Synovial Fluid Regulates the Gene Expression of a Pattern of microRNA via the NF-κB Pathway: An In Vitro Study on Human Osteoarthritic Chondrocytes

Abstract

Synovial fluid (SF) represents the primary source of nutrients of articular cartilage and is implicated in maintaining cartilage metabolism. We investigated the effects of SF, from patients with osteoarthritis (OA), rheumatoid arthritis (RA), and controls, on a pattern of microRNA (miRNA) in human OA chondrocytes. Cells were stimulated with 50% or 100% SF for 24 h and 48 h. Apoptosis and superoxide anion production were detected by cytometry; miRNA (34a, 146a, 155, 181a), cytokines, metalloproteinases (MMPs), type II collagen (Col2a1), antioxidant enzymes, B-cell lymphoma (BCL)2, and nuclear factor (NF)-κB by real-time PCR. The implication of the NF-κB pathway was assessed by the use of NF-κB inhibitor (BAY-11-7082). RA and OA SF up-regulated miR-34a, -146a, -155, -181a, interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, MMP-1, MMP-13, and ADAMTs-5 gene expression, while it down-regulated Col2a1. Pathological SF also induced apoptosis, reduced viability, and decreased BCL2 mRNA, whereas it increased superoxide anions, the expression of antioxidant enzymes, p65 and p50 NF-κB. Opposite and positive results were obtained with 100% control SF. Pre-incubation with BAY-11-7082 counteracted SF effects on miRNA. We highlight the role of the SF microenvironment in regulating some miRNA involved in inflammation and cartilage degradation during OA and RA, via the NF-κB pathway.

Keywords: cartilage metabolism; chondrocytes; cytokines; inflammation; microRNA; osteoarthritis; rheumatoid arthritis; synovial fluid.

Figure 1

SF regulates viability and apoptosis. Human osteoarthritic (OA) chondrocytes were incubated for 24 h and 48 h with 50% and 100% SF derived from patients with OA, rheumatoid arthritis (RA), or controls. (A,B) Evaluation of cell viability by MTT assay. (C,D) Apoptosis detection performed by flow cytometry analysis and measured with annexin Alexa fluor 488 assay. Data were expressed as the percentage of positive cells for annexin-V and propidium iodide (PI) staining. (E,F) Expression levels of B-cell lymphoma (BCL2) analyzed by quantitative real-time PCR. The percentage of survival cells, the ratio of apoptosis, and the gene expression were referenced to the ratio of the value of interest and the value of basal condition, reported equal to 100 or 1. Data were represented as mean ± SD of triplicate values. * p < 0.05, ** p < 0.01, *** p < 0.001 versus basal condition. ° p < 0.05, °° p < 0.01 versus control SF.

Figure 2

SF regulation on inflammation. Human osteoarthritic (OA) chondrocytes were incubated for 24 h and 48 h with 50% and 100% SF derived from patients with OA, rheumatoid arthritis (RA), or controls. (A–F) Expression levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α analyzed by quantitative real-time PCR. The gene expression was referenced to the ratio of the value of interest and the value of basal condition, reported equal to 1. Data were represented as mean ± SD of triplicate values. * p < 0.05, ** p < 0.01, *** p < 0.001 versus basal condition. ° p < 0.05, °° p < 0.01, °°° p < 0.001 versus control SF.

Figure 3

SF regulates chondrocyte metabolism. Human osteoarthritic (OA) chondrocytes were incubated for 24 h and 48 h with 50% and 100% SF derived from patients with OA, rheumatoid arthritis (RA), or controls. (A–H) Expression levels of metalloproteinase (MMP)-1, -13, metalloproteinase with thrombospondin motif (ADAMTS-5), and type II collagen (Col2a1) analyzed by quantitative real-time PCR. The gene expression was referenced to the ratio of the value of interest and the value of basal condition, reported equal to 1. Data were represented as mean ± SD of triplicate values. * p < 0.05, ** p < 0.01, *** p < 0.001 versus basal condition. ° p < 0.05, °° p < 0.01, °°° p < 0.001 versus control SF.

Figure 4

SF modulates oxidative stress balance. Human osteoarthritic (OA) chondrocytes were incubated for 24 h and 48 h with 50% and 100% SF derived from patients with OA, rheumatoid arthritis (RA), or controls. (A,B) Mitochondrial superoxide anion production evaluated by MitoSox Red staining at flow cytometry. (C–F) Expression levels of superoxide dismutase (SOD)-2 and nuclear factor erythroid-derived 2-like 2 (NRF2) analyzed by quantitative real-time PCR. The production of superoxide anion and the gene expression were referenced to the ratio of the value of interest and the value of basal condition, reported equal to 1. Data were represented as mean ± SD of triplicate values. * p < 0.05, ** p < 0.01, *** p < 0.001 versus basal condition. ° p < 0.05, °° p < 0.01 versus control SF.

Figure 5

SF regulation on miRNA. Human osteoarthritic (OA) chondrocytes were incubated for 24 h and 48 h with 50% and 100% SF derived from patients with OA, rheumatoid arthritis (RA), or controls. (A–H) Expression levels of microRNA (miR)-34a, miR-146a, miR-155, and miR-181a analyzed by quantitative real-time PCR. The gene expression was referenced to the ratio of the value of interest and the value of basal condition, reported equal to 1. Data were represented as mean ± SD of triplicate values. * p < 0.05, ** p < 0.01 versus basal condition. ° p < 0.05, °° p < 0.01 versus control SF.

Figure 6

SF modulates the NF-κB signaling pathway. Human osteoarthritic (OA) chondrocytes were incubated for 3 h with 50% and 100% SF derived from patients with OA, rheumatoid arthritis (RA), or controls. (A,B) Expression levels of p50 and p65 subunits analyzed by quantitative real-time PCR. The gene expression was referenced to the ratio of the value of interest and the value of the basal condition, reported equal to 1. Data were represented as mean ± SD of triplicate values. * p < 0.05, ** p < 0.01, *** p < 0.001 versus basal condition. ° p < 0.05, °° p < 0.01 versus control SF.

Figure 7

SF modulates the NF-κB pathway. Human osteoarthritic (OA) chondrocytes were pre-incubated for 3 h with a specific nuclear factor (NF)-κB inhibitor (BAY 11-7082, IKKα/β, 1 μM), and then for 48 h with 100% SF derived from patients with OA, rheumatoid arthritis (RA), or controls. (A–D) Expression levels of microRNA (miR)-34a, miR-146a, miR-155, and miR-181a analyzed by quantitative real-time PCR. The gene expression was referenced to the ratio of the value of interest and the value of basal condition, reported equal to 1. Data were represented as mean ± SD of triplicate values. * p < 0.05, ** p < 0.01, versus basal condition. °° p < 0.01, °°° p < 0.001 versus control SF.