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Upregulating miR-27a-3p inhibits cell proliferation and inflammation of rheumatoid arthritis synovial fibroblasts through targeting toll-like receptor 5.

ABSTRACT: Rheumatoid arthritis (RA) is a serious chronic inflammatory disease and synovial fibroblasts (SFs) serve a vital role in the pathogenesis and progression of RA. Current studies have demonstrated that dysregulation of microRNAs is involved in RA etiopathogenesis. The present study aimed to investigate the role of microRNA (miR)-27a-3p in RASFs, as well as its molecular mechanism. RASFs were isolated from synovial tissues from patients with RA. Expression of miR-27a-3p and toll-like receptor 5 (TLR5) was detected using reverse transcription-quantitative polymerase chain reaction and western blotting. Cell proliferation, apoptosis and inflammatory response were measured with MTT assay, flow cytometry and ELISA kits, respectively. The target binding between miR-27a-3p and TLR5 was predicted on DIANA TOOLS software, and confirmed by dual-luciferase reporter assay and Biotin-coupled miRNA pull-down assay. Expression of miR-27a-3p was downregulated and TLR5 was upregulated in synovial tissues and RASFs isolated from patients with RA. Functionally, upregulating miR-27a-3p may promote the apoptosis rate of RASFs and suppress cell proliferation and secretions of interleukin (IL)-1β, IL-6 and tumor necrosis factor-α. TLR5 was validated as a downstream target for miR-27a-3p in RASFs, and its expression was negatively regulated by miR-27a-3p. Silencing TLR5 in RASFs may exert similar effects to miR-27a-3p-overexpression; whereas, restoring TLR5 counteracted the suppression of miR-27a-3p-overexpression on RASF proliferation and inflammation, as well as the promotion on apoptosis. miR-27a-3p upregulation may suppress RA progression by inhibiting RASFs proliferation and inflammation through targeting TLR5.

SUBMITTER: Chen L

PROVIDER: S-EPMC8438689 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundAntcin K, an extract of Antrodia cinnamomea (a medicinal mushroom endemic to Taiwan commonly used in Chinese medicine preparations), inhibits proinflammatory cytokine production and angiogenesis in human rheumatoid arthritis synovial fibroblasts (RASFs), major players in RA disease. Antcin K also inhibits disease activity in mice with collagen-induced arthritis (CIA). Up until now, the effects of Antcin K upon cell adhesion molecules (CAMs) were unknown.MethodsRA and healthy synovial tissue samples (n = 10 in each group) were retrieved from the Gene Expression Omnibus (GEO) database (accession code: GDS5401) to compare CAM and monocyte marker expressions. In addition, synovial tissue samples from six RA patients and six patients undergoing arthroscopy for trauma/joint derangement (healthy controls) were subjected to immunohistochemical (IHC) analysis. mRNA and protein expression levels were analyzed in RASFs using RT-qPCR (Reverse transcription-quantitative polymerase chain reaction) and Western blot. RASFs were incubated with Antcin K and examined for monocyte adherence by fluorescence microscopy. Ankle joint tissue specimens from a CIA mouse model and healthy controls were stained with hematoxylin and eosin (H&E) and Safranin-O/Fast Green to examine histological changes and evidence of bone loss. IHC analysis determined levels of vascular cell adhesion molecule 1 (VCAM-1) and CD11b in CIA ankle tissue and clinical synovial tissue.ResultsLevels of VCAM-1 expression were higher in the GEO database specimens and the study's clinical samples of RA synovial tissue compared with the healthy specimens. Antcin K dose-dependently inhibited VCAM-1 expression and monocyte adhesion in RASFs. Antcin K also significantly inhibited levels of VCAM-1 and monocyte CD11b expression in CIA tissue. These effects appeared to be mediated by MEK1/2-ERK, p38, and AP-1 signaling.ConclusionsAntcin K seems promising for the treatment of RA and deserves further investigations.

Project description:Cannabidiol (CBD) is a non-intoxicating phytocannabinoid from cannabis sativa that has demonstrated anti-inflammatory effects in several inflammatory conditions including arthritis. However, CBD binds to several receptors and enzymes and, therefore, its mode of action remains elusive. In this study, we show that CBD increases intracellular calcium levels, reduces cell viability and IL-6/IL-8/MMP-3 production of rheumatoid arthritis synovial fibroblasts (RASF). These effects were pronounced under inflammatory conditions by activating transient receptor potential ankyrin (TRPA1), and by opening of the mitochondrial permeability transition pore. Changes in intracellular calcium and cell viability were determined by using the fluorescent dyes Cal-520/PoPo3 together with cell titer blue and the luminescent dye RealTime-glo. Cell-based impedance measurements were conducted with the XCELLigence system and TRPA1 protein was detected by flow cytometry. Cytokine production was evaluated by ELISA. CBD reduced cell viability, proliferation, and IL-6/IL-8 production of RASF. Moreover, CBD increased intracellular calcium and uptake of the cationic viability dye PoPo3 in RASF, which was enhanced by pre-treatment with TNF. Concomitant incubation of CBD with the TRPA1 antagonist A967079 but not the TRPV1 antagonist capsazepine reduced the effects of CBD on calcium and PoPo3 uptake. In addition, an inhibitor of the mitochondrial permeability transition pore, cyclosporin A, also blocked the effects of CBD on cell viability and IL-8 production. PoPo3 uptake was inhibited by the voltage-dependent anion-selective channel inhibitor DIDS and Decynium-22, an inhibitor for all organic cation transporter isoforms. CBD increases intracellular calcium levels, reduces cell viability, and IL-6/IL-8/MMP-3 production of RASF by activating TRPA1 and mitochondrial targets. This effect was enhanced by pre-treatment with TNF suggesting that CBD preferentially targets activated, pro-inflammatory RASF. Thus, CBD possesses anti-arthritic activity and might ameliorate arthritis via targeting synovial fibroblasts under inflammatory conditions.

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Upregulating miR-27a-3p inhibits cell proliferation and inflammation of rheumatoid arthritis synovial fibroblasts through targeting toll-like receptor 5.

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