Metabolomics Analysis Reveals That Anoectochilus roxburghii Extract Enhances Immunity in Immunosuppressed Rats by Modulating Lipid Metabolism and Amino Acid Metabolism

Anoectochilus roxburghii (AR) has been reported to exhibit immunomodulatory activity. However, the underlying mechanism has not yet been fully elucidated. The present study employed a gas chromatography-mass spectrometry-based metabolomics approach, combined with histopathological and biochemical analyses, to investigate the metabolomic changes and therapeutic effects of AR in cyclophosphamide-induced immunosuppressed rats. The rats were divided into four groups: control group, model group, positive group, and AR-treated groups. The immunosuppressed model was established by intraperitoneal injection of cyclophosphamide (CTX, 150 mg/kg/day). The model group showed significant reductions in spleen and thymus indices, along with elevated levels of tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2) but decreased interleukin-10 (IL-10). AR treatment restored spleen and thymus indices and normalized TNF-α, IL-2, and IL-10 levels compared to the model group. Pattern recognition was employed to identify potential biomarkers. Pattern recognition analysis identified potential biomarkers: 26, 25, and 28 differential endogenous metabolites in the model, positive control, and AR-treated groups, respectively. Among these, 15 endogenous metabolites were validated using standard compounds, which demonstrated excellent linearity (R2 > 0.9991) and precision (intraday RSD < 4.82%, interday RSD < 4.39%). Recovery rates for the standards ranged from 95.40% to 104.91%, with RSD values below 5.97%. Pearson correlation analysis revealed that lactic acid, threonine, 5-oxoproline, palmitic acid, and arachidonic acid are positively correlated with spleen and thymus indices, whereas valine, urea, octaethylene glycol, and 2-palmitoyl glycerol are negatively correlated with these indices. Collectively, these findings demonstrate that AR extract enhances immunity in immunosuppressed rats by modulating lipid and amino acid metabolism.