2. Academic Validation
  3. HPCAL1-BNIP3 axis promotes mitophagy-ferroptosis feedback loop that exacerbates intestinal ischemia-reperfusion injury

HPCAL1-BNIP3 axis promotes mitophagy-ferroptosis feedback loop that exacerbates intestinal ischemia-reperfusion injury

  • Free Radic Biol Med. 2025 Dec 31:245:223-236. doi: 10.1016/j.freeradbiomed.2025.12.054.
Ximeng Ren 1,  Xiaolong Lu 2,  Yao Song 1,  Meng Li 1,  Tianyu Shao 1,  Meihan Guo 3,  Yang Liu 4,  Dapeng Ding 5
Affiliations

Affiliations

  • 1 Department of Clinical Laboratory, The First Affiliated Hospital of Dalian Medical University, Dalian, 116011, China; Department of Pharmacology, College of Pharmacy, Dalian Medical University, 9 West Section, Lvshun South Road, Lvshunkou District, Dalian, 116044, China.
  • 2 Department of Pharmacology, College of Pharmacy, Dalian Medical University, 9 West Section, Lvshun South Road, Lvshunkou District, Dalian, 116044, China.
  • 3 Department of Clinical Laboratory, The First Affiliated Hospital of Dalian Medical University, Dalian, 116011, China.
  • 4 Department of Clinical Laboratory, The First Affiliated Hospital of Dalian Medical University, Dalian, 116011, China. Electronic address: [email protected].
  • 5 Department of Clinical Laboratory, The First Affiliated Hospital of Dalian Medical University, Dalian, 116011, China. Electronic address: [email protected].
PMID: 41482082 DOI: 10.1016/j.freeradbiomed.2025.12.054
Abstract

Background: Intestinal ischemia and reperfusion (I/R) injury is a critical pathological condition characterized by the complex interactions among various cell death mechanisms. This study aims to systematically elucidate the regulatory interplay between Ferroptosis and Mitophagy in intestinal I/R injury, particularly highlighting the pivotal role of the neuronal calcium-binding protein HPCAL1.

Methods: Using a mouse intestinal ischemia-reperfusion (I/R) model and a rat small intestinal epithelial cell (IEC-6) hypoxia/reoxygenation (H/R) model, we performed histopathological analysis; Western blotting; co-immunoprecipitation; qPCR; fluorescent probe-based detection; assays for Reactive Oxygen Species and lipid peroxidation; and assessments of mitochondrial membrane potential and autophagic flux. Using these techniques, we examined the time course and features of injury and Ferroptosis, mitochondrial dysfunction, and associated molecular regulatory mechanisms.

Results: The study demonstrated that intestinal ischemia-reperfusion (I/R) injury is significantly time-dependent, peaking at 60 min of reperfusion in vivo or 3 h of reoxygenation in vitro. Key Ferroptosis indicators, including increased ACSL4, decreased GPX4 and XCT, GSH depletion, and Fe2+ accumulation, were markedly altered at this peak. Mitophagy inhibition alleviated tissue injury and Ferroptosis, indicating excessive Mitophagy activation is detrimental. Mechanistically, HPCAL1 was highly expressed at the injury peak. It bound to the Mitophagy receptor BNIP3 in a calcium-dependent manner, enhancing BNIP3's stability and interaction with LC3-II, thereby excessively activating Mitophagy. This process promoted Ferroptosis via a burst of Reactive Oxygen Species (ROS), independent of GPX4 expression changes. Concurrently, the ROS burst activated an Nrf2-mediated compensatory antioxidant response. Disrupting HPCAL1 or BNIP3 effectively broke this cycle, improving cell survival and mitochondrial function.

Conclusion: This study identifies a CA2+ -mediated HPCAL1-BNIP3 signaling pathway that promotes Ferroptosis through ROS-dependent Mitophagy activation. It offers novel insights into the mechanisms underlying intestinal ischemia-reperfusion injury and supports the development of therapeutic strategies targeting the critical time window of injury progression as well as specific molecular targets.

Keywords

BNIP3; Ferroptosis; HPCAL1; Intestinal ischemia-reperfusion injury; Mitophagy; Reactive oxygen species.

Figures
Products