Background/Purpose: Epidemiological findings support the hypothesis that type 2 diabetes (DB) is an independent risk factor of osteoarthritis (OA). Likewise, we have recently reported a higher responsiveness to inflammatory stress of OA cartilage from DB patients and enhanced IL-1β-induced inflammation in cultured chondrocytes in a high glucose (HG) environment¹. To investigate whether the antioxidant defense system may participate in this dysregulation, we investigated the involvement of nuclear factor-erythroid 2-related factor (Nrf-2), the master transcriptional regulator of antioxidant responses, and heme oxygenase-1 (HO-1), one of its main target gene, in OA cartilage from DB and non-DB patients, and in normal murine chondrocytes subjected to HG exposure.

Methods: Human cartilage was obtained at the time of knee replacement of DB and non-DB OA patients (with similar age and body mass index). Cartilage explants were incubated for 24h and Nrf-2 and HO-1 tissue levels were assayed by western blot (WB). Articular chondrocytes in primary culture isolated from newborn C57Bl6 mice were stimulated at 72h w/o IL-1β (5 ng/mL) under a normal (5.5 mM; NG) or a high (25 mM; HG) glucose environment. Nuclear translocation of Nrf-2 (30 min) was analyzed by WB. Gene and protein expression of Nrf-2, HO-1 and IL-6 were assessed by RT-qPCR and WB, respectively. ROS production was measured using DCFDA. To determine the mechanistic pathways involved in cell activation by HG ± IL1β, a HO-1 inducer (CoPP; 10µM) and sulforaphane (SFN; 5mM), a natural Nrf-2 translocation activator, were used, and experiments were also performed in Nrf2^−/− mice generated from inbred C57BL/6J background Nrf2 heterozygous mice.

Results: Ex vivo experiments indicated that Nrf-2 and HO-1 expressions were reduced in OA cartilage from DB patients (n=7, 0.57-fold [Nrf-2] and 0.34-fold [HO-1]) compared to non-DB patients (n=8, p<0.05). HO-1 expression was positively correlated with Nrf-2 levels (p<0.05).

In vitro, IL-1β-stimulated chondrocytes exposed to HG had lower Nrf-2 nuclear levels (0.61-fold) than those incubated in NG (n=5, p<0.05). In agreement, total protein expression of Nrf-2 by IL-1ß-stimulated cells was also reduced in presence of HG versus NG (0.82-fold; n=5, p<0.05). A decreased HO-1 protein levels (0.49-fold) was also observed (n=5, p<0.05). HO-1 expression was positively correlated with Nrf-2 (r = 0.35; p<0.05) and negatively with IL-6 levels (r= -0.35; p<0.05) using PCR.

CoPP reduced ROS production (-75%) and IL-6 expression (-74%) detected in HG + IL-1ß condition (p<0.05). The decrease of HO-1 and increase of IL-6 induced by HG was reversed in IL-1ß-stimulated chondrocytes from Nrf-2^−/−mice. SFN rescued HO-1 levels and reduced ROS production (-20%) triggered by HG + IL-1ß stimulation (p<0.05).

Conclusion: Nrf2/HO-1 axis is disturbed in diabetic OA cartilage and is a critical pathway involved in the hyperglucidic-mediated dysregulation of articular chondrocytes. The impairment of this anti-oxydant system could explain the higher inflammatory responsiveness observed in cartilage from DB patients and may open new opportunities for treating patients with a diabetes-related OA phenotype.

¹Laiguillon MC et al Osteoarthritis &Cartilage 2015 in press

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/downregulation-of-nuclear-factor-erythroid-2-related-factor-nrf2heme-oxygenase-1-axis-in-diabetic-cartilage-towards-a-better-phenotyping-of-diabetes-related-osteoarthritis/