Objective To investigate the therapeutic effects and potential mechanisms of tetramethylpyrazine (TMP) in a high humidity osteoarthritis (HHOA) rat model. Methods The HHOA rat model was established, and rats were given TMP for eight weeks. Safranin O-fast green staining was used to observe morphological changes in cartilage tissue. The levels of matrix metalloproteinase-9 (MMP-9), tissue inhibitors of metalloproteinase-1 (TIMP-1), prostaglandin-endoperoxide synthase 2 (PTGS2), glutathione peroxidase 4 (GPX4), glutathione (GSH), malondialdehyde (MDA) and superoxide dismutase (SOD) in serum were measured. Prussian blue staining was performed to detect iron deposition in cartilage. Immunohistochemistry was used to assess the expressions of GPX4 and acyl-CoA synthetase long-chain family member 4 (ACSL4) in cartilage. Serum untargeted metabolomics was performed to analyze changes in metabolites and their enriched pathways. In vitro, a ferroptosis model of chondrocytes was established and treated with main metabolite N-acetylserotonin. GSH/oxidized glutathione (GSSG) value, MDA and SOD levels were measured, the expressions of GPX4 and ACSL4 were measured by Western blotting. Results Compared with HHOA group, levels of MMP-9, PTGS2 and MDA in serum of rats in TMP group were significantly reduced (P < 0.05, 0.01), while the levels of TIMP-1, GPX4, SOD and GSH in serum were significantly increased (P < 0.05, 0.01). The pathological damage to the articular cartilage was significantly improved, and the deposition of iron ions in articular cartilage was reduced, GPX4 protein expression was increased, and ACSL4 protein expression was reduced. Metabolomics analysis showed that 2 695 differential metabolites were screened between HHOA group and sham group, with significantly more down-regulated metabolites than up-regulated metabolites; A total of 2 846 differential metabolites were screened between TMP high-dose group and HHOA group, with up-regulated metabolites being the main ones. KEGG pathway enrichment analysis results showed that the differential metabolites between HHOA group and sham group were mainly enriched in multiple metabolic pathways such as carbon metabolism and amino acid metabolism, the differential metabolites between TMP group and HHOA group were mainly enriched in multiple pathways closely related to amino acid metabolism and substance transport. Compared with sham group, the levels of γ-glutamylleucine, 3-indoolepropionic acid and N-acetylseletonin in serum of HHOA group rats were significantly reduced (P < 0.05). Compared with HHOA group, the levels of γ-glutamylleucine, 3-indoolepropionic acid and N-acetylseletonin in TMP high-dose group were significantly increased (P < 0.05). The in vitro experimental results showed that the main metabolite N-acetylserotonin significantly increased the GSH/GSSG value and SOD activity in ferroptosis model of chondrocytes (P < 0.05), decreased MDA level (P < 0.05), up-regulated GPX4 protein and down-regulated ACSL4 protein expressions (P < 0.05, 0.01). Conclusion TMP could improve the metabolomic profile of HHOA rats, regulate the tryptophan metabolism pathway represented by N-acetylserotonin, alleviate GPX4/ASCL4 axis mediated lipid peroxidation and iron ion deposition, thereby reducing ferroptosis in chondrocytes and delaying the progression of HHOA.

osteoarthritis  /  high humidity environment  /  tetramethylpyrazine  /  non-targeted metabolomics  /  ferroptosis  /  GPX4/ACSL4 axis  /  N-acetylserotonin