| Summary: | Bawei Chenxiang Wan (BWCX) is a classical Tibetan medicinal formula originating from the canonical Tibetan medical text The Four Tantras. It is traditionally used to activate qi circulation, nourish the heart, tranquilize the mind and enhance intelligence. It has been applied for over a thousand years on the Qinghai–Tibet Plateau to treat cardio-cerebral hypoxic syndromes such as high-altitude (HA) adverse qi flow and mental trance, which are attributed to an imbalance among the three humors (rlung, mkhris-pa, and bad-kan). The traditional applications of BWCX closely align with the clinical manifestations of modern Chronic Mountain Sickness (CMS). However, its effective components and mechanisms of action anti-CMS remain unclear. This study aims to systematically elucidate the material basis of BWCX anti-CMS and its underlying mechanism of cardio-cerebral protection through the regulation of oxidative stress. Chemical constituents of BWCX were characterized by UHPLC-MS. To identify core therapeutic targets, we employed network pharmacology and analyzed CMS patient transcriptomes (GSE145774, GSE103940) by combining weighted gene co-expression network analysis (WGCNA) with the random forest (RF) algorithm. Molecular docking was used to validate interactions between active components and predicted targets. A physiologically relevant CMS model was established by exposing 60 mice to a natural HA environment (Lhasa, 3650 m) for 8 weeks. Hematological parameters were measured; oxidative stress markers (malondialdehyde [MDA], Catalase [CAT], Total Superoxide Dismutase [T-SOD], glutathione peroxidase [GSH-Px], total antioxidant capacity [T-AOC]) and energy metabolism (adenosine triphosphate [ATP]) in cardiac tissues were assessed by ELISA; and the expression of core targets was analyzed using RT-qPCR and Western blot to verify the regulatory effect of BWCX on the AKT1/FOXO3a/CAT signaling axis, and the binding situation of FOXO3a to the promoter region of CAT was analyzed using ChIP-qPCR to verify the regulatory relationship of FOXO3a on CAT. A total of 78 chemical components were identified, including 18-β-glycyrrhetinic acid, β-asarone and quassin. Integrative bioinformatics analysis pinpointed AKT1, FOXO3 and CAT as core targets of BWCX anti-CMS, with significant enrichment in oxidative stress-related pathways. Molecular docking results showed that the binding energies of costunolide with AKT1, FOXO3a, CAT and ESR2 were −9.5, −8.8, −11.1 and −9.9 kcal/mol, respectively; while those of 18-β-glycyrrhetinic acid with the above targets were −9.1, −8.2, −10.2 and −9.2 kcal/mol, respectively, indicating their strong potential for target binding. Animal experiments demonstrated that BWCX intervention significantly reversed CMS-induced hematological abnormalities [increased red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT) and platelets (PLT)], ameliorated cardiac oxidative stress (reduced MDA; elevated T-AOC, T-SOD, GSH-Px, CAT, ATP), and corrected the dysregulated molecular expression: it suppressed the upregulation of AKT1 mRNA/protein and p-AKT1/AKT1 ratio, while restoring the downregulated mRNA/protein expression of FOXO3a and CAT and the p-FOXO3a/FOXO3a ratio in cardiac and hippocampal tissues (P < 0.05). RT-qPCR analysis of AKT1, FOXO3a, and CAT mRNA expression in vitro revealed trends consistent with those observed in the in vivo experiments. It was also verified that significant enrichment of FOXO3a was detected in the CAT promoter region under hypoxic stress (P < 0.05). FOXO3a binds to the CAT promoter in H9C2 cells, thereby confirming that CAT is a direct transcriptional target of FOXO3a. This study provides the first evidence that BWCX alleviates CMS-induced cardio-cerebral hypoxic injury by modulating the AKT1/FOXO3a/CAT signaling axis, thereby rebalancing oxidative stress and improving hemorheology. These findings provide a modern pharmacological interpretation of its traditional cardiocerebral protective effect, offer a paradigm for ethnomedicine research modernization, and lay a scientific foundation for its clinical application and quality control. [Display omitted] • The AKT1/FOXO3a/CAT axis has been identified as the pivotal pathway mediating the therapeutic effects of BWCX against CMS. • Reveals the dual function of CAT upregulation in CMS as both a transcriptional target and a systemic repair effector. • Establishes a research paradigm integrating clinical data, computational prediction, and validation in a CMS model.
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