Proliferation Of Synovial Cells Research Articles

Mid1 promotes synovitis in rheumatoid arthritis via ubiquitin-dependent post-translational modification

IntroductionCurrent anti-rheumatic drugs are primarily modulating immune cell activation, yet their effectiveness remained suboptimal. Therefore, novel therapeutics targeting alternative mechanisms, such as synovial activation, is urgently needed. ObjectivesTo explore the role of Midline-1 (Mid1) in synovial activation. MethodsNOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were used to establish a subcutaneous xenograft model. Wild-type C57BL/6, Mid1-/-, Dpp4-/-, and Mid1-/-Dpp4-/- mice were used to establish a collagen-induced arthritis model. Cell viability, cell cycle, qPCR and western blotting analysis were used to detect MH7A proliferation, dipeptidyl peptidase-4 (DPP4) and Mid1 levels. Co-immunoprecipitation and proteomic analysis identified the candidate protein of Mid1 substrates. Ubiquitination assays were used to determine DPP4 ubiquitination status. ResultsAn increase in Mid1, an E3 ubiquitin ligase, was observed in human RA synovial tissue by GEO dataset analysis, and this elevation was confirmed in a collagen-induced mouse arthritis model. Notably, deletion of Mid1 in a collagen-induced arthritis model completely protected mice from developing arthritis. Subsequent overexpression and knockdown experiments on MH7A, a human synoviocyte cell line, unveiled a previously unrecognized role of Mid1 in synoviocyte proliferation and migration, the key aspects of synovial activation. Co-immunoprecipitation and proteomic analysis identified DPP4 as the most significant candidate of Mid1 substrates. Mechanistically, Mid1 promoted synoviocyte proliferation and migration by inducing ubiquitin-mediated proteasomal degradation of DPP4. DPP4 deficiency led to increased proliferation, migration, and inflammatory cytokine production in MH7A, while reconstitution of DPP4 significantly abolished Mid1-induced augmentation of cell proliferation and activation. Additionally, double knockout model showed that DPP4 deficiency abolished the protective effect of Mid1 defect on arthritis. ConclusionOverall, our findings suggest that the ubiquitination of DPP4 by Mid1 promotes synovial cell proliferation and invasion, exacerbating synovitis in RA. These results reveal a novel mechanism that controls synovial activation, positioning Mid1 as a promising target for therapeutic intervention in RA.

Rapamycin suppresses rheumatoid arthritis fibroblast synovial cell proliferation and induces apoptosis via the AKT/mTORC1 pathway

AbstractBackgroundRheumatoid arthritis (RA) is an autoimmune disease characterized by the overproliferation of synovial fibroblast‐like synoviocytes (FLSs) in the lining, leading to chronic inflammation and progressive joint damage. RA pathogenesis involves lymphocyte infiltration, increased synovial cell proliferation, and impaired cell death. This study investigated the effects of rapamycin on RA‐FLSs and explored the underlying molecular mechanisms.MethodsThe optimal drug concentration and time for rapamycin administration were determined using a cell counting kit‐8 assay. The concentration of rapamycin varied from 1 to 25 nmol/L. The mRNA expression levels of protein kinase B (AKT), mammalian target of rapamycin (mTOR), B cell lymphoma 2 (Bcl‐2), and Bcl‐2‐associated X protein (Bax) were quantified by real‐time polymerase chain reaction. Protein expression (p‐AKT, AKT, p‐mTOR, mTOR, pS6 kinase [S6K], S6K, p‐4E‐binding protein 1 [4EBP1], 4EBP1, Bcl‐2, Bax, caspase 3, caspase 9, cyclin‐dependent kinase 2 [CDK2], and CD1) were detected by Western blotting analysis.ResultsRapamycin suppresses RA‐FLS proliferation and induces apoptosis in a dose‐dependent manner. Rapamycin significantly elevated the protein expression of Bax (p < 0.01), caspase 3 (p < 0.05), and caspase 9 (p < 0.001), and downregulated Bcl‐2 expression (p < 0.01). Rapamycin increased Bax mRNA expression (p < 0.01) and decreased Bcl‐2 (p < 0.05), AKT (p < 0.05), and mTOR (p < 0.05) expression. Additionally, there was a marked reduction in the expression levels of p‐AKT(Ser473) (p < 0.01), p‐mTOR (Ser2448) (p < 0.01), p‐S6K1 (p < 0.01), and p‐4EBP1, CDK2, and CD1 (p < 0.01).ConclusionsRapamycin effectively inhibited proliferation and induced apoptosis of RA‐FLSs by targeting the AKT/mTORC1 pathway. These findings underscore the potential of rapamycin as a therapeutic candidate for addressing the dysregulated proliferation and inflammatory characteristics of RA. Further clinical investigations are required to validate its application in the management of RA.

FAM83D acts as an oncogene by regulating cell cycle progression via multiple pathways in synovial sarcoma: a potential novel downstream target oncogene of anlotinib

ObjectiveSynovial Sarcoma (SS), a highly malignant mesenchymal neoplasm, typically carries a grim prognosis for patients presenting with high-grade or metastatic disease. Although Anlotinib, a new agent for treating soft tissue sarcomas, holds promise, its underlying mechanism remains incompletely understood. This investigation aims to delineate Anlotinib’s anticancer effectiveness and potential mechanistic underpinnings in patients suffering from advanced, refractory SS.Materials and methodsEmploying microarray assay, we examined the potential downstream targets of Anlotinib in SS therapy. A shRNA-based high-content screening was performed to identify candidate genes with the greatest influence on SW982 cell proliferation. The knockdown efficacy of selected genes within SW982 cells was confirmed using RT-qPCR as well as western blot analysis. To assess the effect of putative downstream elimination of genes with synovial sarcoma cells, cell proliferation, and apoptotic assays were carried out. Gene chip microarray as well as bioinformatics techniques were utilized to scrutinize potential signaling networks associated with the candidate downstream gene.ResultsQPCR verified high expression of FAM83D in SW982 cells, shRNA was designed to silence FAM83D by lentivirus transfection, apoptosis assay, and cell cycle arrest showing that FAM83D downregulation augments apoptosis in SW982 cells and arrests cell cycle progression in the S stage. Inhibition of FAM83D expression upregulated STAT1 while downregulated BIRC5, MCM2, and CDK1 genes in vitro.ConclusionsThis experimental study identified FAM83D as a critical regulator that contributes to the proliferation and progression of SS, suggesting that FAM83D-regulated signaling pathway may serve as a prospective target in SS management.

The Hippo-YAP Signaling Pathway in Osteoarthritis and Rheumatoid Arthritis.

Arthritis is the most prevalent joint disease and is characterized by articular cartilage degradation, synovial inflammation, and changes in periarticular and subchondral bone. Recent studies have reported that Yes-associated protein (YAP) and the transcriptional coactivator with PDZ-binding motif (TAZ) have significant effects on the proliferation, migration, and survival of chondrocytes and fibroblast-like synovial cells (FLSs). YAP/TAZ signaling pathway, as well as the related Hippo-YAP signaling pathway, are responsible for the condition of cells and articular cartilage in joints. They are tightly regulated to maintain metabolism in chondrocytes and FLSs because abnormal expression may result in cartilage damage. However, the roles and mechanisms of the Hippo-YAP pathway in arthritis remain largely unknown. This review summarizes the roles and key functions of YAP/TAZ and the Hippo-YAP signaling pathway in FLSs and chondrocytes for the induction of proliferation, migration, survival, and differentiation in rheumatoid arthritis (RA) and osteoarthritis (OA) research. We also discuss the therapeutic strategies involving YAP/TAZ and the related Hippo-YAP signaling pathway involved in OA.

FNDC4 reduces inflammation, proliferation, invasion and migration of rheumatoid synovial cells by inhibiting CCL2/ERK signaling

BackgroundRheumatoid arthritis (RA) is an autoimmune disease characterized by chronic joint inflammation. Fibronectin type III domain-containing protein 4 (FNDC4) is a secretory factor that can regulate inflammatory diseases. However, the role of FNDC4 in RA has not been reported so far. MethodsThe expression of FNDC4 in synovial tissues of RA was analyzed by GEO database (GSE55235 dataset). Then, the expression of FNDC4 in RA fibroblast-like synoviocytes (RA-FLSs) was detected by RT-qPCR and western blot. After constructing FNDC4 overexpression plasmid, cell proliferation and apoptosis were detected. Wound healing and transwell tests were used to detect cell migration and invasion. Then we examined the expression of cytokines related to cell inflammation. Subsequently, the regulatory mechanism of FNDC4 was further discussed. We detected the expression of CCL2 and ERK signaling pathway related proteins downstream of FNDC4. Finally, the mechanism was discussed through the overexpression of FNDC4 and CCL2 and the addition of ERK pathway activator tBHQ. ResultsGEO database showed that FNDC4 expression decreased in synovial tissues of RA. And FNDC4 expression also decreased in RA-FLSs. Overexpression of FNDC4 inhibited the proliferation, invasion and migration of RA-FLSs and promoted its apoptosis. Overexpression of FNDC4 inhibited the release of RA-FLSs inflammatory factors. The regulatory effect of FNDC4 is achieved by inhibiting the CCL2/ERK signaling pathway. ConclusionFNDC4 reduces inflammation, proliferation, invasion and migration of RA-FLSs in RA by inhibiting CCL2/ERK signaling.

Elucidating the mechanism of the Tibetan medicine sanguotang in treating gouty arthritis through network pharmacology and in vivo experiments

BackgroundWe explored the mechanisms of Sanguotang (SGT), a Tibetan medicine, in treating gout arthritis (GA). MethodsThe main active components, action targets, and disease targets of SGT were identified through TCMSP databases. The gene functions were analyzed using protein interaction (PPI) networks, Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and molecular docking. A GA model induced by monosodium urate was established in rats. The ankle joint swelling was observed. The levels of uric acid (UA) and albumin (ALB) in rat serum were measured. Hematoxylin and eosin (HE) staining was conducted to examine the pathological changes in rat ankle joints. ResultsTwenty-nine active components of SGT with proven efficacy and 66 intersection targets were identified, primarily involved in inflammation and immune regulation pathways. The PPI results revealed that the key targets of SGT against GA included ALB, IL6, TNF, TP53, and PTGS. Molecular docking showed favorable binding energy between the ALB protein and the active components. The results from animal experiments demonstrated that SGT effectively alleviated the inflammatory reaction in ankle joints, and decreased UA and ALB levels. Furthermore, SGT effectively inhibited the proliferation of synovial cells in the ankle joint cavity, prevented infiltration of inflammatory cells, and protected synovial tissue, thereby improving GA. ConclusionsSGT comprehensively contributes to the treatment of GA by regulating UA metabolism, reducing the release of inflammatory factors, and modulating immune and inflammatory pathways.

Live macrophages loaded with Fe3O4 and sulfasalazine for ferroptosis and photothermal therapy of rheumatoid arthritis

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by the infiltration of inflammatory cells and proliferation of synovial cells. It can cause cartilage and bone damage as well as disability and is regarded as an incurable chronic disease. Available therapies cannot prevent the development of diseases due to the high toxicity of the therapeutic agents and the inefficient drug delivery. Ferroptosis, an iron-dependent manner of lipid peroxidative cell death, indicates great potential for RA therapy due to ability to damage the infiltrated inflammatory cells and proliferated fibroblast-like synoviocytes. Here, we use macrophages as vector to deliver Fe3O4 nanoparticles and sulfasalazine (SSZ) for ferroptosis and photothermal therapy of RA. The inherent property of migration towards the inflamed joints under the guidance of inflammatory factors enables macrophages to targetedly deliver the payload into the RA. Upon the irradiation of the near infrared light, the Fe3O4 nanoparticles convert the light into heat to damage the proliferated synovium. Meanwhile, the iron released from Fe3O4 nanoparticles works with SSZ to generate synergetic ferroptosis effect. The resident inflammatory cells and proliferated synovium are efficiently damaged by the ferroptosis and photothermal effect, showing pronounced therapeutic effect for RA.

Network Pharmacology and Molecular Docking Explore the Mechanism of Mubiezi-Yinyanghuo Herb Pair in the Treatment of Rheumatoid Arthritis.

Our previous studies have shown that the Mubiezi-Yinyanghuo (MBZ-YYH) herb pair inhibits rheumatoid arthritis (RA) cell proliferation and glycolysis, promising results with an obscure mechanism of action. Therefore, it is necessary to explore the main components of MBZ-YYH and unravel the potential mechanism in RA based on network pharmacology and molecular docking methods. Components and targets of MBZ-YYH were retrieved from the TCMSP. Relevant targets of RA were searched in GeneCards, therapeutic target database (TTD), and DisGeNET databases; the common targets of the MBZ-YYH compounds and RA were obtained by comparison; and a component-target interaction network was established by Cytoscape 3.9.1. Gene ontology (GO) analysis and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway enrichment analysis were performed through the David database. Molecular docking was performed by PyMoL2.3.0 and AutoDock Vina1.1.2 software. 7 active ingredients and 58 putatively identified target genes were screened from MBZ, and 16 effective components of YYH and 230 potential targets were identified. There were 29 mutual targets between the two herbs and RA. Through the PPI network, 9 hub targets which contain JUN, CASP3, PPARG, PTGS2, GSK3B, CASP8, HMOX1, ICAM1, and HK2 were screened out. GO term enrichment analysis indicated that positive regulation of the apoptotic process, response to drugs, and response to hypoxia were significantly enriched. Based on KEGG analysis, it was mainly associated with the IL-17 signaling pathway, the TNF signaling pathway, and the p53 signaling pathway. The docking analysis revealed that the effective components showed strong binding activity with the receptors. The effects of the MBZ-YYH herb pair on RA were coordinated by the interaction of diverse components, which may be through the IL-17 signaling pathway and the TNF signaling pathway, which target GSK3B, HK2, caspase 3, and caspase 8, inhibiting the proliferation and glycolysis of rheumatoid arthritis fibroblast-like synovial cells (RA-FLS) and tending towards an increasing efficacy and decreasing toxicity effect on RA.

Design, synthesis and anti-rheumatoid arthritis activity of target TLR4 inhibitors

A series of 1-(2-oxocyclohexyl)butane-1, 3-dione derivatives were designed and synthesized as TLR4 inhibitors by modifying the core structure of the lead compound ((6, 8-dioxo-1, 2, 3, 4, 6, 7, 8, 8a-octahydronaphthalen-2-yl) carbamate)). In vitro, compound 3p significantly inhibited the proliferation of rat synovial cells, inhibited the proliferation of LPS-induced RAW264.7 cells, and inhibited TLR4 activity, with IC50 values of 1.21 ± 0.09 μM, 0.73 ± 0.05 μM and 0.43 ± 0.03 μM, respectively, which was superior to the positive control methotrexate. In vivo anti-rheumatoid arthritis evaluation, compound 3p can significantly inhibit the inflammatory factors TNF-α, IL-1β and IL-6, so as to achieve the therapeutic purpose. In the preliminary mechanism study, compound 3p has obvious regulatory effects on the abnormal increase of TLR4, JAK2 and STAT3 protein and gene expression related to inflammatory signaling pathway in RAW264.7 cells. In summary, this study aims to develop more effective candidates for rheumatoid arthritis.

Enhancement of Chondrogenic Markers by Exosomes Derived from Cultured Human Synovial Fluid-Derived Cells: A Comparative Analysis of 2D and 3D Conditions.

The goal of this pilot study was to investigate the effects of exosomes derived from synovial fluid-derived cells (SFDCs) cultured under normoxic conditions in a two-dimensional (2D) monolayer or encapsulated within a three-dimensional (3D) matrix for chondrogenic differentiation in vitro and cartilage defect repair in vivo. Synovial fluid samples were obtained from three patients, and SFDCs were isolated and expanded either in a 2D monolayer culture or seeded within a transglutaminase cross-linked gelatin (Col-Tgel) to create a 3D gel culture. Exosomes derived from each environment were isolated and characterized. Then, their effects on cartilage-cell proliferation and chondrogenic differentiation were assessed using an in vitro organoid model, and their potential for enhancing cartilage repair was evaluated using a rat cartilage defect model. SFDCs obtained from different donors reached a state of senescence after four passages in 2D culture. However, transferring these cells to a 3D culture environment mitigated the senescence and improved cell viability. The 3D-cultured exosomes exhibited enhanced potency in promoting chondrogenic differentiation, as evidenced by the increased expression of chondrogenic genes and greater deposition of cartilage-specific extracellular matrix. Furthermore, the 3D-cultured exosomes demonstrated superior effectiveness in enhancing cartilage repair and exhibited better healing properties compared to exosomes derived from a 2D culture. The optimized 3D culture provided a more favorable environment for the proliferation of human synovial cells and the secretion of exosomes compared to the 2D culture. The 3D-cultured exosomes exhibited greater potential for promoting chondrogenic gene expression in vitro and demonstrated improved healing properties in repairing cartilage defects compared to exosomes derived from the 2D culture.

Hederagenin inhibits proliferation, angiogenesis and inflammation of fibroblast-like synovial cells in rheumatoid arthritis

Purpose: To determine the effect of Hederagenin (Hed) on rheumatoid arthritis (RA) in a cell model, and to elucidate the mechanism of action of Hed.
 Methods: MTT, EDU, and Immunoblot assays were used to determine the effects of Hed on the viability of fibroblast-like synovial cells, while the effects of Hed on inflammation were examined by enzymelinked immunosorbent assay (ELISA) and immunoblot assay. The influence of Hed on cell motility angiogenesis was evaluated by Transwell and tube formation assays, while immunoblot analysis was used to determine the mechanism of action of Hed.
 Results: Hed inhibited the viability of RA-FLS cells and suppressed the inflammation of RA-FLS cells (p < 0.05). Furthermore, Hed suppressed the migration and angiogenesis of RA-FLSl cells, as well as regulated MAPK pathway (p < 0.05).
 Conclusion: Hed inhibits the proliferation, angiogenesis and inflammation of fibroblast-like synovial cells in RA by regulating MAPK pathway. Therefore, Hed is a drug for the treatment of RA, However, in vivo studies to validate these findings are recommended

Angelica sinensis polysaccharide inhibits inflammation of collagen-induced arthritis rat fibroblast-like synoviocytes by inhibiting JAK2/STAT3 and MAPK signaling

A. sinensis polysaccharide (ASP), one of the effective components of A. sinensis, has been used to treat inflammatory diseases in China. However, its effect on rheumatoid arthritis (RA) is unknown. The purpose of this study was to explore the anti-inflammatory effects and mechanisms of ASP on RA using a rat model of collagen-induced arthritis (CIA). For evaluation of the therapeutic effects of ASP in vitro, the CIA model was used. Our study showed that ASP (100 and 200 μg/mL) dose-dependently inhibited tumor necrosis factor (TNF)-α-induced (10 ng/mL) proliferation, migration, and invasion of fibroblast-like synovial (FLS) cells, promoted apoptosis, and arrested the cell cycle in G0/G1 phase (P < 0.05). In vivo, ASP increased body weight in CIA rats while decreasing paw swelling, arthritis score, and synovial tissue proliferation (P < 0.05). ASP also reduced the expression of pro-inflammatory cytokines (interleukin (IL) −6, IL-1β, inducible nitric oxide synthetase (iNOS), matrix metalloproteinase (MMP)-1 and MMP-3) (P < 0.05). ASP inhibited the phosphorylation of JAK2/STAT3 and MAPK signaling pathway components (P < 0.05) induced by TNF-α in CIA-FLS cells. Using JAK and p38 inhibitors, we found that JAK2/STAT3 might be an upstream pathway of MAPK. In conclusion, our study investigated the therapeutic effects of ASP in RA in vitro and in vivo. Our research suggests that ASP can inhibit the invasiveness and secretion of inflammatory cytokines of FLS cells in CIA rats via JAK2/STAT3 and MAPK signaling. Elucidation of the underlying mechanism will provide a theoretical basis for clinical application of ASP.

Integrated Bioinformatic Analysis and Experimental Validation to Reveal the Mechanisms of Xinfeng Capsule against Rheumatoid Arthritis.

Xinfeng capsule (XFC) is a well-known drug against rheumatoid arthritis (RA). However, the combination mechanisms of XFC on RA remain unclear. The purpose of this study is to explore the mechanisms of XFC against RA in terms of compounds, targets, and signaling pathways via network pharmacology. The bioactive compounds and potential targets of XFC were extracted from TCMSP and BATMAN-TCM database, and the putative RA-related targets were determined from the DisGeNET, PHGKB, PharmGKB, and CTD database. The approach of protein-protein interaction, gene ontology analysis, and kyoto encyclopedia of genes and genomes pathway enrichment analysis were constructed, respectively. In animal experiments, we evaluated the expression of core targets. We found that XFC handled 30 active compounds and 131 common target genes. Among them, mairin, folic acid, cholesterol, and triptolide in XFC were selected as the central active compounds against RA. The mechanisms of XFC on RA which concerned critical targets were protein kinase B (AKT1) and tumor necrosis factor (TNF). In vivo, we found that the expression levels of AKT1 and TNF in the modeling group were significantly increased but reversed by XFC. The combination mechanisms of XFC were elucidated in terms of components and targets and signaling pathways, which may be related to inhibiting the proliferation of synovial cells and inflammation.

Chinese herbal formula, modified Xianfang Huoming Yin, alleviates the inflammatory proliferation of rat synoviocytes induced by IL-1β through regulating the migration and differentiation of T lymphocytes.

Xianfang Huoming Yin (XFH) is a traditional Chinese herbal formula, which has the effect of clearing heat and detoxifying toxins, dispersing swellings, activating blood circulation, and relieving pain. It is usually applied to treat various autoimmune diseases, including Rheumatoid arthritis (RA). The migration of T lymphocytes plays an indispensable role in the pathogenesis of RA. Our previous studies demonstrated that modified Xianfang Huoming Yin (XFHM) could modulate the differentiation of T, B, and NK cells, and contribute to the restoration of immunologic balance. It also could downregulate the production of pro-inflammatory cytokines by regulating the activation of NF-κ B and JAK/STAT signaling pathways in the collagen-induced arthritis mouse model. In this study, we want to investigate whether XFHM has therapeutic effects on the inflammatory proliferation of rat fibroblast-like synovial cells (FLSs) by interfering with the migration of T lymphocytes in vitro experiments. High performance liquid chromatography-electrospray ionization/mass spectrometer system was used to identify the constituents of the XFHM formula. A co-culture system of rat fibroblast-like synovial cells (RSC-364cells) and peripheral blood lymphocytes stimulated by interleukin-1 beta (IL-1β) was used as the cell model. IL-1β inhibitor (IL-1βRA) was used as a positive control medicine, and two concentrations (100μg/mL and 250μg/mL) of freeze-dried XFHM powder were used as intervention measure. The lymphocyte migration levels were analyzed by the Real-time xCELLigence analysis system after 24h and 48h of treatment. The percentage of CD3+CD4+ T cells and CD3+CD8+ T cells, and the apoptosis rate of FLSs were detected by flow cytometry. The morphology of RSC-364cells was observed by hematoxylin-eosin staining. The protein expression of key factors for T cell differentiation and NF-κ B signaling pathway-related proteins in RSC-364cells were examined by western-blot analysis. The migration-related cytokines levels of P-selectin, VCAM-1, and ICAM-1 in the supernatant were measured by enzyme-linked immunosorbent assay. Twenty-one different components in XFHM were identified. The migration CI index of T cells was significantly decreased in treatment with XFHM. XFHM also could significantly downregulate the levels r of CD3+CD4+T cells and CD3+CD8+T cells that migrated to the FLSs layer. Further study found that XFHM suppresses the production of P-selectin, VCAM-1, and ICAM-1. Meanwhile, it downregulated the protein levels of T-bet, ROR γ t, IKKα/β, TRAF2, and NF-κ B p50, upregulated the expression of GATA-3 and alleviated synovial cells inflammation proliferation, contributing to the FLSs apoptosis. XFHM could attenuate the inflammation of synovium by inhibiting T lymphocyte cell migration, regulating differentiation of T cells through modulating the activation of the NF-κ B signaling pathway.

Effect of heat-reinforcing needling on the inflammation and necrotizing apoptosis of synovial cells in synovial tissues of knee joint in rabbits with cold syndrome rheumatoid arthritis

To observe the effect of heat-reinforcing needling (HRN) on inflammatory factors and necrotizing apoptosis of synovial cells in synovial tissues of knee joint in rabbits with cold syndrome rheumatoid arthritis (RA), so as to explore its underlying mechanisms in treating RA. By using the random number table method, 40 New Zealand rabbits were randomly divided into normal, model, antagonist(AG), twist-reinforceing needling (TRN) and HRN groups, with 8 rabbits in each group. The model of cold syndrome RA was established by ovalbumin induction combined with Freund's complete adjuvant injection and cryogenic freezing method. In the AG group, the antagonist TAK-632 (25 mg/kg) was administered intragastrically, once every 2 days, for a total of 7 times. Rabbits of TRN and HRN groups were treated with corresponding acupuncture techniques on bilateral "Zusanli" (ST36) for 30 min, once a day for 14 days. After intervention, the changes of knee skin temperature and circumference were measured. Color Doppler ultrasound was used to observe the joint cavity effusion, synovial thickness and internal blood flow signal. The histomorphological changes of synovial tissues were observed after HE staining. ELISA was used to detect the contents of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 in serum. Transmission electron microscope was used to observe the ultrastructure, necrosis and apoptosis of synovial cells. Western blot was used to detect the protein expressions of receptor-interacting protein kinase1 (RIPK1), RIPK3, mixed lineage kinase domain-like protein (MLKL), and phosphorylation （p）-MLKL in synovial tissues. Compared with the normal group, the synovial was diffusely hyperplasia, joint cavity effusion and abnormal blood flow signal were obvious, inflammatory cells were clustered, arranged closely and disordered in the model group. The findings of transmission electron microscopy showed disruption of cell membrane integrity, swollen or ruptured mitochondria, obviously ruptured nucleus, condensed and pyknotic chromatin and nucleolus in the model group. Also, the skin temperature of the knee joint was significantly decreased (P<0.01), while the circumference of the knee joint, the contents of TNF-α, IL-1β and IL-6 in serum, the protein expressions of RIPK1, RIPK3, p-MLKL and MLKL in synovial tissues were significantly increased (P<0.01) in the model group. Compared with the model group, synovial tissue hyperplasia, joint cavity effusion, abnormal blood flow signals, synovial cell proliferation, inflammatory cell infiltration, disruption of cell membrane integrity, cell swelling, cell rupture, and nuclear pyknosis were reduced to different degrees in the AG, TRN and HRN groups. Additionally, the skin temperature of the knee joint was increased (P<0.01, P<0.05), while the circumference of the knee joint, the contents of TNF-α, IL-1β and IL-6 in serum, the expressions of RIPK1, RIPK3, p-MLKL and MLKL in synovial tissues were decreased (P<0.01, P<0.05) in the AG, TRN and HRN groups. The effects of HRN and AG were notably superior to that of TRN in up-regulating skin temperature of the knee joint, and down-regulating the circumference of the knee joint, the contents of TNF-α, IL-1β and IL-6 in serum, the expressions of RIPK1, RIPK3, p-MLKL and MLKL in synovial tissues (P<0.01, P<0.05). HRN can reduce synovial inflammation of knee joint in rabbits with cold syndrome RA, which may be related to its function in inhibiting the necrotizing apoptosis of synovial cells.

Alkaloids from the stems of Fissistigma maclurei Merr. inhibit the proliferation of synoviocytes

Two new aporphine-derived alkaloids, aporaloids C and D (1 and 2), along with eight known biogenetically related alkaloids (3–10) were isolated from the stems of Fissistigma maclurei Merr. Their structures were elucidated by detailed analysis of NMR, HRESIMS, MS, IR, UV and Optical rotations data. Compounds 1 and 2 represent a rare example of N-methylol aporphine-derived alkaloids from natural sources. The inhibitory effect of all compounds on the proliferation of primary synovial cells was evaluated. Compound 3 showed potent inhibitory effect on the proliferation of synoviocytes with an IC50 value of 4.8 μM. Compounds 1, 2, 6–9 and 10 exhibited moderate inhibitory activity on synoviocytes, with IC50 values of 36.8, 37.1, 31.2 μM, 32.5, 36.3, 36.8 and 18.2 μM, respectively.

Infusion of GMSCs relieves autoimmune arthritis by suppressing the externalization of neutrophil extracellular traps via PGE2-PKA-ERK axis

IntroductionRheumatoid arthritis (RA) is a systemic autoimmune disease with limited treatment success, characterized by chronic inflammation and progressive cartilage and bone destruction. Accumulating evidence has shown that neutrophil extracellular traps (NETs) released by activated neutrophils are important for initiating and perpetuating synovial inflammation and thereby could be a promising therapeutic target for RA. K/B × N serum transfer-induced arthritis (STIA) is a rapidly developed joint inflammatory model that somehow mimics the inflammatory response in patients with RA. Human gingival-derived mesenchymal stem cells (GMSCs) have been previously shown to possess immunosuppressive effects in arthritis and humanized animal models. However, it is unknown whether GMSCs can manage neutrophils in autoimmune arthritis. ObjectivesTo evaluate whether infusion of GMSCs can alleviate RA by regulating neutrophils and NETs formation. If this is so, we will explore the underlying mechanism(s) in an animal model of inflammatory arthritis. MethodsThe effects of GMSCs on RA were assessed by comparing the symptoms of the K/B × N serum transfer-induced arthritis (STIA) model administered either with GMSCs or with control cells. Phenotypes examined included clinical scores, rear ankle thickness, paw swelling, inflammation, synovial cell proliferation, and immune cell frequency. The regulation of GMSCs on NETs was examined through immunofluorescence and immunoblotting in GMSCs-infused STIA mice and in an in vitro co-culture system of neutrophils with GMSCs. The molecular mechanism(s) by which GMSCs regulate NETs was explored both in vitro and in vivo by silencing experiments. ResultsWe found in this study that adoptive transfer of GMSCs into STIA mice significantly ameliorated experimental arthritis and reduced neutrophil infiltration and NET formation. In vitro studies also showed that GMSCs inhibited the generation of NETs in neutrophils. Subsequent investigations revealed that GMSCs secreted prostaglandin E2 (PGE2) to activate protein kinase A (PKA), which ultimately inhibited the downstream extracellular signal-regulated kinase (ERK) pathway that is essential for NET formation. ConclusionOur results demonstrate that infusion of GMSCs can ameliorate inflammatory arthritis mainly by suppressing NET formation via the PGE2-PKA-ERK signaling pathway. These findings further support the notion that the manipulation of GMSCs is a promising stem cell-based therapy for patients with RA and other autoimmune and inflammatory diseases.

Mechanism underlying the action of Duanteng-Yimu Tang in regulating Treg/Th17 imbalance and anti-rheumatoid arthritis

BackgroundRheumatoid arthritis (RA) is a chronic immune disease characterised by synovitis and cartilage destruction. Currently, many patients experience poor remission after new antirheumatic drug treatments. Duanteng-Yimu Tang (DTYMT), a traditional Chinese medicine, is effective in the treatment of RA. In this research, we designed to investigate the anti-RA effects of DTYMT and explore its potential mechanisms. MethodsNetwork pharmacology was adopted to explore the main pathways of DTYMT in patients with RA. Collagen-induced arthritis models of male DBA/1 mice were established, and their histopathological changes were observed by hematoxylin-eosin staining and micro-CT. qRT-PCR was performed to detect the expression of Foxp3 and RORγt in the serum and synovial tissue and IL-17, IL-1β, TNF-α, and IL-10 mRNA in vivo. The proliferation and invasion of synovial cells were analyzed using Cell Counting Kit-8 and transwell assays, respectively. The ratio of T helper 17 (Th17) to regulatory T (Treg) cells was analyzed by flow cytometry. ResultsNetwork pharmacology analysis revealed that Th17 cell differentiation may be the key pathway of DTYMT in RA. DTYMT ameliorated joint damage, inhibited RORγt expression, and increased Foxp3 expression in CIA mice. DTYMT significantly decreased IL-1β, IL-17, and TNF-α mRNA levels, and increased IL-10 mRNA levels in IL-6-induced cells. Additionally, DTYMT inhibited Th17 cell differentiation and promoted Treg cell production, thus improving the Treg/Th17 imbalance. DTYMT also inhibited the proliferation, migration, and invasion of RA fibroblast-like synovial cells. ConclusionsThese results indicate that DTYMT could regulate the Treg/Th17 cell balance, which is a possible mechanism of DTYMT in treating RA.

Guaiane-Type Sesquiterpenes from the Stems of Fissistigma oldhamii.

Two new guaiane-type sesquiterpenes dysodensiols J and L, one new natural product dysodensiol K together with four known biogenetically related guaiane-type sesquiterpenes were isolated from the stems of Fissistigma oldhamii. Their structures were elucidated by detailed analysis of NMR, HR-ESI-MS, IR and Optical rotations data. Compound 1 contains an uncommon five-membered ether ring. The inhibitory effect of all compounds on the proliferation of primary synovial cells was evaluated. Compound 3 showed inhibitory activity with an IC50 value of 6.8 μM. Compounds 5-7 exhibited moderate inhibitory activity with IC50 values of 23.8, 26.6, and 27.1 μM, respectively.

Morphological Features of the Synovial Membrane and Articular Cartilage in Rats under Simulated Adjuvant Arthritis and Application of Allogeneic Biomaterial

The aim of research was to study morphological features of the synovial membrane and articular cartilage of rats under simulated adjuvant arthritis and application of allogeneic biomaterial.Material and methods. To simulate rheumatoid arthritis, 12 white outbred female rats were injected 100 μl of complete Freund's adjuvant into the plantar surface of the hind paw. In 7 days, 5 mg of the allogeneic biomaterial “Regeneration stimulator” dissolved in physiological solution was injected into the periarticular bursae of the right knee joint and surrounding tissues in the rats of the main experimental group (n=6). Saline solution was injected into animals of the main control group (n=6). Intact rats (n=6) were included in the intact control group. All the animals were withdrawn from the experiment in 37 days after the onset of the experiment. The knee joints of rats were examined using standard histological, morphometric and statistical methods.Results. Animals of the main control group manifested signs of degenerative changes of varying degrees in the articular apparatus. Proliferation of the synovial cells and tissue hyperplasia were detected in the synovial membrane, these leading to the formation of pannus. There was disorganization of the connective tissue: they were thinned, the fibrous layer was separated, the cartilage and bone tissue were destructed. In animals of the experimental group, there were no signs of tissue hyperplasia and edema, pronounced pannus were not detected after the introduction of the allogeneic biomaterial in the synovial membrane. Mild signs of arthritis manifested as slightly increased processes of the synovial cell proliferation and moderate perivascular infiltration with cellular elements were detected in 2 rats. There were no changes in the structure of the cartilage and bone in the knee joint.Conclusion. Intraarticular injection of the allogeneic biomaterial "Regeneration Stimulator" in rats with simulated autoimmune rheumatoid arthritis is accompanied by a decreased external manifestations of a generalized inflammatory process in the knee joints, and by a decreased inflammation and degenerative changes in the articular apparatus at the tissue level. Notably, the structures of the synovial membrane, the articular cartilage of the knee joint and the underlying subchondral bone are preserved compared with animals of the control group.