Amelioration of nitroglycerin-induced migraine in mice via Wuzhuyu decoction: Inhibition of the MZF1/PGK1 pathway and activation of NRF2 antioxidant response.

ETHNOPHARMACOLOGICAL RELEVANCE: Migraine, a chronic and intricate disorder, manifests as recurrent episodic headaches accompanied by various neurological symptoms. Wuzhuyu Decoction (WZYD) is a traditional Chinese medical formula with promising effects in treating migraines; however, its underlying mechanisms have not yet been clarified. AIM OF STUDY: The study aimed to evaluate WZYD's effectiveness in migraine treatment and investigate the potential mechanism of WZYD's effects on migraine and oxidative stress. MATERIALS AND METHODS: Behavior tests and immunofluorescence assay for the intensity of migraine markers to assess the migraine-relieving effect of WZYD after chronic migraine model induced by nitroglycerin in mice. The impacts of WZYD on oxidative stress-related markers, including reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), nuclear factor erythroid 2-related factor 2 (NRF2), heme oxygenase 1 (HO1), and NAD (P)H quinone oxidoreductase 1 (NQO1) in brain tissue were examined. In addition, protein expression or mRNA levels of the MZF1/PGK1 were detected using Western blot or PCR, respectively. Finally, the MZF1 overexpression vector was constructed to the higher level of MZF1. The MZF1/PGK1 signaling pathway expression was evaluated by markers of oxidative stress including NRF2 and others in this series of experiments. RESULTS: Through murine model experimentation, we observed that WZYD effectively alleviates migraine symptoms, signifying its therapeutic efficacy. Mechanistically, WZYD emerges as a potent activator of the NRF2, acting as a robust defense against oxidative stress. In vitro investigations demonstrated that WZYD combats oxidative stress and curbs cell apoptosis induced by these detrimental conditions. Furthermore, by suppressing the transcriptional expression of PGK1, an influential player in the NRF2 pathway, WZYD effectively activates NRF2 signaling. Intriguingly, we have identified MZF1 as the mediator orchestrating the regulation of the PGK1/NRF2 pathway by WZYD. CONCLUSION: The study confirms the effectiveness of WZYD in alleviating migraine symptoms. Mechanistically, WZYD activated the NRF2 signaling pathway; moreover, the action of WZYD involved the down-regulation of PGK1 mediated by MZF1, which promoted the activation of the NRF2 pathway. This study advances our understanding of the intricate mechanisms driving WZYD's efficacy, paving the way for novel treatments in migraine management.

Safety assessment of Wuzhuyu decoction extract: acute and subacute oral toxicity studies in rats.

Wuzhuyu decoction (WZYD) is a well-known classic traditional Chinese medicine prescription and has been widely used to treat headache, nausea, vomiting, insomnia, etc. However, little published information is available about its safety. Our aim was to investigate the acute and subacute oral toxicity of WZYD extract in rats following the technical guidelines from China's National Medical Products Administration (NMPA) for single and repeated doses toxicity studies of drugs. Acute oral toxicity was assessed in rats via oral administration of WZYD extract at 4 g/kg three times within a day followed by a 14-day observation period. To evaluate the subacute toxicity, rats were orally administered with WZYD extract at doses of 0, 0.44, 1.33, and 4 g/kg for 28 days. The items examined included clinical signs, body weight, food consumption, hematological and biochemical parameters, bone marrow smear, organ index, and histopathology. After the rats were administered with 12 g/kg (3 × 4 g/kg) WZYD extract, no mortality and toxic effects were observed during the observation period. In the subacute toxicity study, WZYD extract did not cause any significant treatment-related abnormality in each examined item of rats, so the no observed adverse effect level (NOAEL) of WZYD extract for 28 days orally administered to rats is considered to be 4 g/kg, which is approximately 80-fold of its clinical proposed dosage.

Metabolomics Profiles Reveal the Efficacy of Wuzhuyu Decoction on Patients with Chronic Non-Atrophic Gastritis.

Objective: Chronic non-atrophic gastritis (CNAG) is a common clinical gastrointestinal disease with a long and recurrent course. In China, Wuzhuyu decoction (WZYD) has been used for centuries to treat gastrointestinal disorders. To unravel the efficacy and mechanism of WZYD for CNAG, a clinical study was conducted. And metabolomics was used to explore the mechanism of WZYD for CNAG patients. Methods: Twenty patients in total were recruited in this study (Nos. ChiCTR2200062296) and the protocol was approved by the Ethics Committee (Approval number: KY-2022-2-6-1) and complied with the Declaration of Helsinki. The formula granule of WZYD were assessed by UHPLC-QQQ-TOF to discern the main potential active compounds. The endoscopy evaluation and histopathological changes were detected as effective indicators. Serum samples from patients were used for metabolomics. Inflammatory factors in patients' serum were determined by ELISA. Metabolomics revealed a series of differential metabolites and signaling pathways. Results: WZYD was capable to prevent CNAG by ameliorating score of endoscopy evaluation including erosion, hemorrhage, as well as chronic inflammation and active chronic inflammation score after treatment were decreased. The results indicated that 10 core metabolic components were associated with the treatment of WZYD. Moreover, these metabolic components proved that pyrimidine metabolism and thiamine metabolism were critically responsible for CNAG. In addition, WZYD treatment effectively reduced serum levels of TNF-α, IL-10, and COX-2. Conclusion: Altogether, WZYD can effectively alleviate CNAG by inhibiting inflammation and regulating related metabolic processes, which might be the molecular mechanism of WZYD treatment of CNAG. More studies are warranted to be conducted in this area. Trial Registration: ChiCTR, ChiCTR2200062296. Registered 1 August 2022, https://www.chictr.org.cn/com/25/showprojen.aspx?proj=174027.

Exploring the pharmacological mechanism of Wuzhuyu decoction on hepatocellular carcinoma using network pharmacology.

BACKGROUND: Wuzhuyu decoction, a traditional Chinese medicinal formula, is effective in treating hepatocellular carcinoma (HCC). AIM: To explore the potential mechanism of action of Wuzhuyu decoction against HCC. METHODS: The active components of each Chinese herbal medicinal ingredient in Wuzhuyu decoction and their targets were obtained from the Traditional Chinese Medicine Database and Analysis Platform. HCC was used as a search query in GeneCards, Online Mendelian Inheritance in Man, Malacards, DisGeNET, Therapeutic Target Database, and Comparative Toxicogenomics Database. The overlapping targets of the Wuzhuyu decoction and HCC were defined, and then protein-protein interaction, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed. CytoHubba was used to select hub genes, and their binding activities and key active components were verified using molecular docking. RESULTS: A total of 764 compounds, 77 active compounds, and 204 potential target genes were identified in Wuzhuyu decoction. For HCC, 9468 potential therapeutic target genes were identified by combining the results from the six databases and removing duplicates. A total of 179 overlapping targets of Wuzhuyu decoction and HCC were defined, including 10 hub genes (tumor necrosis factor, interleukin-6, AKT1, TP53, caspase-3, mitogen-activated protein kinase 1, epidermal growth factor receptor, MYC, mitogen-activated protein kinase 8, and JUN). There were six main active components (quercetin, kaempferol, ginsenoside Rh2, rutaecarpine, ß-carotene, and ß-sitosterol) that may act on hub genes to treat HCC in Wuzhuyu decoction. Kyoto Encyclopedia of Genes and Genomes enrichment analysis mainly involved the mitogen-activated protein kinase, p53, phosphatidylinositol-4,5-bisphosphate 3-kinase-Akt, Janus kinase-signal transducer of activators of transcription, and Hippo signaling pathways. Further verification based on molecular docking results showed that the small molecule compounds (quercetin, kaempferol, ginsenoside Rh2, rutaecarpine, ß-carotene, and ß-sitosterol) contained in Wuzhuyu decoction generally have excellent binding affinity to the macromolecular target proteins encoded by the top 10 genes. CONCLUSION: This study revealed that Wuzhuyu decoction may be a latent multicomponent, multitarget, and multipathway treatment for HCC. It provided novel insights for verifying the mechanism of Wuzhuyu decoction in the treatment of HCC.

[Rapid identification of components in Wuzhuyu Decoction using UHPLC Q-Exactive Orbitrap MS~n and molecular network technology].

Wuzhuyu Decoction, the classical formula recorded in the Treatise on Febrile Diseases(Shang Han Lun), has been included in the Catalogue of Ancient Classic Prescriptions(the First Batch). Consisting of Euodiae Fructus, Ginseng Radix et Rhizoma, Zingiberis Rhizoma Recens, and Jujubae Fructus, it is effective in warming the middle, tonifying deficiency, dispelling cold, and descending adverse Qi, and is widely applied clinically with remarkable efficacies. For a classical formula, the chemical composition is the material basis and an important premise for quantity value transfer. This study aimed to establish a rapid identification method of chemical components in Wuzhuyu Decoction by high-resolution mass spectrometry(HR-MS) and molecular network. AQUITY UPLC BEH C_(18) column(2.1 mm×100 mm, 1.7 µm) was used for sample separation, and acetonitrile-0.1% formic acid in water was used as mobile phases for gradient elution. Q-Exactive Orbitrap MS data were collected in positive and negative ion modes, and GNPS molecular network was plotted according to the similarity of MS/MS fragmentation modes. Cytoscape 3.6.1 was used to screen molecular clusters with similar structures. Finally, the chemical components of Wuzhuyu Decoction were rapidly identified according to the controls, as well as the information of retention time, accurate relative molecular weight of HR-MS, and MS/MS multistage fragments. A total of 105 chemical components were identified in Wuzhuyu Decoction. This study can provide data for the follow-up quality control, standard substance research, and pharmacodynamic material research on Wuzhuyu Decoction, as well as references for the rapid qualitative analysis of the chemical components of Chinese medicine.

Mechanism of Wuzhuyu decoction on alcohol-induced gastric ulcers using integrated network analysis and metabolomics.

Background: Gastric ulcers (GUs) are prevalent digestive disorders worldwide. Wuzhuyu Decoction (WZYT) is a traditional Chinese medicine that has been employed for centuries to alleviate digestive ailments like indigestion and vomiting. This study aims to explore the potential effects and underlying mechanisms of WZYT on alcohol induced gastric ulcer treatment. Methods: We employed macroscopic assessment to evaluate the gastric ulcer index (UI), while the enzyme-linked immunosorbent assay (ELISA) was utilized for detecting biochemical indicators. Pathological tissue analysis involved hematoxylin-eosin (H&E) staining and Periodic Acid-Schiff (PAS) staining to assess gastric tissue damage. Additionally, the integration of network analysis and metabolomics facilitated the prediction of potential targets. Validation was conducted using Western blotting. Results: The research revealed that WZYT treatment significantly reduced the gastric ulcer index (UI) and regulation of alcohol-induced biochemical indicators levels. Additionally, improvements were observed in pathological tissue. Network analysis results indicated that 62 compounds contained in WZYT modulate alcohol-induced gastric ulcers by regulating 183 genes. The serum metabolomics indicated significant changes in the content of 19 metabolites after WZYT treatment. Two pivotal targets, heme oxygenase 1 (HMOX1) and albumin (ALB), are believed to assume a significant role in the treatment of gastric ulcers by the construction of "compounds-target-metabolite" networks. Western blot analysis confirmed that WZYT has the capacity to elevate the expression of HMOX1 and ALB targets. Conclusion: The integration of network analysis and metabolomics provides a scientific basis to propel the clinical use of WZYT for GUs. Our study provides a theoretical basis for the use of Wuzhuyu decoction in the treatment of gastric ulcers.

[Study on characteristic chromatogram and content determination of Wuzhuyu Decoction reference sample].

In this study, the critical quality attributes of Wuzhuyu Decoction reference sample were explored by using characteristic chromatogram, index component content and dry extract rate as indexes.The dissemination relationship of quantity value between medicinal materials-decoction pieces-reference sample was investigated to preliminarily formulate the quality standard of the reference sample.The characteristic chromatogram of 15 batches of Wuzhuyu Decoction was established by high performance liquid chromatography(HPLC) and the similarity analysis was conducted.Common peaks were demarcated and assigned to medicinal materials.Moreover, quantitative determination of limonin, evodiamine, rutaecarpine and ginsenoside Rb_1 of Wuzhuyu Decoction were performed.The dissemination of quantity value was explored combined with dry extract rate, similarity of characteristic chromatogram and transfer rate of index component content.A total of 18 common peaks were identified in the corresponding materials of Wuzhuyu Decoction reference sample, with the similarity of characteristic chromatogram greater than 0.9, and Fructus Evodiae, Radix Ginseng, Rhizoma Zingiberis Recens and Fructus Jujubae contributed 9, 5, 8 and 2 chromatographic peaks, respectively.The index component content of corresponding materials and the transfer rates of medicinal materials-decoction pieces and decoction pieces-reference sample of different batches of Wuzhuyu Decoction reference sample were as follows: the content of limonin was 0.16%-0.51%, and the transfer rates were 83.66%-115.60% and 38.54%-54.58%, respectively; the content of evodiamine was 0.01%-0.11%, the transfer rated were 80.80%-116.15% and 3.23%-12.93%, respectively; the content of rutaecarpine was 0.01%-0.05%, the transfer rates were 84.33%-134.53% and 5.72%-21.24%, respectively; the content of ginsenoside Rb_1 was 0.06%-0.11%, and the transfer rates were 90.00%-96.92% and 32.45%-67.24%, respectively.The dry extract rate of the whole prescription was 22.58%-29.89%.In this experiment, the dissemination of quantity value of Wuzhuyu Decoction reference sample was analyzed by the combination of characteristic chromatogram, index component content and dry extract rate.A scientific and stable quality evaluation method of the reference sample was preliminarily established, which provided basis for the subsequent development of Wuzhuyu Decoction and the quality control of related preparations.

Wuzhuyu Decoction relieves hyperalgesia by regulating central and peripheral 5-HT in chronic migraine model rats.

BACKGROUND: Chronic migraine (CM) is a highly disabling and burdensome disease. Wuzhuyu decoction (WZYD), a clinical used formula to treat and prevent episodic migraine and CM, has been reported to relieve the hyperalgesia of CM and increase brainstem and blood serotonin (5-hydroxytryptamine, 5-HT) in migraine model rats in previous studies; yet the mechanism is unclear. PURPOSE: This study aimed to observe the hyperalgesia relief effect of WZYD and investigate the mechanistic association with the regulation on central and peripheral 5-HT. METHODS: WZYD with different doses (3.372, 1.686 and 0.843 g/kg∙d) and the positive drug - sumatriptan (5.83 mg/kg∙3 d) were intragastrically administered in inflammatory soup (IS)-induced CM model rats, respectively. Hyperalgesia was assessed by facial mechanical withdrawal threshold and tail-flick latency. 5-HT was determined by ELISA. Western blot analysis, immunohistochemistry and immunofluorescence determination, and 16S rRNA gene sequencing were performed. RESULTS: WZYD significantly relieved the hyperalgesia by elevating the facial mechanical withdrawal threshold and tail-flick latency. In WZYD groups, increased 5-HT and decreased calcitonin gene-related peptide in both the brainstem and plasma, downregulated TNF-α, IL-1ß, and c-fos expression in the brainstem were observed in dose-dependent manner. Interestingly, 5-HT in colon tissues were also observed, which is associated with upregulating tryptophan hydroxylase, serotonin transporter and Piezo1 expression and increasing 5-HT and chromogranin A in enterochromaffin cells. Disorder of the microbiota, function and metabolism was correlated with 5-HT synthesis. WZYD could regulate the abundance of Anaerostipes and Acidifaciens. CONCLUSION: WZYD has the pharmacological effect on relieving hyperalgesia in CM model rats, possibly by affecting central and peripheral 5-HT.

[Difference in intestinal absorption of Wuzhuyu decoction between normal and migraine model rats].

To compare the intestinal absorption of Wuzhuyu decoction(WZYD) between normal rats and migraine model rats, and investigate the optimized WZYD from aspect of absorption. The rat single pass intestinal perfusion test(SPIP) was adopted for optimized sample and un-optimized sample in normal and migraine model rats induced by nitroglycerin and reserpine. The contents of 8 ingredients were determined by high performance liquid chromatography(HPLC), and 4 absorption parameters for each ingredient were calculated and compared: unit area absorption(Mper area), absorption rate constant(Ka), apparent coefficient(Papp) and relative absorption rate(RA). The results showed that there was a great difference between normal rats and model rats in the intestinal absorption of the same WZYD. As compared with normal rats, the absorption parameters of most ingredients in optimized sample were increased in migraine model rats induced by nitroglycerin; Similar phenomena were also found in migraine model rats induced by reserpine. However, the absorption parameters of most ingredients were decreased in un-optimized sample. Therefore, pathological model rats shall be used for effective ingredient recognition based on the correlation between intestinal absorption spectra and pharmacological effects. As compared with the un-optimized samples, the absorption of effective ingredients was faster, easier and more adequate in the optimized samples, revealing their mechanism on better efficacy from the aspect of absorption.

Efficacy of Modified Wuzhuyu Decoction Granule ( ) for Migraine Patients with Cold and Stasis Obstructing Meridian Syndrome: A Randomized, Double-Blind, Placebo-Controlled Trial.

OBJECTIVE: To study the efficacy of modified Wuzhuyu Decoction Granule (, MWDG) in the treatment of migraine patients with cold and stasis obstructing meridian syndrome. METHODS: This study was a randomized, double-blind, placebo-controlled trial. A total of 78 migraine patients with cold and stasis obstructing meridian syndrome were recruited and randomly assigned by a ratio of 2:1 into a treatment group (51 cases) and a placebo group (27 cases). Patients in the treatment group were treated with MWDG while placebo granules were applied in the control group. The treatment course lasted for 12 weeks with a follow-up of 4 weeks. The primary outcome measures included frequency and days of migraine attacks and the secondary outcome measures were analgesics consumption and visual analogue scale (VAS) scores. All outcome assessments were conducted respectively at baseline, the 4th, 8th and 12th week, and the end of follow-up. RESULTS: In the treatment group, significant decrease in frequency of migraine attacks were observed since the 4th week and that of analgesics consumption since the 8th week (both P<0.05). While, in the placebo group, significant decrease in frequency of migraine attacks were observed since the 8th week and that of analgesics consumption since the 12th week (both P<0.05). No significant decrease in days of migraine attacks and VAS scores of migraine pain were observed in both groups. Between the two groups, there were significant differences in VAS scores and intensity of pain appeared in the 8th week (P<0.05). However, no significant differences were found in days and frequency of migraine attacks and analgesics consumption (P>0.05). CONCLUSIONS: MWDG was probably effective in the treatment of migraine especially for alleviating pain intensity. Furthermore, MWDG could reduce the frequency of migraine attacks and analgesics consumption sooner than the placebo.