Fibroblast-like Synoviocytes Cells Research Articles

Immunological Roles of Fibroblast-Like Synoviocyte Cells in Rheumatoid Arthritis

Introduction: Fibroblast-like synoviocytes (FLS), also known as synovial fibroblasts or type B synoviocytes, are the primary cells responsible for the structure of the synovial lining. They are crucial for the formation of a healthy, organized synovial lining. In rheumatic synovium affected by inflammation, the typical three-layered synovial lining transforms into a pannus-like structure. Various pro-inflammatory conditions in the joints of rheumatoid arthritis (RA) patients, characterized by elevated levels of cytokines, growth factors, and infiltration of inflammatory cells, strongly activate FLS cells. Moreover, environmental conditions in the joints of RA patients, such as high pressure and hypoxia, induce changes that further contribute to FLS activation and the development of aggressive characteristics. These changes include increased proliferation, reduced apoptosis, and enhanced cell migration, collectively referred to as a tumor-like phenotype. Additionally, FLS cells release inflammatory cytokines, amplifying inflammation and attracting immune cells to the joint. They also play a role in degrading the extracellular matrix and causing cartilage and bone damage through the production of enzymes like matrix metalloproteinases (MMPs), collagenase, aggrecans, and cathepsins. Recent therapeutic approaches have been directed at targeting the signaling pathways that activate FLS cells and inhibiting factors and cytokines produced by these cells to alleviate inflammatory symptoms and reduce joint damage. It is anticipated that these treatment strategies will complement existing therapies in the near future.
 Conclusion: FLS cells are the main components of maintaining the health and nutrition of joints. These cells produce various cytokines, chemokines, angiogenic factors, as well as factors that contribute to the breakdown of matrix and cartilage. The main drivers of significant changes in inflamed joints are proliferation and resistance to apoptosis. Treatment strategies have been developed to target the signaling pathways that activate these cells, with a focus on improving inflammatory symptoms. It is expected that these treatment strategies will be incorporated into existing therapies in the near future.

Dual drug nanoparticle synergistically induced apoptosis, suppressed inflammation, and protected autophagic response in rheumatoid arthritis fibroblast-like synoviocytes

Rheumatoid arthritis (RA) is a chronic immune-mediated joint inflammatory disorder associated with aberrant activation of fibroblast-like synoviocytes (FLS). Recently, FLS gained importance due to its crucial role in RA pathogenesis, and thus, targeting FLS is suggested as an attractive treatment strategy for RA. FLS-targeted approaches may be combined with disease-modifying antirheumatic drugs (DMARDs) and natural phytochemicals to improve efficacy in RA control and negate immunosuppression. In this study, we assessed the therapeutic effectiveness of DD NP HG in primary RA-FLS cells isolated from the synovial tissue of FCA-induced RA rats. We observed that DD NP HG had good biosafety for healthy FLS cells and, at higher concentrations, a mild inhibitory effect on RA-FLS. The combination therapy (DD NP HG) of MTX NP and PEITC NE in RA-FLS showed a higher rate of apoptosis with significantly reduced LPS-induced expression of pro-inflammatory cytokines (TNF-α, IL-17A, and IL-6) in arthritic FLS. Further, the gene expression studies showed that DD NP HG significantly down-regulated the mRNA expression of IL-1β, RANKL, NFATc1, DKK1, Bcl-xl, Mcl-1, Atg12, and ULK1, and up-regulated the mRNA expression of OPG, PUMA, NOXA and SQSTM1 in LPS-stimulated RA-FLS cells. Collectively, our results demonstrated that DD NP HG significantly inhibited the RA-FLS proliferation via inducing apoptosis, down-regulating pro-inflammatory cytokines, and further enhancing the expression of genes associated with bone destruction in RA pathogenesis. A nanotechnology approach is a promising strategy for the co-delivery of dual drugs to regulate the RA-FLS function and achieve synergistic treatment of RA.

Tofacitinib restores psoriatic arthritis fibroblast-like synoviocytes function via autophagy and mitochondrial quality control modulation

ObjectivesTo evaluate the in vitro effect of tofacitinib on autophagy activity of psoriatic arthritis (PsA) fibroblast-like synoviocytes (FLS), and to confirm its activity on inflammatory and invasive properties of FLS and synovial cells, deepening the impact on mitochondrial function. MethodsFLS, peripheral blood mononuclear cells (PBMCs), and synovial cells from active PsA patients were cultured with tofacitinib 1 μM or vehicle control for 24 h. Autophagy was measured by Western blot and by fluorescence microscopy. Chemokines/cytokines released into culture supernatants were quantified by ELISA, while invasive properties of FLS by migration assays. Specific mitochondrial probes were adopted to measure intracellular reactive oxygen species (ROS), mitochondrial potential, morphology, turnover and mitophagy. Oxygen consumption rate (OCR), reflecting oxidative phosphorylation, was quantified using the Seahorse technology. Differences were determined by adopting the non-parametric Wilcoxon signed rank test. Results18 patients with moderately-to-severely active PsA were enrolled. Tofacitinib significantly increased the levels of the autophagy markers LC3-II and ATG7 in PsA FLS compared to vehicle control, suggesting an increase in spontaneous autophagy activity; no effect was highlighted in PBMCs and synovial cells cultures. Tofacitinib reduced migration properties of PsA FLS, and reduced MCP-1 and IL-6 release into FLS and synovial cells cultures supernatants. Furthermore, tofacitinib decreased intracellular ROS production, increased basal OCR, ATP production and maximal respiratory capacity, and enhanced mitophagy and mitochondrial turnover. ConclusionsThe JAK inhibitor tofacitinib reduces the pro-invasive and pro-inflammatory properties of PsA FLS. Autophagy induction and mitochondrial quality control modulation by tofacitinib might contribute to FLS function restoration.

TNF-α stimulated exosome derived from fibroblast-like synoviocytes isolated from rheumatoid arthritis patients promotes HUVEC migration, invasion and angiogenesis by targeting the miR-200a-3p/KLF6/VEGFA axis

The pathogenesis of rheumatoid arthritis (RA) is heavily impacted by the inflammation and activation of fibroblast-like synoviocytes (FLS). The objective of this investigation is to clarify the involvement of exosomes derived from FLS stimulated by tumour necrosis factor α (TNF-α) in angiogenesis and the underlying mechanisms. FLS cells were obtained from synovial fluid of RA patients and exosomes were obtained from FLS cell supernatant with TNF-α stimulation by ultracentrifugation. Exosomes were subsequently analysed using transmission electron microscopy, nanoparticle tracking analysis, and western blotting. The functional effects of exosomes with TNF-α stimulation on human umbilical vein endothelial cells (HUVEC) migration, invasion, and angiogenesis was evaluated using wound scratch healing test, transwell invasion assay, and tube formation assay. DNA nanoball-seq (DNBSEQ) sequencing platform was utilised to analysis different expression miRNA from exosomes, miRNA and mRNA from HUVEC. The expression level of miR-200a-3p was determined through quantitative real-time polymerase chain reaction (qRT-PCR). The quantification of KLF6 and VEGFA expression levels were performed by qRT-PCR and western blot analysis. The validation of the association between miR-200a-3p and KLF6 was established through a fluorescence enzyme reporting assay. In comparison to exosome induced by PBS, exosome induced by TNF-α exhibited a substantial exacerbation of invasion, migration, and angiogenesis in HUVEC. 4 miRNAs in exosomes and HUVEC cells, namely miR-1246, miR-200a-3p, miR-30a-3p, and miR-99b-3p was obtained. MiR-200a-3p maintained high consistency with the sequencing results. We obtained 5 gene symbols, and KLF6 was chose for further investigation. The expression of miR-200a-3p in exosomes induced by TNF-α and in HUVEC treated with these exosomes demonstrated a significantly increase. Additionally, HUVEC cells displayed a notable decrease in KLF6 expression and a significant elevation in VEGFA expression. This was further confirmed by the fluorescence enzyme report assay, which provided evidence of the direct targeting of KLF6 by miR-200a-3p. Exosomes induced by TNF-α have the ability to enhance the migration, invasion, and angiogenesis of HUVEC cells via the miR-200a-3p/KLF6/VEGFA axis.

Methyl Canthin-6-one-2-carboxylate Restrains the Migration/Invasion Properties of Fibroblast-like Synoviocytes by Suppressing the Hippo/YAP Signaling Pathway.

Rheumatoid arthritis (RA) is an inflammatory condition that causes severe cartilage degradation and synovial damage in the joints with multiple systemic implications. Previous studies have revealed that fibroblast-like synoviocytes (FLSs) play a pivotal role in the pathogenesis of RA. The appropriate regulation of FLS function is an efficient approach for the treatment of this disease. In the present study, we explored the effects of methyl canthin-6-one-2-carboxylate (Cant), a novel canthin-6-one alkaloid, on the function of FLSs. Our data showed that exposure to Cant significantly suppressed RA-FLS migration and invasion properties in a dose-dependent manner. Meanwhile, pre-treatment with Cant also had an inhibitory effect on the release of several pro-inflammatory cytokines, including IL-6 and IL-1β, as well as the production of MMP1 and MMP3, which are important mediators of FLS invasion. In further mechanistic studies, we found that Cant had an inhibitory effect on the Hippo/YAP signaling pathway. Treatment with Cant suppressed YAP expression and phosphorylation on serine 127 and serine 397 while enhancing LATS1 and MST1 levels, both being important upstream regulators of YAP. Moreover, YAP-specific siRNA or YAP inhibition significantly inhibited wound healing as well as the migration and invasion rate of FLS cells, an impact similar to Cant treatment. Meanwhile, the over-expression of YAP significantly reversed the Cant-induced decline in RA-FLS cell migration and invasion, indicating that YAP was required in the inhibitory effect of Cant on the migration and invasion of RA-FLS cells. Additionally, supplementation of MMP1, but not MMP3, in culture supernatants significantly reversed the inhibitory effect of Cant on RA-FLS cell invasion. Our data collectively demonstrated that Cant may suppress RA-FLS migration and invasion by inhibiting the production of MMP1 via inhibiting the YAP signaling pathway, suggesting a potential of Cant for the further development of anti-RA drugs.

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

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

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

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

The effect of tranexamic acid on synovium of patients undergoing arthroplasty and anterior cruciate ligament reconstruction surgery.

Preoperative hemorrhage can be reduced using anti-fibrinolytic medicine tranexamic acid (TXA). During surgical procedures, local administration is being used more and more frequently, either as an intra-articular infusion or as a perioperative rinse. Serious harm to adult soft tissues can be detrimental to the individual since they possess a weak ability for regeneration. Synovial tissues and primary fibroblast-like synoviocytes (FLS) isolated from patients were examined using TXA treatment in this investigation. FLS is obtained from rheumatoid arthritis (RA), osteoarthritis (OA), and anterior cruciate ligament (ACL)-ruptured patients. The in vitro effect of TXA on primary FLS was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assays for cell death, annexin V/propidium iodide (PI) staining for apoptotic rate, real-time PCR for p65 and MMP-3 expression, and enzyme-linked immunosorbent assay (ELISA) for IL-6 measurement. MTT assays revealed a significant decrease in cell viability in FLS of all groups of patients following treatment with 0.8-60 mg/ml of TXA within 24 h. There was a significant increase in cell apoptosis after 24 h of exposure to TXA (15 mg/ml) in all groups, especially in RA-FLS. TXA increases the expression of MMP-3 and p65 expression. There was no significant change in IL-6 production after TXA treatment. An increase in receptor activator of nuclear factor kappa-Β ligand (RANK-L) production was seen only in RA-FLS. This study demonstrates that TXA caused significant synovial tissue toxicity via the increase in cell death and elevation of inflammatory and invasive gene expression in FLS cells.

Astragalosides inhibit proliferation of fibroblast-like synoviocytes in experimental arthritis by modulating LncRNA S56464.1/miR-152-3p/Wnt1 signaling axis.

Astragalus membranaceus (Fisch.) Bunge., the dried root of the plant A. membranaceus, is widely used in the treatment of rheumatoid arthritis (RA) in many Chinese herbal remedies. Astragalosides (AST) is the primary medicinal ingredient of A. membranaceus and has a therapeutic effect on RA, but the specific mechanism of this effect has yet to be elucidated. In this study, MTT and flow cytometry were used to determine the effects of AST on fibroblast-like synoviocyte (FLS) proliferation and cell cycle progression. Additionally, real-time quantitative polymerase chain reaction and Western blotting were used to determine the effects of AST on the LncRNA S56464.1/miR-152-3p/Wnt1 signaling axis and on critical genes that are essential to the Wnt pathway. The data showed that after the administration of AST, FLS proliferation and LncRNA S56464.1, β-catenin, C-myc, Cyclin D1, and p-GSK-3β(Ser9)/GSK-3β expression were significantly reduced, and miR-152 and SFRP4 expression was notably increased. These results suggest that AST can inhibit FLS proliferation by modulating the LncRNA S56464.1/miR-152-3p/Wnt1 signaling axis and that AST may be a potential therapeutic drug for RA.

MicroRNA-27a-3p enhances the inflammatory phenotype of Juvenile Idiopathic Arthritis fibroblast-like synoviocytes

BackgroundJuvenile Idiopathic Arthritis (JIA) is the most prevalent chronic pediatric rheumatic disorder. In joints of JIA patients, aggressive phenotypic changes in fibroblast-like synoviocytes (FLS) of the synovial lining play a key role in inflammation. MicroRNAs are dysregulated in rheumatoid arthritis and JIA, including miR-27a-3p. However, it is not understood if miR-27a-3p, enriched in JIA synovial fluid (SF) and leukocytes, alters FLS function.MethodsPrimary JIA FLS cells were transfected with a miR-27a-3p mimic or a negative control microRNA (miR-NC) and stimulated with pooled JIA SF or inflammatory cytokines. Viability and apoptosis were analyzed by flow cytometry. Proliferation was evaluated using a 3H-thymidine incorporation assay. Cytokine production was assessed by qPCR and ELISA. Expression of TGF-β pathway genes was determined using a qPCR array.ResultsMiR-27a-3p was constitutively expressed in FLS. Overexpression of miR-27a-3p caused increased interleukin-8 secretion in resting FLS, and interleukin-6 was elevated in SF-activated FLS compared to miR-NC. Furthermore, stimulation with pro-inflammatory cytokines augmented FLS proliferation in miR-27a-3p-transfected FLS relative to miR-NC. Expression of multiple TGF-β pathway genes was modulated by overexpression of miR-27a-3p.ConclusionsMiR-27a-3p significantly contributes to FLS proliferation and cytokine production, making it a potential candidate for epigenetic therapy that targets FLS in arthritis.

Integrated Multi-Omics Techniques and Network Pharmacology Analysis to Explore the Material Basis and Mechanism of Simiao Pill in the Treatment of Rheumatoid Arthritis.

The Simiao pill (SMP) is a classic prescription that has shown anti-inflammatory, analgesic, and immunomodulatory effects and is clinically used to treat inflammatory diseases, such as rheumatoid arthritis (RA) and gouty arthritis, for which the effects and mechanism of action remain largely unknown. In this study, serum samples from RA rats were analyzed using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry based metabolomics technology and liquid chromatography with tandem mass spectrometry proteomics technology together with network pharmacology to explore the pharmacodynamic substances of SMP. To further verify the above results, we constructed a fibroblast-like synoviocyte (FLS) cell model and administered phellodendrine for the test. All these clues suggested that SMP can significantly reduce the level of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in complete Freund's adjuvant rat serum and improve the degree of foot swelling; combined with metabolomics, proteomics, and network pharmacological technology, it is determined that SMP plays a therapeutic role through the inflammatory pathway, and phellodendrine is found to be one of the pharmacodynamic substances. By constructing an FLS model, it is further determined that phellodendrine could effectively inhibit the activity of synovial cells and reduce the expression level of inflammatory factors by downregulating the expression level of related proteins in the TLR4-MyD88-IRAK4-MAPK signal pathway to alleviate joint inflammation and cartilage injury. Overall, these findings suggested that phellodendrine is an effective component of SMP in the treatment of RA.

68Ga-FAPI PET/CT for Rheumatoid Arthritis: A Prospective Study.

Background In rheumatoid arthritis (RA), fibroblast-like synoviocyte cells, which are involved in inflammation of the articular cartilage and bone, overexpress fibroblast activation protein (FAP). This is a feature that could be leveraged to improve imaging assessment of disease. Purpose To determine the performance of gallium 68 (68Ga)-labeled FAP inhibitor (FAPI) in assessing joint disease activity of RA and to compare with fluorine 18 (18F) fluorodeoxyglucose (FDG) imaging. Materials and Methods Twenty participants with RA (15 women; mean age, 55 years ± 10 [SD]) were prospectively enrolled from September 2020 to December 2021 and underwent clinical and laboratory assessment of disease activity and dual-tracer PET/CT (68Ga-FAPI and 18F-FDG) imaging. Imaging-derived variables of PET joint count (the number of joints positive for RA at PET) and PET articular index (a sum of the points of the joints using a three-point scale) were correlated to clinical and laboratory variables of disease activity. Results The combined output of both PET/CT techniques helped detect 244 affected joints, all of which showed positive results at 68Ga-FAPI PET/CT. However, fifteen of 244 (6.1%) FAPI-avid joints in six of 20 (30%) participants were not detected at 18F-FDG PET/CT. The maximum standardized uptake value of the most affected joint in each participant was higher in 68Ga-FAPI than in 18F-FDG PET/CT (9.54 ± 4.92 vs 5.85 ± 2.81, respectively; P = .001). The maximum standardized uptake values of the joints at both 68Ga-FAPI and 18F-FDG PET/CT were positively correlated with laboratory evaluation of C-reactive protein levels (r = 0.49 [P = .03] and 0.54 [P = .01], respectively). The PET joint count and PET articular index scores at 68Ga-FAPI PET/CT were also positively correlated with most clinical disease activity variables and radiographic progression of joint damage (P < .05). Conclusion In participants with rheumatoid arthritis who underwent gallium 68 fibroblast activation protein inhibitor PET/CT, the extent of joint involvement correlated with clinical and laboratory variables of disease activity and showed a greater amount and degree of affected joints than at fluorine 18 fluorodeoxyglucose PET/CT. Clinical trial registration no. NCT04514614 © RSNA, 2023 Supplemental material is available for this article. See also the editorial by Williams and Ahlman in this issue.

Isochlorogenic Acid C Restrains Erk/JNK/NF-κB Signaling to Alleviate Inflammatory Response and Promote Cell Apoptosis

Isochlorogenic acid C (ICAC) is found in a variety of natural foods and medicinal plant materials. This study aims to validate the biological activity of isochlorogenic acid C in limiting inflammation by the Erk/JNK/NF-κB pathway. In this study, TNF-α-induced human fibroblast-like synoviocytes and collagen-induced arthritis animal models were used to perceive the potential anti-inflammation mechanism of ICAC. The role of isochlorogenic acid C was evaluated by observing the migration, invasion ability, and apoptotic activity of TNF-α-induced fibroblast-like synoviocytes cells in humans and analysing foot swelling, joint index, and histopathological changes in a collagen-induced RA animal model. The results reveal that ICAC inhibited the proliferation of human fibroblast-like synoviocytes and promoted their apoptosis. ICAC also blocked the nuclear transfer of NF-κB, Erk, and JNK. It was observed that ICAC significantly inhibited the degree of posterior foot swelling in CIA, reducing arthritis scores, bone tissue injury, and articular synovitis. ICAC may promote cell apoptosis and inhibit the hyperactivation of inflammatory cells to alleviate inflammation-induced synovial proliferation through the Erk/JNK/NF-κB pathway.

Berberine inhibits RA-FLS cell proliferation and adhesion by regulating RAS/MAPK/FOXO/HIF-1 signal pathway in the treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a common chronic immune disease. Berberine, as its main active ingredient, was also contained in a variety of medicinal plants such as Berberaceae, Buttercup, and Rutaceae, which are widely used in digestive system diseases in traditional Chinese medicine with anti-inflammatory and antibacterial effects. The aims of this article were to explore the therapeutic effect and mechanism of berberine on rheumatoid arthritis. Cell Counting Kit-8 was used to evaluate the effect of berberine on the proliferation of RA fibroblast-like synoviocyte (RA-FLS) cells. The effect of berberine on matrix metalloproteinase (MMP)-1, MMP-3, receptor activator of nuclear factor kappa-Β ligand (RANKL), tumour necrosis factor alpha (TNF-α), and other factors was determined by enzyme-linked immunoassay (ELISA) kit. Transcriptome technology was used to screen related pathways and the potential targets after berberine treatment, which were verified by reverse transcription-polymerase chain reaction (RT-qPCR) and Western blot (WB) technology. Berberine inhibited proliferation and adhesion of RA-FLS cells, and significantly reduced the expression of MMP-1, MMP-3, RANKL, and TNF-α. Transcriptional results suggested that berberine intervention mainly regulated forkhead box O (FOXO) signal pathway, prolactin signal pathway, neurotrophic factor signal pathway, and hypoxia-inducible factor 1 (HIF-1) signal pathway. The effect of berberine on RA was related to the regulation of RAS/mitogen-activated protein kinase/FOXO/HIF-1 signal pathway in RA-FLS cells.Cite this article: Bone Joint Res2023;12(2):91-102.

Osthole: A potential AMPK agonist that inhibits NLRP3 inflammasome activation by regulating mitochondrial homeostasis for combating rheumatoid arthritis

BackgroundOsthole (OST), a characteristic coumarin compound in Angelicae pubescentis radix (APR), has shown potent efficacy in the treatment of rheumatoid arthritis (RA), but its specific targets and potential mechanism are limited. PurposeThis study aimed to explore the potential targets and molecular mechanisms of OST against RA using computer-assisted techniques in combination with RA fibroblast-like synoviocytes (FLS) inflammation model and CIA rat model. MethodsNetwork pharmacology and molecular docking were applied to initially predict the potential targets of OST for the treatment of RA. Thereafter, TNFα was used to stimulate FLS to build an in vitro model of inflammation, combined with RNA-seq technology and molecular biology such as qPCR to investigate the anti-inflammatory effects and related mechanisms of OST. Finally, the anti-RA effect of OST was demonstrated by establishing a CIA rat model. ResultsThe network model results showed that the anti-RA effect of OST was mainly related to its anti-inflammatory effect, and AMPK was identified as a potential target for the potency of OST. In the TNFα-induced FLS cells, OST inhibited the secretion of FLS inflammatory factors, which was attributed to the ability of OST to activate AMPK to inhibit the activation of the NLRP3 inflammasome. Further, it was observed that the activation of AMPK by OST facilitated mitochondrial biogenesis, and corrected abnormal mitochondrial dynamics in FLS, which was favoured to the restoration of mitochondrial homeostasis, and further promoted the occurrence of apoptosis and the decrease of ROS in FLS. Consistent with in vivo studies, administration of OST significantly improved joint deformity and toe erythema, reduced arthritis index scores and inhibited synovial inflammation in CIA rats. ConclusionOur study proposed for the first time that AMPK, served as a potential target of OST, positively participated in the anti-RA therapeutic effect of OST. By regulating mitochondrial homeostasis and function, OST can effectively inhibit the activation of inflammasome and the secretion of inflammatory factors in vitro and in vivo, and finally achieve beneficial effects in the treatment of RA, which provides support and greater possibility to make further efforts on pharmacological research and clinical application of OST.

Systems-biology analysis of rheumatoid arthritis fibroblast-like synoviocytes implicates cell line-specific transcription factor function

Rheumatoid arthritis (RA) is an immune-mediated disease affecting diarthrodial joints that remains an unmet medical need despite improved therapy. This limitation likely reflects the diversity of pathogenic pathways in RA, with individual patients demonstrating variable responses to targeted therapies. Better understanding of RA pathogenesis would be aided by a more complete characterization of the disease. To tackle this challenge, we develop and apply a systems biology approach to identify important transcription factors (TFs) in individual RA fibroblast-like synoviocyte (FLS) cell lines by integrating transcriptomic and epigenomic information. Based on the relative importance of the identified TFs, we stratify the RA FLS cell lines into two subtypes with distinct phenotypes and predicted active pathways. We biologically validate these predictions for the top subtype-specific TF RARα and demonstrate differential regulation of TGFβ signaling in the two subtypes. This study characterizes clusters of RA cell lines with distinctive TF biology by integrating transcriptomic and epigenomic data, which could pave the way towards a greater understanding of disease heterogeneity.

Imperatorin Inhibits Proliferation, Migration, and Inflammation via Blocking the NF-κB and MAPK Pathways in Rheumatoid Fibroblast-like Synoviocytes

Rheumatoid arthritis (RA) is a chronic joint inflammatory disease associated with the aberrant activation of fibroblast-like synoviocytes (FLSs). Searching for natural compounds that may suppress the activation of FLSs has become a complementary approach for RA treatment. Here, we investigated the effects and mechanisms of imperatorin (IPT) on proliferation, migration, and inflammation in primary cultured arthritic FLSs. We found that IPT significantly suppressed TNFα-induced proliferation and migration of arthritic FLSs, but showed little effect on survival and apoptosis. In addition, IPT treatment significantly reduced the TNFα-induced expression of pro-inflammatory cytokines (IL-1β, TNFα, IL-6, and IL-8) in arthritic FLSs. Further mechanism studies suggested that IPT inhibited the activations of p38 and extracellular signal-regulated kinase (ERK). Also, IPT blocked the nuclear factor of κB (NF-κB) activation by suppressing the phosphorylation and degradation of IκBα, thereby preventing the translocation of p65. Collectively, our results demonstrated that IPT could inhibit the over-activated phenotypes of arthritic FLSs via the mitogen-activated protein kinase (MAPK) (p38 and ERK) and NF-κB pathways leading to the down-regulation of pro-inflammatory cytokines, which might be beneficial to the anti-proliferative and anti-migratory activities of FLS cells. These findings suggest that IPT has the potential to be developed as a novel agent for RA treatment.

Carnosine Alleviates Knee Osteoarthritis and Promotes Synoviocyte Protection via Activating the Nrf2/HO-1 Signaling Pathway: An In-Vivo and In-Vitro Study.

The most common joint disease in the elderly is knee osteoarthritis (OA). It is distinguished by cartilage degradation, subchondral bone loss, and a decrease in joint space. We studied the effects of carnosine (CA) on knee OA in male Wistar rats. OA is induced by anterior cruciate ligament transection combined with medial meniscectomy (ACLT+MMx) method and in vitro studies are conducted in fibroblast-like synoviocyte cells (FLS). The pain was assessed using weight-bearing and paw-withdrawal tests. CA supplementation significantly reduced pain. The enzyme-linked immunosorbent assay (ELISA) method was used to detect inflammatory proteins in the blood and intra-articular synovial fluid (IASF), and CA reduced the levels of inflammatory proteins. Histopathological studies were performed on knee-tissue samples using toluidine blue and hematoxylin and eosin (H and E) assays. CA treatment improved synovial protection and decreased cartilage degradation while decreasing zonal depth lesions. Furthermore, Western blotting studies revealed that the CA-treated group activated nuclear factor erythroid 2-related factor (Nrf2) and heme oxygenase (HO-1) and reduced the expression of cyclooxygenase-2 (COX-2). FLS cells were isolated from the knee joints and treated with IL-1β to stimulate the inflammatory response and increase reactive oxygen species (ROS). The matrix metalloproteinase protein (MMP’s) levels (MMP-3, and MMP-13) were determined using the reverse transcription-polymerase chain reaction (RT-PCR), and CA treatment reduced the MMP’s expression levels. When tested using the 2′,7′-dicholorodihydrofluroscene diacetate (DCFDA) assay and the 5,5′,6,6′-tetracholoro-1,1′,3,3′-tertraethylbenzimidazolcarboc janine iodide (JC-1) assay in augmented ROS FLS cells, CA reduced the ROS levels and improved the mitochondrial membrane permeability. This study’s investigation suggests that CA significantly alleviates knee OA both in vitro and in vivo.

Anti-inflammatory effects of aucubin in cellular and animal models of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease. It is known that aucubin (AU) exerts anti-inflammatory activity, but its effects and mechanisms in RA are unclear. This study investigated the anti-inflammatory effects and mechanisms of AU in vivo and in vitro. Human fibroblast-like synoviocyte cells from patients with RA (HFLS-RA), RAW264.7 cells, and MC3T3-E1 cells were used to evaluate the effects of AU on migration, invasion, apoptosis, osteoclast differentiation and production. Immunofluorescence was used to observe nuclear translocation of nuclear factor (NF)-κB, the double luciferase reporter gene method was used to observe NF-κB-p65 activity in AU-treated MC3T3-E1 cells. RT-qPCR was used to measure expression of bone metabolism and inflammation-related genes, and western blot was used to measure bone metabolism and NF-κB protein expression levels. Collagen-induced arthritis (CIA) rat model was used for pharmacodynamics study. Arthritis indexes were measured in the ankle and knee, histological staining and Micro-computed tomography were performed on the ankle joints. Also, inflammatory factor gene expression and the levels of NF-κB-related proteins were detected as in vitro. AU effectively inhibited HFLS-RA cell migration and invasion, promoted apoptosis, and inhibited RAW264.7 cell differentiation into osteoclasts, as well as inhibited NF-κB-p65 activity in MC3T3-E1 cells. Notably, AU significantly reduced the gene expression levels of three cell-related inflammatory factors and bone metabolism factors, effectively inhibited the expression of p-Iκκα β, p-IκBα, and p-p65 proteins. In vivo, AU relieved joint inflammation, reduced related inflammatory factors, and inhibited NF-κB signaling. It could be used to treat RA-related synovial inflammation and bone destruction through the NF-κB pathway.

Chelerythrine ameliorates rheumatoid arthritis by modulating the AMPK/mTOR/ULK-1 signaling pathway

BackgroundRheumatoid arthritis (RA) is a long-term, progressive, and disabling autoimmune disease. It causes inflammation, swelling and pain in and around the joints and other body organs. Currently, no cure is available for RA. Clinical interventions can only relieve the condition, and at least 30% of RA patients do not respond to first‑line therapy. This means that the development of more effective therapies against RA is urgently needed. ObjectiveThis study aimed to assess the anti-rheumatoid arthritis effect of chelerythrine (CLT) and explore its mechanism of action. MethodsThe cytotoxic effect of CLT on human rheumatoid arthritis fibroblast-like synoviocyte (HFLS-RA) cells and HFLS-normal cells were measured by MTT assay. The growth and migration of HFLS-RA cells were determined by colony-formation and wound-healing assay. The level of intracellular reactive oxygen species (ROS) was detected using the DCFH-DA reagent. Cell apoptosis was measured by flow cytometry, TUNEL staining, caspase 3 activity, as well as the activation of apoptosis related proteins. In addition, the levels of autophagy related markers such as LC3B and P62 were determined by immunocytochemistry and western blotting. Lastly, the anti-RA effect of CLT was evaluated in an Adjuvant-Induced Arthritis(AIA) rat model and the severity of arthritis was detected and quantified using macroscopic inspection and X‑ray imaging. ResultsWe discovered that treatment with CLT effectively inhibited the migration and colony-formation of the HFLS-RA cells and resulted in cell death. Moreover, CLT increased the intracellular level of ROS and the apoptotic rate of HFLS-RA by activating the AMPK/mTOR/ULK-1 signaling pathways. In vivo study showed CLT effectively ameliorated AIA in rats, protecting them from inflammation and bone damage. ConclusionOur study shows CLT is an effective agent for ameliorating RA in vitro and in vivo by modulation of the AMPK/mTOR/ULK-1 signaling pathway. These findings indicate that CLT is a great potential candidate for development as a therapeutic agent for the prevention and treatment of RA.