Sanmiao San Attenuates D-galactose-induced Chondrocyte Senescence by Regulating the FoxM1-p21 Signaling Pathway.

Abstract

ETHNOPHARMACOLOGICAL RELEVANCE: Sanmiao San (SMS), a traditional Chinese medicine formula, is commonly used to treat Bi syndrome and has been widely applied in the clinical management of knee osteoarthritis (KOA) in China. However, its protective effects against cartilage degeneration and chondrocyte senescence, as well as the underlying molecular mechanisms, remain to be fully elucidated. AIM OF THE STUDY: To investigate the inhibitory effects of SMS on D-galactose (D-gal)-induced chondrocyte senescence and to elucidate the underlying molecular mechanisms and primary active components of SMS. MATERIALS AND METHODS: D-gal-induced senescence models were established in a human chondrocyte cell line and rat primary chondrocytes. Cellular senescence was assessed by senescence-associated β-galactosidase (SA-β-gal) staining. Colony formation assays were performed to evaluate the clonogenic capacity of chondrocytes. Proteoglycan secretion was evaluated by toluidine blue staining. γ-H2A.X expression was assessed by immunofluorescence. Cell cycle arrest was analyzed by flow cytometry. Transcriptomic analysis was performed to identify differentially expressed genes. These genes were annotated using the CellAge database to screen for senescence-related genes and senescence-associated secretory phenotype (SASP) factors. Key SASP factors were further validated by enzyme-linked immunosorbent assay (ELISA). Intracellular reactive oxygen species (ROS) levels were measured by fluorescence assay. The expression of Forkhead box M1 (FoxM1) and p21 was analyzed by quantitative real-time PCR (qRT-PCR), Western blotting and immunofluorescence. The prototype constituents and metabolites of SMS in rat serum were identified by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). RESULTS: SMS-containing serum (SMS-CS) attenuated D-gal-induced chondrocyte senescence by reducing the proportion of SA-β-gal-positive cells, downregulating p21 and γ-H2A.X expression, alleviating cell cycle arrest and promoting colony formation. Transcriptomic analysis revealed that SMS-CS upregulated most genes annotated as downregulated during senescence and downregulated a subset of those annotated as upregulated during senescence in the CellAge database. SMS-CS also modulated most SASP factors, particularly interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), as further confirmed by ELISA. KEGG and GO enrichment analysis of specific drug targets revealed significant enrichment in cell cycle, mitotic processes and cellular senescence, while comprehensive bioinformatics analysis predicted FoxM1 as a core target. FoxM1 inhibitor intervention showed that SMS-CS significantly counteracted the FoxM1 downregulation-induced increases in SA-β-gal-positive cell percentage, p21 mRNA levels, γ-H2A.X expression, ROS levels and SASP secretion, as well as the FoxM1 downregulation-induced decrease in proteoglycan secretion, thereby attenuating chondrocyte senescence progression. UPLC-MS analysis identified the following major prototype components and metabolites of SMS in rat serum: Ferulic acid, Esculetin, Atractyloside A, Inokosterone, 20,26-Dihydroxyecdysone, trans-Ferulic acid + hydroxy, Obacunone + hydroxy, and Atractylenolide III + hydroxy. CONCLUSIONS: These findings suggest that the FoxM1-p21 axis may be involved in the anti-senescence effects of SMS-CS in chondrocytes and could represent a potential therapeutic target for KOA.

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