The central paradox of ischemia–reperfusion injury: Reperfusion as the primary driver of tissue damage

Ischemia–reperfusion (IR) injury reflects secondary pathological processes arising after reperfusion of hypoperfused organs. The central paradox is that reperfusion itself drives much of the tissue damage. In the lungs, reperfusion-induced damage is associated with structural and functional impairment. This acute phase is characterized by excessive release of reactive oxygen species (ROS) and inflammatory mediators, with consequent loss of membrane integrity and dysregulation of immune responses, which can result in microvascular hyperpermeability (pulmonary edema) and acute lung injury. Mechanistically, the cathepsin C axis in alveolar macrophages is an important modulator of this process; cathepsin C has been shown to regulate p38 mitogen-activated protein kinase-mediated p47 phosphorylation for nicotinamide adenine dinucleotide phosphate oxidase activation and is associated with increased pro-inflammatory cytokines [1].

Beyond the lungs, the heart exhibits a similar paradoxical response. This cascade, driven by ROS-mediated oxidative stress, advances toward proteomic modifications which compromise membrane integrity and provoke programmed cell death (apoptosis) and contribute to myocyte loss. Importantly, IR is characterized by the selective release of lysine crotonylation-modified proteins—predominantly from the contractile apparatus—with kinetic profiles that correlate with injury severity and suggest autonomous cross-reactivity of post-translational modifications. Despite this, there remains a significant gap in our understanding of the mechanisms at play and the exact molecular signatures that can be exploited for precision intervention.

Underlying these organ-specific manifestations is a common molecular hub: iron dysregulation. ROS generation is the initiator, with the buildup of unstable Fe(II) as a central molecular hub. The overwhelming intracellular Fe(II), in addition to directly oxidizing membrane structures, lipids, and nuclear DNA, also elicits a systemic inflammatory cascade. Interestingly, iron overload inhibits [4Fe-4S] cluster biogenesis, leading to the destabilization of lipoic acid synthase. As a result, the depletion of [4Fe-4S] inhibits protein lipoylation—a metabolic contributor to IR injury in the kidney [2]. Thus, exploring how Fe(II) interacts with the mitochondrial cofactors provides a theoretical basis to identify specific drugs against reperfusion injury  in clinical practice.

2.1 The interplay of ferroptosis, apoptosis, and necrosis

Iron-overload-activated ferroptosis is an important contributor to cell death in IR injury. Ischemia produces an energetic catastrophe, while reperfusion triggers a synergistic cascade—manifesting as bursts of ROS generation and inflammatory signaling, along with Ca²⁺ dysregulation—that acutely augments cellular demise. The ALOX15–15-HpETE axis, involving arachidonate 15-lipoxygenase and its metabolite 15-hydroperoxyeicosatetraenoic acid, is specifically involved in cardiomyocyte pathology. The pathological outcome is determined by a balance between regulated apoptosis and uncontrolled necrosis. Apoptosis is immunologically “silent”, whereas gross tissue injury tends to yield necrosis. This transition liberates damage-associated molecular patterns, generating a positive feedback loop of tertiary inflammation and  tissue destruction.

2.2 Initiation and persistence of the inflammatory cascade

Overwhelming inflammation is the major pathogenetic mechanism in IR. The major immunological stimuli that recruit immune effectors to the affected organ are fluctuations in oxygen levels and metabolic substrates after reperfusion, which trigger the release of damage-associated molecular patterns and cytokines. These leukocytes release ROS and proteases, which induce a self-perpetuating inflammatory loop and promote lesion expansion. Li et al. found that NOD-like receptor family pyrin domain containing 3 inflammasome activation and astrocyte pyroptosis after cerebral IR are dependent on the lipocalin 2–24p3 receptor axis [3]. This neurodegeneration-promoting autocrine and paracrine feedback is accelerated in the final stages of programmed cell death, when functional recovery becomes unattainable.

2.3 The mitochondrial–redox nexus: From bioenergetic failure to cell death

Mitochondrial dysfunction-induced oxidative stress is the key initiator of the IR injury cascade. This pathology, instead of being a binary phenomenon, is actually a continuum between sublethal bioenergetic failure and programmed cell death execution. Ischemia modifies the intracellular milieu through accumulation of metabolic intermediates; then reoxygenation produces an explosive burst of ROS. This oxidative stress propagates through ROS-induced ROS release and converges on the mitochondrial permeability transition pore—a decisive checkpoint for cell fate. Moreover, mitochondrial collapse leads to adenosine triphosphate depletion and calcium dysregulation that drive a pathophysiologic feedback loop of dysregulated mitophagy and inflammation that exacerbates tissue damage (Figure 1).

3.1 Myocardial IR injury: Clinical insights and therapeutic innovations

Herbal mixtures have long served as a source of pharmacological agents against IR injury after acute myocardial infarction (AMI), and are increasingly used as adjuvants in the clinical management of myocardial ischemia–reperfusion injury (MIRI). Emerging evidence indicates that the Yiqi Liangxue Shengji decoction improves cardiac function and attenuates MIRI, thereby relieving clinical symptoms of AMI and reducing the incidence of major adverse cardiovascular (CV) events [4]. Progress in the prevention of MIRI offers transformative approaches for improving AMI outcomes and advancing integrated CV care models. In addition, in-depth mechanistic studies and novel interventional approaches have the potential to attenuate or even reverse myocardial injury, providing a robust basis for early diagnosis and precision medicine in CV care.

3.2 Neurological sequelae of reperfusion injury

The neurological sequelae of reperfusion injury appear as a secondary event after restoration of blood flow to the brain. This process is characterized by the rapid disruption of cellular homeostasis; although flow is restored, neurons often irreversibly transition into a state of metabolic and structural dysfunction mediated by an intense inflammatory response. One important contributor to this neuroinflammatory cascade is the HDAC3–p65–cGAS–STING signaling pathway, which can potentiate innate immunity and contribute to focal neurological deficits [5]. In addition, disrupted neurotransmitter release in the damaged areas prevents synaptic stability, resulting in a loss of function. Finally, cerebral reperfusion injury not only amplifies the primary ischemic insult but also triggers sustained—and in many cases permanent—neurological dysfunction, which constitutes a critical barrier to clinical recovery.

3.3 Challenges of reperfusion injury in organ transplantation

IR injury is a nearly unavoidable pathological phenomenon in organ transplantation that profoundly damages early graft function and long-term graft survival, and further narrows the available donor pool [6]. In the presence of reperfusion, reflow and a sudden increase in oxygen level induce a massive spike of ROS overproduction, provoking oxidative damage to cellular membranes, proteins, and DNA. At the same time, this response activates damage-associated innate immune signaling and amplifies inflammatory cascades, both of which contribute to tissue injury and dysfunction. Such multistep insults impair graft viability and increase susceptibility to transplant rejection. This rejection remains a significant clinical barrier to successful transplantation outcomes.

4.1 Preconditioning: Priming the endogenous defense

Ischemic preconditioning is a classical cytoprotective strategy against reperfusion injury that involves exposing cells to brief sublethal hypoxia, thereby equipping cardiomyocytes with an intrinsic protective mechanism against subsequent stressors. Recent studies demonstrate that stanniocalcin 1 is induced by ischemic preconditioning and contributes to cellular defense against metabolic and reperfusion-induced oxidative stress, reducing necrosis and apoptosis [7]. This protective effect is primarily mediated by the upregulation of antioxidant defense systems, repression of pro-inflammatory signaling, and downregulation of stress-responsive pathways. Ultimately, these adaptive responses allow for better functional maintenance and protection of the heart in response to the imposed IR insult.

4.2 Pharmacological modulation of reperfusion injury

Pharmacological approaches to reperfusion injury focus mainly on oxidative stress, inflammatory pathways, and programmed cell death. Ligustilide (LIG) is an emerging candidate with potent therapeutic efficacy by virtue of its dual antioxidant and anti-inflammatory properties. LIG exerts cytoprotective effects by inhibiting ROS bursts, limiting inflammatory responses, and preventing microvascular perfusion loss. In cerebral IR models, LIG protects mitochondrial function and protects against both apoptosis and necrosis. Mechanistically, these effects are mediated by crosstalk with the PTEN-induced kinase 1 axis, which both prevents functional deterioration and promotes recovery. As such, a stratified phase-based intervention paradigm is proposed, targeting tissue resilience during ischemia, ROS scavenging and mitochondrial stabilization during reperfusion, and structural remodeling modulation during prolonged recovery (Figure 2).

4.3 Frontiers in targeted precision therapy

Targeted interventions have also emerged as a focus of IR research. One such agent is complement receptor 2–complement receptor 1-related protein y (CR2–Crry), a molecular species well-positioned for targeted intervention. CR2–Crry is a high-affinity, domain-specific inhibitor of the complement cascade that constrains pathogenic immune activation and thereby prevents tissue injury. CR2–Crry provides strong local protection with limited systemic coverage when administered prophylactically, a beneficial feature in the context of brain death-induced IR injury potentiation [8]. Indeed, the significant free radical scavenging activity, Hedgehog signaling inhibition, and anti-inflammatory properties of this compound suggest broad applicability across a spectrum of CV and cerebrovascular disorders. In fact, CR2–Crry is a prototype of the vectorial specificity needed to modulate reperfusion injuries that can best be applied using a site-directed mechanism of action and where expectations for a high-efficacy, low-toxicity paradigm can be realized.

Current biomarkers for IR injury have notable limitations, prompting the search for more sensitive and early indicators. The most widely used circulating biomarkers include lactate dehydrogenase and creatine kinase-MB, which are commonly applied to support the clinical assessment of IR injury. In this context, experimental modeling—including studies of epigenetic and metabolic remodeling—demonstrates systemic alterations in the levels of these enzymes, reflecting membrane disruption and leakage of cytosolic proteins, thereby contributing to their diagnostic relevance in cardiac injury assessment [9]. There is a shift away from traditional diagnostic markers toward mechanism-based biomarkers and next-generation multi-parameter panels. Therefore, future studies should explore ultra-early injury biomarkers with high sensitivity, such as microRNAs, selective post-translational modifications, or changes in metabolic intermediates that allow quantification of therapeutic effects.

Another promising direction is research on solute carrier family 26 member 6 (Slc26a6), which highlights its importance in modulating the IR environment. This mechanism has two components: modulation of acid-base balance in the myocardium via the Cl^-/HCO₃^- exchanger, and modulation of ischemia-induced acidosis. It has been demonstrated that ablation of Slc26a6 protects against ischemic metabolic acidosis through elevation of myocardial pH. Importantly, a key role of Slc26a6 has been described in the regulation of transmembrane ionic balance, which is a major determinant of myocardial susceptibility to IR injury [10]. Elucidation of these complex Slc26a6 functions should augment our understanding of cardiovascular pathophysiology with potential relevance to novel therapeutic frontiers and interdisciplinary science.

Finally, inadequate blinding in trial designs represents a significant barrier to the translation of IR-based interventions. Trials lacking double-blinding—where either the experimenter or the subjects remain unblinded—are susceptible to experimenter bias and subject expectancy effects. These confounders act to cloud therapeutic assessment and mask the actual effect of therapies directed toward reperfusion. Thus, future studies should be double-blind randomized controlled trials with rigorous methodological quality and greater clinical relevance. Here, we emphasize the importance of a strict blinding process to ensure objectivity and accurate assessment of treatment effects, thereby facilitating the bench-to-bedside translation of IR therapies.

AMI, Acute myocardial infarction; CV, Cardiovascular; HDAC3, Histone deacetylase 3; cGAS, cyclic GMP-AMP synthase; STING, stimulator of interferon genes; IR, Ischemia–reperfusion; LIG, Ligustilide; MIRI, Myocardial ischemia–reperfusion injury; RIPC, Remote ischemic preconditioning; ROS, Reactive oxygen species; CR2–Crry, complement receptor 2–complement receptor 1-related protein y; Slc26a6, solute carrier family 26 member 6.

Author contributions

Haotian Wu contributed to manuscript writing and figure preparation; Huan Zhang designed the work; Siyi Yan supervised the work. All authors have read and approved the final manuscript.

Funding

This research received no external funding.

Data availability

Data sharing not applicable to this article as no datasets were generated or analyzed during the current study. All information is derived from publicly available articles and datasets.

Ethics approval and consent to participate

Not applicable. This manuscript does not contain any studies with human participants or animals performed by any of the authors.

Consent for publication

Not applicable. This manuscript does not include details, images, or videos relating to an individual person.

Competing interests

The authors declare that they have no competing interests.

Acknowledgements

Not applicable.

[1] Yu J, Fu Y, Gao J, Zhang Q, Zhang N, Zhang Z, et al. Cathepsin C from extracellular histone-induced M1 alveolar macrophages promotes NETosis during lung ischemia-reperfusion injury. Redox Biol. 2024 Aug;74:103231. https://doi.org/10.1016/j.redox.2024.103231
[2] Chen S, Chen T, Xu C, Yu X, Shi J, Yang C, et al. Iron overload exaggerates renal ischemia-reperfusion injury by promoting tubular coproptosis via interrupting function of LIAS. Redox Biol. 2025 Oct;86:103795. https://doi.org/10.1016/j.redox.2025.103795
[3] Li J, Xu P, Hong Y, Xie Y, Peng M, Sun R, et al. Lipocalin-2-mediated astrocyte pyroptosis promotes neuroinflammatory injury via NLRP3 inflammasome activation in cerebral ischemia/reperfusion injury. J Neuroinflammation. 2023 Jun 23;20(1):148. https://doi.org/10.1186/s12974-023-02819-5
[4] Li Y, Li Y, Wang W, Cui X, Wan J, Zhou K, et al. Clinical study of Yiqi Liangxue Shengji prescription for improving cardiac function after myocardial ischemia reperfusion injury in patients with acute myocardial infarction: a randomized, double-blind, placebo-controlled trial. J Tradit Chin Med. 2025 Aug;45(4):836-844. https://doi.org/10.19852/j.cnki.jtcm.2025.04.012
[5] Liao Y, Cheng J, Kong X, Li S, Li X, Zhang M, et al. HDAC3 inhibition ameliorates ischemia/reperfusion-induced brain injury by regulating the microglial cGAS-STING pathway. Theranostics. 2020 Jul 29;10(21):9644-9662. https://doi.org/10.7150/thno.47651
[6] Guo Z, Zhao Q, Jia Z, Huang C, Wang D, Ju W, et al. A randomized-controlled trial of ischemia-free liver transplantation for end-stage liver disease. J Hepatol. 2023 Aug;79(2):394-402. https://doi.org/10.1016/j.jhep.2023.04.010
[7] Huang H, Ruan Y, Li C, Zheng H, Tang Y, Chen Y, et al. Hypoxia microenvironment preconditioning attenuated myocardial ischemia-reperfusion injury via Stc1-mediating cardiomyocyte self-protection and neutrophil polarization. Adv Sci (Weinh). 2025 Feb;12(6):e2411880. https://doi.org/10.1002/advs.202411880
[8] Lei B, Sleiman MM, Cheng Q, Tu Z, Zhu P, Goddard M, et al. In situ pre-treatment of vascularized composite allografts with a targeted complement inhibitor protects against brain death and ischemia reperfusion induced injuries. Front Immunol. 2021 Jul 29;12:630581. https://doi.org/10.3389/fimmu.2021.630581
[9] Boovarahan SR, AlAsmari AF, Ali N, Khan R, Kurian GA. Targeting DNA methylation can reduce cardiac injury associated with ischemia reperfusion: One step closer to clinical translation with blood-borne assessment. Front Cardiovasc Med. 2022 Sep 28;9:1021909. https://doi.org/10.3389/fcvm.2022.1021909
[10] Thai PN, Ren L, Diloretto DA, Trinh P, Timofeyev V, Zong N, et al. Ablation of Slc26a6 mitigates myocardial ischemia/reperfusion injury. Biomedicines. 2025 Nov 25;13(12):2874. https://doi.org/10.3390/biomedicines13122874