Heterophyllin B ameliorates diabetic lower limb ischemia by inhibiting SMOX to activate the Nrf2 antioxidant pathway

Diabetic lower limb ischemia (DLLI) is a serious complication of diabetes with limited therapeutic options. Heterophyllin B (HET-B), a bioactive cyclopeptide from Pseudostellaria heterophylla, possesses antioxidant properties. However, its therapeutic mechanism in DLLI remains unclear.

This study aims to investigate the protective effects of HET-B against DLLI and elucidate the underlying metabolic and molecular mechanisms.

A murine DLLI model was established in streptozotocin-induced diabetic mice via femoral artery ligation. Therapeutic efficacy was assessed by laser Doppler imaging, histopathology, and immunofluorescence. A high glucose-induced endothelial injury model was established using HUVECs. Endothelial function was evaluated by tube formation, migration, and wound healing assays. Single-cell RNA sequencing data (GSE165816) were analyzed to identify key metabolic targets. Mechanistic validation was performed using Spermine oxidase (SMOX) gene silencing and pharmacological activation with spermine.

HET-B significantly improved hindlimb blood flow recovery, promoted angiogenesis with increased CD31, α-SMA, VEGF, and eNOS, and attenuated inflammation with reduced TNF-α, IL-6, IL-1β, and TGF-β in ischemic muscles. In HUVECs, HET-B restored high glucose-impaired endothelial function, promoted Nrf2 nuclear translocation with NQO1 upregulation, suppressed TNF-α expression, and subsequently reduced Caspase-1/3 activation. Bioinformatic analysis identified SMOX as a key dysregulated gene in diabetic endothelium, and HET-B reversed its overexpression both in vitro and in vivo. Immunofluorescence co-staining confirmed SMOX and Nrf2 localization in CD31-positive endothelial cells, with HET-B reversing SMOX upregulation while restoring Nrf2 activation. SMOX knockdown mimicked HET-B effects, whereas SMOX activation with spermine abrogated HET-B-mediated protection, Nrf2 activation, and NF-κB suppression, confirming that HET-B acts through functional inhibition of SMOX.

HET-B alleviates DLLI by inhibiting SMOX to activate Nrf2-mediated antioxidant defense and suppress inflammatory signaling, suggesting that the SMOX-Nrf2 axis may represent a potential therapeutic target for DLLI.