Icariside II attenuates renal fibrosis through mTOR signaling modulation: an integrated approach combining network pharmacology, molecular dynamics, and in vitro validation.

BACKGROUND

Renal fibrosis (RF), the end-stage progression of chronic kidney disease (CKD), remains a challenge due to limited effective therapies. Icariside II (ICA-II), a metabolite of icariin from the Chinese herb Epimedium, has shown therapeutic promise in treating diabetic CKD. However, the underlying molecular mechanisms remain elusive. This study sought to elucidate these mechanisms using an integrated approach: network pharmacology, molecular dynamics (MD) simulations, and in vitro experiments.

METHODS

Network pharmacology was integrated with public databases to identify ICA-II targets and RF-associated genes. Key targets were prioritized using protein-protein interaction (PPI) networks. Molecular docking and MD simulations assessed ICA-II-target interactions. In vitro, transforming growth factor β1 (TGF-β1)-induced normal rat kidney-49 fibroblast (NRK-49F) and human kidney 2 (HK2) cells were treated with ICA-II, with Cell Counting Kit-8 (CCK-8) assays, western blotting, and immunofluorescence evaluating cell viability, fibrotic markers and signaling proteins.

RESULTS

Network pharmacology revealed ten key targets: AKT1, mTOR, EGFR, ESR1, BCL2, CASP3, TP53, CTNNB1, HSP90AA1, and HSP90AB1. Critical pathways included PI3K/Akt/mTOR, lipid and atherosclerosis, and EGFR tyrosine kinase inhibitor resistance. Molecular docking showed stable ICA-II binding to mTOR (docking energy: -12 kcal/mol), confirmed by MD simulations over 100 ns. In vitro studies in NRK-49F and HK2 cells demonstrated that ICA-II directly binds to mTOR, as evidenced by cellular thermal shift assay (CETSA). This interaction potentially inhibits mTOR activity, leading to a significant downregulation of TGF-β1-induced expression of type I collagen and α-smooth muscle actin (α-SMA).

CONCLUSIONS

ICA-II demonstrates anti-fibrotic effects through a multi-target, multi-pathway regulatory network. Its beneficial role in RF may involve direct inhibition of mTOR activity, thereby attenuating excessive extracellular matrix (ECM) accumulation. These findings provide a theoretical and experimental basis for ICA-II as a promising candidate for the treatment of RF.