Proliferation Of Synovial Fibroblasts Research Articles

Endogenous Melanin and Hydrogen-Based Specific Activated Theranostics Nanoagents: A Novel Multi-Treatment Paradigm for Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disorder characterized by excessive proliferation of rheumatoid arthritis synovial fibroblasts (RASFs) and accumulation of inflammatory cytokines. Exploring the suppression of RASFs and modulation of the RA microenvironment is considered a comprehensive strategy for RA. In this work, specifically activated nanoagents (MAHI NGs) based on the hypoxic and weakly acidic RA microenvironment are developed to achieve a second near-infrared fluorescence (NIR-II FL)/photoacoustic (PA) dual-model imaging-guided multi-treatment. Due to optimal size, the MAHI NGs passively accumulate in the diseased joint region and undergo rapid responsive degradation, precisely releasing functionalized components: endogenous melanin-nanoparticles (MNPs), hydrogen gas (H2), and indocyanine green (ICG). The released MNPs play a crucial role in ablating RASFs within the RA microenvironment through photothermal therapy (PTT) guided by accurate PA imaging. However, the regional hyperthermia generated by PTT may exacerbate reactive oxygen species (ROS) production and inflammatory response following cell lysis. Remarkably, under the acidic microenvironment, the controlled release of H2 exhibits precise synergistic antioxidant and anti-inflammatory effects with MNPs. Moreover, the ICG, the second near-infrared dye currently approved for clinical use, possesses excellent NIR-II FL imaging properties that facilitate the diagnosis of deep tissue diseases and provide the right time-point for PTT.

Efficient delivery of the lncRNA LEF1-AS1 through the antibody LAIR-1 (CD305)-modified Zn-Adenine targets articular inflammation to enhance the treatment of rheumatoid arthritis

BackgroundsRheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by synovial hyperplasia. Maintaining a balance between the proliferation and apoptosis of rheumatoid arthritis synovial fibroblasts (RASFs) is crucial for preventing the erosion of bone and cartilage and, ultimately, mitigating the progression of RA. We found that the lncRNA LEF1-AS1 was expressed at low levels in the RASFs and inhibited their abnormal proliferation by targeting PIK3R2 protein and regulating the PI3K/AKT signal pathway through its interaction with miR-30-5p. In this study, we fabricated a nano-drug delivery system for LEF1-AS1 using Zn-Adenine nanoparticles (NPs) as a novel therapeutic strategy against RA.MethodsThe expression levels of LEF1-AS1, miR-30-5p, PIK3R2, p-PI3K, and p-AKT were detected in the primary RASFs and a human fibroblast-like synovial cell line (HFLS). Zn-Adenine nanoparticles (NPs) were functionalized with anti-CD305 antibody to construct (Zn-Adenine)@Ab. These NPs were then loaded with LEF1-AS1 to form (Zn-Adenine)@Ab@lncRNA LEF1-AS1. Finally, the (Zn-Adenine)@Ab@lncRNA LEF1-AS1 NPs were locally injected into a rat model with collagen-induced arthritis (CIA). The arthritic injuries in each group were evaluated by HE staining and other methods.ResultsLEF1-AS1 was expressed at low levels in the primary RASFs. High expression levels of LEF1-AS1 were detected in the HFLS cells, which corresponded to a significant downregulation of miR-30-5p. In addition, the expression level of PIK3R2 was significantly increased, and that of p-PI3K and p-AKT were significantly downregulated in these cells. The (Zn-Adenine)@Ab@lncRNA LEF1-AS1 NPs significantly inhibited the proliferation of RASFs and decreased the production of inflammatory cytokines (IL-1β, IL-6, TNF-α). Intra-articular injection (IAI) of (Zn-Adenine)@Ab@lncRNA LEF1-AS1 NPs significantly alleviated cartilage destruction and joint injury in the CIA-modeled rats.ConclusionsLEF1-AS1 interacts with miR-30-5p to inhibit the abnormal proliferation of RASFs by regulating the PI3K/AKT signal pathway. The (Zn-Adenine)@Ab NPs achieved targeted delivery of the loaded LEF1-AS1 into the RASFs, which improved the cellular internalization rate and therapeutic effects. Thus, LEF1-AS1 is a potential target for the treatment of RA.

Highly Efficient Oral Iguratimod/Polyvinyl Alcohol Nanodrugs Fabricated by High-Gravity Nanoprecipitation Technique for Treatment of Rheumatoid Arthritis.

Rheumatoid arthritis (RA), a systemic autoimmune disease, poses a significant human health threat. Iguratimod (IGUR), a novel disease-modifying antirheumatic drug (DMARD), has attracted great attention for RA treatment. Due to IGUR's hydrophobic nature, there's a pressing need for effective pharmaceutical formulations to enhance bioavailability and therapeutic efficacy. The high-gravity nanoprecipitation technique (HGNPT) emerges as a promising approach for formulating poorly water-soluble drugs. In this study, IGUR nanodrugs (NanoIGUR) are synthesized using HGNPT, with a focus on optimizing various operational parameters. The outcomes revealed that HGNPT enabled the continuous production of NanoIGUR with smaller sizes (ranging from 300 to 1000 nm), more uniform shapes, and reduced crystallinity. In vitro drug release tests demonstrated improved dissolution rates with decreasing particle size and crystallinity. Notably, in vitro and in vivo investigations showcased NanoIGUR's efficacy in inhibiting synovial fibroblast proliferation, migration, and invasion, as well as reducing inflammation in collagen-induced arthritis. This study introduces a promising strategy to enhance and broaden the application of poorly water-soluble drugs.

ATF3 Modulates the Proliferation, Migration, and Apoptosis of Synovial Fibroblasts after Arthroscopy by Promoting RGS1 Transcription.

Osteoarthritis (OA) is a degenerative joint disease involving both cartilage and synovium. Activating transcription factor 3 (ATF3) and regulator of G protein signaling 1 (RGS1) have been reported to be up-regulated in OA. However, little is known regarding the relationship between these two genes and the mechanism of this relationship in OA development. Therefore, the present study explores the mechanism of ATF3-mediated RGS1 in the proliferation, migration, and apoptosis of synovial fibroblasts. After the OA cell model was constructed with TGF-β1 induction, human fibroblast-like synoviocytes (HFLSs) were transfected with ATF3 shRNA or RGS1 shRNA alone or co-transfected with ATF3 shRNA and pcDNA3.1-RGS1. Then, proliferation, migration, apoptosis, and the expression of ATF3, RGS1, α-SMA, BCL-2, caspase3, and cleaved-caspase3 were measured. Meanwhile, the potential relationship between ATF3 and RGS1 was predicted and validated. Analysis of the GSE185059 dataset suggested that RGS1 was up-regulated in OA synovial fluid exosomes. Moreover, ATF3 and RGS1 were both highly expressed in TGF-β1-induced HFLSs. Transfection of ATF3 shRNA or RGS1 shRNA significantly reduced proliferation and migration and promoted apoptosis of TGF- β1-induced HFLSs. Mechanistically, ATF3 bound to the RGS1 promoter and elevated RGS1 expression. Silencing ATF3 repressed proliferation and migration and enhanced apoptosis of TGF-β1-induced HFLSs by down-regulating RGS1. ATF3 binds to the RGS1 promoter and enhances RGS1 expression to accelerate cell proliferation and block cell apoptosis in TGF-β1-induced synovial fibroblasts.

Circ_0000396 suppresses the proliferation and inflammation of rheumatoid arthritis synovial fibroblasts by targeting miR-574-5p/RSPO1 axis

BackgroundCircular RNAs (circRNAs) are important regulators on the onset and progression of rheumatoid arthritis (RA). Our purpose is to explore the role and underpin mechanism of circ_0000396 in RA progression.MethodsRA patients (n = 39) and healthy volunteers (n = 33) were recruited from the Affiliated Hospital of Shaanxi University of Chinese Medicine for the present work. Circ_0000396, microRNA-574-5p (miR-574-5p) and R-spondin 1 (RSPO1) RNA levels were analyzed by reverse transcription-quantitative polymerase chain reaction. Cell proliferation was analyzed by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay, and 5-ethynyl-2′-deoxyuridine (EDU) assay. Cell apoptosis was assessed by flow cytometry. Protein expression levels of proliferating cell nuclear antigen (PCNA), Cyclin D1, Cyclin E1, BCL2-associated × protein (Bax), B-cell lymphoma-2 (Bcl2), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and RSPO1 were detected by western blot assay. Enzyme-linked immunosorbent assay (ELISA) was conducted to analyze the secretion of pro-inflammatory cytokines including IL-1β and TNF-α. The interaction between miR-574-5p and circ_0000396 or RSPO1 was confirmed by dual-luciferase reporter assay and RNA-pull down assay.ResultsCirc_0000396 expression was notably down-regulated in RA patients compared with healthy controls. Circ_0000396 overexpression suppressed the proliferation and inflammatory response and triggered the apoptosis of RA synovial fibroblasts (RASFs), accompanied by decreases in PCNA, Cyclin D1, Cyclin E1, Bcl2, IL-1β and TNF-α protein expression and an increase in Bax protein expression. Circ_0000396 acted as a molecular sponge for miR-574-5p, and circ_0000396 overexpression-mediated protective effects on RASFs dysfunction were largely reversed by the introduction of miR-574-5p mimics. miR-574-5p interacted with the 3’ untranslated region (3’UTR) of RSPO1, and miR-574-5p negatively regulated RSPO1 expression in RASFs. Circ_0000396 could up-regulate the expression of RSPO1 by sponging miR-574-5p in RASFs. RSPO1 interference largely overturned circ_0000396 overexpression-mediated effects in RASFs.ConclusionCirc_0000396 restrained the proliferation and inflammation and induced the apoptosis of RASFs by mediating miR-574-5p/RSPO1 axis, which provided novel potential targets for RA treatment.

Knockdown of BUB1 inhibits tumor necrosis factor-α-induced proliferation and migration of rheumatoid arthritis synovial fibroblasts by regulating PI3K/Akt pathway.

Rheumatoid arthritis (RA) is a common disease with joint cartilage destruction. BUB1 Mitotic Checkpoint Serine/Threonine Kinase (BUB1) is abnormally expressed in synovial tissues of RA patients, but its effect on RA remains unclear. In this study, we explored the role of BUB1 in RA. An RA cell model was constructed by treating MH7A cells with tumor necrosis factor-α (TNF-α). The levels of BUB1, GAPDH, phosphorylated phosphatidylinositol 3 kinase (p-PI3K)/PI3K, and phosphorylated serine/threonine kinase (p-Akt)/Akt in MH7A cells were examined by Western blot. The MH7A cell proliferation was examined by colony formation assay. Wound healing assay and transwell assay were carried out to detect MH7A cell migration and invasion. The mRNA levels of proinflammatory cytokines were assessed by quantitative reverse transcription polymerase chain reaction. The results showed that knockdown BUB1 inhibited TNF-α-induced MH7A cell proliferation, migration, and invasion. Silencing BUB1 repressed the PI3K/Akt pathway in TNF-α-induced MH7A cells. We also found that the TNF-α-induced MH7A cell proliferation, migration, and invasion were repressed by si-BUB1 transfection, whereas these effects were attenuated by 740Y-P (an activator of the PI3K pathway) co-treatment. Knockdown of BUB1 reduced the expression of the proinflammatory cytokines. Knockdown BUB1 repressed TNF-α-induced MH7A cell proliferation, migration and invasion through the PI3K/Akt pathway.

CD5L aggravates rheumatoid arthritis progression via promoting synovial fibroblasts proliferation and activity.

Rheumatoid arthritis (RA) is a chronic inflammatory disease with progressive cartilage erosion and joint destruction. Synovial fibroblasts (SFs) play a crucial role in the pathogenesis of RA. This study aims to explore the function and mechanism of CD5L during RA progression. We examined the levels of CD5L in synovial tissues and SFs. The collagen-induced arthritis (CIA) rat models were used to investigate the effect of CD5L on RA progression. We also investigated the effects of exogenous CD5L on the behavior and activity of RA synovial fibroblasts (RASFs). Our results showed that CD5L expression was significantly upregulated in synovium of RA patients and CIA-rats. Histology and Micro-CT analysis showed that synovial inflammation and bone destruction were more severe in CD5L-treated CIA rats compared with control rats. Correspondingly, CD5L blockade alleviated bone damage and synovial inflammation in CIA-rats. The exogenous CD5L treatment promoted RASFs proliferation invasion and proinflammatory cytokine production. Knockdown of CD5L receptor by siRNA significantly reversed the effect of CD5L treatment on RASFs. Moreover, we observed that CD5L treatment potentiated PI3K/Akt signaling in the RASFs. The promoted effects of CD5L on IL-6 and IL-8 expression were significantly reversed by PI3K/Akt signaling inhibitor. In conclusion, CD5L promote RA disease progression via activating RASFs. CD5L blocking is a potential therapeutic approach for RA patients.

Qinteng Tongbi Decoction Medicated Serum Exerts Regulates the Proliferation, Migration, and Apoptosis of Synovial Fibroblasts in Adjuvant-Induced Arthritis Rats Model via Phosphatidylinositol 3-Kinase/Protein Kinase B Signaling Pathway

Background Qinteng Tongbi decoction (QTTBD) was an empirical prescription that could effectively prevent and treat rheumatoid arthritis (RA), but there was no report of pharmacological studies on this prescription. The purpose of this paper was to report the effects of QTTBD on the proliferation, migration, and apoptosis of synovial fibroblasts in adjuvant arthritis model rats, and to reveal its anti-RA regulatory mechanism. Methods To divide the fibroblast-like synovial (FLS) cells of experimental rats into 6 groups (blank control group, model control group, positive drug group, QTTBD high, medium, and low dose groups) and cultured with serum-containing drugs. And using Cell Counting Kit-8 to detect the proliferation rate of FLS cells, flow cytometry to detect the apoptosis of FLS cells, the enzyme-linked immunosorbent assay method to detect the levels of interleukin (IL)-6, IL-1β, and tumor necrosis factor-α (TNF-α), Western Blot to detect phosphatidylinositol 3-kinase (PI3K), AKT1, p-AKT1, Bax and Bcl-2 gene and protein expression. Results Experimental results showed that QTTBD-containing serum could effectively inhibit the proliferation of FLS cells ( p <0 .05), induce the apoptosis of FLS cells, reduce the expression levels of inflammatory factors such as IL-6, IL-1β, and TNF-α ( p <0 .05), reduce expression of PI3K, AKT1, p-AKT1, and Bax ( p <0 .05), while the Bcl-2 expression increased ( p <0 .05). Conclusion QTTBD could effectively regulate the proliferation, migration, and apoptosis of FLS cells in adjuvant-induced arthritis (AIA) rats, and its mechanism might be related to regulating the level of inflammatory factors and intervening in the PI3K/protein kinase B (AKT) signaling pathway.

Potential Anti-Rheumatoid Arthritis Activities and Mechanisms of Ganoderma lucidum Polysaccharides

Rheumatoid arthritis (RA) is a chronic and autoimmune disease characterized by inflammation, autoimmune dysfunction, and cartilage and bone destruction. In this review, we summarized the available reports on the protective effects of Ganoderma lucidum polysaccharides (GLP) on RA in terms of anti-inflammatory, immunomodulatory, anti-angiogenic and osteoprotective effects. Firstly, GLP inhibits RA synovial fibroblast (RASF) proliferation and migration, modulates pro- and anti-inflammatory cytokines and reduces synovial inflammation. Secondly, GLP regulates the proliferation and differentiation of antigen-presenting cells such as dendritic cells, inhibits phagocytosis by mononuclear macrophages and nature killer (NK) cells and regulates the ratio of M1, M2 and related inflammatory cytokines. In addition, GLP produced activities in balancing humoral and cellular immunity, such as regulating immunoglobulin production, modulating T and B lymphocyte proliferative responses and cytokine release, exhibiting immunomodulatory effects. Thirdly, GLP inhibits angiogenesis through the direct inhibition of vascular endothelial cell proliferation and induction of cell death and the indirect inhibition of vascular endothelial growth factor (VEGF) production in the cells. Finally, GLP can inhibit the production of matrix metalloproteinases and promote osteoblast formation, exerting protective effects on bone and articular cartilage. It is suggested that GLP may be a promising agent for the treatment of RA.

Anti-proliferation and anti-migration effects of Yishen Tongbi decoction in experimental rheumatoid arthritis by suppressing SLC3A2/integrin β3 signaling pathways

BackgroundYishen Tongbi (YSTB) decoction is a patented herbal formula that is used in China to treat rheumatoid arthritis (RA); however, the exact mechanism of its anti-synovial hyperplasia efficacy has not been fully elucidated. PurposeBased on our previous proteomics study, we aimed to reveal whether YSTB inhibits the proliferation and migration of RA-FLSs through the SLC3A2/integrin β3 pathway in vivo and in vitro. Study designThe study design consists of three parts, a comparison of the expression of SLC3A2 and integrin β3 in synovial tissues of RA and OA patients; an animal experiment to verify the pharmacodynamic effect of YSTB, and in vitro experiment to elucidate the specific mechanism of YSTB. MethodsThe expression of SLC3A2 and integrin β3 in the synovial tissues of patients with RA and osteoarthritis (OA) patients were detected by immunohistochemistry (IHC). In vitro, firstly, the proliferation and migration abilities of HFLS (human fibroblast-like synoviocytes) and HFLS-RA (human fibroblast-like synoviocytes-RA) cells were compared by EdU staining and wound healing assays, respectively, and the differences in the expression and localization of SLC3A2, integrin β3, p-FAK and p-Src between HFLS and HFLS-RA cells were detected by IF and WB. In vivo, DBA/1 mice were injected with bovine collagen II to construct a CIA mouse model. Paw swelling, body weight and the arthritis index (AI) were used as basic treatment evaluation indicators for YSTB. Micro-CT and histopathological analyses of the knee and ankle joints were also performed. In addition, the expression of SLC3A2, integrin β3, p-FAK and p-Src in the synovial tissue of mice was detected by IHC. Subsequently, CCK-8 was used to screen for suitable concentrations of YSTB for use in HFLS-RA cells. EdU staining and transwell migration assays were performed to evaluate the inhibitory effect of YSTB on cell proliferation and migration, and WB was conducted to assess whether YSTB inhibited HFLS-RA migration through downregulation of the SLC3A2/integrin β3 pathways. ResultsIHC showed that the expression of SLC3A2 and integrin β3 was higher in RA synovial tissues than in OA tissues. In vivo experiments showed that YSTB inhibited synovial hyperplasia, prevented bone destruction, and reduced the expression of SLC3A2, integrin β3, p-FAK and p-Src. In vitro experiments showed that YSTB inhibited HFLS-RA migration and proliferation by inhibiting the expression of SLC3A2/integrin β3 and downstream signaling molecules. ConclusionYSTB inhibits the proliferation and migration of synovial fibroblasts in RA by downregulating the SLC3A2/integrin β3 pathways.

SMSCs-derived sEV overexpressing miR-433-3p inhibits angiogenesis induced by sEV released from synoviocytes under triggering of ferroptosis

BackgroundFerroptosis is characterized by accumulation of lipid peroxides that leads to oxidative stress. In progressive rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) suffered from oxidative stress induced by generation of excess reactive oxygen species (ROS) and survived from elevated lipid oxidation. However the phenomenon of abnormal synovial fibroblasts proliferation under ferroptotic stress remain to be explained and the effects of this event on disease progression of RA need to be investigated. MethodsFLS from RA patients (RA-FLS) were stimulated with LPS as an inflammatory model in vitro, and simultaneously treated with ferroptosis inducer Erastin/RSL3 or inhibitor ferrostatin-1. Besides, small extracellular vesicles (sEV) from the supernatant of RA-FLS culture under Erastin/RSL3 management were isolated. The degree of ferroptosis in cells were evaluated by Lipid-ROS detection via flowcytometry and ferroptosis marker protein expression determined by western bloting. The expression of core component of ESCRT-III CHMP4A and CHMP5 was determined by western bloting, and knockdown of CHMP4A was further performed to detect the influence of ESCRT-III complex on ferroptosis as well as LPS/Erastin induced sEV (LPS/Erastin-sEV) releasing. Moreover, miR-433-3p level in the isolated sEV was evaluated by RT-qPCR and interaction of miR-433-3p with FOXO1/VEGF axis were evaluated. MiR-433-3p was overexpressed in synovial mesenchymal stem cells (SMSCs) via miR-433-3p mimics transfection. RA-FLS was co-cultured with human dermal microvascular endothelial cells (HDMECs). LPS/Erastin-sEV or sEV derived from miR-433-3p-overexpressing SMSCs (miR-433-3p-SMSCs-sEV) were added to the co-culture system, and supernatants from co-culture without sEV were given to HDMECs. Angiogenic activity of HDMECs were identified by transwell test and endothelial tube formation analysis. Erastin-sEV and miR-433-3p-SMSCs-sEV were also administrated in collagen-induced arthritis (CIA) mouse model respectively, and progression of arthritis were evaluated. ResultsFerroptosis of RA-FLS was triggered by LPS/Erastin and accompanied with increased expression of ESCRT-III core components as well as elevated release of sEV from RA-FLS. HDMECs’ migration and tube formation in vitro was significantly induced/suppressed by supernatants from co-culture under management of Erastin-sEV/miR-433-3p-SMSCs-sEV due to varied VEGF expression regulated by miR-433-3p targeting FOXO1. MiR-433-3p-SMSCs-sEV could inhibit the Erastin-sEV promoted VEGF expression and mitigated arthritis severity. ConclusionErastin-sEV could aggravate synovial angiogenesis and promote arthritis progression. Administration of miR-433-3p-SMSCs-sEV may be a potential novel therapeutic method as significant antagonism to Erastin-sEV for RA treatment.

[Chemical constituents from fruits of Morinda citrifolia and their inhibitory effects on proliferation of synoviocytes in vitro].

The chemical constituents from the fruits of Morinda citrifolia were systematically explored by chromatographic fractionation methods including silica gel, octadecylsilyl(ODS) gel, Sephadex LH-20 gel, and preparative high performance liquid chromatography(pre-HPLC). The chemical structures of all isolated compounds were identified on the basis of their physicochemical properties, spectroscopic analyses, as well as the comparisons of their physicochemical and spectroscopic data with the reported data in literature. As a result, 22 isolated compounds from the 90% ethanol extract of the fruits of M. citrifolia were identified, which were moricitritone(1), 2'-deoxythymidine(2), cyclo-(L-Pro-L-Tyr)(3), methyl-5-hydroxy-2-pyridinecarboxylate(4), methyl pyroglutamate(5), bisbenzopyran(6), epipinoresinol(7), 3, 3'-bisdemethyl pinoresinol(8), 3, 3'-bisdemethyltanegool(9), trimesic acid(10), crypticin B(11), kojic acid(12), vanillic acid(13), protocatechoic acid(14), 5-hydroxymethyl furfural(15), blumenol A(16), 1-O-(9Z, 12Z-octadecadienoyl) glycerol(17), mucic acid dimethylester(18), methyl 2-O-β-D-glucopyranosylbenzoate(19), 2-phenylethyl-O-β-D-glucoside(20), scopoletin(21), and quercetin(22). Among them, compound 1 was a new pyrone derivative, compounds 2, 4-7, 10-12, and 17 were isolated from the plants belonging to Morinda genus for the first time, and compound 18 was obtained from M. citrifolia for the first time. Moreover, on the basis of testing the activities of all isolated compounds on inhibiting the proliferation of synovial fibroblasts in vitro by MTS assay, the anti-rheumatoid arthritis activities of all isolated compounds were initially evaluated. The results showed that compounds 1-6, 9, 19, and 20 exhibited remarkable anti-rheumatoid arthritis activities, which displayed the inhibitory effects on the proliferation of MH7A synovial fibroblast cells with the IC_(50) values in the range of(3.69±0.08) to(168.96±0.98) μmol·L~(-1).

Inhibition of NUCB2 suppresses the proliferation, migration, and invasion of rheumatoid arthritis synovial fibroblasts from patients with rheumatoid arthritis in vitro

Rheumatoid arthritis (RA) is an autoimmune polyarthritis in which synovial fibroblasts (SF) play a major role in cartilage and bone destruction through tumorlike proliferation, migration, and invasion. Nesfatin-1, an 82-amino-acid-long peptide discovered by Oh-I in 2006, is derived from the precursor protein nucleobindin-2 (NUCB2). NUCB2/nesfatin-1 promotes cell proliferation, migration, and invasion in various tumors. We have previously shown that increased nesfatin-1 levels in the synovium may be associated with disease severity in patients with RA. However, the effect of NUCB2 on the tumorlike transformation of RASF has not yet been reported. The expression of NUCB2 mRNA in the synovium of RA and non-RA patients was further confirmed using three individual datasets from the NCBI GEO database. Gene set enrichment analysis (GSEA) was employed to explore the association between NUCB2 mRNA and RA-related gene signatures or signaling pathways in the GSE77298 dataset. Cell proliferation, migration, and invasion abilities were determined using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), wound healing, and transwell assays, respectively. The results showed that the levels of NUCB2 mRNA in the synovium were significantly elevated in patients with RA. Moreover, GSEA showed that high expression of NUCB2 mRNA was related to gene signatures, including those involved in the cell cycle, DNA replication, extracellular matrix–receptor interaction, and focal adhesion. Furthermore, the results of CCK-8 and EdU assays indicated that inhibition of NUCB2 markedly repressed RASF proliferation. Additionally, the results of wound healing and transwell assays demonstrated that inhibition of NUCB2 significantly suppressed the migratory and invasive abilities of RASFs. Our findings are the first to demonstrate that the inhibition of NUCB2 suppresses the proliferation, migration, and invasion of RASFs in vitro.

LncRNA RNA XIST binding to GATA1 contributes to rheumatoid arthritis through its effects on proliferation of synovial fibroblasts and angiogenesis via regulation of CCN6

This study explored the role of the long non-coding RNA (lncRNA) XIST (X-inactive specific transcript) as a driver of RA pathogenesis, with a particular focus on the ability of this lncRNA to interact with GATA1 and CCN6. The GSE83147and GSE181614 datasets were downloaded for analysis. XIST and CCN6 expression were assessed in synovial fibroblasts (SFs) and in both normal cartilage samples and those from RA patients, with the relationship between XIST and CCN6 additionally being examined. XIST and CCN6 were respectively knocked down or overexpressed in SFs to establish their regulatory roles in these cells in the context of RA. Further studies of the regulatory interplay between XIST, GATA1, and CCN6 were then performed through RNA immunoprecipitation, RNA pull-down, gain-of-function, loss-of-function, and luciferase reporter assays. In addition, RA model rats were established and used to measure the production of TNF-α, IL-6, and IL-8 and to subject tissues from these animals to histopathological examination. RA patient synovial tissues and SFs exhibited XIST and CCN6 upregulation. The knockdown of XIST suppressed SF migratory, proliferative, invasive, and angiogenic activity, while CCN6 knockdown partially reversed the ability of XIST to influence these phenotypic outcomes in vitro and in vivo. XIST bound to GATA1 within SFs, thus promoting enhanced CCN6 transcription. Knocking down XIST alleviated RA-related pathological damage, synovial injury, and inflammatory response induction in rats. The binding of XIST to GATA1 leads to CCN6 upregulation, driving RA pathogenesis by altering SF proliferation and angiogenic activity, suggesting that this pathway may represent a viable target for therapeutic intervention.

Wenhua Juanbi Recipe Attenuates Rheumatoid Arthritis via Inhibiting miRNA-146a-Mediated Autophagy.

Wenhua Juanbi Recipe (WJR) is widely used for the treatment of rheumatoid arthritis (RA) in China. However, its mechanism of action remains unclear. This study was designed to investigate the potential therapeutic effects of WJR on the proliferation and apoptosis of synovial fibroblasts in RA and its efficacy in inhibiting miRNA-146a-mediated cellular autophagy. A collagen-induced arthritis (CIA) Wistar rat model was established. The model rats were administered WJR or methotrexate (MTX) to assess the therapeutic effect of the drugs. The chemical components of WJR were analyzed using UPLC-Q/TOF-MS. Histological changes; miRNA-146a, ATG5, ATG7, ATG12, Beclin1, LC3II, Bax, and Bcl2 expression; synovial apoptosis; and cellular proliferation were assessed. Primary synovial fibroblasts (FLS) were cultured in vitro using tissue block and transfected with miRNA-146a; an autophagy inducer was added to FLS, inhibiting the PI3K/AKT/mTOR pathway. FLS were cocultured with WJR-containing serum to observe the effects of miRNA-146a-mediated autophagy via the PI3K/AKT/mTOR pathway on CIA-affected rats. Forty and thirty-one compounds were identified in WJR in the positive and negative ion modes, respectively. WJR significantly reduced toe swelling, arthritis scores, and expression of miRNA-146a and autophagy genes (ATG5, ATG7, ATG12, Beclin1, LC32, and Bcl2). Moreover, Bax expression, apoptosis, and attenuated proliferation were observed in rats. WJR could, therefore, regulate autophagy by influencing the miRNA-146a-mediated PI3K/AKT/mTOR pathway, which induces apoptosis and proliferation of FLS. WJR can inhibit autophagy, apoptosis, and proliferation in a CIA rat model by inhibiting the miRNA-146a-mediated PI3K/AKT/mTOR pathway.

KLF9 positively regulates TRIM33 to inhibit abnormal synovial fibroblast proliferation, migration as well as inflammation in rheumatoid arthritis.

BackgroundRheumatoid arthritis (RA) can cause irreversible joint injury and serious disability. This study aimed to investigate how TRIM33 regulated by KLF9 affects the aggressive behaviors of synovial fibroblasts induced by tumor necrosis factor‐α (TNF‐α).Materials and MethodsTNF‐α‐induced MH7A cells were used to simulate the in vitro model of RA. TRIM33 and KLF9 expression in TNF‐α‐challenged MH7A cells and transfection efficiency were analyzed via real‐time reverse transcription polymerase chain reaction together with western blot. The viability, proliferation, invasion, and migration of TNF‐α‐induced MH7A cells after transfection was respectively detected by CCK‐8, EdU staining, transwell, and wound‐healing assays. The expression of invasion and migration‐related proteins and inflammation‐related proteins was determined by western blot and the levels of inflammatory factors were detected by enzyme‐linked immunosorbent assay. The combination between TRIM33 and KLF9 was substantiated through dual‐luciferase reporter assay and chromatin immunoprecipitation.ResultsTRIM33 and KLF9 expression in TNF‐α‐challenged MH7A cells was downregulated. TRIM33 elevation inhibited TNF‐α‐elicited proliferation, metastasis as well as inflammation of MH7A cells. Moreover, KLF9 was combined with TRIM33 and KLF9 promoted transcription of TRIM33. The inhibitory effect of TRIM33 overexpression on proliferation, invasion and migration and inflammation of MH7A cells induced by TNF‐α was alleviated by the downregulation of KLF9.ConclusionKLF9 positively regulates TRIM33 to suppress the abnormal MH7A cell proliferation, migration, and reduce inflammation upon exposure to TNF‐α, which was reversed by inhibiting KLF9.

Targeting Neuroinflammation in Osteoarthritis with Intra-Articular Adelmidrol.

Neuroinflammation is an emerging therapeutic target in chronic degenerative and autoimmune diseases, such as osteoarthritis (OA) and rheumatoid arthritis. Mast cells (MCs) play a key role in the homeostasis of joints and the activation of MCs induces the release of a huge number of mediators, which fuel the fire of neuroinflammation. Particularly, synovial MCs release substances which accelerate the degradation of the extra-cellular matrix causing morphological joint changes and cartilage damage and inducing the proliferation of synovial fibroblasts, angiogenesis, and the sprouting of sensory nerve fibers, which mediate chronic pain. Palmitoylethanolamide (PEA) is a well-known MCs modulator, but in osteoarthritic joints, its levels are significantly reduced. Adelmidrol, a synthetic derivate of azelaic acid belonging to the ALIAmides family, is a PEA enhancer. Preclinical and clinical investigations showed that the intra-articular administration of Adelmidrol significantly reduced MC infiltration, pro-inflammatory cytokine release, and cartilage degeneration. The combination of 1% high molecular weight hyaluronic acid and 2% Adelmidrol has been effectively used for knee osteoarthritis and, a significant improvement in analgesia and functionality has been recorded.

Curcumin-Loaded Mesenchymal Stem Cell-Derived Exosomes Efficiently Attenuate Proliferation and Inflammatory Response in Rheumatoid Arthritis Fibroblast-Like Synoviocytes.

This study aimed to evaluate the potential of mesenchymal stem cell-derived exosomes loaded with curcumin (Curc-Exos) as an effective therapeutic strategy for rheumatoid arthritis through modulation of proliferation and inflammatory response in HIG-82 synovial cells. For this purpose, Exos were isolated and characterized with BCA protein assay, DLS, FE-SEM, and TEM. The Curc was embedded by mixing it with Exos in a 1:4 ratio. It was found that the Curc stability has improved after loading on Exos compared to the free Curc. Besides, the in vitro studies using LPS-stimulated HIG-82 synovial cells indicated the efficiency of Curc-Exos in enhancing cytotoxicity and apoptosis compared to the free Curc treatment. It was also revealed that Curc-Exos significantly could reduce the expression levels of anti-apoptotic proteins IAP1 and IAP2 and inflammatory mediators including IL-6, TNF-α, MMP1, and PGE2. This preliminary study confirmed the suitability of Curc-Exos in counteracting the proliferation and inflammatory response of rheumatoid arthritis synovial fibroblasts in vitro.

Immunomodulatory role of T helper cells in rheumatoid arthritis : a comprehensive research review.

Rheumatoid arthritis (RA) is an autoimmune disease that involves T and B cells and their reciprocal immune interactions with proinflammatory cytokines. T cells, an essential part of the immune system, play an important role in RA. T helper 1 (Th1) cells induce interferon-γ (IFN-γ), tumour necrosis factor-α (TNF-α), and interleukin (IL)-2, which are proinflammatory cytokines, leading to cartilage destruction and bone erosion. Th2 cells primarily secrete IL-4, IL-5, and IL-13, which exert anti-inflammatory and anti-osteoclastogenic effects in inflammatory arthritis models. IL-22 secreted by Th17 cells promotes the proliferation of synovial fibroblasts through induction of the chemokine C-C chemokine ligand 2 (CCL2). T follicular helper (Tfh) cells produce IL-21, which is key for B cell stimulation by the C-X-C chemokine receptor 5 (CXCR5) and coexpression with programmed cell death-1 (PD-1) and/or inducible T cell costimulator (ICOS). PD-1 inhibits T cell proliferation and cytokine production. In addition, there are many immunomodulatory agents that promote or inhibit the immunomodulatory role of T helper cells in RA to alleviate disease progression. These findings help to elucidate the aetiology and treatment of RA and point us toward the next steps.Cite this article: Bone Joint Res 2022;11(7):426–438.

SLAMF8 promotes the proliferation and migration of synovial fibroblasts by regulating the ERK/MMPs signalling pathway

Rheumatoid arthritis is troublesome to treat effectively and often requires concomitant long-term treatment. Meanwhile, synovial fibroblasts could induce inflammation response and lead to joint erosion, finally causing progressive joint destruction, disability, and increased mortality. This study focussed on the role of SLAM family member 8 (SLAMF8) in mediating cell function from rheumatoid arthritis synovial fibroblasts stimulated with TNF-α. Cell Counting Kit-8 (CCK-8) and colony-forming unit assay were used to evaluate cell proliferation. SLAMF8 expression was analysed by reverse transcription-quantitative PCR (RT-qPCR) and western blot. Annexin V‐FITC/PI double staining was used to measure the apoptosis rate. The cell migration and invasion in TNF-α-stimulated MH7A (human rheumatoid arthritis synovial cell line) and HFLS-RA cells (human fibroblast-like synoviocytes: rheumatoid arthritis) were tested via wound healing assay and transwell migration assay. In the present study, after TNF-α treatments, the SLAMF8 mRNA and protein expression in both MH7A and HFLS-RA cell lines have a time-dependent increase. The attenuation of SLAMF8 ameliorated TNF-α-induced proliferation, invasion and migration in MH7A and HFLS-RA cells. Simultaneously, when SLAMF8 was silenced, the expression of p-ERK, MMP-1, and MMP-13 was suppressed significantly. In summary, these results indicated that the knockdown of the SLAMF8 significantly attenuated TNF-α-induced proinflammatory responses in MH7A and HFLS-RA cells. Therefore, SLAMF8 exhibits therapeutic potential for the management of inflammation in rheumatoid arthritis.