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

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.

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.

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.

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.

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.