Proteomic and metabolomic profiling of acupuncture for migraine reveals a correlative link via energy metabolism

Abstract

Migraine is a neurovascular disease with a high disability rate. Acupuncture treatment has emerged as a safe and viable alternative prophylactic therapy that can effectively alleviate the duration and frequency of migraine attacks. However, the therapeutic mechanisms underlying the effects of acupuncture are yet to be systematically elucidated. In this study, we enrolled female patients with migraine without aura (n = 20) and healthy controls (n = 10). Patients received acupuncture treatment on DU20, DU24, bilateral GB13, GB8, and GB20, applied three times per week over the course of 4 weeks for 12 sessions in total. Blood samples were collected from the median cubital vein before and after acupuncture treatment. Proteomic and metabolomic profiling was performed using liquid chromatography-mass spectrometry to determine the characteristics of differentially expressed molecules and expression of their corresponding biological pathways as well as to elucidate the pathogenesis of migraine and the biological effects underlying the treatment of migraine with acupuncture. Proteomic and metabolomic profiling of plasma samples from patients with migraine without aura before and after acupuncture treatment revealed enrichment of immune-related pathway functions and the arginine synthesis pathway. Joint pathway analyses revealed significant enrichment of the pentose phosphate and glycolysis/gluconeogenesis pathways in patients with migraine. The glycolysis/gluconeogenesis and riboflavin metabolism pathways were significantly enriched after acupuncture treatment. The expression levels of various key proteins and metabolites, including α-D-glucose, flavin adenine dinucleotide, biliverdin reductase B, and L-glutamate, were significantly differentially expressed before and after acupuncture treatment in patients with migraine without aura. Treatment of migraine with acupuncture was associated with significant changes in key molecules and pathways, indicative of physiological changes in the trigeminovascular system, glutamate neurotoxicity, and other migraine-related physiological changes. Overall, our comprehensive analysis using proteomic and metabolomic profiling demonstrates that energy metabolism may serve as a key correlative link in the occurrence of migraine and the therapeutic effects of acupuncture treatment. Our findings may facilitate the identification of diagnostic and therapeutic modalities in the ongoing search for effective treatments for migraine attacks.

Keywords: acupuncture; energy metabolism pathways; metabolomics; migraine; proteomics.

FIGURE 1

Study design and workflow. Overview patterns of blood sample collection from migraine without aura patients, including M group (migraine patients before acupuncture treatment) (n = 20), A group (migraine patients after acupuncture treatment) (n = 20), and H group (healthy controls) (n = 10). A total of 1,354 proteins and 2,828 metabolites were identified by proteomics and metabolomics. Differentially expressed biomarkers (DEPs and DEMs) are involved in pathways including a variety of immune responses and changes in energy metabolism. This may help us to understand the pathogenesis of migraine and the potential biological effects of acupuncture in the treatment of migraine. Furthermore, this may allow the identification of potential biomarker combinations for the classification of migraine without aura patients and healthy controls by using a machine learning strategy.

FIGURE 2

Gene Ontology (GO) terms and KEGG pathways enrichment analysis of DEPs. GO enrichment analysis (A) and KEGG pathway analysis (B) of DEPs in M group and H group. GO enrichment analysis (C) and KEGG pathway analysis (D) of DEPs in M group and A group.

FIGURE 3

Pathway analysis for DEMs. (A) The metabolic pathway analysis between H group and M group. (B) The metabolic pathway analysis between M group and A group. The size and color of each bubble is based on the pathway impact value and P-value, respectively.

FIGURE 4

Dysregulated proteins and metabolites in H group and M group. (A) Heatmap of DEPs and DEMs between H group and M group. (B) The expression level change of the key proteins and metabolites with significant difference between H group and M group. Asterisks indicate statistical significance based on unpaired two-sided Welch’s t-test. Significance levels: *P < 0.05, **P < 0.01, ***P < 0.001.

FIGURE 5

Dysregulated proteins and metabolites between M group and A group. (A) Heatmap of DEPs and DEMs between M group and A group. (B) The expression level change of the key proteins and metabolites with significant difference between M group and A group. Asterisks indicate statistical significance based on unpaired two-sided Welch’s t-test. Significance levels: *P < 0.05, **P < 0.01, ***P < 0.001.

FIGURE 6

Identification of H group and M group by machine learning of proteomic and metabolomic features. (A) Top 25 characteristic molecules prioritized by SVM model by the average importance. (B) Receiver operating characteristic (ROC) of the SVM model can classify H group and M group perfectly; AUC: 0.998.

FIGURE 7

Key proteins and metabolites profiling in migraine patients after acupuncture treatment could reveal the correlative link between acupuncture and energy metabolism in the context of trigeminovascular system. Proteomics and metabolomics profiles indicated that acupuncture treatments may decrease oxidative stress levels (FAD, BLVRB, L-glutamate) while increasing glucose availability (α-D-glucose), thereby aiding in restoring energy homeostasis.