To investigate the effects of modified Shenling Baizhu powder on succinate-induced mitochondrial oxidative stress injury in osteoarthritis (OA) chondrocytes and its potential downstream mechanisms.

The effects of different concentrations of diethyl succinate (DS) and medicated serum containing modified Shenling Baizhu powder on chondrocyte viability were assessed using the cell counting kit-8 (CCK-8) assay, followed by Dunnett’s test. Subsequently, an OA-like chondrocyte model was established by lipopolysaccharide (LPS) stimulation. The cells were divided into the control group, LPS group, DS group, and DS+JWSLBZS group (DS plus 5% medicated serum containing modified Shenling Baizhu powder). Extracellular matrix changes were evaluated by toluidine blue and alcian blue staining, and the expression levels of type II collagen (COL2) and matrix metalloproteinase (MMP)13 were detected by immunofluorescence. Oxidative stress was assessed by measuring reactive oxygen species (ROS) levels and superoxide dismutase (SOD) activity, with all pairwise comparisons among groups performed using Tukey’s multiple-comparison test. Mitochondrial ultrastructure was examined by transmission electron microscopy. Transcriptome sequencing was performed to identify differentially expressed genes and potential target genes of modified Shenling Baizhu powder, followed by gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), and gene set enrichment analysis (GSEA).

The CCK-8 assay showed that DS inhibited chondrocyte viability in a dose-dependent manner, with a half-maximal inhibitory concentration (IC₅₀) of 15.5 mmol/L. Cell viability decreased when the concentration of medicated serum reached 10% (q=3.47, P<0.05). Therefore, 10 mmol/L DS and 5% medicated serum were selected for subsequent experiments. Compared with the control group, the LPS and DS groups showed weaker extracellular matrix staining, decreased COL2 expression, and increased MMP13 expression; these abnormalities were improved in the DS+JWSLBZS group compared with the DS group. Compared with the control group, the LPS and DS groups showed increased ROS levels and decreased SOD activity, accompanied by mitochondrial swelling, reduced cristae, and other structural abnormalities. Tukey’s multiple-comparison test for SOD activity showed significant differences between the LPS and CON groups (q=28.88, P<0.001) and between the DS and control groups (q=18.71, P<0.001). Following intervention with medicated serum, these indices showed an improving trend, and mitochondrial structural damage was alleviated. Transcriptomic analysis identified 138 differentially expressed genes shared by the LPS and DS groups, which were enriched in pathways including nuclear factor-κB (NF-κB) signaling. Further directional screening showed that the “abnormally upregulated–intervention downregulated” gene set was mainly enriched in mitogen-activated protein kinase (MAPK) signaling and transient receptor potential (TRP) channel-related signaling axes, whereas the “abnormally down regulated–intervention up regulated” gene set was mainly enriched in matrix homeostasis-related pathways, including transforming growth factor-β (TGF-β) signaling. The GSEA results were consistent with these enrichment patterns.

Succinate accumulation may induce a metabolically imbalanced phenotype in chondrocytes characterized by reduced matrix synthesis and enhanced matrix degradation, accompanied by increased oxidative stress and mitochondrial ultrastructural damage. Modified Shenling Baizhu powder may alleviate succinate accumulation-induced mitochondrial oxidative stress injury by regulating MAPK signaling and related networks.