Duhuo jisheng decoction alleviates osteoarthritis progression by mitigating ferroptosis in chondrocytes via the nuclear factor erythroid 2-related factor 2/glutathione peroxidase 4 axis

Ethnopharmacological relevance: Osteoarthritis (OA) is a leading cause of disability worldwide, imposing a substantial burden on global public health and healthcare systems. Currently, there are no viable strategies to halt the progression or reverse the course of this disease. Duhuo Jisheng Decoction (DHJSD) has been extensively employed in treating OA. Although its clinical efficacy is well-established, the precise mechanisms underlying its therapeutic effects remain poorly understood.

Aim of the study: This study explored whether DHJSD attenuates OA progression by inhibiting chondrocyte Ferroptosis via the nuclear factor erythroid 2-related factor 2 (Nrf2)/GPX4 (Glutathione Peroxidase 4) axis.

Materials and methods: Chondrocyte damage was elicited to replicate an iron overload-induced osteoarthritis (IOOA) environment in vitro by utilizing interleukin-1 beta (IL-1β) and ferric ammonium citrate (FAC). An IOOA mouse model was established by intraperitoneal iron dextran injection and medial meniscus destabilization. The effects of DHJSD-medicated serum on chondrocyte viability were evaluated with Cell Counting Kit-8 (CCK-8) and toluidine blue staining. The impact on intracellular Ferroptosis markers was assessed using fluorescent staining to detect Reactive Oxygen Species (ROS), mitochondrial membrane potential, and labile ferrous iron via Calcein-AM; and biochemical assays to quantify Fe²⁺ levels, malondialdehyde (MDA), and glutathione (GSH/GSSG). Transmission electron microscopy (TEM) was also used to evaluate mitochondrial morphology. The changes in protein expression were identified using western blotting and immunofluorescence (IF). The therapeutic effects of DHJSD were further evaluated using micro-computed tomography (micro-CT), histopathological staining, and IF to examine its impact on Ferroptosis in the IOOA mouse model.

Results: In chondrocytes stimulated with IL-1β and FAC, ROS production, MDA levels, and intracellular iron accumulation were markedly increased, while cell viability, and GSH/GSSG ratio were significantly reduced. These changes were effectively attenuated by treatment with DHJSD-medicated serum. Moreover, DHJSD-medicated serum promoted the nuclear translocation of Nrf2 and upregulated the expression of Nrf2, heme oxygenase-1 (HO-1), GPX4, and cystine/glutamate transporter (xCT). TEM and JC-1 staining revealed that DHJSD-medicated serum ameliorated mitochondrial dysfunction. ML385, a selective Nrf2 inhibitor, partially reversed these protective effects. In vivo, IOOA mice exhibited increased cartilage degeneration and Ferroptosis, both alleviated by DHJSD treatment.

Conclusions: DHJSD may suppress Ferroptosis via activation of the Nrf2/GPX4 axis, thereby attenuating cartilage degeneration and slowing the progression of OA. However, as only a single batch of DHJSD was used, further studies are needed to confirm the reproducibility of these findings.

Duhuo jisheng decoction; Ferroptosis; Nrf2/GPX4 axis; Osteoarthritis.