Figure 4. Wogonin (WG) modulated the cellular redox status of human OA chondrocytes.

(A, B) Wogonin elevated basal ROS levels in OA chondrocytes. Human OA chondrocytes were stained with H₂DCF-DA (20 μM) for 0.5 h at 37°C, and then (A) treated with Wogonin (10–50 μM) for 1 h or (B) treated with Wogonin (10–50 μM) for indicated time points at 37°C. ROS generation was estimated by measuring fluorescence emission at 535 nm. Bar graph shows arbitrary fluorescence units indicating ROS levels. (C–E) Wogonin induced production of H₂O₂ and O₂⁻ in OA chondrocytes. (C) OA chondrocytes were incubated with catalase (1000 unit/ml) or SOD (200 unit/ml) for 1 h, stained with H₂DCF-DA (20 μM) for 0.5 h at 37°C and then treated with Wogonin (50 μM) for 1 h at 37°C. Fluorescence emission was measured at 535 nm. Bar graph shows arbitrary fluorescence units indicating ROS levels. (D) The generation of H₂O₂ in the supernatant of OA chondrocytes treated with Wogonin (25, 50 μM, 1 h) was estimated using Amplex red assay as described in method section. (E) The production of O₂⁻ in OA chondrocytes stained with MistoSOX red (5 μM) and then treated with Wogonin (25, 50 μM, 1 h) was estimated by measuring the fluorescence emission at 580 nm following the excitation at 510 nm. (F) Wogonin depleted cellular GSH levels in OA chondrocytes. OA chondrocytes were treated with Wogonin (10–50μM, 1h) and then stained with mBCl (40μM, 0.5 h) and GSH levels were estimated by measuring fluorescence emission at 490 nm, following the excitation at 394 nm. Bar graph represents mean±SD from two subjects. *p≤0.01, as compared to control, #≤0.01, as compared to Wogonin treated cells.