OBJECTIVE: To analyze the differences in the effects of different mechanical stimuli on cells, cytokines, and proteins in synovial fluid of osteoarthritis joints, and to elucidate the indirect mechanism by which mechanical signals remodel the synovial fluid microenvironment through tissue cells. METHODS: Systematically integrate recent literature, focusing on the regulatory effects of different mechanical stimuli on the physicochemical properties of synovial fluid. Analyze the dynamic process by which mechanical stimuli regulate secretory and metabolic activities through tissue cells, thereby altering the physicochemical properties of cytokines and proteins. RESULTS: Appropriate mechanical stimuli activate mechanical signals in chondrocytes, macrophages, and synovial cells, thereby influencing cellular metabolic activities, including inhibiting the release of pro-inflammatory factors and promoting the secretion of anti-inflammatory factors, and regulating the expression of matrix and inflammation-related proteins such as cartilage oligomeric matrix protein, peptidoglycan recognition protein 4, and matrix metalloproteinases. CONCLUSION Mechanical stimuli act on tissue cells, indirectly reshaping the synovial fluid microenvironment through metabolic activities, thereby regulating the pathological process of osteoarthritis.
Humans ; Osteoarthritis/physiopathology* ; Synovial Fluid/chemistry* ; Chondrocytes/metabolism* ; Cytokines/metabolism* ; Macrophages/metabolism* ; Stress, Mechanical ; Cartilage Oligomeric Matrix Protein/metabolism* ; Matrix Metalloproteinases/metabolism* ; Synovial Membrane/cytology*

OBJECTIVE: To investigate the effects of 4-methylcatechol (4MC) on the migration and inflammatory response in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS), as well as its underlying mechanisms of action. METHODS: RA-FLS was isolated from synovial tissue donated by RA patients, and the optimal concentration of 4MC was determined by cell counting kit 8 method for subsequent experiments, and the effect of 4MC on the migratory ability of RA-FLS was evaluated via a cell scratch assay. An inflammation model of RA-FLS was induced by tumor necrosis factor α (TNF-α). Real-time fluorescence quantitative PCR and ELISA were employed to detect the gene and protein expression levels of interleukin-1β (IL-1β) and IL-6 in RA-FLS and their culture supernatants, respectively, thereby investigating the anti-inflammatory effects of 4MC. Western blot was used to examine the expressions of nuclear factor κB (NF-κB) signaling pathway-related proteins, including inhibitor of NF-κB-α (IKBα), phosphorylated (P)-IκBα, NF-κB-inducing kinase α (IKKα), P-IKKαβ, P-p65, and p65. Cellular immunofluorescence was utilized to detect the expression and localization of p65 in RA-FLS, exploring whether 4MC exerts its anti-inflammatory effects by regulating the NF-κB signaling pathway. Finally, a collagen-induced arthritis (CIA) mouse model was established. The anti-RA effect of 4MC in vivo was evaluated by gross observation and histological examination. RESULTS: 4MC inhibited RA-FLS migration in a concentration-dependent manner. In the TNF-α-induced RA-FLS inflammation model, 4MC significantly decreased the gene and protein expression levels of IL-1β and IL-6. Furthermore, 4MC markedly reduced the ratios of P-IΚBα/IΚBα, P-IKKαβ/IKKα, and P-p65/p65, thereby blocking the transcriptional activity of p65 by inhibiting its nuclear translocation. This mechanism effectively suppressed the activation of the TNF-α-mediated NF-κB signaling pathway. Animal studies demonstrated that 4MC [10 mg/(kg·day)] significantly lowered serum levels of IL-1β, IL-6, and TNF-α, and alleviated arthritis severity and bone destruction in CIA mice. CONCLUSION 4MC not only inhibits the migration of RA-FLS but also mitigates their inflammatory response by suppressing the NF-κB signaling pathway, thereby effectively exerting its anti-RA effects.
Synoviocytes/metabolism* ; Arthritis, Rheumatoid/metabolism* ; Animals ; Cell Movement/drug effects* ; Humans ; Catechols/therapeutic use* ; Fibroblasts/drug effects* ; Mice ; Tumor Necrosis Factor-alpha/pharmacology* ; Interleukin-1beta/metabolism* ; Interleukin-6/metabolism* ; Signal Transduction/drug effects* ; NF-kappa B/metabolism* ; Transcription Factor RelA/metabolism* ; Synovial Membrane/cytology* ; Cells, Cultured ; Male ; Arthritis, Experimental ; Anti-Inflammatory Agents/pharmacology* ; NF-KappaB Inhibitor alpha ; Inflammation

Objective To investigate the effects of hyaluronic acid and proteoglycan-linked protein 1 (HAPLN1) secreted by synovial fibroblasts (FLS) on the polarization of macrophages (Mϕ) in rheumatoid arthritis (RA). Methods Human monocytic leukemia cells (THP-1) were differentiated into Mϕ, which were subsequently exposed to recombinant HAPLN1 (rHAPLN1). RA-FLS were transfected separately with HAPLN1 overexpression plasmid (HAPLN1^OE) or small interfering RNA targeting HAPLN1 (si-HAPLN1), and then co-cultured with Mϕ to establish a co-culture model. The viability of Mϕ was assessed using the CCK-8 assay, and the proportions of pro-inflammatory M1-type and anti-inflammatory M2-type Mϕ were analyzed by flow cytometry. Additionally, the expression levels of inflammatory markers, including interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), and inducible nitric oxide synthase (iNOS), were quantified using quantitative real-time PCR and Western blot analysis. Results The viability of Mϕ was increased in the rHAPLN1 group compared to the control group. Furthermore, both the M1/Mϕ ratio and inflammatory factor levels were elevated in the rHAPLN1 and HAPLN1^OE groups. In contrast, the si-HAPLN1 group exhibited a decrease in the M1/Mϕ ratio and inflammatory factor expression. Notably, the introduction of rHAPLN1 in rescue experiments further promoted Mϕ polarization towards the M1 phenotype. Conclusion HAPLN1, secreted by RA fibroblast-like synoviocytes (RA-FLS), enhances Mϕ polarization towards the M1 phenotype.
Humans ; Arthritis, Rheumatoid/genetics* ; Macrophages/immunology* ; Fibroblasts/metabolism* ; Phenotype ; Extracellular Matrix Proteins/genetics* ; Proteoglycans/genetics* ; Synovial Membrane/cytology* ; Tumor Necrosis Factor-alpha/genetics* ; Interleukin-1beta/genetics* ; Nitric Oxide Synthase Type II/genetics* ; Cell Differentiation ; Coculture Techniques ; THP-1 Cells

OBJECTIVES: To investigate the role of the BNIP3-PI3K/Akt signaling pathway in mediating the inhibitory effect of Buyang Huanwu Decoction (BYHWT) on mitochondrial autophagy in human synovial fibroblasts from rheumatoid arthritis patients (FLS-RA) cultured under a hypoxic condition. METHODS: Forty normal Wistar rats were randomized into two groups (n=20) for daily gavage of BYHWT or distilled water for 7 days to prepare BYHWT-medicated or control sera. FLS-RA were cultured in routine condition or exposed to hypoxia (10% O₂) for 24 h wigh subsequent treatment with IL-1β, followed by treatment with diluted BYHWT-medicated serum (5%, 10% and 20%) or control serum. AnnexinV-APC/7-AAD double staining and T-AOC kit were used for detecting apoptosis and total antioxidant capacity of the cells, and the changes in ROS, ATP level, mitochondrial membrane potential and Ca²⁺ homeostasis were analyzed. The changes in mRNA and protein expressions of BNIP3, PI3K and AKT and mRNA expressions of LC3, Beclin-1 and P62 were detected using RT-qPCR and Western blotting. RESULTS: Treatment with BYHWT-medicated serum dose-dependently lowered apoptosis rate of IL-1β-induced FLS-RA with hypoxic exposure. The treatment significantly decreased T-AOC concentration, increased ROS production, autophagosome formation and ATPase levels, and lowered mitochondrial membrane potential and Ca²⁺ level in the cells. In IL-1β-induced FLS-RA with hypoxic exposure, treatment with BYHWT-medicated serum significantly increased BNIP3 protein expression, decreased the protein expressions of PI3K and AKT, increased the mRNA expressions of BNIP3 and P62, and lowered the mRNA expressions of PI3K, AKT, LC3 and Beclin-1 without significantly affecting Beclin-1 protein expression. The cells treated with 5% and 10% BYHWT-medicated serum showed no significant changes in LC3 expression. CONCLUSIONS BYHWT inhibits mitochondrial autophagy in IL-1β-induced FLS-RA with hypoxic exposure possibly by inhibiting BNIP3-mediated PI3K/AKT signaling pathway.
Drugs, Chinese Herbal/pharmacology* ; Arthritis, Rheumatoid/pathology* ; Animals ; Signal Transduction/drug effects* ; Proto-Oncogene Proteins c-akt/metabolism* ; Autophagy/drug effects* ; Humans ; Fibroblasts/cytology* ; Rats, Wistar ; Membrane Proteins/metabolism* ; Rats ; Phosphatidylinositol 3-Kinases/metabolism* ; Mitochondria/metabolism* ; Cells, Cultured ; Proto-Oncogene Proteins/metabolism* ; Apoptosis/drug effects* ; Cell Hypoxia ; Synovial Membrane/cytology* ; Male ; Mitochondrial Proteins

The ambiguity of etiology makes temporomandibular joint osteoarthritis (TMJOA) "difficult-to-treat". Emerging evidence underscores the therapeutic promise of exosomes in osteoarthritis management. Nonetheless, challenges such as low yields and insignificant efficacy of current exosome therapies necessitate significant advances. Addressing lower strontium (Sr) levels in arthritic synovial microenvironment, we studied the effect of Sr element on exosomes and miRNA selectively loading in synovial mesenchymal stem cells (SMSCs). Here, we developed an optimized system that boosts the yield of SMSC-derived exosomes (SMSC-EXOs) and improves their miRNA profiles with an elevated proportion of beneficial miRNAs, while reducing harmful ones by pretreating SMSCs with Sr. Compared to untreated SMSC-EXOs, Sr-pretreated SMSC-derived exosomes (Sr-SMSC-EXOs) demonstrated superior therapeutic efficacy by mitigating chondrocyte ferroptosis and reducing osteoclast-mediated joint pain in TMJOA. Our results illustrate Alix's crucial role in Sr-triggered miRNA loading, identifying miR-143-3p as a key anti-TMJOA exosomal component. Interestingly, this system is specifically oriented towards synovium-derived stem cells. The insight into trace element-driven, site-specific miRNA selectively loading in SMSC-EXOs proposes a promising therapeutic enhancement strategy for TMJOA.
MicroRNAs/metabolism* ; Mesenchymal Stem Cells/drug effects* ; Osteoarthritis/drug therapy* ; Exosomes/drug effects* ; Strontium/pharmacology* ; Synovial Membrane/cytology* ; Humans ; Animals ; Temporomandibular Joint Disorders/therapy* ; Temporomandibular Joint

Objective To explore the role of multidrug resistance gene-1(MDR1)gene in methotrexate(MTX)resistance in patients with rheumatoid arthritis(RA).Methods Fibroblast-like synoviocytes(FLS)from RA patients were infected with recombinant adenovirus Ad-EGFP-MDR1 to obtain MDR1 over-expressed RA FLS.The transcription level of MDR1 gene and the expression level of its coding product P-glycoprotein(P-gp) rotein were detected by real-time PCR and Western blot analysis.The efflux function was verified by rhodamine 123 efflux assay.The resistance to MTX was detected by MTT assay.Results RA FLS were infected with recombinant adenovirus Ad-EGFP-MDR1;72 hours later,the particles size in MDR1 over-expressed RA FLS increased,the cell volume became larger,and the growth rate decreased.The transcription level of MDR1(1.4325±0.3924 0.0650±0.0070;=6.035,=0.004),the expression level of P-gp protein(1.8667±0.2857 0.9367±0.0551;=5.536,=0.005),and the ability of extracellular rhodamine 123(979.43±196.81 1680.06±147.04;=-4.940,=0.008) in MDR1 over-expressed RA FLS were significantly higher than those of negative virus control RA-FLS,and the survival rate of MDR1 over-expressed RA FLS was significantly increased at each concentration of MTX(<0.05).Conclusion The high expression of MDR1 can affect the efflux ability to MTX by up-regulating the expression of P-gp,thus enhancing the drug resistance to MTX in RA FLS.
ATP Binding Cassette Transporter, Subfamily B ; genetics ; Arthritis, Rheumatoid ; drug therapy ; genetics ; Cells, Cultured ; Drug Resistance ; Fibroblasts ; drug effects ; Humans ; Methotrexate ; pharmacology ; Synovial Membrane ; cytology

OBJECTIVETo study the mechanism underlying the inhibitory effect of Qingluo Tongbi Granule (, QTG) on osteoclast differentiation in rheumatoid arthritis in rats.

METHODSFibroblast and monocyte co-culture were used to induce osteoclast differentiation in adjuvant-induced arthritic (AIA) rats. Serum containing QTG was prepared and added to the osteoclasts, and activation of the tumor necrosis factor receptor-associated factor 6/mitogen-activated protein kinase/nuclear factor of activated T cells, cytoplasmic1 (TRAF6/MAPK/NFATc1) pathways was examined.

RESULTSThe induced osteoclasts were multinucleated and stained positive for tartrate-resistant acid phosphatase (TRAP) staining. Serum containing QTG at 14.4, 7.2 or 3.6 g/kg inhibited the activation of TRAF6, extracellular regulated protein kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and p38 and decreased the percentage of cells with nuclear NFATc1 in a dose-dependent manner, the high and middle doses exhibited clear inhibitory activity (P<0.01 and P<0.05, respectively). After the addition of MAPK inhibitors, the NFATc1 expression showed no significant difference compared with the control group (P>0.05).

CONCLUSIONSSerum containing QTG could generally inhibit the TRAF6/MAPK pathways and possibly inhibit the NFATc1 pathway. In addition, QTG may regulate other signaling pathways that are related to osteoclast differentiation and maturation.

Adjuvants, Immunologic ; adverse effects ; Animals ; Arthritis, Experimental ; pathology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Coculture Techniques ; Down-Regulation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Fibroblasts ; pathology ; Male ; Monocytes ; pathology ; Osteoclasts ; cytology ; drug effects ; physiology ; Rats ; Rats, Sprague-Dawley ; Synovial Membrane ; pathology

OBJECTIVETo investigate the effect of Flavonoids extracted from Echinps latifolius Tausch(FELT) on rheumatoid arthritis (RA) in rat model.

METHODFifty SD rats were randomly divided into model group, control group, and low, medium, and high-dose FELT groups (n=10 in each group). Complete Freund's adjuvant (0.1 mL) was used to induce RA in rats. FELT in doses of 50 mg/kg, 100 mg/kg, 150 mg/kg was given to rats in low, medium and high-dose FELT groups by gavage, and same volume of PBS was given to rats in control group. The arthritis score and the paw swelling score were measured to evaluate the therapeutic effect of FELT. Real time qPCR was used to detect the mRNA expression of fibronectin and MMP3 in synovial tissue and the mRNA expression of caspase 3, Bcl-2 and Bcl-2 associated X protein (Bax) in fibroblast-like synoviocytes (FLS).

RESULTSThe arthritis score and the paw swelling score were significantly decreased in three FELT groups compared to RA model rats (P <0.05). The relative expression levels of FN and MMP3 mRNA in synovium of three FELT-treatment groups were significantly lower than those in model group (1.80, 1.76 and 1.67 vs 2.53; 1.69, 1.46 and 1.45 vs 2.67, respectively, all P <0.05). The relative expression levels of Bax and caspase 3 mRNA in FLSs of three FELT groups were higher than those in model group (0.56, 0.58 and 0.60 vs 0.30; 0.54, 0.56 and 0.59 vs 0.29, respectively, all P <0.05); while the relative expression levels of Bcl-2 mRNA in FELT groups were lower than that in model group (2.20, 2.08 and 2.08 vs 4.04, respectively, P <0.05).

CONCLUSIONFELT may inhibit the synovium proliferation in RA model rats through promoting the FLS apoptosis.

Animals ; Apoptosis ; Arthritis, Rheumatoid ; drug therapy ; Caspase 3 ; metabolism ; Disease Models, Animal ; Echinops Plant ; chemistry ; Fibroblasts ; metabolism ; Flavonoids ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Synovial Membrane ; cytology ; bcl-2-Associated X Protein ; metabolism

Eupatilin is the main active component of DA-9601, an extract from Artemisia. Recently, eupatilin was reported to have anti-inflammatory properties. We investigated the anti-arthritic effect of eupatilin in a murine arthritis model and human rheumatoid synoviocytes. DA-9601 was injected into collagen-induced arthritis (CIA) mice. Arthritis score was regularly evaluated. Mouse monocytes were differentiated into osteoclasts when eupatilin was added simultaneously. Osteoclasts were stained with tartrate-resistant acid phosphatase and then manually counted. Rheumatoid synoviocytes were stimulated with TNF-alpha and then treated with eupatilin, and the levels of IL-6 and IL-1beta mRNA expression in synoviocytes were measured by RT-PCR. Intraperitoneal injection of DA-9601 reduced arthritis scores in CIA mice. TNF-alpha treatment of synoviocytes increased the expression of IL-6 and IL-1beta mRNAs, which was inhibited by eupatilin. Eupatilin decreased the number of osteoclasts in a concentration dependent manner. These findings, showing that eupatilin and DA-9601 inhibited the expression of inflammatory cytokines and the differentiation of osteoclasts, suggest that eupatilin and DA-9601 is a candidate anti-inflammatory agent.
Animals ; Anti-Inflammatory Agents/pharmacology/*therapeutic use ; Arthritis, Experimental/chemically induced/*drug therapy ; Arthritis, Rheumatoid/drug therapy/pathology ; Cell Differentiation/*drug effects ; Cells, Cultured ; Collagen Type II ; Cytokines/biosynthesis ; Disease Models, Animal ; Drugs, Chinese Herbal/therapeutic use ; Female ; Flavonoids/pharmacology/*therapeutic use ; Humans ; Inflammation/drug therapy/immunology ; Interleukin-1beta/genetics/metabolism ; Interleukin-6/genetics/metabolism ; Lymph Nodes/cytology ; Mice ; Mice, Inbred DBA ; Monocytes/cytology ; Osteoclasts/*cytology ; Plant Extracts/pharmacology ; RNA, Messenger/biosynthesis ; Synovial Membrane/cytology ; T-Lymphocytes, Regulatory/cytology/immunology ; Tumor Necrosis Factor-alpha/pharmacology

Tetrandrine (Tet), the main active constituent of Stephania tetrandra root, has been demonstrated to alleviate adjuvant-induced arthritis in rats. The present study was designed to investigate the effects of Tet on the migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) and explore the underlying mechanisms. By using cultures of primary FLS isolated from synoviums of RA patients and cell line MH7A, Tet (0.3, 1 μmol·L(-1)) was proven to significantly impede migration and invasion of RA-FLS, but not cell proliferation. Tet also greatly reduced the activation and expressions of matrix degrading enzymes MMP-2/9, the expression of F-actin and the activation of FAK, which controlled the morphologic changes in migration process of FLS. To identify the key signaling pathways by which Tet exerts anti-migration effect, the specific inhibitors of multiple signaling pathways LY294002, Triciribine, SP600125, U0126, SB203580, and PDTC (against PI3K, Akt, JNK, ERK, p38 MAPK and NF-κB-p65, respectively) were used. Among them, LY294002, Triciribine, and SP600125 were shown to obviously inhibit the migration of MH7A cells. Consistently, Tet was able to down-regulate the activation of Akt and JNK as demonstrated by Western blotting assay. Moreover, Tet could reduce the expressions of migration-related proteins Rho GTPases Rac1, Cdc42, and RhoA in MH7A cells. In conclusion, Tet can impede the migration and invasion of RA-FLS, which provides a plausible explanation for its protective effect on RA. The underlying mechanisms involve the reduction of the expressions of Rac1, Cdc42, and RhoA, inhibition of the activation of Akt and JNK, and subsequent down-regulation of activation and/or expressions of MMP-2/9, F-actin, and FAK.
Animals ; Arthritis ; Arthritis, Rheumatoid ; metabolism ; prevention & control ; Benzylisoquinolines ; pharmacology ; therapeutic use ; Cell Movement ; drug effects ; Cell Proliferation ; Cells, Cultured ; Disease Models, Animal ; Down-Regulation ; Fibroblasts ; drug effects ; metabolism ; Humans ; MAP Kinase Signaling System ; Phosphatidylinositol 3-Kinases ; metabolism ; Phytotherapy ; Plant Extracts ; pharmacology ; therapeutic use ; Plant Roots ; Protein-Serine-Threonine Kinases ; metabolism ; Signal Transduction ; Stephania ; chemistry ; Synovial Membrane ; cytology ; drug effects ; metabolism ; rac1 GTP-Binding Protein ; metabolism ; rhoA GTP-Binding Protein ; metabolism