targets the 3ʹ untranslated region of MMP13 and downregulates MMP13 expression. After overexpression of miR-4423-3p, cell proliferation, migration, and invasion were inhibited, the cell cycle was prevented and cell apoptosis was promoted. Overexpression of MMP13 promoted cell proliferation, migration, and invasion, while accelerating the cell cycle process and suppressing apoptosis. The findings indicate that in HFLS-RA cells, overexpression of miR-4423-3p inhibited proliferation, migration, and invasion, and promoted apoptosis by negatively regulating MMP13. The overexpression of miR-4423-3p might be a novel strategy for the treatment of RA.

Phospholipase A1 Member A Activates Fibroblast-like Synoviocytes through the Autotaxin-Lysophosphatidic Acid Receptor Axis.

Lysophosphatidylserine (lysoPS) is known to regulate immune cell functions. Phospholipase A1 member A (PLA1A) can generate this bioactive lipid through hydrolysis of sn-1 fatty acids on phosphatidylserine (PS). PLA1A has been associated with cancer metastasis, asthma, as well as acute coronary syndrome. However, the functions of PLA1A in the development of systemic autoimmune rheumatic diseases remain elusive. To investigate the possible implication of PLA1A during rheumatic diseases, we monitored PLA1A in synovial fluids from patients with rheumatoid arthritis and plasma of early-diagnosed arthritis (EA) patients and clinically stable systemic lupus erythematosus (SLE) patients. We used human primary fibroblast-like synoviocytes (FLSs) to evaluate the PLA1A-induced biological responses. Our results highlighted that the plasma concentrations of PLA1A in EA and SLE patients were elevated compared to healthy donors. High concentrations of PLA1A were also detected in synovial fluids from rheumatoid arthritis patients compared to those from osteoarthritis (OA) and gout patients. The origin of PLA1A in FLSs and the arthritic joints remained unknown, as healthy human primary FLSs does not express the PLA1A transcript. Besides, the addition of recombinant PLA1A stimulated cultured human primary FLSs to secrete IL-8. Preincubation with heparin, autotaxin (ATX) inhibitor HA130 or lysophosphatidic acid (LPA) receptor antagonist Ki16425 reduced PLA1A-induced-secretion of IL-8. Our data suggested that FLS-associated PLA1A cleaves membrane-exposed PS into lysoPS, which is subsequently converted to LPA by ATX. Since primary FLSs do not express any lysoPS receptors, the data suggested PLA1A-mediated pro-inflammatory responses through the ATX-LPA receptor signaling axis.

Human fibroblast-like synoviocyte isolation matter: a comparison between cell isolation from synovial tissue and synovial fluid from patients with rheumatoid arthritis.

Cell culture technology has become a popular method in the field of cell biology, pharmacology, and medical researches. Primary cells represent the normal physiological condition of human cells. Fibroblasts are the most common native cells of connective tissue that play a crucial role in the entire pathogenesis of various disorders, such as rheumatoid arthritis (RA). Fibroblast-like synoviocytes (FLSs), which overlie the loose connective tissue of the synovial sublining, are known to be the central mediators of joint damage. The most routine approach for the isolation of FLS is an enzymatic digestion of synovial tissue. This experimental study is designed to introduce an easy, fast, and high-throughput method compared with enzymatic digestion for isolation of FLS. The synovial tissue and synovial fluid (SF) samples were collected from eight patients with RA who underwent routine knee replacement surgery. Synovial tissue was incubated with collagenase VIII enzyme, while SF was washed with a similar volume of phosphate-buffered saline. The cells were further subcultured and stored based on the standard protocols. The purity of isolated synoviocytes was confirmed using flow cytometry analysis. Isolation of FLS from SF was more successful with a faster rate, 3-5 days after culture. The morphological assessment and flow cytometry analysis confirmed the purity of SF-derived cells in passage 4. SF could be a more accessible source of FLS than synovial tissue. Obtaining primary FLS from SF is a simple, fast, and cost-effective way to have a large-scale cell during a short time.

Icariin arrests cell cycle progression and induces cell apoptosis through the mitochondrial pathway in human fibroblast-like synoviocytes

Rheumatoid arthritis (RA) is a highly disabling autoimmune disorder, characterized by the proliferation of Fibroblast-like Synoviocytes (FLSs). Icariin is a prenylated flavonol glycoside extracted from the medical plant Epimedium, which can inhibit the proliferation and migration of FLSs. However, the potential mechanism of icariin to alleviate RA remains unclear. In this study, icariin inhibited the migration and proliferation of FLSs in a concentration-dependent manner, by inducing G2/M phase arrest and apoptosis. Icariin reduced the mitochondrial transmembrane potential, upregulated cytosolic cytochrome c and increased the level of intracellular reactive oxygen species (ROS). In conclusion, icariin inhibited the proliferation of FLSs by interfering with the cell cycle process and inducing cell apoptosis, suggesting its potential use for the treatment of RA.

Gaultheria leucocarpa var. yunnanensis for Treating Rheumatoid Arthritis-An Assessment Combining Machine Learning-Guided ADME Properties Prediction, Network Pharmacology, and Pharmacological Assessment.

Background: Dianbaizhu (Gaultheria leucocarpa var. yunnanensis), a traditional Chinese/ethnic medicine (TC/EM), has been used to treat rheumatoid arthritis (RA) for a long time. The anti–rheumatic arthritis fraction (ARF) of G. yunnanensis has significant anti-inflammatory and analgesic activities and is mainly composed of methyl salicylate glycosides, flavonoids, organic acids, and others. The effective ingredients and rudimentary mechanism of ARF remedying RA have not been elucidated to date. Purpose: The aim of the present study is to give an insight into the effective components and mechanisms of Dianbaizhu in ameliorating RA, based on the estimation of the absorption, distribution, metabolism, and excretion (ADME) properties, analysis of network pharmacology, and in vivo and in vitro validations. Study design and methods: The IL-1β–induced human fibroblast-like synoviocytes of RA (HFLS-RA) model and adjuvant-induced arthritis in the rat model were adopted to assess the anti-RA effect of ARF. The components in ARF were identified by using UHPLC-LTQ-Orbitrap-MSn. The quantitative structure–activity relationship (QSAR) models were developed by using five machine learning algorithms, alone or in combination with genetic algorithms for predicting the ADME properties of ARF. The molecular networks and pathways presumably referring to the therapy of ARF on RA were yielded by using common databases and visible software, and the experimental validations of the key targets conducted in vitro. Results: ARF effectively relieved RA in vivo and in vitro. The five optimized QSAR models that were developed showed robustness and predictive ability. The characterized 48 components in ARF had good biological potency. Four key signaling pathways were obtained, which were related to both cytokine signaling and cell immune response. ARF suppressed IL-1β–induced expression of EGFR, MMP 9, IL2, MAPK14, and KDR in the HFLS-RA . Conclusions: ARF has good druggability and high exploitation potential. Methyl salicylate glycosides and flavonoids play essential roles in attuning RA. ARF may partially attenuate RA by regulating the expression of multi-targets in the inflammation–immune system. These provide valuable information to rationalize ARF and other TC/EMs in the treatment of RA.

WTD Attenuating Rheumatoid Arthritis via Suppressing Angiogenesis and Modulating the PI3K/AKT/mTOR/HIF-1α Pathway.

Background: Wutou Decoction (WTD), as a classic prescription, has been generally used to treat rheumatoid arthritis (RA) for two thousand years in China. However, the potential protective effects of WTD on rheumatoid arthritis and its possible mechanism have rarely been reported. Purpose: The aim of this study was to explore the possible mechanism of WTD against RA and a promising alternative candidate for RA therapy. Methods: A model of collagen-induced arthritis (CIA) was constructed in rats to assess the therapeutic effects of WTD. Histopathological staining, immunofluorescence, and western blotting of synovial sections were conducted to detect the antiangiogenic effects of WTD. Then, cell viability assays, flow cytometry, scratch healing assays, and invasion assays were conducted to explore the effects of WTD on MH7A human fibroblast-like synoviocyte (FLS) cell proliferation, apoptosis, migration, and invasion in vitro. The ability of WTD to induce blood vessel formation after MH7A cell and human umbilical vein endothelial cell line (HUVEC) coculture with WTD intervention was detected by a tube formation assay. The mechanisms of WTD were screened by network pharmacology and confirmed by in vivo and in vitro experiments. Results: WTD ameliorated the symptoms and synovial pannus hyperplasia of CIA rats. Treatment with WTD inhibited MH7A cell proliferation, migration, and invasion and promoted MH7A apoptosis. WTD could inhibit MH7A cell expression of proangiogenic factors, including VEGF and ANGI, to induce HUVEC tube formation. Furthermore, the PI3K-AKT-mTOR-HIF-1α pathway was enriched as a potential target of WTD for the treatment of RA through network pharmacology enrichment analysis. Finally, it was confirmed in vitro and in vivo that WTD inhibits angiogenesis in RA by interrupting the PI3K-AKT-mTOR-HIF-1α pathway. Conclusion: WTD can inhibit synovial hyperplasia and angiogenesis, presumably by inhibiting the migration and invasion of MH7A cells and blocking the production of proangiogenic effectors in MH7A cells. The possible underlying mechanism by which WTD ameliorates angiogenesis in RA is the PI3K-AKT-mTOR-HIF-1α pathway.

Cannabinoid receptor type 2 is upregulated in synovium following joint injury and mediates anti-inflammatory effects in synovial fibroblasts and macrophages

Joint injury-induced perturbations to the endocannabinoid system (ECS), a regulator of both inflammation and nociception, remain largely uncharacterized. We employed a mouse model of ACL rupture to assess alterations to nociception, inflammation, and the ECS while using in vitro models to determine whether CB2 agonism can mitigate inflammatory signaling in macrophages and fibroblast-like synoviocytes (FLS). Mice underwent noninvasive ACL rupture (ACLR) via tibial compression-based loading. Nociception was measured longitudinally using mechanical allodynia and knee hyperalgesia testing. Synovitis was assessed using histological scoring and histomorphometry. Gene and protein markers of inflammation were characterized in whole joints and synovium. Immunohistochemistry assessed injury-induced alterations to CB1+, CB2+, and F4/80+ cells in synovium. To assess whether CB2 agonism can inhibit pro-inflammatory macrophage polarization, murine bone marrow-derived macrophages (mBMDM) were stimulated with IL-1β or conditioned medium from IL-1β-treated FLS and treated with vehicle (DMSO), the CB2 agonist HU308, or cannabidiol (CBD). Macrophage polarization was assessed as the ratio of M1-associated (IL1b, MMP1b, and IL6) to M2-associated (IL10, IL4, and CD206) gene expression. Human FLS (hFLS) isolated from synovial tissue of OA patients were treated with vehicle (DMSO) or HU308 following TNF-α or IL-1β stimulation to assess inhibition of catabolic/inflammatory gene expression. ACLR induces synovitis, progressively-worsening PTOA severity, and an immediate and sustained increase in both mechanical allodynia and knee hyperalgesia, which persist beyond the resolution of molecular inflammation. Enrichment of CB2, but not CB1, was observed in ACLR synovium at 3d, 14d, and 28d, and CB2 was found to be associated with F4/80 (+) cells, which are increased in number in ACLR synovium at all time points. The CB2 agonist HU308 strongly inhibited mBMDM M1-type polarization following stimulation with either IL-1β or conditioned medium from IL-1β-treated mFLS, which was characterized by reductions in Il1b, Mmp1b, and Il6 and increases in Cd206 gene expression. Cannabidiol similarly inhibited IL-1β-induced mBMDM M1 polarization via a reduction in Il1b and an increase in Cd206 and Il4 gene expression. Lastly, in OA hFLS, HU308 treatment inhibited IL-1β-induced CCL2, MMP1, MMP3, and IL6 expression and further inhibited TNF-α-induced CCL2, MMP1, and GMCSF expression, demonstrating human OA-relevant anti-inflammatory effects by targeting CB2. Joint injury perturbs the intra-articular ECS, characterized by an increase in synovial F4/80(+) cells, which express CB2, but not CB1. Targeting CB2 in murine macrophages and human FLS induced potent anti-inflammatory and anti-catabolic effects, which indicates that the CB2 receptor plays a key role in regulating inflammatory signaling in the two primary effector cells in the synovium. The intraarticular ECS is therefore a potential therapeutic target for blocking pathological inflammation in future disease-modifying PTOA treatments.

Effect of key enzyme inhibition of glycolysis on synovial fibroblasts in Rheumatoid Arthritis

Objective: To observe effects of glycolysis on human rheumatoid arthritis Fibroblast-like synoviocytes (HFLS-RA) by inhibiting glycolysis. Methods: Hexokinase inhibitor (3-bromopyruvate, 3-BrPa), 6-phosphofructokinase 1 inhibitor citric acid and pyruvate kinase inhibitor shikonin were applied to HFLS-RA respectively. Cell count 8 Kit detects cell proliferation activity, the activity of hexokinase, 6-phosphofructokinase 1, and pyruvate kinase, as well as the cellular glucose, lactate and ATP content were detected by kits, and the ELISA kit detects the expression of cellular inflammatory factors TNF-α and TGF-β. Results: 10 μg/mL 3-BrPa, 160 μg/mL citric acid and 5 μg/mL shikonin significantly inhibited cell proliferation activity (P&lt;0.001); and significantly inhibited HFLS-RA hexokinase and fructose 6-phosphate Kinase 1 and pyruvate kinase activity; Glucose, lactate and ATP content decreased; TNF-α expression decreased, while TGF-β expression increased. Conclusion: This study explored the changes in glucose metabolism and the expression of inflammatory factors in HFLS-RA by inhibiting the key enzymes of glycolysis, further confirming the important role of glycolysis in HFLS-RA, and laying a theoretical basis for a deep understanding of the pathogenesis of RA.

Spontaneously Resolving Joint Inflammation Is Characterised by Metabolic Agility of Fibroblast-Like Synoviocytes.

Fibroblast-like synoviocytes (FLS) play an important role in maintaining joint homeostasis and orchestrating local inflammatory processes. When activated during injury or inflammation, FLS undergo transiently increased bioenergetic and biosynthetic demand. We aimed to identify metabolic changes which occur early in inflammatory disease pathogenesis which might support sustained cellular activation in persistent inflammation. We took primary human FLS from synovial biopsies of patients with very early rheumatoid arthritis (veRA) or resolving synovitis, and compared them with uninflamed control samples from the synovium of people without arthritis. Metabotypes were compared using NMR spectroscopy-based metabolomics and correlated with serum C-reactive protein levels. We measured glycolysis and oxidative phosphorylation by Seahorse analysis and assessed mitochondrial morphology by immunofluorescence. We demonstrate differences in FLS metabolism measurable after ex vivo culture, suggesting that disease-associated metabolic changes are long-lasting. We term this phenomenon ‘metabolic memory’. We identify changes in cell metabolism after acute TNFα stimulation across disease groups. When compared to FLS from patients with early rheumatoid arthritis, FLS from patients with resolving synovitis have significantly elevated mitochondrial respiratory capacity in the resting state, and less fragmented mitochondrial morphology after TNFα treatment. Our findings indicate the potential to restore cell metabotypes by modulating mitochondrial function at sites of inflammation, with implications for treatment of RA and related inflammatory conditions in which fibroblasts play a role.

Circ-Sirt1 inhibits proliferation, induces apoptosis, and ameliorates inflammation in human rheumatoid arthritis fibroblast-like synoviocytes

Rheumatoid arthritis (RA) is a chronic inflammatory disease related to abnormal activation of fibroblast-like synovium cells (FLS) with apoptosis, inflammation, and oxidative damage. Circular RNA Sirt1 (circ-Sirt1) is an abundant circRNA, exerts the function in inhibiting inflammation. However, little is known about the roles of circ-Sirt1, if any, in RA. The present study aimed to investigate the biological roles and mechanism of circ-Sirt1 on cell inflammation in RA-FLS MH7A cell line. This study showed circ-Sirt1 inhibited the proliferation and induced apoptosis of MH7A cells. Overexpression of circ-Sirt1 decreased of the levels of interleukin (IL)-1β and IL-6, tumour necrosis factor (TNF)-α, and matrix matalloproteinases (MMP)-1 and MMP-3 in MH7A cells. In addition, overexpression of circ-Sirt1 increased the expression of Sirt1, Nrf2, HO-1, IκBα, GCLC and GCLM, and decreased the ratio of acetylated NF-κB to normal NF-κB, and the expression of AP-1, COX-2 and HMGB1. Moreover, the expression of Keap1 and the ratio of acetylated NF-κB to normal NF-κB were partially increased and the Nrf2 and Sirt1 were partially reduced by siSirt1. Additionally, circ-Sirt1 overexpression promoted the activation of Sirt1 signal pathways by upregulating miR-132. In conclusion, the protective effect of Circ-Sirt1 on MH7A depends on inhibiting cell proliferation, promoting apoptosis and miR-132-mediated Sirt1 pathway to reduce inflammation.

Bioactive fractions and compound of Ardisia crispa roots exhibit anti-arthritic properties mediated via angiogenesis inhibition in vitro

BackgroundArdisia crispa (Thunb.) A.DC (Primulaceae), is a medicinal herb traditionally used by Asian people as remedies to cure inflammatory related diseases, including rheumatism. The plant roots possess various pharmacological activities including antipyretic, anti-inflammation and antitumor. Previous phytochemical studies of the plant roots have identified long chain alkyl-1,4-benzoquinones as major constituents, together with other phytochemicals. Hexane fraction of the plant roots (ACRH), was previously reported with anti-angiogenic and anti-arthritic properties, while its effect on their anti-arthritic in vitro, is yet unrevealed. Considering the significance of angiogenesis inhibition in developing new anti-arthritic agent, thus we investigated the anti-arthritic potential of Ardisia crispa roots by suppressing angiogenesis, in vitro.MethodsArdisia crispa roots hexane extract (ACRH) was prepared from the plant roots using absolute n-hexane. ACRH was fractionated into quinone-rich fraction (QRF) and further isolated to yield benzoquinonoid compound (BQ), respectively. In vitro experiments using VEGF-induced human umbilical vein endothelial cells (HUVECs) and IL-1β-induced human fibroblast-like synoviocytes for rheumatoid arthritis (HFLS-RA) were performed to evaluate the effects of these samples on VEGF-induced HUVECs proliferation and tube formation, and towards IL-1β-induced HFLS-RA proliferation, invasion, and apoptosis, respectively. Therapeutic concentrations (0.05, 0.5, and 5 μg/mL) tested in this study were predetermined based on the IC50 values obtained from the MTT assay.ResultsACRH, QRF, and BQ exerted concentration-independent antiproliferative effects on VEGF-induced HUVECs and IL-1β-induced HFLS-RA, with IC50 values at 1.09 ± 0.18, 3.85 ± 0.26, and 1.34 ± 0.16 μg/mL in HUVECs; and 3.60 ± 1.38, 4.47 ± 0.34, and 1.09 ± 0.09 μg/mL in HFLS-RA, respectively. Anti-angiogenic properties of these samples were verified via significant inhibition on VEGF-induced HUVECs tube formation, in a concentration-independent manner. The invasiveness of IL-1β-induced HFLS-RA was also significantly inhibited in a concentration-independent manner by all samples. ACRH and BQ, but not QRF, significantly enhanced the apoptosis of IL-1β-induced HFLS-RA elicited at their highest concentration (5 μg/mL) (P < 0.05).ConclusionsThese findings highlight the bioactive fractions and compound from Ardisia crispa roots as potential anti-arthritic agents by inhibiting both HUVECs and HFLS-RA’s cellular functions in vitro, possibly mediated via their anti-angiogenic effects.

Sclerostin-Mediated Impaired Osteogenesis by Fibroblast-Like Synoviocytes in the Particle-Induced Osteolysis Model.

Engineered biomaterials are envisioned to replace, augment, or interact with living tissues for improving the functional deformities associated with end-stage joint pathologies. Unfortunately, wear debris from implant interfaces is the major factor leading to periprosthetic osteolysis. Fibroblast-like synoviocytes (FLSs) populate the intimal lining of the synovium and are in direct contact with wear debris. This study aimed to elucidate the effect of Ti particles as wear debris on human FLSs and the mechanism by which they might participate in the bone remodeling process during periprosthetic osteolysis. FLSs were isolated from synovial tissue from patients, and the condition medium (CM) was collected after treating FLSs with sterilized Ti particles. The effect of CM was analyzed for the induction of osteoclastogenesis or any effect on osteogenesis and signaling pathways. The results demonstrated that Ti particles could induce activation of the NFκB signaling pathway and induction of COX-2 and inflammatory cytokines in FLSs. The amount of Rankl in the conditioned medium collected from Ti particle–stimulated FLSs (Ti CM) showed the ability to stimulate osteoclast formation. The Ti CM also suppressed the osteogenic initial and terminal differentiation markers for osteoprogenitors, such as alkaline phosphate activity, matrix mineralization, collagen synthesis, and expression levels of Osterix, Runx2, collagen 1α, and bone sialoprotein. Inhibition of the WNT and BMP signaling pathways was observed in osteoprogenitors after the treatment with the Ti CM. In the presence of the Ti CM, exogenous stimulation by WNT and BMP signaling pathways failed to stimulate osteogenic activity in osteoprogenitors. Induced expression of sclerostin (SOST: an antagonist of WNT and BMP signaling) in Ti particle–treated FLSs and secretion of SOST in the Ti CM were detected. Neutralization of SOST in the Ti CM partially restored the suppressed WNT and BMP signaling activity as well as the osteogenic activity in osteoprogenitors. Our results reveal that wear debris–stimulated FLSs might affect bone loss by not only stimulating osteoclastogenesis but also suppressing the bone-forming ability of osteoprogenitors. In the clinical setting, targeting FLSs for the secretion of antagonists like SOST might be a novel therapeutic approach for preventing bone loss during inflammatory osteolysis.

HIF-1α Dependent Upregulation of ZIP8, ZIP14, and TRPA1 Modify Intracellular Zn2+ Accumulation in Inflammatory Synoviocytes.

Intracellular free zinc ([Zn2+]i) is mobilized in neuronal and non-neuronal cells under physiological and/or pathophysiological conditions; therefore, [Zn2+]i is a component of cellular signal transduction in biological systems. Although several transporters and ion channels that carry Zn2+ have been identified, proteins that are involved in Zn2+ supply into cells and their expression are poorly understood, particularly under inflammatory conditions. Here, we show that the expression of Zn2+ transporters ZIP8 and ZIP14 is increased via the activation of hypoxia-induced factor 1α (HIF-1α) in inflammation, leading to [Zn2+]i accumulation, which intrinsically activates transient receptor potential ankyrin 1 (TRPA1) channel and elevates basal [Zn2+]i. In human fibroblast-like synoviocytes (FLSs), treatment with inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin-1α (IL-1α), evoked TRPA1-dependent intrinsic Ca2+ oscillations. Assays with fluorescent Zn2+ indicators revealed that the basal [Zn2+]i concentration was significantly higher in TRPA1-expressing HEK cells and inflammatory FLSs. Moreover, TRPA1 activation induced an elevation of [Zn2+]i level in the presence of 1 μM Zn2+ in inflammatory FLSs. Among the 17 out of 24 known Zn2+ transporters, FLSs that were treated with TNF-α and IL-1α exhibited a higher expression of ZIP8 and ZIP14. Their expression levels were augmented by transfection with an active component of nuclear factor-κB P65 and HIF-1α expression vectors, and they could be abolished by pretreatment with the HIF-1α inhibitor echinomycin (Echi). The functional expression of ZIP8 and ZIP14 in HEK cells significantly increased the basal [Zn2+]i level. Taken together, Zn2+ carrier proteins, TRPA1, ZIP8, and ZIP14, induced under HIF-1α mediated inflammation can synergistically change [Zn2+]i in inflammatory FLSs.

Survivin promotes rheumatoid arthritis fibroblast-like synoviocyte cell proliferation, and the expression of angiogenesis-related proteins by activating the NOTCH pathway.

Survivin have been shown to play a crucial role in rheumatoid arthritis (RA); however, the regulatory mechanism of survivin in RA has not been fully elucidated. This study aims to investigate the effect of survivin on the proliferation and apoptosis of human RA fibroblast-like synoviocyte (RA-HFLS) cells in RA and its underlying mechanism through the NOTCH pathway. The RA synovial tissues of 65 RA patients with partial resection of synovium of knee joint by arthroscopy were collected. The expression of survivin in synovial tissue was detected by immunohistochemistry, and the correlation of survivin expression and clinical-pathological parameters of patients was analyzed. In vitro, the proliferation of HFLS and RA-HFLS were detected by MTT; the apoptosis of HFLS and RA-HFLS were detected by flow cytometry; the expression of survivin proteins, key protein factors (Notch1, Jagged1, Hes1) in the Notch pathway, and angiogenesis-related proteins (vascular endothelial growth factor receptor 1 [VEGFR1], Ang1, Ang2) were determined by western blot. We found that survivin was highly expressed in RA synovial tissues and RA-HFLS cells, and was positively correlated with erythrocyte sedimentation rate, cyclic citrullinated peptide, C-reactive protein, Disease Activity Score of 28 joints and other pathological indexes. Knockdown survivin induces RA-HFLS cell apoptosis, suppresses proliferation and the expression of VEGFR1, Ang1, Ang2. In addition, blocking Notch pathway using FLI-06 significantly down-regulated survivin expression. When survivin is up-regulated, it promotes RA-HFLS cell proliferation, the expression of VEGFR1, Ang1, Ang2 and suppresses apoptosis by activating the NOTCH. This study confirmed that survivin promotes RA-HFLS cell proliferation, the expression of angiogenesis-related proteins and suppresses apoptosis by activating the NOTCH pathway.

Hydroquinone Exposure Worsens Rheumatoid Arthritis through the Activation of the Aryl Hydrocarbon Receptor and Interleukin-17 Pathways.

Rheumatoid arthritis (RA) development is strongly associated with cigarette smoke exposure, which activates the aryl hydrocarbon receptor (AhR) as a trigger for Th17 inflammatory pathways. We previously demonstrated that the exposure to hydroquinone (HQ), one of the major compounds of cigarette tar, aggravates the arthritis symptomatology in rats. However, the mechanisms related to the HQ-related RA still remain elusive. Cell viability, cytokine secretion, and gene expression were measured in RA human fibroblast-like synoviocytes (RAHFLS) treated with HQ and stimulated or not with TNF-α. Antigen-induced arthritis (AIA) was also elicited in wild type (WT), AhR −/− or IL-17R −/− C57BL/6 mice upon daily exposure to nebulized HQ (25ppm) between days 15 to 21. At day 21, mice were challenged with mBSA and inflammatory parameters were assessed. The in vitro HQ treatment up-regulated TNFR1, TNFR2 expression, and increased ROS production. The co-treatment of HQ and TNF-α enhanced the IL-6 and IL-8 secretion. However, the pre-incubation of RAHFLS with an AhR antagonist inhibited the HQ-mediated cell proliferation and gene expression profile. About the in vivo approach, the HQ exposure worsened the AIA symptoms (edema, pain, cytokines secretion and NETs formation) in WT mice. These AIA effects were abolished in HQ-exposed AhR −/− and IL-17R −/− animals though. Our data demonstrated the harmful HQ influence over the onset of arthritis through the activation and proliferation of synoviocytes. The HQ-related RA severity was also associated with the activation of AhR and IL-17 pathways, highlighting how cigarette smoke compounds can contribute to the RA progression.