Rheumatoid Arthritis Synovial Tissue Research Articles

Oxygen supplementation liposomes for rheumatoid arthritis treatment via synergistic phototherapy and repolarization of M1-to-M2 macrophages

Rheumatoid arthritis (RA) is one of the most common systemic autoimmune diseases, with no current, clinically effective treatment. Activation of pro-inflammatory M1 macrophages is known to play a vital role in the pathogenesis of RA. Accumulating evidence suggests that phototherapy, especially photodynamic therapy (PDT), could apply as a new therapeutic method for the treatment of RA. However, due to the anoxic environment of RA synovial tissues and the oxygen consumption required for PDT process, the phototherapy efficiency was always reduced. Moreover, surviving and newly invading macrophages will be induced to polarize towards the pro-inflammatory M1-type, resulting in a relapse of the disease. To compensate for these deficits, we designed and developed novel phototherapeutic liposomal nanoparticles (CyI&Hb/FA-LPs) by incorporating heavy atom modified, cyanine dyes (CyI) as photosensitizers, and loading hemoglobin (Hb) into the inner core for relieving the hypoxic microenvironments and repolarizing M1-to-M2 macrophages. Meanwhile, folic acid was attached to the liposome surface to achieve active targeting of M1 macrophages. In vitro and in vivo studies demonstrate that near infrared light irradiation of CyI&Hb/FA-LPs reduces M1 macrophage populations and reduces the expression of hypoxia-inducible factor-1α that drives the remaining M1 macrophages to transition to the anti-inflammatory M2 phenotype. Moreover, intra-articular treatment of CyI&Hb/FA-LPs in a RA mouse model inhibited synovial cell infiltration, and alleviated inflammation and pathological features in the affected joint. This study highlights the potential of CyI-based oxygenated liposomes as a new and versatile strategy for implementing RA targeting phototherapy and M1-to-M2 repolarization. The dual mode phototherapy and O2 supplementation agent appears to function synergistically by reducing M1 macrophage populations through direct killing and induced apoptosis, as well as by inducing M2 phenotype proliferation.

Preparation of mesoporous silica nanocarriers targeting glucose-6-phosphate isomerase inhibition and application in the treatment of rheumatoid arthritis.

Glucose 6-phosphate isomerase (G6PI) is an indicator to assist in diagnosis of rheumatoid arthritis (RA) and monitor the disease. It also plays a key role in proliferating RA synovial tissues, pannus formation, and invasion and destruction of articular cartilage. In this study, we synthesized nanoparticles targeting G6PI (siG6PI-MSN) using mesoporous silica nanocarriers (MSN) and small interfering RNA (siRNA), followed by identifying the characteristics and functions, and preliminarily exploring their application in the treatment of RA in vivo with a type II collagen-induced arthritis (CIA) rat model. It showed that the synthetic functionalized carrier had a regular pore structure and a specific volume and surface area. No obvious hemolysis or toxicity of the carrier was found when its concentration was below 100 µg/ml. Cytological results in vitro suggested that siG6PI-MSN significantly inhibited G6PI expression and reduced the ability of proliferation, migration, and invasion of FLSs, compared with the siNC-MSN group. In vivo results in the CIA rat model showed that the arthritis index and degree of joint swelling among rats in the siG6PI-MSN-treatment group were significantly lower than those in the control group. Moreover, the number of FLSs in Synovium and the levels of TNF α and IL-1 β were also significantly decreased in the siG6PI-MSN group. Histopathology of the synovial tissue and cartilage revealed siG6PI-MSN treatment significantly reduced the pathological manifestations of arthritis. In conclusion, siG6PI-MSN effectively suppresses the proliferation and invasive growth of synovial tissue and improve joint swelling and inflammatory infiltration, thereby preventing joint damage in RA. This carrier may be a new therapeutic measure for RA, with potential social and economic benefits.

Screening and identification of potential hub genes and immune cell infiltration in the synovial tissue of rheumatoid arthritis by bioinformatic approach

BackgroundRheumatoid arthritis (RA) is an autoimmune disease that affects individuals of all ages. The basic pathological manifestations are synovial inflammation, pannus formation, and erosion of articular cartilage, bone destruction will eventually lead to joint deformities and loss of function. However, the specific molecular mechanisms of synovitis tissue in RA are still unclear. Therefore, this study aimed to screen and explore the potential hub genes and immune cell infiltration in RA. MethodsThree microarray datasets (GSE12021, GSE55457, and GSE55235), from the Gene Expression Omnibus (GEO) database, have been analyzed to explore the potential hub genes and immune cell infiltration in RA. First, the LIMMA package was used to screen the differentially expression genes (DEGs) after removing the batch effect. Then the clusterProfiler package was used to perform functional enrichment analyses. Second, through weighted coexpression network analysis (WGCNA), the key module was identified in the coexpression network of the gene set. Third, the protein-protein interaction (PPI) network was constructed through STRING website and the module analysis was performed using Cytoscape software. Fourth, the CIBERSORT and ssGSEA algorithm were used to analyze the immune status of RA and healthy synovial tissue, and the associations between immune cell infiltration and RA-related diagnostic biomarkers were evaluated. Fifth, we used the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to validate the expression levels of the hub genes, and ROC curve analysis of hub genes for discriminating between RA and healthy tissue. Finally, the gene-drug interaction network was constructed using DrugCentral database, and identification of drug molecules based on hub genes using the Drug Signature Database (DSigDB) by Enrichr. ResultsA total of 679 DEGs were identified, containing 270 downregulated genes and 409 upregulated genes. DEGs were primarily enriched in immune response and chemokine signaling pathways, according to functional enrichment analysis of DEGs. WGCNA explored the co-expression network of the gene set and identified key modules, the blue module was selected as the key module associated with RA. Seven hub genes are identified when PPI network and WGCNA core modules are intersected. Immune infiltration analysis using CIBERSORT and ssGSEA algorithms revealed that multiple types of immune infiltration were found to be upregulated in RA tissue compared to normal tissue. Furthermore, the levels of 7 hub genes were closely related to the relative proportions of multiple immune cells in RA. The results of the qRT-PCR demonstrated that the relative expression levels of 6 hub genes (CD27, LCK, CD2, GZMB, IL7R, and IL2RG) were up-regulated in RA synovial tissue, compared with normal tissue. Simultaneously, ROC curves indicated that the above 6 hub genes had strong biomarker potential for RA (AUC >0.8). ConclusionsThrough bioinformatics analysis and qRT-PCR experiment, our study ultimately discovered 6 hub genes (CD27, LCK, CD2, GZMB, IL7R, and IL2RG) that closely related to RA. These findings may provide valuable direction for future RA clinical diagnosis, treatment, and associated research.

Bone Marrow Mesenchymal Stem Cells Release miR-378a-5p-Carried Extracellular Vesicles to Alleviate Rheumatoid Arthritis

This study investigates whether bone marrow mesenchymal stem cell (BMSC)-derived extracellular vesicles (EVs) can affect rheumatoid arthritis (RA) by delivering microRNA (miR)-378a-5p to regulate the interferon regulatory factor 1/signal transducer and transcription 1 (IRF1/STAT1) axis. We identified RA-associated miRNAs using the GEO microarray dataset GSE121894. We found the most important miRNAs in RA synovial tissues using RT-qPCR. BMSC-derived EVs were ultracentrifuged and cocultured with human synovial microvascular endothelial cells (HSMECs) in vitro. Dual-luciferase and RNA immunoprecipitation studies examined miR-378a-5p’s specific binding to IRF1. We also measured angiogenesis, migration, and proliferation using CCK-8, Transwell, and tube formation assays. Collagen-induced arthritis (CIA) mice models were created by inducing arthritis and scoring it. RA synovial tissues had low miR-378a-5p expression, whereas BMSC-derived EVs had high levels. The transfer of miR-378a-5p by BMSC-derived EVs to HSMECs boosted proliferation, migration, and angiogenesis. miR-378a-5p inhibited IRF1. MiR-378a-5p-containing BMSC-derived EVs decreased STAT1 phosphorylation and HSMEC IRF1 expression. EVs with miR-378a-5p mimic promoted HSMEC proliferation, migration, and angiogenesis, whereas dexmedetomidine inhibited STAT1 phosphorylation. In CIA mice, BMSC-derived EVs containing miR-378a-5p enhanced synovial vascular remodeling and histopathology. Thus, miR-378a-5p from BMSC-derived EVs promotes HSMEC proliferation, migration, and angiogenesis, inactivating the IRF1/STAT1 axis and preventing RA.

Nesfatin-1 Stimulates CCL2-dependent Monocyte Migration And M1 Macrophage Polarization: Implications For Rheumatoid Arthritis Therapy.

Rheumatoid arthritis (RA) is a prototypic inflammatory disease, characterized by the infiltration of proinflammatory cytokines into the joint synovium and the migration of mononuclear cells into inflammatory sites. The adipokine nesfatin-1 is linked to inflammatory events in various diseases, although its role in RA pathology is uncertain. Analysis of the Gene Expression Omnibus GSE55235 dataset revealed high levels of expression of the adipokine nesfatin-1 in human RA synovial tissue. Similarly, our human synovial tissue samples exhibited increasing levels of nesfatin-1 expression and Ccl2 mRNA expression. Nesfatin-1-induced stimulation of CCL2 expression and monocyte migration involved the MEK/ERK, p38, and NF-κB signaling pathways. Notably, nesfatin-1-induced increases in CCL2 expression favored M1 macrophage polarization, which increased the expression of proinflammatory cytokines IL-1β, IL-6, and TNF-α. Finally, nesfatin-1 shRNA ameliorated the severity of inflammatory disease and reduced levels of M1 macrophage expression in CIA mice. Our studies confirm that nesfatin-1 appears to be worth targeting in RA treatment.

Circ_0083964 knockdown impedes rheumatoid arthritis progression via the miR-204-5p-dependent regulation of YY1

BackgroundRheumatoid arthritis (RA) is a chronic inflammatory disease. Abnormal proliferation and inflammation of fibroblast-like synoviocytes (FLSs) are the main pathological features of the disease. Accumulating studies have identified that circular RNAs (circRNAs) were involved in the progression of RA. Our study was to assess the function and mechanism of circ_0083964 in RA.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) and western blot were utilized to test the level of circ_0083964, miR-204-5p and YY1. Counting Kit-8 (CCK-8) assay, EdU assay, flow cytometry, transwell assay and wound-healing assay were utilized to test cell viability, proliferation, apoptosis, invasion and migration. Cell inflammation was estimated with enzyme-linked immunosorbent assay (ELISA) kits. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were employed to identify the target relationship between miR-204-5p and circ_0083964 or YY1.ResultsCirc_0083964 was highly expressed in RA synovial tissues and RA-FLSs. Circ_0083964 downregulation constrained proliferation, metastasis and inflammation and facilitated apoptosis in RA-FLSs. Furthermore, circ_0083964 served as a sponge of miR-204-5p, and rescue experiments proved that miR-204-5p deficiency overturned the suppressive impacts of circ_0083964 silencing on RA-FLSs progression. Additionally, we also verified that YY1 could be targeted by miR-204-5p, and its overexpression rescued the repressive impact of miR-204-5p introduction on RA-FLSs development. Besides that, we revealed that circ_0083964 mediated YY1 expression by regulating miR-204-5p.ConclusionCirc_0083964 inhibition confined RA development by sponging miR-204-5p to hamper the YY1 level, which will provide a theoretical basis for the treatment of RA.

Epstein-Barr virus-induced gene 3 commits human mesenchymal stem cells to differentiate into chondrocytes via endoplasmic reticulum stress sensor.

Mesenchymal stem cells (MSC) can differentiate into chondrocytes. Epstein-Barr virus-induced gene 3 (EBI3) is differentially expressed during chondrogenic differentiation and can be produced by MSC. EBI3 is also a subunit of interleukin (IL)-27 and IL-35, and it accumulates in the endoplasmic reticulum (ER) when its partners, such as IL-27 p28 and IL-35 p35, are insufficient. ER stress induced by protein accumulation is responsible for chondrogenic differentiation. However, the role of EBI3 and its relevance to the ER stress in chondrogenic differentiation of MSC have never been addressed. Here, we demonstrate that EBI3 protein is expressed in the early stage of chondrogenic differentiation of MSC. Additionally, knockdown, overexpression, or induction of EBI3 through IL-1β inhibits chondrogenesis. We show that EBI3 localizes and accumulates in the ER of MSC after overexpression or induction by IL-1β and TNF-α, whereas ER stress inhibitor 4-phenylbutyric acid decreases its accumulation in MSC. Moreover, EBI3 modulates ER stress sensor inositol-requiring enzyme 1 α (IRE1α) after induced by IL-1β, and MSC-like cells coexpress EBI3 and IRE1α in rheumatoid arthritis (RA) synovial tissue. Altogether, these data demonstrate that intracellular EBI3 commits to chondrogenic differentiation by regulating ER stress sensor IRE1α.

SMOC2 promotes aggressive behavior of fibroblast-like synoviocytes in rheumatoid arthritis through transcriptional and post-transcriptional regulating MYO1C

Fibroblast-like synoviocytes (FLSs), play a key role in perpetuating synovial inflammation and bone erosion in rheumatoid arthritis (RA), however, the underlying mechanism(s) of RA FLSs activation and aggression remain unclear. Identifying endogenous proteins that selectively target FLSs is urgently needed. Here, we systematically identified that secreted modular calcium-binding protein 2 (SMOC2), was significantly increased in RA FLSs and synovial tissues. SMOC2 knockdown specifically regulated cytoskeleton remodeling and decreased the migration and invasion of RA FLSs. Mechanistically, cytoskeleton-related genes were significantly downregulated in RA FLSs with reduced SMOC2 expression, especially the motor protein myosin1c (MYO1C). SMOC2 controlled MYO1C expression by SRY-related high-mobility group box 4 (SOX4) and AlkB homolog 5 (ALKHB5) mediated-m6A modification through transcriptional and post-transcriptional regulation. Furthermore, intra-articular Ad-shRNA-SMOC2 treatment attenuated synovial inflammation as well as bone and cartilage erosion in rats with collagen-induced arthritis (CIA). Our findings suggest that increased SMOC2 expression in FLSs may contribute to synovial aggression and joint destruction in RA. SMOC2 may serve as a potential target against RA.SMOC2-mediated regulation of the synovial migration and invasion in RA FLSs. In RA FLSs, SMOC2 is significantly increased, leading to the increased level of MYO1C via SOX4-mediated transcriptional regulation and ALKBH5-mediated m6A modification, thereby causing cytoskeleton remodeling and promoting RA FLSs migration and invasion. The Figure was drawn by Figdraw.

Increased serum IL-41 is associated with disease activity in rheumatoid arthritis

BackgroundInterleukin (IL)-41 is upregulated in the synovial tissue of rheumatoid arthritis (RA) patients, but its serum level has not been reported. The present study aimed to determine IL-41 expression in serum from RA patients and to clarify the relationships between IL-41 and disease-related parameters in RA patients. MethodsThe study included 46 RA patients and 32 healthy controls (HC). Baseline data were obtained by routine physical examinations and immune-related parameters were measured by an automated chemiluminescent immunoassay analyzer. The correlations between IL-41 and disease activity score in 28 joints (DAS28) and serum clinical data were analyzed using the Pearson correlation test. ResultsSerum IL-41 concentrations were higher in RA patients than in HC. Serum IL-41 was positively correlated with DAS28 based on C-reactive protein (DAS28-CRP), CRP, erythrocyte sedimentation rate (ESR), mean platelet volume (MPV), and CRP-to-albumin ratio (CAR) and negatively correlated with platelet count, while rheumatoid factor was significantly correlated with ESR, CRP, and CAR. Receiver operating characteristic curve analysis showed that IL-41 had diagnostic value for RA, especially when combined with MPV. ConclusionsThe present findings suggest that IL-41 is increased in the serum of RA patients and may be a potential new diagnostic biomarker for RA.

NFIL3 and its immunoregulatory role in rheumatoid arthritis patients.

Nuclear-factor, interleukin 3 regulated (NFIL3) is an immune regulator that plays an essential role in autoimmune diseases. However, the relationship between rheumatoid arthritis (RA) and NFIL3 remains largely unknown. In this study, we examined NFIL3 expression in RA patients and its potential molecular mechanisms in RA. Increased NFIL3 expression levels were identified in peripheral blood mononuclear cells (PBMCs) from 62 initially diagnosed RA patients and 75 healthy controls (HCs) by quantitative real-time PCR (qRT-PCR). No correlation between NFIL3 and disease activity was observed. In addition, NFIL3 expression was significantly upregulated in RA synovial tissues analyzed in the Gene Expression Omnibus (GEO) dataset (GSE89408). Then, we classified synovial tissues into NFIL3-high (≥75%) and NFIL3-low (≤25%) groups according to NFIL3 expression levels. Four hundred five differentially expressed genes (DEGs) between the NFIL3-high and NFIL3-low groups were screened out using the "limma" R package. Enrichment analysis showed that most of the enriched genes were primarily involved in the TNF signaling pathway via NFκB, IL-17 signaling pathway, and rheumatoid arthritis pathways. Then, 10 genes (IL6, IL1β, CXCL8, CCL2, PTGS2, MMP3, MMP1, FOS, SPP1, and ADIPOQ) were identified as hub genes, and most of them play a key role in RA. Positive correlations between the hub genes and NFIL3 were revealed by qRT-PCR in RA PBMCs. An NFIL3-related protein-protein interaction (PPI) network was constructed using the STRING database, and four clusters (mainly participating in the inflammatory response, lipid metabolism process, extracellular matrix organization, and circadian rhythm) were constructed with MCODE in Cytoscape. Furthermore, 29 DEGs overlapped with RA-related genes from the RADB database and were mainly enriched in IL-17 signaling pathways. Thus, our study revealed the elevated expression of NFIL3 in both RA peripheral blood and synovial tissues, and the high expression of NFIL3 correlated with the abnormal inflammatory cytokines and inflammatory responses, which potentially contributed to RA progression.

Identification and Validation of Hub Genes for Predicting Treatment Targets and Immune Landscape in Rheumatoid Arthritis.

Background Rheumatoid arthritis (RA) is recognized as a chronic inflammatory disease featured by pathological synovial inflammation. Currently, the underlying pathophysiological mechanisms of RA remain unclear. In the study, we attempted to explore the underlying mechanisms of RA and provide potential targets for the therapy of RA via bioinformatics analysis. Methods We downloaded four microarray datasets (GSE77298, GSE55235, GSE12021, and GSE55457) from the GEO database. Firstly, GSE77298 and GSE55457 were identified DEGs by the “limma” and “sva” packages of R software. Then, we performed GO, KEGG, and GSEA enrichment analyses to further analyze the function of DEGs. Hub genes were screened using LASSO analysis and SVM-RFE analysis. To further explore the differences of the expression of hub genes in healthy control and RA patient synovial tissues, we calculated the ROC curves and AUC. The expression levels of hub genes were verified in synovial tissues of normal and RA rats by qRT-PCR and western blot. Furthermore, the CIBERSORTx was implemented to assess the differences of infiltration in 22 immune cells between normal and RA synovial tissues. We explored the association between hub genes and infiltrating immune cells. Results CRTAM, CXCL13, and LRRC15 were identified as RA's potential hub genes by machine learning and LASSO algorithms. In addition, we verified the expression levels of three hub genes in the synovial tissue of normal and RA rats by PCR and western blot. Moreover, immune cell infiltration analysis showed that plasma cells, T follicular helper cells, M0 macrophages, M1 macrophages, and gamma delta T cells may be engaged in the development and progression of RA. Conclusions In brief, our study identified and validated that three hub genes CRTAM, CXCL13, and LRRC15 might involve in the pathological development of RA, which could provide novel perspectives for the diagnosis and treatment with RA.

Protein arginine methyltransferase 1 upregulates matrix metalloproteinase-2/9 expression via Zeste Homolog 2 to promote human rheumatoid arthritis fibroblast-like synovial cell survival and metastasis.

To explore the role of protein arginine methyltransferase 1 (PRMT1) in the development of rheumatoid arthritis (RA). Fibroblast-like synoviocytes (FLS) were isolated from synovial tissues, cultured and transfected with plasmid vector or short hairpin RNA (shRNA). The morphology and surface markers of FLS were investigated by light microscopy and flow cytometry. The expression levels of PRMT1, Zeste Homolog 2 (EZH2), matrix metalloproteinase-2 (MMP-2) and MMP-9 were detected by real-time polymerase chain reaction and Western blotting. Cell viability was tested by MTT assay, cell proliferation was assessed by EdU assay, cell invasion was evaluated by Transwell invasion test, cell migration was detected by wound healing assay, and cell apoptosis was detected by flow cytometry. The expression of PRMT1 was elevated in RA synovial tissues compared with control tissues. FLS from control tissues showed a lower level of PRMT1 than FLS from RA tissues, and FLS from RA tissues had a stronger ability of cell survival and metastasis than those from control tissues. When silencing PRMT1 expression, FLS from RA tissues showed a decreased ability of cell survival and metastasis. Additionally, FLS from RA tissues expressed a higher level of MMP-2 and MMP-9. When silencing PRMT1 expression, the expression of MMP-2 and MMP-9 of FLS was suppressed. Furthermore, the effect of PRMT1 on FLS was mediated by EZH2. We found that PRMT1 had an overall effect on FLS via EZH2, which contributed to the development of RA. Hence, PRMT1 and EZH2 provide potential targets for RA treatment.

Identifying hub circadian rhythm biomarkers and immune cell infiltration in rheumatoid arthritis.

BackgroundRheumatoid arthritis (RA) is a chronic systemic autoimmune disease with symptoms characterized by typical circadian rhythmic changes. This study aimed to identify the hub circadian rhythm genes (CRGs) in RA and explore their association with immune cell infiltration and pathogenesis of RA.MethodsThe differentially expressed CRGs (DECRGs) between RA and normal control samples were screened from Datasets GSE12021 and GSE55235. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Gene Set Enrichment Analysis were used to explore the potential functional mechanisms of DECRGs in RA. Weighted Gene Co-expression Network Analysis and Least Absolute Shrinkage and Selection Operator regression analysis were performed to identify hub CRGs of RA. CIBERSORT was conducted to compare the infiltration level of immune cells in RA and control synovial tissue and their relationship with hub genes. In addition, the diagnostic value of hub biomarkers was evaluated by the area under the receiver operator characteristic curve. Further, a nomogram prediction model was constructed and its significance for clinical decision-making was evaluated.ResultsThe green module was identified as the hub module associated with RA. Four hub CRGs (EGR1, FOSL2, GADD45B, and NFIL3) were identified and showed that they had the highest specificity and sensitivity for RA diagnosis, respectively. The expression levels and diagnostic values of these genes were externally validated in the dataset GSE55457. A nomogram prediction model based on the four hub CRGs was constructed and proved to have a certain clinical decision value. Additionally, the correlation analysis of immune cells with hub genes showed that all hub genes were significantly positively correlated with activated mast cells, resting memory CD4+ T cells, and monocytes. Whereas, all hub genes were negatively correlated with plasma cells, CD8+ T cells, and activated memory CD4+ T cells. Meanwhile, FOSL2 and GADD45B were negatively correlated with Tfh cells.ConclusionFour hub CRGs were identified and showed excellent diagnostic value for RA. These genes may be involved in the pathological process of RA by disrupting the rhythmic oscillations of cytokines through immune-related pathways and could be considered molecular targets for future chronotherapy against RA.

Effect of heat-reinforcing needling on expression of serum inflammatory factors and autophagy of knee joint synovial tissue in rheumatoid arthritis rabbits with cold syndrome

To observe the effect of heat-reinforcing needling on the expression of serum inflammatory factors and autophagy of knee synovial tissue in rheumatoid arthritis (RA) rabbits with cold syndrome, so as to explore its mechanism of anti-inflammatory in the treatment of RA. Fifty rabbits were randomly divided into normal, model, heat-reinforcing needling, inhibitor and agonist groups (n=10 rabbits in each group). The model of RA with cold syndrome was established by Freund's adjuvant and ovalbumin mixed solution injection combined with freezing and wind-cold dampness method. Heat-reinforcing needling was applied at "Zusanli" (ST36) for 30 min, once a day for 14 days. Rabbits of the inhibitor and agonist groups were given intraperitoneally injected with autophagy inhibitor 3-methyladenine (3-MA) or autophagy agonist rapamycin, once every 2 days for 7 days. The knee circumference and skin temperature of the rabbits in each group were measured. Color doppler ultrasonography was applied to examine the synovial membrane, joint effusion and blood flow signals in the knee joints of the rabbits in each group. Serum tumor necrosis factor (TNF) -α, interleukin (IL)-1β, IL-6 and C-creactive protein (CRP) were detected by ELISA. Transmission electron microscopy was applied to observe the ultrastructure and autophagosomes of synovial cells. The protein expressions of autophagy-related protein Atg5, serine/threonine protein kinase-dysregulated 51-like kinase 1 (ULK1), microtubule-associated protein light chain 3B (LC3B), and Beclin-1 were detected by Western blot. Fluorescence quantitative PCR was used to detect the mRNA expressions of NOD-like receptor 3 (NLRP3) and nuclear factor-κB (NF-κB). Compared with the normal group, the circumference of the knee joint was increased (P<0.01), the skin temperature was decreased (P<0.01), the knee joint synovium was thickened and the blood flow signal was abundant, the contents of serum TNF-α, IL-1β, IL-6, and CRP were increased (P<0.01), the protein expressions of Atg5, ULK1, Beclin-1 and LC3BⅡ/LC3BⅠof synovial tissue were significantly decreased (P<0.01), the mRNA expressions of NLRP3 and NF-κB were increased (P<0.01) in the model group. In comparison with the model and inhibitor groups, the circumference of the knee joint was decreased (P<0.01), whlie the skin temperature was increased (P<0.01), the synovial membrane became thinner and the blood flow signal was wea-kened, the contents of TNF-α, IL-1β, IL-6 and CRP were decreased (P<0.01), the protein expressions of Atg5, ULK1, Beclin-1 and LC3B Ⅱ/LC3B Ⅰ were increased (P<0.01), and the mRNA expressions of NLRP3 and NF-κB were decreased (P<0.01) in the heat-reinforcing needling and agonist groups. Heat-reinforcing needling can alleviate the inflammatory response of the knee joint synovium in RA rabbits with cold syndrome, which may be related to its function in enhancing the autophagy activity of synovial cells and inhibiting the synthesis and release of inflammatory factors TNF-α, IL-1β, IL-6 and CRP.

Circ_0004712 Silencing Suppresses the Aggressive Changes of Rheumatoid Arthritis Fibroblast-Like Synoviocytes by Targeting miR-633/TRAF6 Axis.

Circular RNA_0004712 (circ_0004712) is reported to be up-regulated in rheumatoid arthritis (RA) patients. Nevertheless, its role and mechanism in RA pathology remain to be clarified. RNA and protein expression was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot assay. Cell viability, proliferation, apoptosis, migration, and inflammation were assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, 5-ethynyl-20-deoxyuridine assay, flow cytometry, scratch test, and enzyme-linked immunosorbent assay. The target correlation between microRNA-633 (miR-633) and circ_0004712 or TNF receptor associated factor 6 (TRAF6) was verified by dual-luciferase reporter assay and RNA immunoprecipitation assay. Circ_0004712 was up-regulated in RA synovial tissues and RA fibroblast-like synoviocytes (RA-FLSs). Circ_0004712 silencing suppressed the viability, proliferation, migration and inflammatory response and facilitated the apoptosis of RA-FLSs. miR-633 was confirmed to be a direct target of circ_0004712, and miR-633 knockdown reversed circ_0004712 silencing-mediated protective effects on the dysfunction and inflammation of RA-FLSs. TRAF6 was a direct target of miR-633, and miR-633 overexpression suppressed the aggressive changes of RA-FLSs by down-regulating TRAF6. Circ_0004712 could up-regulate TRAF6 expression by sponging miR-633 in RA-FLSs. Circ_0004712 interference inactivated nuclear factor (NF)-κB signaling by targeting miR-633/TRAF6 axis. Circ_0004712 silencing inhibited the aggressive changes of RA-FLSs by targeting miR-633/TRAF6 axis and NF-κB signaling, which provided new targets for RA therapy.

Assessment of disease activity in patients with rheumatoid arthritis using plasma tumour M2-pyruvate kinase test.

BackgroundPyruvate kinase M2 (PKM2) is an enzyme that regulates the final process of glycolysis and exists in tetrameric and dimeric forms. The dimeric form of PKM2, also known as tumour M2-PK, increases when aerobic glycolysis is augmented, a feature observed in rheumatoid arthritis (RA). We investigated whether plasma tumour M2-PK is elevated in patients with RA and whether its levels correlate with disease activity.MethodsPlasma levels of tumour M2-PK were measured for patients with RA (n=151), those with osteoarthritis (OA) (n=37), and controls (n=37). We evaluated the association between plasma tumour M2-PK and continuous variables using Pearson’s correlation analysis, and multivariate logistic regression analysis to determine the association between plasma tumour M2-PK and disease activity status. Knee synovial tissue blocks from patients with RA and OA were subjected to real-time quantitative PCR (qPCR) using two different primers for PKM2 and tumour M2-PK immunohistochemical (IHC) staining.ResultsThe tumour M2-PK level significantly correlated with the disease activity score in 28 joints (DAS28)-erythrocyte sedimentation rate (ESR) (r=0.546, p<0.001) and DAS28-C-reactive protein (CRP) (r=0.589, p<0.001). Moreover, repeat testing of tumour M2-PK levels in 20 patients revealed a significant decline in tumour M2-PK levels after reduction in inflammation (p<0.001). Area under the receiver operating characteristic curve (AUROC) analysis demonstrated that upon incorporation of tumour M2-PK, ESR, and CRP, the area under the curve was 0.962 for distinguishing moderate/high from remission/low disease activity. Adjusted logistic regression also revealed that a tumour M2-PK >43.9 U/mL (OR 3.672, p=0.042) independently predicted moderate/high disease activity status. Furthermore, tumour M2-PK levels in patients with RA were significantly higher than in those with OA and controls (all p<0.001). However, no differences were found in PKM2 expression in RA and OA synovial tissues as assessed by qPCR, and IHC analysis revealed negligible tumour M2-PK expression in the synovial tissues.ConclusionCirculating plasma tumour M2-PK levels may be a clinically useful indicator for evaluating disease activity and RA diagnosis.

Elevated expression of TAM receptor tyrosine kinase in synovial fluid and synovial tissue of rheumatoid arthritis.

To investigate the expression and roles of TAM (Tyro3/Axl/Mer) receptor tyrosine kinases (TK) in synovial fluid and synovial tissue of patients with rheumatoid arthritis (RA). The expression of TAM TKs in the synovial fluid and synovial tissues of RA and osteoarthritis (OA) patients was measured by ELISA and immunohistochemistry. The relationships between soluble TAM TKs (sTAM TKs) levels and the clinical features, laboratory parameters and disease activity were analyzed in RA. The concentrations of sTAM TK in the synovial fluids of RA patients were increased in comparison to those of OA patients. Compared with OA patients, the expression of membrane Tyro3 TK (mTyro3 TK) and mMer TK in RA patient synovial tissue were significantly increased, which may partly explain the possible mechanism of elevated levels of sTAM TK in RA patient synovial fluid. sAxl TK levels were decreased in RA patients under sulfasalazine treatment and elevated in patients under Iguratimod treatment. Furthermore, sTyro3 TK levels were positively correlated with erythrocyte sedimentation rate (ESR) and negatively correlated with white blood cells (WBCs), red blood cells (RBCs), and hemoglobin (HB) in RA patients. The levels of sMer TK were positively associated with disease duration and rheumatoid factor (RF) and negatively correlated with HB, complement 3 (C3), and C4. Taken together, TAM TKs might be involved in RA synovial tissue inflammation.

Novel isatin-based hybrids as potential anti-rheumatoid arthritis drug candidates: Synthesis and biological evaluation

Rheumatoid arthritis (RA) is an autoimmune disease accompanied with serious symptoms, such as joint destruction and chronic synovitis. Though many anti-RA drugs could improve the outcome of RA patients to a certain extent, about 40% inefficient rate, severe side effects, and high costs have become urgent problems. Therefore, exploring new alternative drugs for RA therapy is still an urgent need so far. Isatin is an important structural motif found in numerous biologically active compounds and therapeutic agents. Herein, we aim to synthesize several novel isatin analogues for RA therapy and further explore the mechanism of the most potential anti-RA drug candidate in suppressing the pathological progress of RA in vitro and in vivo. We found that the most therapeutic potential compound, a novel small molecule isatin-honokiol hybrid named CT5-2 inhibited the viability of RA-fibroblast-like synoviocytes (FLSs), an effector cell of synovial hyperplasia in the RA synovial tissue with IC50 ranging from 8.54 to 10.66 μM. In addition, CT5-2 reduced the DNA replication and triggered cell cycle arrest and apoptosis of RA-FLSs. Moreover, differential analyses of RNA-sequencing and the mechanistic studies demonstrated that CDCA7 is a key gene correlated with RA progression, and CT5-2 could inhibit the c-Myc/CDCA7/p65 pathway to regulate CDK1, Bcl-2, and vimentin in RA-FLSs. Furthermore, CT5-2 relieved collagen-induced arthritis (CIA) and reduced the level of CDCA7, CDK1, Bcl-2, and vimentin of synovial tissue in CIA mice. Taken together, the novel small molecule isatin-honokiol hybrid CT5-2 exhibits a potential anti-RA drug candidate that inhibits proliferation and triggers cell cycle arrest and apoptosis of RA-FLSs by regulating the c-Myc/CDCA7/p65 pathway. Our study lays a good foundation for further clinical research and structuralmodification of CT5-2.

Grape seed proanthocyanidin extract induces apoptotic and autophagic cell death in rheumatoid arthritis fibroblast-like synoviocytes.

In this study, we aimed to evaluate the association between grape seed proanthocyanidin extract (GSPE) and rheumatoid arthritis-fibroblast-like synoviocytes (RA-FLSs) and to investigate whether GSPE induces cell death in RA-FLSs. The FLSs were isolated from RA synovial tissues. Cell viability and cell cycle staging were analyzed using a hemocytometer and flow cytometry. Caspase 3 and poly (ADP-ribose) polymerase (PARP) proteins were analyzed using Western blotting with z-VAD-fmk. Protein LC3 and polyubiquitin-binding protein p62 that were degraded by autophagy were evaluated using Western blotting with 3-methyladenine and chloroquine. Reactive oxygen species (ROS) were also evaluated. When RA-FLSs were treated with GSPE, cell viability decreased, the number of cells in sub-G1 and G2/M phases increased, and the expression of pro-PARP and pro-caspase 3 proteins decreased in a concentration-dependent manner. This result was offset, when the cells were co-treated with the pan-caspase inhibitor z-VAD-fmk. The reduced cell viability, increased expression of LC3-II protein, and reduced expression of p62 protein with GSPE treatment were offset, when RA-FLSs were co-treated with GSPE and autophagy inhibitors 3-methyladenine and chloroquine. The level of ROS in RA-FLSs treated with GSPE was significantly lower than treatment with N-acetyl-cysteine, a ROS inhibitor. Our study results show that GSPE induces apoptotic and autophagic cell death and inhibites reactive oxygen species in RA-FLSs.

Two Main Cellular Components in Rheumatoid Arthritis: Communication Between T Cells and Fibroblast-Like Synoviocytes in the Joint Synovium.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that endangers the health of approximately 1% of the global population. Current RA medications on the market mainly include non-steroidal anti-inflammatory drugs, biological agents, and disease-modifying drugs. These drugs aim to inhibit the overactivated immune response or inflammation of RA, but they cannot cure RA. A better understanding of the pathogenesis of RA will provide a new understanding to search for RA targets and for drug development. The infiltration of T cells and hyper-proliferation of fibroblast-like synoviocytes (FLS) in the synovium of patients with RA are significantly upregulated. Furthermore, the abnormal activation of these two types of cells has been confirmed to promote development of the course of A by many studies. This article systematically summarizes the interactions between T cells and FLS in RA synovial tissues, including one-way/mutual regulation and direct/indirect regulation between the two. It further aims to investigate the pathogenesis of RA from the perspective of mutual regulation between T cells and FLS and to provide new insights into RA research.