Senkyunolide A from Danggui Buxue Decoction Protects Podocytes Against Diabetic Nephropathy via the miR-223-3p/NLRP3 Inflammasome Axis

Ethnopharmacological relevance Danggui Buxue Decoction (DBD), a classical Traditional Chinese Medicine (TCM) formula comprising Astragalus membranaceus (Fisch.) Bunge (Astragali Radix) and Angelica sinensis (Oliv.) Diels (Angelicae Sinensis Radix) in a 5:1 ratio, has been traditionally used to treat “Xiaoke” (wasting-thirst syndrome) and is increasingly applied as an adjunctive therapy for diabetic nephropathy (DN). DN is a major microvascular complication of diabetes mellitus for which effective curative therapies remain limited. Although DBD has demonstrated promising clinical efficacy in the prevention and management of DN, its pharmacologically active constituents and underlying mechanisms of action have yet to be fully elucidated. Aim of the study This study aimed to comprehensively profile the blood-entering bioactive constituents of DBD, identify the core active compound through integrated UPLC-Q-TOF-MS/MS and network pharmacology analyses, and elucidate the molecular mechanisms by which the core constituent protects against high glucose-induced podocyte injury, with particular focus on the miR-223-3p/NLRP3 inflammasome axis. Materials and methods UPLC-Q-TOF-MS/MS was employed to globally characterize the chemical constituents of Danggui Buxue Decoction (DBD), and the absorbed components and in vivo metabolites were identified in a DN rat model. Network pharmacology combined with target intersection analysis was used to identify the core active constituents. Subsequently, PPI network construction, GO functional annotation, and KEGG pathway enrichment analyses were performed to elucidate the underlying molecular mechanisms. In a high glucose–induced MPC5 podocyte model, CCK-8, TUNEL, and JC-1 assays were conducted to evaluate the cytoprotective effects of the major active constituents. Furthermore, Western blotting, qRT-PCR, and dual-luciferase reporter assays were performed to clarify the regulatory mechanism of the miR-223-3p/NLRP3 axis. Results UPLC-Q-TOF-MS/MS identified a total of 1,584 chemical constituents in DBD, among which 249 absorbed components and 155 in vivo metabolites were detected in the plasma of DN model rats. Network pharmacology analysis identified senkyunolide A (SA) as the core active constituent, which targeted 33 NLRP3 inflammasome–related proteins, covering the entire activation cascade of this pathway. PPI network construction and enrichment analyses further revealed that SA-associated targets were involved in key biological processes at the systems level, including oxidative stress and PI3K-Akt/NF-κB signaling pathways. In vitro experiments demonstrated that SA at a concentration of 50 μmol/L significantly ameliorated high glucose–induced podocyte injury, apoptosis, and mitochondrial dysfunction. Mechanistically, SA upregulated miR-223-3p expression, thereby directly inhibiting NLRP3 inflammasome activation, which in turn suppressed Caspase-1 cleavage and GSDMD-mediated pyroptosis. Meanwhile, SA maintained mitochondrial dynamic homeostasis and energy metabolism by regulating DRP1 phosphorylation, upregulating OPA1 and MFN2, and activating the PGC-1α/MnSOD pathway, ultimately exerting multi-level protective effects on podocytes. Conclusions SA is identified as the core bioactive constituent of DBD responsible for its therapeutic effects in DN. SA exerts multi-level renoprotective effects by upregulating miR-223-3p, suppressing NLRP3 inflammasome activation and GSDMD-mediated pyroptosis, and maintaining mitochondrial dynamic homeostasis via the PGC-1α/MnSOD pathway. These findings provide a scientific basis for the rational application of DBD in the clinical prevention and treatment of diabetic nephropathy and highlight the miR-223-3p/NLRP3 axis as a promising therapeutic target.