Chuanxiong Rhizoma and Cyperi Rhizoma (CRCR), an ancient and classic formula comprised of Chuanxiong Rhizoma and Cyperi Rhizoma in a weight ratio of 1:2, has long been used for curing migraine. This study aimed to explore their anti-migraine effect and active constituents. A nitroglycerin (NTG)-induced migraine model in rats was established to evaluate pharmacological effects. Cerebral blood flow was detected by a laser Doppler perfusion monitor. The levels of endothelin-1 (ET-1), γ-aminobutyric acid (GABA), nitric oxide synthase (NOS), nitric oxide (NO), 5-hydroxytryptamine (5-HT), 5-hydoxyindoleacetic acid (5-HIAA), calcitonin gene-related peptide (CGRP) and β-endorphin (β-EP) were quantified with enzyme-linked immunosorbent assay. CGRP and c-Fos mRNA expression were quantified with quantitative real-time polymerase chain reaction. A UPLC-MS/MS method was developed and validated for the simultaneous quantification of active constituents in rat serum and cerebral cortex. CRCR significantly increased cerebral blood flow, decreased the levels of ET-1, GABA and NOS, and increased the levels of 5-HT, 5-HIAA and β-EP in NTG-induced migraine rats. CGRP levels and CGRP mRNA expression, as well as c-Fos mRNA expression in the brainstem were markedly down-regulated with the treatment of CRCR. After oral administration of CRCR, ferulic acid (FA), senkyunolide A (SA), 3-n-butylphthalide (NBP), Z-ligustilide (LIG), Z-3-butylidenephthalide (BDPH), cyperotundone (CYT), nookatone (NKT) and α-cyperone (CYP) were qualified in rat serum and cerebral cortex. The above results suggested that CRCR showed powerfully therapeutic effects on migraine via increasing the cerebral blood flow, decreasing the expression of CGRP and c-Fos mRNA, and regulating the releasing of ET-1, GABA, NOS, 5-HT, 5-HIAA, CGRP and β-EP in the serum and brainstem, consequently relieving neurogenic inflammation. The active constituents in CRCR for treating migraine were FA, SA, NBP, LIG, BDPH, CYT, NKT and CYP. These findings contributed for the further use of CRCR as a combinational and complementary phytomedicine for migraine treatment.

Migraine is a common neurological disorder that manifests as recurrent attacks of unilateral and throbbing headache. Conioselinum anthriscoides “Chuanxiong” (Apiaceae; Chuanxiong rhizoma) and Cyperus rotundus L. (Cyperaceae; Cyperi rhizoma) (CRCR), is a classic prescription for treating migraine. This study aimed to reveal the potential mechanisms of CRCR extract against migraine using integrated analysis of metabolomics and network pharmacology. Behavioral changes in the nitroglycerin rat migraine model were determined from von Frey withdrawal response. Untargeted serum metabolomics was used to identify the differentially expressed metabolites and metabolic pathways. The differentially expressed metabolites were analyzed to obtain the corresponding targets by a compound–reaction–enzyme–gene network. Network pharmacology was used to construct a compound–target–pathway network. The common targets of metabolomics and network pharmacology were further analyzed. Metabolomics analysis identified 96 differentially expressed metabolites and 77 corresponding targets. Network pharmacology analysis identified 201 potential targets for CRCR against migraine. By intersecting 77 targets with 201 targets, monoamine oxidase A (MAO-A), monoamine oxidase B (MAO-B), and catechol-O-methyltransferase (COMT) were identified as the common targets, and MAO-A, MAO-B, and COMT were involved in the tyrosine metabolism pathway. Further experiments demonstrated that the contents of MAO-A and COMT were significantly increased in serum and brainstem tissue of the migraine rats. CRCR extract significantly decreased the contents of MAO-A and COMT, while no significant difference was found in MAO-B. Metabolomics analysis indicated that the contents of 3,4-dihydroxyphenylacetate (DOPAC) and 3-(4-hydroxyphenyl)pyruvate (HPP) were significantly increased in the migraine rats, and CRCR extract caused significant decreases in DOPAC and HPP. Interestingly, DOPAC and HPP were two differentially expressed metabolites involved in the tyrosine metabolism pathway. Correlation analysis showed that DOPAC and HPP were highly positively correlated with MAO-A and COMT. Taken together, two key differentially expressed metabolites (DOPAC and HPP), two key targets (MAO-A and COMT), and one relevant metabolic pathway (tyrosine metabolism) showed great importance in the treatment of migraine. This research could provide a new understanding of the potential mechanism of CRCR against migraine. More attentions should be paid into the tyrosine metabolism pathway in future studies.

Background: Chronic migraine (CM) is a common complex nervous system disease, often accompanied by symptoms of the digestive tract that interact with each other, leading to prolonged and difficult-to-cure migraines. These symptoms are associated with abnormalities in 5-HT and its receptors. Wuzhuyu decoction (WZYD) is a traditional Chinese medicine prescription commonly used in clinics to treat CM; it relieves gastrointestinal symptoms, such as nausea and vomiting; however, its mechanism is still unclear. Investigating the differences in the role of WZYD compared to existing drugs targeting 5-HT receptors in the treatment of CM not only helps elucidate its pathogenesis but also provides possibilities for the development of new therapeutic approaches. Methods: An inflammation soup (IS)-induced CM male rat model was established. Based on a preliminary experiment, the target of WZYD in treating CM was determined by network pharmacology, and verified by molecular docking. ELISA, immunofluorescence, western blot, and real-time quantitative polymerase chain reaction (RT-qPCR) were used to evaluate the expression levels of CM-related indicators (5-HT, calcitonin gene-related peptide (CGRP), and c-Fos) to ensure the successful establishment of the CM model and the effectiveness of the drug. On this basis, the protein expression levels of 5-HT1A/3A receptors and their cAMP-response element binding protein (CREB) signaling pathway were detected by western blot and immunohistochemistry. The role of 5-HT1A/3A receptors in the treatment of CM by WZYD was validated using a 5-HT1A receptor antagonist (WAY 100635) and a 5-HT3A receptor agonist (SR 57227). Results: The results showed that WZYD increased the expression of 5-HT in the brain, decreased the expression of CGRP, c-Fos, ionized calcium-binding adapter molecule 1 (Iba1), and relieved CM. At the same time, WZYD also increased the expression of the 5-HT1A receptor and decreased the expression of the 5-HT3A receptor in the brain and colon of CM rats. Subsequently, WZYD further exerted its brain-gut integrated therapeutic effects by regulating the CREB signaling pathway mediated by 5-HT1A/3A receptors in the brain and colon of CM rats. Conclusions: WZYD not only regulates neurotransmitters in the brain and colon at the same time, but also specifically regulates 5-HT1A/3A receptors in the brain and colon, which explains the characteristics and advantages of WZYD from a new perspective. While effectively relieving headache symptoms, it also improves related gastrointestinal symptoms, which is more conducive to the treatment of CM.