A novel ingredients recipe derived from Shugan-Hewei Formula targeting chemical carcinogenesis-ROS signaling pathway treated gastroesophageal reflux disease.

Ethnopharmacological Relevance: Gastroesophageal reflux disease (GERD) is a common gastrointestinal disease Shugan-Hewei (SGHW) formula is an extensively used traditional herbal decoction for treating GERD, which can significantly improve the clinical symptoms, quality of life, anxiety and depression status, etc. AIM OF THE STUDY: To elucidate the potential targets and pathways of SGHW in treating GERD by employing an integrative approach involving transcriptomics-based analysis combined with accurate network pharmacology.

Materials And Methods: First, we conducted animal experiments to investigate the effect of SGHW on GERD. Then, transcriptome sequencing was used to reveal the differentially expressed genes (DEGs). Meanwhile, the main ingredients of SGHW were identified by UPLC/Q-TOF MS, and the compound-target network was constructed. We integrated the DEGs with the compound-target network to identify core DEGs, which were used to establish a protein-protein interaction (PPI) network and GO/KEGG pathways. After that, we mapped the core PPI network and the core pathway to pinpoint the critical targets. Similarly, we mapped the critical targets and the compound-target network to discover core compound-target pairs and employed molecular docking techniques to elucidate the interactions between these pairs. Finally, key signaling pathways and their targets were validated by immunoblotting and immunofluorescence.

Results: The application of the SGHW resulted in a notable enhancement of the phenotype in the mixed reflux rat model. Transcriptomic analysis revealed a total of 1388 DEGs, among which 801 were upregulated and 587 were downregulated. According to Lipinski's "rule of 5", 45 compounds were extracted from the SGHW samples using UPLC/Q-TOF MS. Through online database searches, we identified 1131 potential targets for the active compounds and constructed a compound-target network based on these potential targets. Subsequently, we mapped the DEGs associated with the compound-target network, identifying 29 compounds targeting 119 core DEGs. KEGG pathway analysis of these core DEGs highlighted the chemical carcinogenesis-reactive oxygen species (ROS) signaling pathway as one of the most prominent pathways involved. We then established a PPI network based on these core DEGs and mapped the core PPI network alongside ROS pathway-related targets, identifying HMOX1 and CYP1A1 as the critical targets. Further analysis pinpointed key compound-target pairs, including Berberine targeting CYP1A1, and Honokiol, Tangeretin, α-Cyperone, 1-O-Acetylbritannilactone, Rotundine B, Cyperolone targeting HMOX1. These findings were validated through immunoblotting and immunofluorescence assays conducted in vivo.

Conclusion: In this study, we identified the monomers recipe derived from SGHW, including Berberine targeting CYP1A1, and Honokiol, Tangeretin, α-Cyperone, 1-O-Acetylbritannilactone, Rotundine B, Cyperolone targeting HMOX1 to regulate ROS signaling pathway in GERD treatment. Our findings offered a comprehensive insight into GERD treatment and herbal intervention, enhancing our understanding of accurate network pharmacology. It suggested that concentrating on a single pathway, specifically the ROS signaling pathway, could serve as a new therapeutic strategy for herbal medicine in GERD treatment.