Asiaticoside attenuates renal fibrosis by targeting STAT3 to restore Th17/Treg homeostasis

BACKGROUND: Renal fibrosis (RF) is a progressive pathological process driven by chronic inflammation and Th17/Treg imbalance. Asiaticoside (AS), a triterpenoid compound from Centella asiatica (L.) Urb., exhibits anti-inflammatory and antifibrotic activities, though its molecular mechanism remains unclear. OBJECTIVE: This study aimed to investigate whether AS alleviates RF by targeting Signal transducer and activator of transcription 3 (STAT3) through a "bind to destabilize" mechanism to restore Th17/Treg homeostasis. METHODS: An integrated approach combining network pharmacology, transcriptomics, and multimodal experimental validation was applied. UUO mice were treated with AS (10, 50, 100mg/kg/d) for 10 days. Histopathology, RNA‑seq, flow cytometry, immunofluorescence, Luminex, qPCR, DARTS‑LC‑MS/MS, molecular docking/dynamics simulations and SPR were performed. Pharmacological interventions using Stattic (STAT3 inhibitor) and Colivelin (STAT3 agonist) were included to functionally validate the role of STAT3. RESULTS: Network pharmacology identified STAT3 as the core target, with Th17 differentiation as the key pathway. AS treatment significantly attenuated RF, improved renal function, and rebalanced Th17/Treg ratios in UUO mice, accompanied by reduced IL-17A and elevated IL-10. Transcriptomic analysis revealed enriched Th17 cell differentiation genes, validated by qPCR. DARTS-LC-MS/MS confirmed direct binding of AS to STAT3 and identified a peptide derived from the SH2 domain (residues 582-602), indicating conformational destabilization. SPR showed high affinity binding to both human and murine STAT3. Molecular docking and dynamics simulations demonstrated a "local anchoring-allosteric effect" mode within the SH2 domain. qPCR analysis showed that AS significantly inhibited the mRNA expression of both IL-17A and total STAT3 in the renal tissues of UUO mice. Immunofluorescence revealed reduced STAT3 and p-STAT3 expression in kidneys. STAT3 inhibitor Stattic mimicked AS's antifibrotic and Th17 suppressive effects, whereas agonist Colivelin exacerbated fibrosis and was partially rescued by AS. CONCLUSION: AS alleviates RF via a novel "bind to destabilize" allosteric degradation mechanism that directly targets the STAT3 SH2 domain. This interaction induces conformational instability, suppresses STAT3 activation and transcriptional activity, restores Th17/Treg homeostasis, and ultimately mitigates renal inflammation and fibrosis. Collectively, these findings establish a new therapeutic strategy for STAT3-driven fibrotic diseases.