Dendrobine ameliorates diabetic cardiomyopathy by targeting KEAP1 to disrupt KEAP1-NRF2 interaction and activate antioxidant defense

Dendrobium officinale extract DOE ameliorates metabolic disorders and diabetic cardiomyopathy. • DOE reduced myocardial injury, fibrosis, and oxidative stress via activating NRF2 pathway. • Dendrobine was the principal cardioprotective alkaloid in DOE against diabetic cardiomyopathy . • Dendrobine directly targets KEAP1 Kelch domain to disrupt the KEAP1-NRF2 interaction . Background Dendrobium officinale Kimura et Migo is a prestigious traditional Chinese medicine historically used for treating "Xiao-Ke" (diabetes and its complications). However, the specific material basis and direct molecular targets responsible for its efficacy against diabetic cardiomyopathy (DCM) remain largely obscure. This study aimed to systematically investigate whether dendrobine, the characteristic active alkaloid of DO, serves as the principal cardioprotective component by directly targeting the KEAP1-NRF2 signaling axis. Methods The therapeutic effects of D. officinale extract (DOE) and dendrobine were evaluated in vivo using db/db mice and in vitro using high-glucose (HG)-challenged H9c2 cardiomyocytes. Cardiac pathological remodeling, lipid peroxidation, and systemic metabolic profiles were comprehensively assessed. The direct interaction between dendrobine and KEAP1 was definitively characterized using Co-Immunoprecipitation (Co-IP), Cellular Thermal Shift Assay (CETSA), Drug Affinity Responsive Target Stability (DARTS), MicroScale Thermophoresis (MST), and 100-ns Molecular Dynamics (MD) simulations. Reverse pharmacological validation was performed using NRF2-specific siRNA and the inhibitor ML385. Results In vivo , DOE significantly ameliorated systemic insulin resistance, hyperlipidemia, and myocardial pathological remodeling in db/db mice while robustly suppressing myocardial oxidative stress (MDA, 4-HNE) by upregulating NRF2-driven antioxidant enzymes (HO-1, NQO1, GPX4). In vitro , dendrobine dose-dependently replicated these cardioprotective and antioxidative efficacies. Mechanistically, dendrobine promoted NRF2 nuclear translocation and initiated the downstream antioxidant cascade. Crucially, MST and CETSA revealed that dendrobine binds directly to KEAP1 with high affinity ( K d  = 1.18 μM), functioning as a competitive protein-protein interaction (PPI) inhibitor to disrupt the KEAP1-NRF2 complex. MD simulations and mutagenesis validation pinpointed the Kelch domain of KEAP1 (specifically residue Arg415) as the binding pocket. Finally, silencing or inhibiting NRF2 abolished the anti-hypertrophic and cytoprotective effects of dendrobine, and the in vivo administration of dendrobine successfully recapitulated the metabolic regulation and KEAP1/NRF2-mediated cardioprotection of DOE in db/db mice. Conclusion Dendrobine, the principal alkaloid of D. officinale , effectively ameliorates diabetic cardiomyopathy by functioning as a novel, direct competitive inhibitor of the KEAP1-NRF2 interaction. This targeted disruption liberates NRF2 to restore myocardial redox homeostasis, highlighting dendrobine as a promising lead compound for DCM therapy. Download: Download high-res image (395KB) Download: Download full-size image