Nuclear Factor-κB Decoy Oligodeoxynucleotide Attenuates Cartilage Resorption In Vitro

Abstract

Background: Cartilage harvest and transplantation is a common surgery using costal, auricular, and septal cartilage for craniofacial reconstruction. However, absorption and warping of the cartilage grafts can occur due to inflammatory factors associated with wound healing. Transcription factor nuclear factor-κB (NF-κB) is activated by the various stimulation such as interleukin-1 (IL-1), and plays a central role in the transactivation of this inflammatory cytokine gene. Inhibition of NF-κB may have anti-inflammatory effects. The aim of this study was to explore the potential of an NF-κB decoy oligodeoxynucleotide (Decoy) as a chondroprotective agent.

Materials and methods: Safe and efficacious concentrations of Decoy were assessed using rabbit nasal septal chondrocytes (rNSChs) and assays for cytotoxicity, proteoglycan (PG) synthesis, and PG turnover were carried out. The efficacious concentration of Decoy determined from the rNSChs was then applied to human nasal septal cartilage (hNSC) in vitro and analyzed for PG turnover, the levels of inflammatory markers, and catabolic enzymes in explant-conditioned culture medium.

Results: Over the range of Decoy conditions and concentrations, no inhibition of PG synthesis or cytotoxicity was observed. Decoy at 10 μM effectively inhibited PG degradation in the hNSC explant, prolonging PG half-life by 63% and decreasing matrix metalloprotease 3 (MMP-3) by 70.7% (p = 0.027).

Conclusions: Decoy may be considered a novel chondroprotective therapeutic agent in cartilage transplantation due to its ability to inhibit cartilage degradation due to inflammation cytokines.

Keywords: cartilage; decoy oligodeoxynucleotide; nuclear factor-κb; resorption.

Figure 1

Mechanism of Decoy: (a) In the classical pathway, NF-κB is normally present in the cytoplasm in an inactivated state by binding inhibitor proteins including IκB. NF-κB is normally present as an inactive, Igκ-bound complex in cytoplasm. Multi-subunit IκB kinase (IKK) is activated by stimuli such as IL-1 and TNF, and IκB is inducibly degraded by activated IKK. Free NF-κB rapidly enters the nucleus and transactivates target genes such as IL-1, TNF, iNOs, MMPs, COX-2, and IL-6. (b) Under the presence of Decoy, transactivation will not begin because the activated NF-κB is bound to Decoy. Abbreviations: iNOs (inducible nitric oxide synthase), MMPs (matrix metalloproteinases), COX-2 (cyclooxygenase-2).

Figure 2

Cytotoxicity (LDH) assay of rNSChs. Cytotoxicity of Decoy was not demonstrated in the LDH assay (p = 0.44). The plotted values are indicated as mean ± standard error of the mean. (n = 3 biological replicates). N.S., not significant.

Figure 3

PG synthesis assay of rNSCh. IL-1 treatment significantly decreased PG synthesis (p < 0.0001). The 10 μM Decoy group did not inhibit PG synthesis. Decoy did not inhibit the attenuation of PG synthesis by IL-1. Data are expressed as the mean ± standard error of the mean (n = 9, 3 batches in triplicate). One-way ANOVA with Games–Howell as a post hoc test was used. N.S.; not significant.

Figure 4

PG turnover assay of rNSCh. Dots were individual data. Compared with controls, the 10 μM decoy group showed significant inhibition of PG degradation (p < 0.0001). Although PG retention in the IL-1 group was lower than in the control group, there was no significant difference. In the presence of IL-1, Decoy at 1 μM did not inhibit PG degradation, while 10 µM Decoy inhibited degradation of PG significantly (p < 0.0001). The line in the graph shows the average for each group. Data are expressed as the mean ± standard error of the mean (n = 9, 3 batches in triplicate). Two-way repeated ANOVA with Games–Howell as a post hoc test was used.

Figure 5

Efficiency of transfection of Decoy. Live–dead assay was separately performed using human nasal septal cartilage tissues. (a) Green indicates FITC-labeled Decoy 6 h after the transfection of FITC decoy. (b,c) The separate tissues were subjected to live–dead assay. Green indicates viable cells. Red indicates dead cells. Most of cells were viable in the tissues. From these images, it was inferred that Decoy was incorporated into viable cells.

Figure 6

PG turnover assay of tissue culture. Dots were individual data. Decoy significantly inhibited degradation of PG with or without IL-1 (p < 0.01, p < 0.01). There was no significant difference between the control group and the IL-1 group. Data are expressed as the mean ± standard error of the mean (n = 4 patients, in triplicate). Three-way repeated ANOVA with Games–Howell as a post hoc test was used.

Figure 7

ELISA MMP-3. Dots were individual data. MMP-3 was decreased by more than 70% with administration of Decoy compared with the controls (p < 0.05). IL-1 treatment increased MMP-3 to 301.3% of controls (p < 0.001). In the presence of IL-1, MMP-3 was also significantly reduced by 51.1% with Decoy administration (p < 0.001). Data are expressed as the mean ± standard error of the mean (n = 4 patients, in triplicate). Two-way ANOVA with Games–Howell as a post hoc test was used.

Figure 8

ELISA TNF, IL-6, and nitrate assay. Dots were individual data. (a) ELISA TNF. Decoy did not inhibit TNF. Data are expressed as the mean ± standard error of the mean (n = 4 patients, in triplicate). Two-way ANOVA with Games–Howell as a post hoc test was used. (b) ELISA IL-6. Decoy did not inhibit IL-6. Data are expressed as the mean ± standard error of the mean (n = 4 patients, in triplicate). Two-way ANOVA with Games–Howell as a post hoc test was used. (c) Nitrate assay. Decoy did not inhibit nitrate. Data are expressed as the mean ± standard error of the mean (n = 4 patients, in triplicate). Two-way ANOVA with Fisher’s LSD as a post hoc test was used. N.S.; not significant.