Acupuncture-Associated Changes in Migraine-Like Behaviors and Serum Proteomic and Metabolomic Profiles in Nitroglycerin-Induced Rats

Shijie Wei,1,* Huafeng Cui,2,* Zhe Wang,1 Guoliang Jiang,1 Xiaowei Hu,1 Qianxiu Chen,1 Yihang Xu,1 Jing Han21School of Acupuncture and Tuina, Shandong University of Traditional Chinese Medicine, Jinan, Shandong, People’s Republic of China; 2Department of Acupuncture, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, Shandong, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jing Han, Department of Acupuncture, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, No. 16369 Jingshi Road, Jinan, Shandong, People’s Republic of China, Email hanjing0127@163.comPurpose: Acupuncture is widely used as a therapeutic approach for migraine; however, its underlying anti-migraine mechanisms remain incompletely understood. This study aimed to investigate the therapeutic effects of acupuncture on migraine and identify potential molecular mechanisms using integrated multi-omics analyses.Patients and Methods: 32 male Sprague–Dawley rats were allocated to control, model, sham-acupuncture, and acupuncture groups (n = 8 per group). The model was established by repeated nitroglycerin (NTG) administration. The effects of acupuncture were evaluated by assessing periorbital and hind paw withdrawal thresholds. Serum samples were subsequently analyzed using liquid chromatography–mass spectrometry (LC–MS)-based metabolomics and proteomics. The mRNA levels of key targets in serum were further quantified using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Differential metabolites and proteins were identified and subjected to Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, correlation, and protein–protein interaction (PPI) analyses.Results: Acupuncture alleviated the reduction in periorbital and hind paw withdrawal thresholds in NTG-induced migraine model rats. Integrated metabolomic and proteomic analyses identified differential metabolites and proteins associated with the therapeutic effects of acupuncture. Ten candidate hub proteins were ultimately identified. KEGG enrichment analysis indicated that these candidate hub proteins were closely related to immune defense, inflammatory responses, cholesterol metabolism and clearance, and may also be involved in energy metabolism and hormonal regulation. GO analysis further showed that acupuncture primarily modulated biological processes related to lipoprotein metabolism, transport, and clearance.Conclusion: Acupuncture improved migraine-like mechanical hypersensitivity in NTG-induced rats and was accompanied by changes in serum proteins and metabolites. Lipid/lipoprotein metabolism and complement-related pathways emerged as candidate pathways associated with the response. These exploratory findings do not establish molecular mediation or a direct effect on vascular function and require confirmation by targeted functional studies.Keywords: acupuncture, migraine, metabolomics, proteomics, multi-omics profiling