MicroRNA as Possible Mediators of the Synergistic Effect of Celecoxib and Glucosamine Sulfate in Human Osteoarthritic Chondrocyte Exposed to IL-1β

Abstract

This study investigated the role of a pattern of microRNA (miRNA) as possible mediators of celecoxib and prescription-grade glucosamine sulfate (GS) effects in human osteoarthritis (OA) chondrocytes. Chondrocytes were treated with celecoxib (1.85 µM) and GS (9 µM), alone or in combination, for 24 h, with or without interleukin (IL)-1β (10 ng/mL). Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, apoptosis and reactive oxygen species (ROS) by cytometry, nitric oxide (NO) by Griess method. Gene levels of miRNA, antioxidant enzymes, nuclear factor erythroid (NRF)2, and B-cell lymphoma (BCL)2 expressions were analyzed by quantitative real time polymerase chain reaction (real time PCR). Protein expression of NRF2 and BCL2 was also detected at immunofluorescence and western blot. Celecoxib and GS, alone or in combination, significantly increased viability, reduced apoptosis, ROS and NO production and the gene expression of miR-34a, -146a, -181a, -210, in comparison to baseline and to IL-1β. The transfection with miRNA specific inhibitors significantly counteracted the IL-1β activity and potentiated the properties of celecoxib and GS on viability, apoptosis and oxidant system, through nuclear factor (NF)-κB regulation. The observed effects were enhanced when the drugs were tested in combination. Our data confirmed the synergistic anti-inflammatory and chondroprotective properties of celecoxib and GS, suggesting microRNA as possible mediators.

Keywords: NF-κB; celecoxib; chondrocytes; chondroprotection; glucosamine sulfate; inflammation; microRNA; osteoarthritis.

Figure 1

Chondrocytes were evaluated at control (CTRL) condition, after 24 h of treatment with celecoxib (cel) (1.85 µM) and glucosamine sulfate (GS) (9 µM), in presence or not of interleukin (IL)-1β (10 ng/mL). (a,c,e) Apoptosis detection and reactive oxygen species (ROS) production by flow cytometry. (b,d) Expression levels of B-cell lymphoma 2 (BCL2) and nuclear factor erythroid 2 (NRF2) by quantitative real time PCR. (f) Nitric oxide (NO) release by Griess method. (g) Viability by MTT assay. All the results, except for NO and viability, were expressed as fold change of the value of interest respect to CTRL, reported equal to 1. Data were represented as mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001 versus CTRL. ° p < 0.05, °° p < 0.01, °°° p < 0.001 versus IL-1β. # p < 0.05, ## p < 0.01 versus Cel or GS + IL-1β.

Figure 2

Chondrocytes were evaluated at control (CTRL) condition, after 24 h of treatment with celecoxib (cel) (1.85 µM) and glucosamine sulfate (GS) (9 µM), in presence or not of interleukin (IL)-1β (10 ng/mL). (a–h) Indirect immunofluorescence microscopy of cells incubated with monoclonal anti- B-cell lymphoma 2 (BCL2) (a–d) and anti-nuclear factor erythroid 2 (NRF2) (e–h) primary antibodies. (a,e) CTRL: a fair fluorescence in the cytoplasm is shown; (b,f) IL-1β: an intense signal is evident in the cytoplasm; (c,g) Cel + GS; the label is almost absent. (d,h) Cel + GS + IL-1β: the signal is diffused in the cytoplasm but reduced with respect to IL-1β. Nuclei (blue) were stained with DAPI. Bars: (a–h) 50 µm.

Figure 3

Chondrocytes were evaluated at control (CTRL) condition, after 24 h of treatment with celecoxib (cel) (1.85 µM) and glucosamine sulfate (GS) (9 µM), in presence or not of interleukin (IL)-1β (10 ng/mL). (a–d) Representative immunoblotting image and densitometric analysis of B-cell lymphoma 2 (BCL2) and nuclear factor erythroid 2 (NRF2) protein levels by western blot. The results were expressed as fold change of the value of interest respect to CTRL, reported equal to 1. Data were represented as mean ± standard deviation. * p < 0.05 versus basal CTRL. ° p < 0.05, °° p < 0.01 versus IL-1β.

Figure 4

Chondrocytes were evaluated at control (CTRL) condition, after 24 h of treatment with celecoxib (cel) (1.85 µM) and glucosamine sulfate (GS) (9 µM), in presence or not of interleukin (IL)-1β (10 ng/mL). (a–h) Expression levels of microRNA by quantitative real time PCR. The results were expressed as fold change of the value of interest respect to CTRL, reported equal to 1. Data were represented as mean ± standard deviation. * p < 0.05, ** p < 0.01 versus basal CTRL. ° p < 0.05, °° p < 0.01, °°° p < 0.001 versus IL-1β. # p < 0.05 versus Cel or GS + IL-1β.

Figure 5

Chondrocytes were evaluated at control (CTRL) condition, after 24 h of transient transfection with miR-34a, miR-146a, miR-181a, and miR-210 inhibitors (50 nM) or NC (5 nM), 24 h of incubation with celecoxib (cel) (1.85 µM) and glucosamine sulfate (GS) (9 µM), in presence of interleukin (IL)-1β (10 ng/mL). (a–e) Expression levels of microRNA by quantitative real time PCR. The results were expressed as fold change of the value of interest respect to CTRL, reported equal to 1. Data were represented as mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001 versus CTRL or NC. ° p < 0.05, °° p < 0.01 versus IL-1β. # p < 0.05, ## p < 0.01 versus cel + IL, GS + IL and cel + GS + IL. INIB = Inhibitor, NC = negative control siRNA.

Figure 6

Chondrocytes were evaluated at control (CTRL) condition, after 24 h of transient transfection with miR-34a, miR-146a, miR-181a, and miR-210 inhibitors (50 nM) or NC (5 nM), 24 h of incubation with celecoxib (cel) (1.85 µM) and glucosamine sulfate (GS) (9 µM), in presence of interleukin (IL)-1β (10 ng/mL). (a–d) Apoptosis detection by flow cytometry. The results were expressed as fold change of the value of interest respect to CTRL, reported equal to 1. (e–h) Viability by MTT assay. Data were represented as mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001 versus CTRL or NC. ° p < 0.05, °° p < 0.01 versus IL-1β. # p < 0.05, ## p < 0.01 versus cel + IL, GS + IL and cel + GS + IL. INIB = Inhibitor, NC = negative control siRNA.

Figure 7

Chondrocytes were evaluated at control (CTRL) condition, after 24 h of transient transfection with miR-34a, miR-146a, miR-181a, and miR-210 inhibitors (50 nM) or NC (5 nM), 24 h of incubation with celecoxib (cel) (1.85 µM) and glucosamine sulfate (GS) (9 µM), in presence of interleukin (IL)-1β (10 ng/mL). (a–d) Mitochondrial superoxide anion and (e–h) extracellular ROS production by flow cytometry. The results were expressed as fold change of the value of interest respect to CTRL, reported equal to 1. Data were represented as mean ± standard deviation. ** p < 0.01, *** p < 0.001 versus CTRL or NC. ° p < 0.05, °° p < 0.01 versus IL-1β. # p < 0.05, ## p < 0.01 versus cel + IL, GS + IL and cel + GS + IL. INIB = Inhibitor, NC = negative control siRNA.

Figure 8

Chondrocytes were evaluated at control (CTRL) condition, after 24 h of transient transfection with miR-34a, miR-146a, miR-181a, and miR-210 inhibitors (50 nM) or NC (5 nM), 24 h of incubation with celecoxib (cel) (1.85 µM) and glucosamine sulfate (GS) (9 µM), in presence of interleukin (IL)-1β (10 ng/mL). (a–d) Nitric oxide (NO) release by Griess method. Data were represented as mean ± standard deviation. * p < 0.05, ** p < 0.01 versus CTRL or NC. ° p < 0.05, °° p < 0.01 versus IL-1β. # p < 0.05 versus cel + IL, GS + IL and cel + GS + IL. INIB = Inhibitor, NC = negative control siRNA.

Figure 9

Chondrocytes were evaluated at control (CTRL) condition, after 24 h of incubation with a specific nuclear factor (NF)-κB inhibitor (BAY 11-7082, IKKα/β, 1 μM), 24 h of treatment with celecoxib (cel) (1.85 µM) and glucosamine sulfate (GS) (9 µM), in presence of interleukin (IL)-1β (10 ng/mL). (a–c) Expression levels of microRNA by quantitative real time PCR. The results were expressed as fold change of the value of interest respect to CTRL, reported equal to 1. Data were represented as mean ± standard deviation. * p < 0.05, ** p < 0.01 versus CTRL. ° p < 0.05, °° p < 0.01 versus IL-1β. # p < 0.05 versus cel or GS + IL-1β. ^$ p < 0.05 versus BAY + IL-1β; ^& p < 0.05 versus BAY + Cel or GS + IL-1β.