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

  • Chin Med. 2026 Jun 10;21(1):167. doi: 10.1186/s13020-026-01440-x.
Shudong Lin  #  1 ,  Yunchao Huang  #  1 ,  Yiqiong Wang  1 ,  Yuhui Xue  1 ,  Shuyuan Lou  2 ,  Qiuru Wang  1 ,  Yaoting Wang  1 ,  Yunyun Lu  3 ,  Jiangyue Wu  4 ,  Shujing Zhang  4  5 ,  Houfa Yin  6  7 ,  Ting Zhang  1 ,  Lan Li  8 ,  Ling Zhang  9
Affiliations
  • 1. School of Life Sciences, Zhejiang Chinese Medical University, Hangzhou, 310053, Zhejiang, China.
  • 2. School of Medical Technology and Information Engineering, Zhejiang Chinese Medical University, Hangzhou, 310053, Zhejiang, China.
  • 3. School of Pharmaceutical Science, Zhejiang Chinese Medical University, Hangzhou, 310053, China.
  • 4. College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China.
  • 5. Innovation Institute for Artificial Intelligence in Medicine, Zhejiang University, Hangzhou, 310018, China.
  • 6. Eye Center, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310009, China.
  • 7. Zhejiang Provincial Key Laboratory of Ophthalmology, Zhejiang Provincial Clinical Research Center for Eye Diseases, Zhejiang Provincial Engineering Institute On Eye Diseases, Hangzhou, 310009, China.
  • 8. School of Medical Technology and Information Engineering, Zhejiang Chinese Medical University, Hangzhou, 310053, Zhejiang, China. [email protected].
  • 9. School of Life Sciences, Zhejiang Chinese Medical University, Hangzhou, 310053, Zhejiang, China. [email protected].
  • # Contributed equally.
PMID: 42271493 DOI: 10.1186/s13020-026-01440-x
Abstract

Background: 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.

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

Methods: 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.

Results: 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.

Conclusion: 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.

Keywords
Diabetic lower limb ischemia; Endothelial dysfunction; Heterophyllin B; Nrf2; Oxidative stress; Spermine oxidase (SMOX).
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