Apoptosis Of Synoviocytes Research Articles

YTHDC1 Regulates the Migration, Invasion, Proliferation, and Apoptosis of Rheumatoid Fibroblast-Like Synoviocytes

BackgroundRheumatoid arthritis (RA), a chronic autoimmune condition, is characterized by persistent synovial inflammation, bone degradation, and progressive joint deterioration. Despite considerable research efforts, the precise molecular mechanism underlying RA remains elusive. This investigation aims to elucidate the potential role and molecular mechanism of N6-methyladenosine (m6A) methylation regulators in the pathogenesis of RA.MethodsIn this study, we employed bioinformatics tools to elucidate the association between RA and m6A modifications, aiming to identify potential biological markers. We extracted datasets GSE12021, GSE55235, and GSE55457 from the Gene Expression Omnibus (GEO) database for comprehensive analysis. Utilizing differential expression analysis, protein-protein interaction (PPI) analysis, and single-cell sequencing techniques, we identified pivotal hub genes implicated in the pathogenesis of RA. Subsequently, we assessed the correlation between these hub genes and the pathogenesis of RA using Gene Set Enrichment Analysis (GSEA). Both in vivo and in vitro experiments were performed to confirm the expression and functional roles of the identified key hub gene in RA.ResultsDifferential expression analysis, PPI analysis, and single-cell analysis identified three key hub genes (YTHDC1, YTHDC2, and YTHDF2) associated with RA. GSEA results further revealed that these genes are enriched in pathways associated with inflammatory responses. Subsequent correlation analysis demonstrated a significant negative correlation between YTHDC1 expression and CD8+ T cell levels. Notably, the gene and protein expression levels of YTHDC1 and YTHDF2 were significantly reduced in the synovial tissue of RA patients. Furthermore, silencing YTHDC1 in fibroblast-like synoviocytes (FLSs) significantly inhibited their migration, invasion, proliferation, and induced apoptosis.ConclusionYTHDC1 may potentially be involved in the pathogenesis of RA through its regulation of migration, invasion, proliferation, and apoptosis in FLSs.

Polydatin and chitosan-silver co-loaded nanocomplexes for synergistic treatment of rheumatoid arthritis via repolarizing macrophages and inducing apoptosis of fibroblast-like synoviocytes

Rheumatoid arthritis (RA) is a chronic and refractory autoimmune disease that primarily affects the synovium of diarthrodial joints. Inflammatory macrophages and fibroblast-like synoviocytes (FLS) in the synovial microenvironment produce pathogenic mediators such as cytokines and proteases that perpetuate immune-mediated inflammation and contribute to the destruction of cartilage and bone. Polydatin (PD), a natural active compound, has demonstrated potential anti-inflammatory and anti-arthritic effects. However, drug development and delivery of PD is still a great challenge owing to its low solubility, short half-life, and high dose requirement. In order to overcome these drawbacks, we developed a novel nanodrug system named HA-M@PB@Ag@PD NPs. This system is composed of hybrid membrane (M), hyaluronic acid (HA), Prussian blue nanoparticles (PB NPs), PD, and chitosan-silver (Chi-Ag). In vitro experiments demonstrated that HA-M@PB@Ag@PD NPs effectively cleared ROS, promoted the repolarization of inflammatory macrophages, and induced apoptosis of RA-FLS. Using a rat model of RA, HA-M@PB@Ag@PD NPs markedly suppressed joint inflammation, inhibited synovial hyperplasia, and protected joints against destruction of cartilage and bone. Moreover, HA-M@PB@Ag@PD NPs significantly improved the synovial microenvironment of arthritic rats by reducing the number of RA-FLS and inflammatory macrophages, and facilitating the repolarization of inflammatory macrophages.

Low-Dose Radiotherapy Attenuates Experimental Autoimmune Arthritis by Inducing Apoptosis of Lymphocytes and Fibroblast-Like Synoviocytes.

Low-dose radiotherapy (LDRT) has been explored as a treatment option for various inflammatory diseases; however, its application in the context of rheumatoid arthritis (RA) is lacking. This study aimed to elucidate the mechanism underlying LDRT-based treatment for RA and standardize it. LDRT reduced the total numbers of immune cells, but increased the apoptotic CD4+ T and B220+ B cells, in the draining lymph nodes of collagen induced arthritis and K/BxN models. In addition, it significantly reduced the severity of various pathological manifestations, including bone destruction, cartilage erosion, and swelling of hind limb ankle. Post-LDRT, the proportion of apoptotic CD4+ T and CD19+ B cells increased significantly in the PBMCs derived from human patients with RA. LDRT showed a similar effect in fibroblast-like synoviocytes as well. In conclusion, we report that LDRT induces apoptosis in immune cells and fibro-blast-like synoviocytes, contributing to attenuation of arthritis.

Dual-targeted halofuginone hydrobromide nanocomplexes for promotion of macrophage repolarization and apoptosis of rheumatoid arthritis fibroblast-like synoviocytes in adjuvant-induced arthritis in rats

Rheumatoid arthritis (RA) is a prevalent autoimmune disease characterized by chronic inflammation and excessive proliferation of the synovium. Currently, treatment options focus on either reducing inflammation or inhibiting synovial hyperplasia. However, these modalities are unsatisfactory in achieving the desired therapeutic outcomes. Halofuginone hydrobromide (HF), an herbal active ingredient, has demonstrated pharmacological effects of both anti-inflammation and inhibition of synovial hyperplasia proliferation. However, HF’s medical efficacy is limited due to its poor water solubility, short half-life, and non-target toxicity. In the current study, by using the advantages of nanotechnology, we presented a novel dual-targeted nanocomplex, termed HA-M@P@HF NPs, which consisted of a hyaluronic acid (HA)-modified hybrid membrane (M)-camouflaged poly lactic-co-glycolic acid (PLGA) nanosystem for HF delivery. These nanocomplexes not only overcame the limitations of HF but also achieved simultaneous targeting of inflammatory macrophages and human fibroblast-like synoviocytes-rheumatoid arthritis (HFLS-RA). In vivo experiments demonstrated that these nanocomplexes effectively suppressed immune-mediated inflammation and synovial hyperplasia, safeguarding against bone destruction in rats with adjuvant-induced arthritis (AIA). Remarkable anti-arthritic effects of these nanocomplexes were accomplished through promoting repolarization of M1-to-M2 macrophages and apoptosis of HFLS-RA, thereby offering a promising therapeutic strategy for RA.

Oridonin Promotes Apoptosis in Rheumatoid Arthritis Fibroblast-like Synoviocytes Through PERK/eIF2α/CHOP of Endoplasmic Reticulum Stress Pathway.

Oridonin (ORI), derived from Chinese herbs Rabdosia rubescens, has anti-inflammatory, proapoptotic, anticancer effects. Previous studies have found that ORI induces apoptosis in rheumatoid arthritis fibroblast synovial cells (RA-FLSs), but this mechanism is not clear. We will investigate the apoptosis mechanism of ORI on RA-FLSs. RA-FLSs were treated with various concentrations of ORI (0, 5, 10, 15, 20, 25, and 30 μM) for 24 h. CCK8, LDH, and hochest/PI assay determined the viability, cytotoxicity, and death of ORI on RA-FLSs. The endoplasmic reticulum probe was used to observe structural changes of endoplasmic reticulum in RA-FLSs. RNA expression was detected with RNA sequencing analysis and quantitative real-time PCR. The PERK/eIF2α/CHOP pathway protein of the endoplasmic reticulum was verified with Western Blot. Our results show that ORI induced the apoptosis of RA-FLSs from CCK8, LDH, and Hochest/PI. The endoplasmic reticulum distribution was altered in RA-FLSs after being treated with ORI. Bioinformatics analysis of RNA sequencing data found that 1453 genes were elevated. The PERK/eIF2α/CHOP pathway of the endoplasmic reticulum was regulated from the Gene ontology and KEGG analysis. The results of quantitative real-time PCR and Western blot analysis verified the regulation of PERK/eIF2α/CHOP pathway in RA-FLSs. Our data imply that the endoplasmic reticulum's PERK/eIF2α/CHOP signaling pathway is certainly implicated in the induction of RA-FLS apoptosis by ORI. This study has important implications for the pharmacological effects of ORI and the treatment of RA.

IL-1β stimulated human umbilical cord mesenchymal stem cells ameliorate rheumatoid arthritis via inducing apoptosis of fibroblast-like synoviocytes

Rheumatoid arthritis (RA) is characterized by synovial proliferation and lymphocyte accumulation leading to progressive damage of the periarticular bone and the articular cartilage. The hyperplasia of the synovial intima lining mainly consists of fibroblast-like synoviocytes-rheumatoid arthritis (HFLS-RA) which exhibit apoptosis-resistance, hyper-proliferation, and high invasiveness. The therapeutic efficacy of mesenchymal stem cells (MSCs) treatment in RA has been shown to be due to its immuno-regulatory ability. However, the exact factors and mechanisms involved in MSCs treatment in RA remain unclear. In this study, TRAIL receptor-Death receptor 4 (DR4), DR5, and LFA-1 ligand-intercellular adhesion molecule-1 (ICAM-1) were upregulated in IL-1β-stimulated HFLS-RA. We demonstrated that the total cell number of IL-1β-stimulated hUCMSCs adhering to IL-1β-stimulated HFLA-RA increased via LFA-1/ICAM-1 interaction. Direct co-culture of IL-1β-stimulated hUCMSCs with IL-1β-stimulated HFLS-RA increased the apoptosis of HFLS-RA. RA symptoms in the CIA mouse model improved after administration of IL-1β-stimulated hUCMSCs. In conclusion, IL-1β-stimulated hUCMSCs adhering to HFLS-RA occurred via LFA-1/ICAM-1 interaction, apoptosis of HFLS-RA was induced via TRAIL/DR4, DR5 contact, and RA symptoms and inflammation were significantly improved in a CIA mouse model. The results of this study suggest that IL-1β-stimulated hUCMSCs have therapeutic potential in RA treatment.

Expression of Concern: Tanshinone IIA promotes the apoptosis of fibroblast-like synoviocytes in rheumatoid arthritis by upregulating lncRNA GAS5.

Expression of Concern: Tanshinone IIA promotes the apoptosis of fibroblast-like synoviocytes in rheumatoid arthritis by upregulating lncRNA GAS5.

Exploring the supplementary potential of all-trans retinoic acid with methotrexate in rheumatoid arthritis: modulation of synovial cell apoptosis and autophagy.

The imbalance between apoptosis and proliferation in fibroblast-like synoviocytes (FLSs) plays a key role in the pathogenesis of rheumatoid arthritis (RA). This study aims to investigate the potential of all-trans retinoic acid (ATRA) as a supplementary therapeutic agent alongside methotrexate (MTX) for RA, by examining its ability to inhibit synovial cell proliferation and enhance apoptosis through the ROS-JNK signalling pathway. The viability, apoptosis, and autophagy levels of human rheumatoid arthritis fibroblast-like synovial cells (HFLS-RA) were evaluated, while ROS generation was measured through the DCFH-DA fluorescence microplate assay. Western blotting was used to analyse the expression levels of JNK signalling pathway-related proteins. To assess therapeutic potential in vivo, a collagen-induced arthritis (CIA) model was established in Wistar rats. Small doses of MTX did not significantly affect the viability of HFLS-RAs or induce apoptosis. However, when ATRA was added to the treatment, the therapy markedly inhibited cell proliferation and induced apoptosis and excessive autophagy. Mechanistically, ATRA activated the ROS/JNK signalling pathway in HFLS-RAs. ROS scavengers and JNK inhibitors significantly attenuated ATRA-induced apoptosis and autophagy. In vivo, the combination therapy demonstrated a remarkable enhancement of the anti-arthritic efficacy in CIA rats. The ability of ATRA to inhibit proliferation in RA FLSs through autophagy and apoptosis underscores its potential as a supplementary therapeutic agent alongside MTX for RA, particularly when compared to the limited impact of MTX on these processes. This combined strategy holds promise for enhancing therapeutic outcomes and warrants further investigation in the management of RA.

Geniposide augments apoptosis in fibroblast-like synoviocytes by restoring hypoxia-enhanced JNK-BNIP3-mediated autophagy.

As the main effector cells of chronic inflammation and hyperplasia of synovium, fibroblast-like synoviocytes (FLSs) show abnormal proliferation and insufficient apoptosis in the hypoxic microenvironment, which is due to the increase of BNIP3-mediated autophagy. This study aimed to explore the mechanism of geniposide (GE) on hypoxia-induced hyper-proliferative FLSs with a focus on autophagy and the JNK-BNIP3 pathway. The dynamic changes of autophagy, apoptosis, and hypoxia-related proteins in adjuvant arthritis (AA) rats were detected by immunohistochemistry and Western blot. The proliferation, autophagy, apoptosis, and mitochondrial state of FLSs were detected by CCK-8, flow cytometry, immunofluorescence, and transmission electron microscopy, respectively. Western blot, qRT-PCR, and co-immunoprecipitation were used to detect the expression of the JNK-BNIP3 pathway. The excessive accumulation of BNIP3 in the synovium of AA rats was accompanied by inhibition of apoptosis and an increase in autophagy. GE inhibited the expression of BNIP3, enhanced apoptosis, decreased autophagy, and improved chronic inflammation and hyperplasia of synovium. The amount of autophagy under different oxygen concentrations was the key to mediating the different survival rates of FLSs, and the inhibition of autophagy triggered apoptosis. GE suppressed the proliferation of FLSs and down-regulated autophagy, leading to the accumulation of ROS and the decrease of mitochondrial membrane potential, induced the increase of apoptosis, and suppressed the accumulation of BNIP3 and the hyperphosphorylation of JNK. GE inhibited autophagy by restoring the hypoxia-induced activated JNK-BNIP3 pathway, inducing mitochondrial oxidative damage, augmented apoptosis, and decreased survival rate of FLSs.

Cold atmospheric pressure plasma: A potential physical therapy for rheumatoid arthritis hyperplastic synovium

The most significant pathological change in rheumatoid arthritis (RA) is synovial hyperplasia within the joint. The production of a series of degrading enzymes and oxidative stress caused by synovial hyperplasia lead to severe bone and cartilage damage in rheumatoid joints. The core effector cell in hyperplastic synovium is fibroblast-like synovium cells, which can invade cartilage, cause inflammation, destroy joints, and show tumor-like anti-apoptosis characteristics. This study focused on the effect of cold atmospheric pressure plasma on proliferative synovium, and the results showed that no synovial hyperplasia, angiogenesis, or inflammatory infiltration was observed after cold atmospheric pressure plasma (CAP) treatment. The molecular and cellular mechanisms also reveal the spontaneous reactive oxygen species (ROS) cascade inducing apoptosis in rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) cells. This study proposes a potential physical therapy method for treating proliferative synovium and also provides ideas for the application of CAP in other types of tumor diseases.

CT2-3 induces cell cycle arrest and apoptosis in rheumatoid arthritis fibroblast-like synoviocytes through regulating PI3K/AKT pathway

Rheumatoid arthritis (RA) is a kind of chronic autoimmune disease. The existing therapies encountered several challenges. Therefore, continued novel anti-RA drug discovery remains necessary for RA therapy. Recently, our group reported a novel compound named CT2-3, which could be realized as a hybrid of the natural product magnolol and phthalimide and exhibited anti-lung cancer activity. However, the effect of CT2-3 on RA is unclear. Here, we aim to explore the effect and potential mechanism of CT2-3 on the abnormal functions of RA-fibroblast-like synoviocytes (RA-FLSs). In this study, we identified the important role of the dysregulated cell cycle and apoptosis of RA-FLSs in RA progression. Interestingly, we found that CT2-3 inhibited the proliferation and DNA replication of primary RA-FLSs and immortalized RA-FLSs namely MH7A. In addition, CT2-3 downregulated the mRNA and protein expression of cyclin-dependent kinase 2 (CDK2), cyclin A2, and cyclin B1, resulting in cell cycle arrest of primary RA-FLSs and MH7A cells. Also, CT2-3 downregulated the level of B-cell lymphoma-2 (Bcl-2), and increased the level of Bcl-2 associated X (Bax), contributing to apoptosis of primary RA-FLSs and MH7A cells. Furthermore, differential analyses of RNA-sequencing, Western blot, and network pharmacological analysis confirmed that CT2-3 inhibited phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway of primary RA-FLSs and MH7A cells. In conclusion, CT2-3 induces cell cycle arrest and apoptosis in RA-FLSs through modulating PI3K/AKT pathway, which may serve as a potential lead compound for further novel small molecule anti-RA drug development.

Cynanchum komarovii extract for the treatment of rheumatoid arthritis by acting on synovial cells in vitro and in vivo

Cynanchum komarovii (CK), the northwest Chinese region's common medicinal herb, was traditionally utilized to treat arthritis, toothache, bald sores and cholecystitis. Various forms of arthritis can be treated with CK, based on "Medicinal Plants of Chinese Desert Areas". However, the exact mechanism of action in rheumatoid arthritis (RA) is uncertain. To evaluate the in vitro and in vivo effects of CK extracts on RA and to preliminarily investigate its anti-RA mechanism of action. The main components of CK extract were analyzed by HPLC method. The effects of CK on the proliferation and apoptosis of human rheumatoid arthritis fibroblast-like synoviocytes (HFLS-RA) cells and the expression of apoptosis-related proteins in HFLS-RA cells were evaluated by CCK8 assay, flow cytometry and WB assay. To verify the anti-RA effect of CK extracts in vivo, a collagen-induced arthritis (CIA) rat model was established. The rats were divided into six groups: normal group, model group, CK high-dose group (1000mg/kg, CK-H), CK medium-dose group (500mg/kg, CK-M), CK low-dose group (250mg/kg, CK-L) and methotrexate-positive drug group (MTX); the drug was administered continuously for 28 days. Body weight changes, joint swelling, arthritis index, bone density, ankle lesions, immune organ index, splenic lesions and inflammatory factor expression were used to evaluate the in vivo anti-RA activity of the extract. The findings of in vitro experiments showed that 10% CK-containing serum decreased the expression level of Bcl-2, increased the expression levels of Bax and Cleaved Caspase-3 in synovial cells, and prevented TNF-α induced aberrant proliferation and apoptotic antagonism in HFLS-RA cells. According to in vivo studies, CK extract at doses above 250mg/kg was effective in controlling the levels of inflammatory factors, lowering the arthritis index, and improving foot swelling in CIA rats. When administered at doses up to 1000mg/kg, CK extract significantly improved synovial lesions, increased bone density, and decreased abnormally elevated immune organ index in CIA rats. CK has significant anti-RA activity, and its anti-RA mechanism of action may be related to the regulation of the expression levels of apoptosis related proteins and the promotion of apoptosis in synovial cells.

Circ_0002984 promotes proliferation, migration and inflammatory cytokine secretion and inhibits apoptosis of rheumatoid arthritis fibroblast-like synoviocytes by inducing PCSK6 through miR-543

BackgroundRheumatoid arthritis (RA) is inflammatory arthritic disease, and circular RNA is involved in RA development. The aim of the present work is to analyze the role of circ_0002984 in the process of RA fibroblast-like synoviocytes (RAFLSs) and the underlying mechanism.MethodsCirc_0002984, miR-543, and proprotein convertase subtilisin/kexin type 6 (PCSK6) expression levels were analyzed by quantitative real-time polymerase chain reaction or western blotting. Cell proliferation, migration, inflammatory response, and apoptosis were investigated through 5-Ethynyl-2′-deoxyuridine assay, wound-healing assay, enzyme-linked immunosorbent assay, and flow cytometry analysis. Dual-luciferase reporter assay and RNA immunoprecipitation assay were performed to assess the binding relationship.ResultsCirc_0002984 and PCSK6 expression were increased, while miR-543 expression was decreased in the synovial tissues of RA patients and RAFLSs. Circ_0002984 introduction facilitated RAFLS cell proliferation, migration and inflammatory response and repressed apoptosis, but circ_0002984 knockdown had an opposite role. Circ_0002984 targeted miR-543, and PCSK6 was targeted by miR-543. MiR-543 downregulation or PCSK6 overexpression restored the effects of circ_0002984 interference on RAFLS phenotypes.ConclusionCirc_0002984 promoted RAFLS proliferation, migration and inflammatory cytokine secretion and inhibited apoptosis by binding to miR-543 to induce PCSK6 production, providing a potential target for RA therapy.

HDAC2 exacerbates rheumatoid arthritis progression via the IL-17-CCL7 signaling pathway.

Histone deacetylases (HDACs) have been reported to regulate the immune response in rheumatoid arthritis (RA). The current study aimed to explore key HDACs and their molecular mechanism in RA. First, the expression of HDAC1, HDAC2, HDAC3 and HDAC8 in RA synovial tissue was determined by qRT-PCR. The effects of HDAC2 on the proliferation, migration, invasion, and apoptosis of fibroblast-like synoviocytes (FLS) in vitro were studied. Furthermore, collagen-induced arthritis (CIA) rat models were established to evaluate the severity of arthritis in joints, and the levels of inflammatory factors were examined by immunohistochemistry staining, ELISA, and qRT-PCR. Transcriptome sequencing was used to screen differentially expressed genes (DEGs) with HDAC2 silencing in the synovial tissue of CIA rat, and downstream signaling pathways were predicted by enrichment analysis. The results showed that HDAC2 was highly expressed in the synovial tissue of RA patients and CIA rats. Overexpressed HDAC2 promoted FLS proliferation, migration, and invasion and inhibited FLS apoptosis in vitro, resulting in secretion of inflammatory factors and RA exacerbation in vivo. There were 176 DEGs, including 57 downregulated and 119 upregulated genes, after silencing HDAC2 in CIA rats. DEGs were primarily enriched in Platinum drug resistance, IL-17 as well as the PI3K-Akt signaling pathways. CCL7, which was implicated in the IL-17 signaling pathway, was downregulated after HDAC2 silencing. Furthermore, CCL7 overexpression aggravated the development of RA, which was demonstrated to be effectively attenuated by HDAC2 suppression. In conclusion, this study demonstrated that HDAC2 exacerbated the progression of RA by regulating the IL-17-CCL7 signaling pathway, suggesting that HDAC2 may be a promising therapeutic target for RA treatment.

Alcohol extract of root and root bark of Toddalia asiatica alleviates CIA in rats through anti-inflammatory and proapoptotic effects

This study aims to investigate the therapeutic effect of alcohol extract of root and root bark of Toddalia asiatica(TAAE) on collagen-induced arthritis(CIA) in rats through phosphatidylinoinosidine-3 kinase/protein kinase B(PI3K/Akt) signaling pathway. To be specific, CIA was induced in rats, and then the rats were treated(oral, daily) with TAAE and Tripterygium Glycoside Tablets(TGT), respectively. The swelling degree of the hind leg joints was scored weekly. After 35 days of administration, the histopathological changes were observed based on hematoxylin and eosin(HE) staining. Enzyme-linked immunosorbent assay(ELISA) was employed to detect the levels of cytokines [tumor necrosis factor-α(TNF-α), interleukin(IL)-6)]. Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) staining was performed to detect the apoptosis of synoviocytes in rats. Western blot was used to detect the expression levels of apoptosis-related proteins B-cell lymphoma 2(Bcl-2)-associated X(Bax), Bcl-2, and caspase-3 and pathway-related proteins phosphoinositide 3-kinase(PI3K), phosphorylated(p)-PI3K, protein kinase B(Akt), and p-Akt. RT-qPCR was conducted to examine the mRNA levels of Bax, Bcl-2, caspase-3, TNF-α, IL-6, and IL-1β and pathway-related proteins PI3K, p-PI3K, Akt, and p-Akt. TAAE can alleviate the joint swelling in CIA rats, reduce serum levels of inflammatory cytokines, improve synovial histopathological changes, promote apoptosis of synoviocytes, and inhibit synovial inflammation. In addition, RT-qPCR and Western blot results showed that TAAE up-regulated the level of Bax, down-regulated the level of Bcl-2, and activated caspase-3 to promote apoptosis in synoviocytes. TAAE effectively down-regulated the protein levels of p-PI3K and p-Akt. In this study, TAAE shows therapeutic effect on CIA in rats and reduces the inflammation. The mechanism is that it suppresses PI3K/Akt signaling pathway and promotes synoviocyte apoptosis. Overall, this study provides a new clue for the research on the anti-inflammatory mechanism of TAAE and lays a theoretical basis for the better clinical application of TAAE in the treatment of inflammatory and autoimmune diseases.

LncRNA HOTTIP impacts the proliferation and differentiation of fibroblast-like synoviocytes in ankylosing spondylitis through the microRNA-30b-3p/PGK1 axis

ObjectiveLong non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have been reported to exert regulatory effects on biological processes. This study intended to assess the role of the lncRNA HOXA transcript at the distal tip (HOTTIP)/miR-30b-3p/phosphoglycerate kinase 1 (PGK1) axis in ankylosing spondylitis (AS).MethodsLevels of HOTTIP, miR-30b-3p and PGK1 in AS synovial tissues and cultured AS fibroblast-like synoviocytes (ASFLSs) were assessed. The ASFLSs were identified and, respectively, treated with altered expression of HOTTIP and miR-30b-3p, and then, the proliferation and differentiation of the ASFLSs were assessed. The AS mouse models were established by injection of proteoglycan and Freund’s complete adjuvant and then were treated with altered expression of HOTTIP and miR-30b-3p, and the pathological changes and apoptosis of synoviocytes in mice’ synovial tissues were measured. The relationship of HOTTIP, miR-30b-3p and PGK1 was verified.ResultsHOTTIP and PGK1 were elevated, while miR-30b-3p was reduced in AS synovial tissues and ASFLSs. Elevated miR-30b-3p or inhibited HOTTIP restrained proliferation and differentiation of ASFLSs and also improved the pathological changes and promoted apoptosis of synoviocytes in mice’s synovial tissues. PGK1 was a target of miR-30b-3p, and miR-30b-3p could directly bind to HOTTIP. Silencing miR-30b-3p or overexpressing PGK1 reversed the improvement of AS by knocking down HOTTIP or up-regulating miR-30b-3p.ConclusionOur study suggests that reduced HOTTIP ameliorates AS progression by suppressing the proliferation and differentiation of ASFLSs through the interaction of miR-30b-3p and PGK1.

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

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

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

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

Hexokinase inhibitor 2-deoxyglucose coordinates citrullination of vimentin and apoptosis of fibroblast-like synoviocytes by inhibiting HK2 /mTORC1-induced autophagy.

High hexokinase 2 (HK2) expression is associated with aberrant activation of fibroblast-like synoviocytes (FLSs) in rheumatoid arthritis (RA). However, the mechanism by which this occurs has not been fully elucidated. To investigate the role of HK2 and its underlying mechanism, adjuvant arthritis (AA) rats were treated with the HK2 inhibitor, 2-deoxyglucose (2-DG). In conjunction with HK2 knockdown experiments in FLSs, we evaluated the effect of HK2 on the citrullination of vimentin (cVIM), autophagy and apoptosis-associated protein expression, including that of cVIM, LC3, p62, Beclin1, Bax, Bcl2, and caspase 3. We further investigated the interaction of HK2 with downstream mTORC1 signaling effectors. Correlation analysis revealed that 2-DG treatment and HK2 knockdown upregulated the expression levels of caspase3, Bax, and p62 and downregulated the expression levels of LC3, Bcl2, and Beclin1, as well as decreasing vimentin citrullination. Furthermore, interactions between HK2 and mTOR decreased, coinciding with mTORC1 pathway activation. These findings suggest that the regulation of apoptosis and cVIM by HK2/mTORC1-dependent autophagy involves the inhibition of aberrant FLSs activation in the rat model of arthritis.

Interleukin-18 binding protein regulates the apoptosis and necroptosis of fibroblast-like synoviocytes and chondrocytes in rheumatoid arthritis.

Interleukin (IL)-18 plays a pro-inflammatory role in rheumatoid arthritis (RA), and its soluble inhibitor IL-18 binding protein (IL-18BP) has a potential therapeutic role. We investigated the role of IL-18BP on the joint destruction process of RA by accessing the effects of IL-18BP on fibroblast-like synoviocytes (FLSs) and chondrocytes. Peripheral blood mononuclear cells (PBMCs) from patients with RA and healthy controls were cultured under T cell proliferative conditions with 10, 50, or 100 ng/mL of IL-18BP. After three days of culture, flow cytometry for CD4+ T cells was performed using various IL-18BP concentrations. The apoptosis and necroptosis of FLSs and chondrocytes were measured by flow cytometry using annexin V and propidium iodide (PI) and western blot under TNF-α stimulation with IL-18BP (10, 50, and 100 ng/mL). Differentiation of CD4+ IL-17A+ and CD4+ IL-4+ cells decreased and that of CD4+ CD25high Foxp3+ and CD4+ interferon (IFN)-γ+ cells increased on addition of IL-18BP to cultured RA patient-driven PBMCs. RA-FLS migration ability was not suppressed by IL-18BP after 12 or 24 h. IL-18BP increased annexin V+ FLS level and reduced annexin V+ chondrocyte level in a dose-dependent manner, whereas PI+ annexin V- FLS and chondrocyte levels were suppressed by 50, 100 ng/mL IL-18BP in culture. The administration of IL-18BP regulated the type 17 helper T cell/ regulatory T cell imbalance and attenuated the production of pro-inflammatory cytokines. IL-18BP further increased FLS apoptosis and decreased the necroptosis of FLS/chondrocytes and apoptosis of chondrocytes suggesting the joint preservative potential of IL-18BP.