Wogonin exerts anti-inflammatory effect by disrupting KEAP-1/NRF2 interactions and activating NRF2 in human OA chondrocytes

Abstract

Purpose: Osteoarthritis (OA), characterized by progressive destruction of articular cartilage, is the most common form of human arthritis. Wogonin is a naturally occurring flavonoid found in root extract of Scutellaria baicalensis, and it has been shown to exhibits diverse biological activities. Here, we evaluated the potential chondroprotective effects of Wogonin in IL-1β stimulated human OA chondrocytes. Methods: OA chondrocytes were prepared by the enzymatic digestion of discarded, deidentified, OA cartilage obtained commercially. In OA chondrocytes, effect of Wogonin on IL-1β-induced mRNA and protein expression of iNOS, COX-2, IL-6, MMP-3, MMP-9, MMP-13, ADAMTS4, ACAN and COL2A1 was examined by TaqMan Gene Expression assays and immunoblotting respectively. Basal and induced ROS levels were measured by using cell permeable fluorogenic probes DHR123 and H2DCF-DA. Chondrocytes viability was determined using MTT assay. H2O2 generation was determined using Amplex Red assay and mitochondrial O2− generation was determined using MitoSOX assay. Estimation of NO in culture supernatant was done using Griess assay and PGE2 production in culture supernatant was quantified by ELISA. Protein levels of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), heme-oxygenase-1 (HO-1), SOD2, NQO1, GCLC and total protein levels and phosphorylation of MAPKs were assayed by immunoblotting using validated antibodies. Nuclear translocation of Nrf2 was visualized using confocal microscopy. siRNA mediated knockdown was used to deplete the expression of target gene. Proteoglycan loss in cartilage explants was measured by Safaranin-O staining. Molecular docking studies were performed using Glide tool in Schrödinger Maestro suite. Results: Treatment with Wogonin completely suppressed the mRNA and protein expression of inflammatory mediators including IL-6, COX-2, and iNOS in IL-1β-stimulated OA chondrocytes. Wogonin significantly inhibited the production of inflammatory mediators such as NO and PGE2 in IL-1β-stimulated OA chondrocytes. Further, Wogonin inhibited the expression and activities of matrix degrading proteases MMP-13, MMP-3, MMP-9, and ADAMTS-4 in IL-1β-stimulated OA chondrocytes, and blocked the release of s-GAG and COL2A1 in IL-1β-stimulated OA cartilage explants in vitro. Furthermore, treatment with Wogonin enhanced the expression of cartilage anabolic factors COL2A1 and ACAN in chondrocytes and restored the IL-1β-mediated depletion of COL2A1 and proteoglycan content of human cartilage explants. The suppressive effect of Wogonin was not mediated through the inhibition of MAPKs and NF-κB activation. Wogonin induced mild oxidative stress through the generation of ROS and depletion of GSH, thereby modulating the cellular redox leading to the activation of Nrf2/ARE pathways in OA chondrocytes. Molecular docking studies revealed that Wogonin binds to the Kelch domain of KEAP1 protein specifically at Arg415 and Asn414 and thus may directly compete and restrict the binding of Nrf2 to KEAP1.Wogonin activated Nrf2 independent of PI3K/AKT/GSK3β signaling axis but through the phosphorylation of ERK1/2 in OA chondrocytes. Treatment with Wogonin upregulated the mRNA and protein levels of Nrf2 regulated genes HO-1, SOD2, NQO1 and GCLC known to be involved in the cellular antioxidant defense mechanism. SiRNA-mediated depletion of Nrf2 significantly abrogated the anti-inflammatory and chondroprotective effects of Wogonin in OA chondrocytes under pathological conditions. Conclusions: Our data demonstrates that Wogonin suppresses molecular events involved in oxidative stress, inflammation and matrix degradation in OA chondrocytes and cartilage explants. The study provides novel insights into the development of Nrf2 as a promising candidate for the management of OA.