康复学报 (Apr 2025)
Mechanism of Duhuo Jisheng Decoction Inhibiting Chondrocyte Ferroptosis Based on GSH/GPX4/ROS Signaling Pathway
Abstract
ObjectiveTo explore the mechanism of Duhuo Jisheng decoction inhibiting erastin-induced chondrocyte ferroptosis through the glutathione/glutathione peroxidase 4/reactive oxygen species (GSH/GPX4/ROS) signaling pathway.MethodsA total of 30 4-week-old SPF-grade male SD rats were selected. Primary chondrocytes were isolated by mechanical type Ⅱ collagenase digestion method and cultured in vitro. Chondrocytes were randomly divided into control group, model group, Duhuo Jisheng Decoction group, and Fer-1 group. The control group was cultured with normal medium; the model group was cultured with medium containing 1 μmol/L erastin; the Duhuo Jisheng Decoction group was cultured with medium containing 1 μmol/L erastin and 300 μg/mL Duhuo Jisheng Decoction; the Fer-1 group was cultured in medium containing 1 μmol/L erastin and 1 μmol/L Ferrostatin-1. All groups were treated for 24 hours. After intervention, transmission electron microscope was used to observe the ultrastructure of the chondrocytes; colorimetric method was used to detect the content of malondialdehyde (MDA); micromethod was used to detect the content of iron ion (Fe2+); microplate assay was used to detect the content of glutathione (GSH); Western blot was used to detect the protein expression levels of glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11) and long-chain acyl-coenzyme A (CoA) synthase 4 (ACSL4); fluorescence microscopy was used to detect the expression level of GPX4 protein; fluorescence probe was used to detect the levels of intracellular ROS and lipid peroxidation.Results(1) Ultrastructure of chondrocytes: compared with the control group, the mitochondrial cristae in the model group decreased or even disappeared, and the outer membranes of mitochondria were broken. Compared with the model group, the mitochondrial membranes of the Duhuo Jisheng Decoction group were relatively complete, the mitochondria became narrow, and the number of cristae increased. (2) Contents of MDA, Fe2+ and GSH: compared with the control group, the contents of MDA and Fe2+ in the model group increased significantly, while the content of GSH decreased significantly (P<0.05). Compared with the model group, the contents of MDA and Fe2+ in the Duhuo Jisheng Decoction group and the Fer-1 group decreased significantly, while the content of GSH increased significantly (P<0.05). (3) Expression level of ferroptosis related proteins in chondrocytes: compared with the control group, the protein expression levels of GPX4 and SLC7A11 in the model group decreased significantly, the protein expression level of ACSL4 increased significantly, and the fluorescence expression level of GPX4 decreased significantly (P<0.05); compared with the model group, the protein expression levels of GPX4 and SLC7A11 in the Duhuo Jisheng Decoction group increased significantly (P<0.05), there was no significant difference in the protein expression levels of GPX4 and SLC7A11 in the Fer-1 group (P>0.05), the protein expression level of ACSL4 decreased significantly in the Duhuo Jisheng Decoction group and the Fer-1 group, and the fluorescence expression level of GPX4 increased significantly, and the differences were statistically significant (P<0.05). (4) Levels of intracellular ROS and lipid peroxidation: compared with the control group, the levels of intracellular ROS and lipid peroxidation in the model group increased significantly (P<0.05); compared with the model group, the levels of intracellular ROS and lipid peroxidation in the Duhuo Jisheng Decoction group and the Fer-1 group decreased significantly (P<0.05).ConclusionDuhuo Jisheng Decoction can inhibit the erastininduced ferroptosis in chondrocytes, and the mechanism may be related to the regulation of GSH/GPX4/ROS pathway.
