Abstract
BACKGROUND: Acupuncture effectively reduces chronic neck pain and plasma Substance P (SP) levels, but upstream molecular mechanisms remain unknown. OBJECTIVES: We aimed to identify circulating microRNAs (miRNAs) associated with acupuncture-induced SP reduction and explore potential neuroplasticity mechanisms. METHODS: We performed longitudinal plasma miRNA profiling (Affymetrix miRNA 4.0 Array, ~4600 miRNAs) in chronic neck pain patients: Acupuncture group (n = 3; 0, 4, 8 weeks) and Control group (n = 3; 0, 4 weeks). Linear Mixed-Effects Models (LMMs) tested associations between each miRNA and SP dynamics (miRNA × Time × Group interaction). Statistical significance was validated using permutation testing (2000 iterations). RESULTS: Screening identified 53 miRNAs significantly associated with SP, validated by permutation testing (p < 0.001). Fourteen high-confidence miRNAs showed significant three-way interactions, indicating treatment-specific SP relationships. The most significant was miR-1302-6 (p = 7.65 × 10(-6)), followed by miR-181b-2. These miRNAs displayed diverse temporal patterns: some (miR-196b, miR-6788) increased during treatment, while others (let-7d, miR-1302-6) decreased parallel to SP. Functional enrichment revealed striking convergence on neuroplasticity pathways: axon guidance (p = 2.61 × 10(-6)), MAPK signaling (p = 4.18 × 10(-5)), neuron projection development (p = 7.52 × 10(-10)), and synaptic structures (p = 9.52 × 10(-12)). CONCLUSIONS: This exploratory study provides first molecular evidence for an acupuncture-miRNA-SP axis in chronic pain. The enrichment of neuroplasticity pathways suggests acupuncture may induce structural remodeling of nociceptive circuits rather than simply suppressing inflammation, offering novel mechanistic insights and potential biomarkers for personalized acupuncture therapy. The trial was registered with the Korean Clinical Trial Registry (KCT0005363).