Supplementary Material for: Urinary Metabolomics Study on Acupuncture plus Wet Cupping Therapy for Post Stroke Depression: A pilot study

Objective: This study aimed to investigate urinary metabolomics alterations associated with acupuncture plus wet cupping (AC) therapy in patients with post-stroke depression (PSD).
Methods: A total of 40 subjects were randomly allocated to either the acupuncture plus wet cupping group (AC group, n=20) or the acupuncture group (Acu group, n=20). Morning urine samples were collected at baseline and after 4 weeks of treatment, yielding a total of 80 samples. An additional 20 healthy volunteers served as a blank control group, from whom urine samples were also obtained. Urinary metabolites were analyzed using gas chromatography-mass spectrometry (GC-MS). Differential metabolites and related metabolic pathways were identified. Clinical efficacy was assessed using the Hamilton Depression Rating Scale-24 items (HAMD-24) at baseline and post-treatment. Correlation analysis was performed to assess the relationship between metabolic changes and clinical improvement.
Results: After 4 weeks of treatment, the HAMD-24 scores in the AC group (median: 10.00, IQR: 9.00-13.00) were significantly lower than those in the Acu group (median: 18.00, IQR: 15.25-19.00), with a statistically significant between-group difference (P<0.01). Metabolomic profiling revealed significant differences between healthy volunteers and untreated PSD patients, with 21 differential metabolites identified. Caffeine metabolism emerged as the most significantly altered pathway (FDR-P<0.05). No significant metabolic changes were detected in the Acu group after treatment. In contrast, the AC group demonstrated marked alterations, with 33 differential metabolites identified and starch and sucrose metabolism emerging as the most affected pathway (FDR-P<0.05). Furthermore, direct comparisons between the AC and Acu groups post-treatment identified 13 differential metabolites, with tyrosine metabolism as the most significantly impacted pathway (FDR-P<0.05). Correlation analysis showed that the differential metabolites in the AC group were significantly correlated with HAMD-24 score changes (P<0.05).
Conclusions: Caffeine metabolism was associated with the metabolic distinction between healthy volunteers and PSD patients. AC therapy was associated with clinical improvement in PSD and alterations in starch and sucrose metabolism as well as tyrosine metabolism. Tyrosine metabolism may be the key metabolic pathway distinguishing AC therapy from acupuncture alone. These findings suggested that AC therapy may exert its effects through associations with alterations in energy metabolism and neurotransmitter synthesis, which was linked to the core pathophysiological mechanisms of PSD, though causal relationships require further investigation.