# Wuzhuyu Decoction Relieves Chronic Migraine by Regulating 5-HT1A and 3A Receptors-Mediated CREB Signaling Pathway in Brain and Intestine

## TL;DR

Wuzhuyu Decoction (WZYD) helps treat chronic migraines by regulating brain and gut serotonin receptors, offering a new approach for managing both headache and digestive symptoms.

## Contribution

This study reveals WZYD's novel mechanism of action via 5-HT1A/3A receptors and CREB signaling in both brain and gut, explaining its dual therapeutic effects.

## Key findings

- WZYD increased 5-HT levels in the brain and decreased CGRP and c-Fos, alleviating chronic migraine symptoms.
- WZYD modulated 5-HT1A and 3A receptors in the brain and colon, improving both headache and gastrointestinal symptoms.
- The CREB signaling pathway mediated by 5-HT1A/3A receptors was regulated by WZYD, contributing to its brain-gut therapeutic effects.

## Abstract

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.