[Paeoniflorin alleviates osteoarthritis by inhibiting chondrocyte ferroptosis via Nrf2/GPX4 signaling pathway]

This study aimed to investigate the effects of paeoniflorin on ferroptosis in chondrocytes in osteoarthritis and to elucidate the underlying mechanism via the nuclear factor E2-related factor 2(Nrf2)/glutathione peroxidase 4(GPX4) signaling pathway. Rat articular chondrocytes were treated with interleukin(IL)-1β, and divided into control, model, ferroptosis inhibitor Ferrostatin-1(Fer-1, 5 μmol·L~(-1)), and paeoniflorin low-, medium-, and high-dose(12.5, 25, and 50 μmol·L~(-1)) groups. Cell viability was measured by the CCK-8 assay. Levels of tumor necrosis factor-α(TNF-α), IL-6, prostaglandin E2(PGE2), and nitric oxide(NO) in cell supernatants were detected by ELISA. Western blot analysis was performed to detect the protein expression of cyclooxygenase-2(COX-2), inducible nitric oxide synthase(iNOS), collagen type Ⅱ(collagen Ⅱ), Aggrecan, matrix metalloproteinase-13(MMP-13), a disintegrin and metalloproteinase with thrombospondin motifs-5(ADAMTS-5), and proteins related to the Nrf2/GPX4 signaling pathway. Intracellular reactive oxygen species(ROS), lipid peroxidation, and ferrous ion(Fe~(2+)) levels were measured using fluorescent probes. Commercial kits were used to determine the levels of iron, malondialdehyde(MDA), and reduced glutathione(GSH), as well as the activities of superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px). In vivo, 60 SD rats were randomly divided into control, model, Fer-1, and paeoniflorin low-, medium-, and high-dose(25, 50, and 100 mg·kg~(-1)) groups. An osteoarthritis model was established by anterior cruciate ligament transection, followed by intragastric administration for 8 weeks. Subchondral bone microstructure was evaluated by micro-CT. Histological assessment was performed using hematoxylin-eosin(HE) and safranin fast green staining. Immunofluorescence staining was used to detect the expression of collagen Ⅱ, MMP-13, Nrf2, and GPX4 in cartilage tissues. Compared with the control group, the model group showed significantly decreased cell viability, increased levels of TNF-α, IL-6, PGE2, and NO, increased protein expression of COX-2, iNOS, MMP-13, and ADAMTS-5, and decreased expression of collagen Ⅱ and Aggrecan. The levels of ROS, lipid peroxidation, Fe~(2+), and MDA were increased, whereas GSH content, GSH/GSSG ratio, and SOD and GSH-Px activities were decreased. The expression of Kelch-like ECH-associated protein 1(Keap1) was increased, while the expression of Nrf2, heme oxygenase-1(HO-1), GPX4, and cystine/glutamate antiporter system Xc-(xCT) was decreased. In vivo, the model group exhibited aggravated subchondral bone microstructural damage and histopathological injury, with decreased expression of collagen Ⅱ, Nrf2, and GPX4 and increased expression of MMP-13 in cartilage tissue. Compared with the model group, Fer-1 and paeoniflorin increased chondrocyte viability, significantly reduced TNF-α, IL-6, PGE2, and NO levels and COX-2 and iNOS expression, upregulated collagen Ⅱ and Aggrecan expression, and downregulated MMP-13 and ADAMTS-5 expression. In addition, both treatments reduced intracellular ROS, lipid peroxidation, Fe~(2+), and MDA levels, increased GSH content, GSH/GSSG ratio, and SOD and GSH-Px activities, downregulated Keap1 expression, and upregulated Nrf2, HO-1, GPX4, and xCT expression. Furthermore, Fer-1 and paeoniflorin markedly improved subchondral bone microstructure and histopathological damage in osteoarthritis rats, upregulated collagen Ⅱ, Nrf2, and GPX4 expression, and downregulated MMP-13 expression in cartilage tissue. Paeoniflorin can delay the progression of osteoarthritis, and its protective effect is attributed to the inhibition of ferroptosis in chondrocytes, which may be mediated by the Nrf2/GPX4 signaling pathway.