The vacuolar-type ATPase (V-ATPase) is a multi-subunit enzyme complex that maintains lysosomal acidification, a critical process for cellular homeostasis. By controlling the pH within lysosomes, V-ATPase contributes to overall cellular homeostasis, helping to maintain a balance between the degradation and synthesis of cellular components. Dysfunction of V-ATPase impairs lysosomal acidification, leading to the accumulation of undigested materials and contributing to various diseases, including cardiovascular diseases (CVDs) like atherosclerosis and myocardial disease. Furthermore, V-ATPase’s role in lysosomal function suggests potential therapeutic strategies targeting this enzyme complex to mitigate cardiovascular disease progression. Understanding the mechanisms by which V-ATPase influences cardiovascular pathology is essential for developing novel treatments aimed at improving outcomes in patients with heart and vascular diseases.

Objective: Shenqi Granules is a traditional Chinese medicine formula derived from the classic prescription Shenqi Formula (SQF), composed of Astragali Radix (Huangqi) and Codonopsis Radix (Dangshen). SQF shows neuroprotection in cerebral ischemia-reperfusion injury (CIRI), but the underlying mechanisms remain unclear. Methods: The rat model of middle cerebral artery occlusion (MCAO) was established using the suture method to evaluate the neuroprotective effect of SQF. Blood-absorbed components were identified via UHPLC-Q-TOF-MS/MS, and potential targets and pathways were predicted through network pharmacology. Non-targeted metabolomics of serum and feces was performed using UHPLC-Orbitrap MS, and core metabolic pathways were selected based on regulation magnitude (RM). Gut microbiota structure was analyzed by 16S rRNA sequencing, with associations between microbiota and metabolites assessed via the Mantel test and redundancy analysis (RDA). Weighted gene co-expression network analysis (WGCNA) was applied to screen core Hub genes for ischemic stroke (IS). Based on single-cell RNA data, a uridine transport/metabolism-associated transcriptional signature score (Uridine_Score) was constructed, and cell type-specific coupling between glucocerebrosidase (GBA) and this transcriptional signature was analyzed. Microglial temporal trajectory analysis was conducted using Monocle3. Molecular docking between core components and GBA was verified by AutoDock, and GBA mRNA expression was confirmed by RT-qPCR. Results: High-dose SQF reduced infarct volume and neuronal damage and improved behavior. A total of 28 absorbed components and 31 shared IS targets were identified. Metabolomics revealed nucleotide metabolism as a core pathway common to both serum and fecal samples. SQF restored gut–blood uridine balance (serum uridine 0.41× Sham → 0.85× post-SQF), reduced fecal dTMP and hypoxanthine. 16S rRNA sequencing demonstrated that the overall correlation between microbial community structure and nucleotide metabolites was statistically significant (Mantel r = 0.307–0.321, p < 0.01). WGCNA highlighted GBA, MMP9, and PYGL. Single-cell analysis showed a microglia-specific uridine transport/metabolism-associated transcriptional signature post-stroke, with GBA most correlated with this signature (ρ = 0.377). Pseudotime revealed microglial state transitions (Homeostatic → Acute_Reactive → DAM), with positive GBA–uridine transport/metabolism-associated transcriptional coupling in the Homeostatic and DAM states. Riboflavin showed the strongest GBA binding (−9.83 kcal/mol), and SQF upregulated GBA mRNA in the penumbra (p < 0.0001). Conclusions: SQF reprograms nucleotide metabolism via the gut–blood axis and enhances microglial GBA–uridine transport/metabolism-associated transcriptional coupling.