Data Sheet 1_Gastric mucosal repair by Men’s Huwei Powder via EGF-NO/PGE2-PI3K-TLR4 in RELISH: Restoring Equilibrium through long-term integration of synergistic health.xlsx

Background:Gastric mucosal injury (GMI) involves inflammation, oxidative stress, and barrier dysfunction. Existing therapies offer limited efficacy with side effects. Men's Huwei Powder (MHWP), a Traditional Chinese Medicine (TCM) formula developed under the RELISH (Restoring Equilibrium through Long-term Integration of Synergistic Health) framework, aims to restore mucosal and systemic equilibrium.
Methods:The experiment was grouped using a uniform design, followed by the construction of an ethanol-induced GMI rat model. The effects of MHWP were assessed through histological examination, ELISA, RT-PCR, 16S rRNA sequencing, and LC-MS-based serum fingerprint analysis. Multivariate modeling techniques, including SPRA, PLSR, and pSEM, were utilized to explore the comprehensive regulatory mechanisms of MHWP.
Results:MHWP promotes activation of the EGF–NO/PGE2 axis and concurrently suppresses key nodes of the PI3K/Akt/NF-κB signaling pathway, leading to downregulation of pro-inflammatory cytokines—including IL-1β, IL-6, and TNF-α—in both serum and gastric tissue. Beyond its localized effects, MHWP exerts systemic benefits by inhibiting hepatic TLR4, MyD88, and NF-κB expression, thereby reducing liver-derived IL-6 and TNF-α and ameliorating ethanol-induced liver injury. This hepatic protection contributes to improved gastric mucosal healing and systemic inflammatory balance. Gut microbiota profiling identified key genera—such as Ligilactobacillus, Acutalibacter, and Lachnospiraceae:CAG_95—as critical mediators of mucosal repair, with MHWP modulating their abundance in a botanicals–dependent manner. These genera were closely linked to the regulation of NO, COX-2, and gastric IL-1β and IL-6, highlighting their critical role in the EGF–NO/PGE2 axis and inflammatory signaling. Serum HPLC fingerprinting identified several bioactive metabolites—including 6-gingerol (P1), atractylenolide II (P10), and dihydrostilbene base + 3O,2Prenyl (P18)—as major contributors to MHWP's efficacy. Closely associated with specific botanical drugs, these metabolites synergistically regulated multiple inflammatory and reparative pathways, underscoring MHWP's holistic therapeutic mechanism.
Conclusion:MHWP exerts multi-targeted effects through integrated modulation of the liver, gut microbiota, and serum metabolites. These findings underscore its potential as a holistic and sustainable TCM-based intervention for GMI, in alignment with the RELISH framework.

背景：胃黏膜损伤（Gastric mucosal injury, GMI）涉及炎症、氧化应激与屏障功能障碍。现有治疗手段疗效有限且伴随不良反应。男士护卫散（Men's Huwei Powder, MHWP）是基于RELISH（Restoring Equilibrium through Long-term Integration of Synergistic Health，即通过协同健康的长期整合恢复平衡）框架开发的中药方剂，旨在恢复黏膜与全身稳态。
方法：本实验采用均匀设计法进行分组，随后构建乙醇诱导的胃黏膜损伤大鼠模型。通过组织学检查、酶联免疫吸附试验（ELISA）、实时荧光定量聚合酶链反应（RT-PCR）、16S rRNA测序以及基于液相色谱-质谱（LC-MS）的血清指纹图谱分析，对MHWP的药效进行评估。本研究采用包括SPRA、偏最小二乘回归（PLSR）、偏最小二乘结构方程模型（pSEM）在内的多变量建模技术，以探究MHWP的全面调控机制。
结果：MHWP可促进表皮生长因子-一氧化氮/前列腺素E2（EGF-NO/PGE2）轴的激活，同时抑制磷脂酰肌醇3-激酶/蛋白激酶B/核因子κB（PI3K/Akt/NF-κB）信号通路的关键节点，从而下调血清与胃组织中包括白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）以及肿瘤坏死因子-α（TNF-α）在内的促炎细胞因子水平。除局部作用外，MHWP还可通过抑制肝脏Toll样受体4（TLR4）、髓系分化因子88（MyD88）以及核因子κB的表达，减少肝脏来源的IL-6与TNF-α，改善乙醇诱导的肝损伤。这种肝脏保护作用有助于促进胃黏膜愈合并维持全身炎症平衡。肠道菌群谱分析鉴定出若干关键菌属，如Ligilactobacillus、Acutalibacter以及毛螺菌科CAG_95（Lachnospiraceae:CAG_95），它们是黏膜修复的关键介导因子；MHWP可通过植物药依赖性方式调节这些菌属的丰度。这些菌属与一氧化氮（NO）、环氧合酶-2（COX-2）以及胃组织IL-1β、IL-6的调控密切相关，凸显了其在EGF-NO/PGE2轴与炎症信号通路中的关键作用。血清高效液相色谱（HPLC）指纹图谱分析鉴定出若干活性代谢物，包括6-姜酚（P1）、苍术内酯II（P10）以及二氢茋类碱+3O,2异戊烯基（P18），它们是MHWP药效的主要贡献成分。这些代谢物与特定植物药成分密切相关，可协同调控多条炎症与修复通路，进一步阐明了MHWP的整体治疗机制。
结论：MHWP通过对肝脏、肠道菌群以及血清代谢物的整合调控，发挥多靶点作用。本研究结果证实，MHWP有望成为符合RELISH框架的、用于胃黏膜损伤的整体化且可持续的中药干预手段。