Protective effect of ischemic preconditioning against hepatic ischemia-reperfusion injury and associated remote organ damage

Abstract

Hepatic ischemia-reperfusion injury is a significant complication of liver surgery, including major hepatectomy, trauma surgery and liver transplantation. It is a key factor in postoperative organ failure, which negatively affects prognosis and overall patient survival. Beyond its localized hepatic effects, ischemia-reperfusion injury is increasingly recognized as a potent trigger of the systemic inflammatory response and remote organ damage. The cellular and molecular mechanisms involved are highly complicated and have yet to be entirely elucidated. The core pathophysiological mechanisms of hepatic ischemia-reperfusion injury include a transition to anaerobic metabolism and adenosine triphosphate depletion; the development of intracellular acidosis and calcium overload; the impairment of mitochondrial function; oxidative stress; the activation and accumulation of distinct cell populations, notably Kupffer cells, neutrophils and platelets; the upregulation and downregulation of microRNAs; increased nitric oxide production; and the triggering of an immune system response with the activation of the complement system and excessive cytokine release. Ischemic preconditioning (IP) is a surgical technique in which brief cycles of controlled ischemia followed by reperfusion are applied directly to an organ, aiming to enhance its tolerance to subsequent prolonged ischemia. Hepatic IP has been demonstrated to reduce ischemia-reperfusion injury by decreasing the release of proinflammatory cytokines and damage-associated molecular patterns; suppressing reactive oxygen species production; activating the antioxidant enzyme heme-oxygenase 1, caspase, heat shock proteins and protein kinase cascades; modulating energy supplies and electrolyte homeostasis; and intervening in cell death pathways. In addition to its local effects on the liver, growing evidence indicates that IP also provides systemic advantages by reducing the inflammatory response and limiting injury to distant organs following major hepatic injury. This review integrates current data on IP, highlighting its role in hepatic protection and prevention of remote organ damage, while exploring the underlying mechanisms and translational potential of this approach in hepatic surgery and transplantation.

Key Words: Hepatic ischemia-reperfusion injury; Remote organ damage; Systemic inflammatory response; Ischemic preconditioning; Liver transplantation; Protective method; Pathophysiological mechanisms

Core Tip: Hepatic ischemia-reperfusion injury remains a critical challenge in liver surgery and transplantation and results in increased postoperative morbidity and mortality. The complexity of the underlying pathophysiological mechanisms makes liver damage difficult to manage. Ischemic preconditioning is a promising protective strategy that limits hepatic injury while providing systemic benefits through the regulation of oxidative stress, the inflammatory response, and programmed cell death pathways. By offering protection to remote organs, ischemic preconditioning has the potential to enhance the outcomes of transplantation and other high-risk procedures, although further studies are needed to establish optimal clinical protocols.

INTRODUCTION

Hepatic ischemia-reperfusion injury (HIRI) is a serious complication that occurs during the restoration of blood supply to the liver following prolonged temporary occlusion of blood flow. This condition commonly occurs during liver surgery, especially in major hepatectomies, trauma management and transplantation. However, several other nonsurgical conditions, including heart failure, severe hypotension and shock, have also been associated with HIRI[1-3]. The incidence of clinically significant surgically induced HIRI is not clearly defined; nonetheless, it is recognized as a significant contributing factor to postoperative liver dysfunction and delayed recovery. The prevalence of end-stage liver disease and associated mortality have increased markedly in recent years, and liver transplantation remains the only therapeutic option for these patients[4]. During liver transplantation, HIRI affects almost all grafts to varying degrees, and its severity is correlated with high morbidity, primary graft dysfunction and even graft loss[5]. Moreover, the growing demand for liver grafts and the urgent need for expansion of the graft pool have led to the acceptance of grafts procured from donors under expanded criteria (grafts) and from donors after cardiac death (grafts)[6,7]. Owing to certain risk factors and prolonged warm ischemia time during the donation process, these grafts are particularly vulnerable to HIRI, leading to a higher incidence of postoperative complications, including biliary stricture, primary nonfunction, and delayed graft function[8,9].

HIRI results from a multimechanistic process involving reactive oxygen species (ROS) production and oxidative stress, mitochondrial dysfunction, anaerobic metabolism and intracellular acidosis, calcium overload and activation of the innate inflammatory response and cell death signaling pathways[10,11]. Furthermore, there is strong evidence of systemic complications, as multiple remote organs appear to be affected during this process, highlighting the extensive pathophysiological effects of HIRI[12]. Various therapeutic interventions, including pharmacological agents and specific surgical techniques, have demonstrated encouraging results in preclinical and clinical settings[13].

Ischemic preconditioning (IP) refers to a surgical procedure in the form of repeated brief cycles of ischemia followed by reperfusion that increase the resistance of organs to ischemia-reperfusion injury. This method was originally described in the myocardium, and its application to the liver was first reported in 1993 by Lloris-Carsν et al[14-16]. Experimental and clinical evidence has revealed that IP enhances the liver’s tolerance for ischemia and ameliorates hepatic injury[17]. This phenomenon involves complex signaling pathways that activate numerous cellular defense mechanisms, which include reducing ROS production, modulating the inflammatory response, stabilizing mitochondrial function and inhibiting apoptosis[18]. However, despite the clinical applications of IP, the precise molecular mechanisms underlying HIRI and IP-induced protection remain to be elucidated.

While the local protective effects of hepatic IP have been studied, emerging evidence suggests that its benefits may extend beyond the liver, contributing to remote organ protection in the context of HIRI. The concept of IP as a modulator of systemic protection has been demonstrated in lung, kidney, heart, intestine, pancreas and brain injury models[19-23]. These beneficial effects are reported to be mediated through complex neurohumoral pathways involving circulating mediators, proinflammatory factors and programmed cell death modulation. These observations suggest that hepatic IP may induce a systemic conditioning response, suggesting promising therapeutic implications in the contexts of shock, multiorgan dysfunction and high-risk surgical interventions. In this narrative review, we discuss current knowledge of the mechanisms whereby hepatic IP protects against HIRI and associated remote organ damage, and we describe its possible translation into clinical practice.

PATHOPHYSIOLOGICAL MECHANISMS OF HIRI

Liver functions

The liver performs a multitude of vital functions essential to the maintenance of physiological balance in humans. Macronutrient metabolism, detoxification and drug metabolism, bile production, regulation of blood volume, support of the immune system and coagulation, vitamin and mineral storage, protein synthesis and maintenance of lipid and cholesterol balance are among the fundamental roles of the liver[24]. The structural organization of the liver and its dual blood supply support its metabolic and secretory functions. The liver consists of multiple cell types derived from different embryological origins, including hepatocytes, cholangiocytes (biliary epithelial cells), hepatic stellate cells, Kupffer cells, and liver sinusoidal endothelial cells. Together, these cells form the liver acinus, the functional microanatomical unit of the liver, which is organized around the terminal branches of the portal triad (hepatic arteriole, portal venule, and bile ductule). Approximately 75% of the liver’s blood inflow originates from the portal vein, which delivers nutrient-rich but oxygen-poor blood from the gastrointestinal tract, pancreas, and spleen. The remaining 25% is supplied by the hepatic artery, which provides oxygen-rich blood from the systemic circulation. The liver acinus is divided into three zones (1, 2, and 3), defined according to their proximity to the portal triad and central vein. Zone 1, also known as the periportal zone, receives the most oxygenated blood; zone 2 has intermediate exposure, and zone 3, or the centrilobular zone, is the most distant and thus the most susceptible to hypoxia (Figure 1). Overall, the liver receives approximately 25% of the cardiac output. The liver’s unique dual vascular system has the capacity to partially compensate for disruptions in organ perfusion[25-27]. However, when the compensatory mechanism is no longer sufficient to maintain adequate oxygen delivery, the liver becomes vulnerable to HIRI.

Figure 1 Schematic view of normal liver acinus.

Transition to anaerobic metabolism and acidosis

HIRI is defined as the phenomenon in which hepatocellular damage during liver ischemia is paradoxically exacerbated during the restoration of blood flow[28]. HIRI can typically be categorized into two main types: Warm and cold. Warm ischemia occurs in the contexts of elective liver surgery, liver transplantation, hemorrhagic shock, trauma and donation after cardiac death, where hepatic blood flow is temporarily interrupted at physiological temperature (about 37 °C). Cold hepatic ischemia is associated with organ preservation before liver transplantation[28-30]. Hepatocytes are predominantly damaged during warm ischemia, whereas liver sinusoidal endothelial cells are more severely injured during cold ischemia. Despite notable variations, especially in their clinical contexts, the two types of HIRI share similar underlying pathophysiological mechanisms based on the induction of the innate immune response[31,32].

A reduction in hepatic blood flow results in a deficient supply of oxygen as well as glucose and other nutrients. The low-oxygen environment forces hepatocytes to shift their metabolic state from aerobic to anaerobic, a change that is associated with decreased adenosine triphosphate (ATP) levels. ATP depletion and anaerobic glycolysis lead to intracellular lactic acid accumulation, which, together with impaired mitochondrial oxidative phosphorylation, results in high-anion-gap metabolic acidosis[33]. Hepatocyte edema and membrane destabilization occur during this phase as a result of compromised ion homeostasis. Specifically, an increased intracellular hydrogen ion concentration forces ion exchange and the influx of sodium into cells. Furthermore, the reduced function of the ATP-dependent sodium-potassium pump contributes to increased intracellular pressure and disruption of cell structure[34,35]. Paradoxically, the acidic intracellular environment may play a protective role, as low-pH conditions suppress the activity of proteolytic enzymes, such as proteases, phosphatases and phospholipases, and limit mitochondrial permeability transition pore (MPTP) function[36].

Similarly, impaired calcium homeostasis has been associated with ischemic cellular injury. Intracellular sodium accumulation induces sodium-calcium exchange, leading to an influx of calcium into the cytoplasm. Increased intracellular calcium ion concentrations have been shown to activate numerous calcium-dependent enzymes, including protein kinase C (PKC), phospholipase C, xanthine dehydrogenase and calcium-dependent proteases[37]. Increased cytosolic calcium triggers the opening of the MPTP, which further disrupts the mitochondrial membrane potential; causes mitochondrial edema; and leads to the release of ions, metabolic compounds and proapoptotic factors[38]. Thus, calcium overload process plays a pivotal role in the mediation of programmed cell death, predominantly through stimulation of the apoptotic signaling cascade[39-41].

Oxidative stress

Reperfusion of previously ischemic liver tissue and restoration of oxygen and nutrient delivery further exacerbate cellular injury through a cascade of events, including activation of Kupffer cells, increased ROS production and oxidative stress, mitochondrial dysfunction, and induction of the inflammatory response[42]. Post-reperfusion injury can be categorized into two distinct phases on the basis of time. The early phase occurs within the first two hours after reperfusion, when the rapid activation of resident Kupffer cells and sinusoidal endothelial cells results in the release of ROS and proinflammatory cytokines, such as tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β). The late phase, characterized by the infiltration of neutrophils and CD4+ T lymphocytes, occurs between approximately 6 hours and 48 hours after reperfusion. These infiltrating cells release proteases and other inflammatory mediators, thereby contributing to cellular damage[28].

ROS are chemically reactive molecules containing oxygen (superoxide radicals, hydrogen peroxide, hydroxyl radicals, and singlet oxygen); they are normally generated as byproducts during physiological processes, such as oxidative phosphorylation; redox regulation of proteins; cell proliferation, differentiation, and apoptosis; activation of signaling factors; and the immune response[43]. To neutralize excess ROS, cells rely on both enzymatic and nonenzymatic antioxidant defense systems to maintain homeostasis. The enzymatic antioxidant network consists of superoxide dismutase, catalase, glutathione peroxidase and thioredoxin, which have effective protective properties, while numerous vitamins, minerals, metabolites and flavonoids have nonenzymatic antioxidant activities[44]. During HIRI, the imbalance between ROS production and removal leads to elevated intracellular ROS levels, known as “oxidative stress”. In the liver, ROS originate primarily in the mitochondria and endoplasmic reticulum of hepatocytes, Kupffer cells and activated neutrophils, which produce them through mechanisms including the respiratory chain, peroxisomes, xanthine oxidases, cytochrome P450 oxidases and nicotinamide adenine dinucleotide phosphate oxidases[45]. ROS can induce damage to vital cellular components, including lipid membranes, proteins, and DNA, ultimately compromising cell viability and function[46-48]. Specifically, the oxidation of proteins may have negative functional consequences, and oxidative damage to nucleic acids may result in genomic instability, while the peroxidation of membrane lipids, particularly polyunsaturated fatty acids, can trigger cell death by activating the apoptosis and ferroptosis pathways[49]. These mechanisms, along with sinusoidal endothelial dysfunction, result in hepatic microcirculation abnormalities and collectively contribute to hepatocyte death[50].

Mitochondria play a central role in the pathogenesis of hepatocyte damage, as HIRI significantly affects mitochondrial function. Mitochondria are crucial for the maintenance of cell energy balance and cell survival. To preserve functional homeostasis, mitochondria have numerous quality control mechanisms, including biogenesis, kinetics, and mitochondrial autophagy (known as mitophagy). The perturbation of these mechanisms during HIRI leads to the suppression of mitophagy and the accumulation of damaged mitochondria[37]. Furthermore, mitochondria are the main site of ROS production, which results in the inhibition of oxidative phosphorylation, the depletion of ATP and the onset of oxidative stress[51]. Factors such as intracellular calcium ions and ROS trigger long-term MPTP opening, inhibit ATP production and activate apoptotic and necrotic pathways. Mitochondrial membrane potential loss and osmotic dysregulation cause massive edema of mitochondria and the activation of outer mitochondrial membrane proteins, such as Bax and Bad. These proteins promote the rupture of the mitochondrial membrane and the release of proapoptotic molecules, such as cytochrome c, into the cytosol[52]. In parallel, mitochondrial DNA (mtDNA) is highly vulnerable to oxidative stress during HIRI, as it lacks histones and has limited DNA repair capacity. Increased intracellular ROS production leads to DNA fragmentation and a high mutation rate. Damaged mtDNA compromises mitochondrial protein synthesis and acts as a damage-associated molecular pattern (DAMP) when it is released into the cytosol or extracellular space. Cytosolic damaged mtDNA triggers signaling pathways of the innate immune system, such as the cGAS-STING signaling pathway, Toll-like receptor 9 and the intracellular polyprotein complex NLRP3 inflammasome, promoting a systemic inflammatory response and cell death[53-56]. Although mitochondrial dysfunction plays a central role in apoptotic cell death, current data demonstrate that mitochondria are also involved in other forms of regulated cell death, including necrosis, pyroptosis and ferroptosis[57].

The early phase of post-reperfusion injury is characterized mainly by the activation of Kupffer cells, the resident macrophages of the liver. Prolonged ischemia causes the death of cells that are particularly sensitive to hypoxia (zone 3 of the liver acinus). DAMPs released from initially dead cells primarily activate Kupffer cells through pattern recognition receptors[58]. Activated Kupffer cells are responsible for the production of a variety of proinflammatory factors, including ROS; the cytokines TNF-α, IL-1β, and IL-6; chemokines, such as monocyte chemoattractant protein-1; and DAMPs, such as high mobility group box 1 (HMGB1) protein, S100 proteins, heat shock proteins (HSPs) and circulating RNA and DNA. These mediators promote hepatocellular damage through the recruitment and activation of circulating immune cells[29,59]. In addition, activated Kupffer cells trigger the expression of the adhesion molecules intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 on sinusoidal endothelial cells, promoting the recruitment of neutrophils and CD4+ T lymphocytes. These cellular interactions facilitate progression from the early phase to the late phase of HIRI, which is characterized mainly by neutrophil accumulation and activation[60,61]. Once activated, neutrophils produce excessive amounts of ROS and myeloperoxidase (MPO). MPO catalyzes the formation of hypochlorous acid from hydrogen peroxide, which, together with ROS, contributes significantly to direct hepatocellular damage[62,63]. In addition to MPO, neutrophils produce proteases such as elastase, cathepsin G, heparanase, collagenase and hydrolytic enzymes that promote cellular injury, edema and microvascular thrombosis[42]. Another notable mechanism of neutrophil-induced damage is the formation of neutrophil extracellular traps, which are web-like structures composed of DNA, histones and other proteins that contribute to inflammatory and thrombotic processes[64]. Notably, Kupffer cells may also play a protective role during HIRI through the expression of anti-inflammatory mediators, such as IL-10, IL-13 and hepatic heme oxygenase-1; the upregulation of vascular endothelial growth factor and hepatocyte growth factor; and the phagocytosis of damaged cells[33,58,65].

Platelets also play a crucial role in the pathogenesis of HIRI by adhering to sinusoidal endothelial cells via P-selectin and integrin αIIbβ3, which contributes to microvascular embolism and impaired sinusoidal perfusion[66,67]. Activated platelets release multiple factors, including serotonin, thromboxane A₂, growth factors (transforming growth factor-β and vascular endothelial growth factor A), and plasminogen activator inhibitor 1, promoting vasoconstriction, oxidative stress, and endothelial dysfunction. Meanwhile, their interactions with Kupffer cells and activated neutrophils increase the production of TNF-α, IL-1β and ROS, thereby enhancing the inflammatory cascade[68-70].

During HIRI, excessive cytokine release and a complex inflammatory cascade play crucial roles in the pathophysiology of liver and remote organ damage. Interestingly, cytokines may play a dual role in HIRI, acting as mediators of both tissue damage and repair processes[33]. In the early post-reperfusion phase, activated Kupffer cells release proinflammatory cytokines, such as TNF-α, IL-1β, IL-6, IL-12, IL-23, interferon γ and chemokines [C-X-C motif ligand 1 (CXCL1), CXCL2, and CXCL5], with TNF-α and IL-1β being identified as major mediators of HIRI[42,64,71]. This cytokine/chemokine storm promotes the recruitment and adhesion of circulating immune cells and further exacerbates the inflammatory response, contributing to hepatocellular injury. By binding to the receptors of hepatocytes, TNF-α increases the production of ROS and epithelial neutrophil-activating protein 78. Furthermore, the activation of the nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) pathways promotes the expression of ICAM-1 and vascular cell adhesion molecule 1 adhesion molecules on sinusoidal endothelial cells and causes direct cellular damage[72]. In the late phase of HIRI, recruited inflammatory cells continue to release proinflammatory cytokines, exacerbating hepatocyte apoptosis and microvascular dysfunction[73]. Conversely, anti-inflammatory cytokines such as IL-10, IL-33, and IL-37 are also released during the late phase, leading to the suppression of NF-κB activation and reductions in neutrophil accumulation and proinflammatory cytokine expression, with the effect of limiting hepatocellular damage[74-76].

Pathogenesis of HIRI

Complement components have been recognized as mediators of both HIRI and liver regeneration. HIRI-induced DAMPs activate the classic, alternative, and lectin complement pathways, leading to the cleavage of C3 and C5. The resulting biologically active fragments C3a and C5a promote neutrophil recruitment and Kupffer cell activation and amplify oxidative stress and cytokine release. Additionally, the membrane attack complex (C5b-9) directly injures hepatocytes and sinusoidal endothelial cells, exacerbating necrosis. Beyond direct cytotoxicity, membrane attack complex also contributes to the inflammatory response by enhancing neutrophil recruitment and adhesion to the endothelium, stimulating the release of ROS and cytokines[77,78].

In the past decade, microRNAs (miRNAs) have been identified as significant mediators of HIRI, playing a role as post-transcriptional gene regulators that affect the expression of proteins involved in HIRI. miRNAs are a group of small endogenous single-stranded noncoding RNAs that are approximately 21-25 nucleotides in length[79]. The upregulation of several miRNAs, including miR-122, miR-450-5b, miR-155, miR-210, miR-191, miR-450-5b, miR-370, miR-34, miR-297, miR-497-5p, and miR-128-3p, promotes the inflammatory response and apoptotic cell death. The downregulation of miR-146a, miR-194, and miR-142-3p during HIRI targets different genes with the common consequence of activating the NF-κB pathway, resulting in increased inflammation[80,81]. Experimental modulation of miRNA expression using either mimics or inhibitors has been demonstrated to reduce hepatocellular damage, highlighting their significance both as biomarkers and as promising therapeutic targets in HIRI[79,82,83].

Nitric oxide (NO) plays an important and contradictory role in the progression of HIRI, as its formation and alterations in its concentration may significantly affect the degree of injury. Endothelial NO synthase (eNOS)-derived NO improves hepatic microcirculation by promoting vasodilation, neutralizing ROS, inhibiting platelet aggregation and limiting leukocyte adhesion, thereby reducing oxidative stress, the inflammatory response and cellular injury. In contrast, NO production by inducible NO synthase (iNOS) during the reperfusion phase exacerbates hepatocellular injury and leads to excessive cytokine and ROS production[28,84]. Moreover, NO reacts with superoxide to form peroxynitrite, a highly reactive oxidant, which causes direct cellular damage through lipid peroxidation and mitochondrial dysfunction[85]. Depending on the context, NO may reduce the production of leukotriene C4 by downregulating C4 synthase through the inhibition of the NF-κB pathway, whereas the regulation of other apoptotic pathways (caspases, the Bcl-2 family, and MAPK) may inhibit or induce hepatocyte apoptotic processes caused by HIRI[86]. NO may also affect neutrophil apoptosis. Specifically, iNOS-derived NO prolongs neutrophil survival by activating the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) pathway, inhibiting caspase-3 and caspase-9 and thereby sustaining inflammatory responses. Reduced eNOS-derived NO similarly promotes neutrophil apoptosis by inducing mitochondrial dysfunction, which activates caspase-dependent cell death and suppresses the NF-κB signaling pathway[87]. Thus, the balance between eNOS- and iNOS-derived NO critically determines whether NO acts as a cytoprotective or cytotoxic factor in HIRI.

The mechanisms involved in the pathogenesis of HIRI, including excessive ROS generation, lipid peroxidation, disruption of calcium homeostasis, mitochondrial dysfunction, activation of Kupffer cells and cytokine release, subsequently lead to a massive inflammatory response that promotes the activation of multiple programmed cell death pathways[88]. Although necrosis appears to be the most significant form of cell death during HIRI, other processes of programmed cell death may be complementarily involved and contribute to complex hepatocellular damage. Apoptosis involves two main pathways, namely, the exogenous pathway activated by death receptors and the endogenous mitochondrial death pathway, both of which integrate signaling from the transforming growth factor-β-activated kinase 1, JNK, p38 MAPK, apoptosis signal-regulating kinase 1, and PI3K-Akt pathways. Pyroptosis is a proinflammatory cytosolic death mechanism mediated by inflammasome activation (NLRP3); caspases 1, 4, and 11; and the gasdermin D family, which additionally exacerbates hepatic inflammation. Ferroptosis is a form of programmed cell death characterized by iron-dependent lipid peroxidation and membrane damage. Other forms of cell death, including autophagy, neutrophil extracellular trap-associated cell death, parthanatos, and copper-induced and lysosome-dependent pathways, may also contribute, but their roles appear less prominent and remain incompletely defined[89,90]. The pathophysiological mechanisms of HIRI are summarized in Table 1.

Table 1 Pathophysiological mechanisms of hepatic ischemia-reperfusion injury.

Mechanism	Description
Anaerobic metabolism	↓ ATP, intracellular metabolic acidosis, abnormal ion homeostasis
Calcium overload	↓ Intracellular calcium, activation of calcium-dependent enzymes, opening of MPTP, mediation of programmed cell death
Immune response	Kupffer cells activation, release of ROS and proinflammatory cytokines, infiltration of neutrophils and CD4+ T-lymphocytes
Kupffer cells activation	Release of ROS, TNF-α, IL-1β, IL-6, MCP-1, DAMPs, HMGB1, S100 proteins, heat shock proteins. Expression of ICAM-1, VCAM-1
Neutrophil activation	Release of ROS and MPO, production of elastase, cathepsin G, heparanase, collagenase and hydrolytic enzymes, NET formation
Oxidative stress	ROS production, damage of proteins and DNA, peroxidation of lipid membranes, activation of cell death pathways
Microcirculatory disturbance	Sinusoidal endothelial dysfunction, microvascular thrombosis
Mitochondrial dysfunction	Suppression of mitophagy, ROS production, ATP depletion, opening of MPTP, edema of mitochondria, rupture of mitochondrial membrane, damage of mtDNA, triggering of inflammatory response, activation of cell death pathways
Platelets accumulation	Microvascular embolism, release of serotonin, thromboxane A2, TGF-β, VEGF-A, PAI-1, enhancement of cytokine production
Complement activation	Promotion of neutrophil recruitment, Kupffer cells activation and cytokine release, direct hepatocytes and sinusoidal endothelial cells damage by MAC
miRNAs modulation	Post-transcriptional gene regulators, promotion of inflammatory response and programmed cell death, activation of NF-κB pathway
NO production	eNOS-derived NO improves hepatic microcirculation, promotes vasodilation, neutralizes ROS, inhibits platelet aggregation, limits leukocyte adhesion. iNOS-derived NO exacerbates hepatocellular injury, leads to excessive cytokine and ROS production. Lipid peroxidation, mitochondrial dysfunction
Cell death pathways	Necrosis, apoptosis, pyroptosis, ferroptosis, autophagy, NET-associated cell death, parthanatos, copper-induced and lysosome-dependent pathways

ATP: Adenosine triphosphate; MPTP: Mitochondrial permeability transition pore; ROS: Reactive oxygen species; TNF-α: Tumor necrosis factor α; IL-1β: Interleukin 1β; IL-6: Interleukin 6; MCP-1: Monocyte chemoattractant protein-1; DAMPs: Damage-associated molecular patterns; HMGB1: High mobility group box 1; ICAM-1: Intercellular adhesion molecule 1; VCAM-1: Vascular cell adhesion molecule 1; MPO: Myeloperoxidase; NET: Neutrophil extracellular trap; mtDNA: Mitochondrial DNA; TGF-β: Transforming growth factor-β; VEGF-A: Vascular endothelial growth factor A; PAI-1: Plasminogen activator inhibitor-1; MAC: Membrane attack complex; miRNAs: MicroRNAs; NF-κB: Nuclear factor kappa B; eNOS: Endothelial nitric oxide synthase; NO: Nitric oxide; iNOS: Inducible nitric oxide synthase.

The progression of HIRI can trigger widespread local and systemic inflammatory reactions, ultimately causing liver failure and distant organ injury, resulting in the development of multiple organ dysfunction syndrome. The massive release of ROS and proinflammatory cytokines into the systemic circulation facilitates immune cell infiltration and platelet activation in distant tissues. In combination with increased vascular permeability and a prothrombotic state, these reactions contribute to the dysfunction of remote organs such as the lungs, kidneys, heart, gastrointestinal tract, pancreas and brain[12,91-93].

IP

IP is an intraoperative maneuver during liver surgery, where controlled repeated brief episodes of ischemia followed by reperfusion are applied to the liver before prolonged ischemia. Accumulating evidence indicates that this technique activates innate cellular and molecular mechanisms, which subsequently have protective effects on hepatocytes during HIRI. The cytoprotective processes are mediated through a network of pathways, including adenosine, NO, prostaglandins, PKC, the PI3K-Akt cascade, heme-oxygenase 1 (HO-1) caspase, HSPs and other pathways, leading to the modulation of the inflammatory response, a reduction in oxidative stress, the stabilization of mitochondrial function, the modulation of cell death pathways and microcirculatory protection[94-96]. In a modification of direct IP, brief cycles of reocclusion and reperfusion are performed immediately at the onset of reperfusion after prolonged ischemia; this procedure is referred to as ischemic postconditioning (IPostC). The application of this method reduces the sudden reintroduction of oxygenated blood to the ischemic organ and attenuates oxidative and inflammatory bursts. Remote IP (RIP) extends this concept by applying short ischemia-reperfusion cycles to a distant organ or limb, which then triggers systemic protective pathways that confer resistance against HIRI[97,98]. The schematic protocols for IP, RIP and IPostC are shown in Figure 2.

Figure 2 Ischemic preconditioning, remote ischemic preconditioning and postconditioning. IP: Ischemic preconditioning.

Early and late IP

Hepatic IP provides protection against HIRI in two distinct phases. The early phase, mediated by preexisting proteins and signaling cascades, arises immediately and persists for 2-3 hours, whereas the late phase, which depends on transcriptional activation and de novo protein synthesis, emerges 12-24 hours post-IP and lasts up to 72 hours[99,100].

Both clinical and experimental studies have shown that IP enhanced portal vein flow and improves microvascular and macrovascular perfusion after reperfusion, thereby contributing to the preservation of liver function[101,102]. Furthermore, IP improved sinusoidal perfusion, which maintained parenchymal function mainly through a reduction in Kupffer cell activation and the preservation of an adequate mitochondrial redox state[103]. On the basis of these mechanisms, IP was correlated with better preservation of liver function, reflected by lower serum transaminase and glutamate dehydrogenase levels and improved bile flow.

Regulation of inflammatory response

One of the major protective mechanisms involves modulation of the inflammatory response, which is a main underlying cause of tissue injury in HIRI. IP was found to significantly reduce the activation of Kupffer cells, which was associated with the attenuation of neutrophil recruitment[103]. Moreover, IP pretreatment in experimental models led to a significant reduction in serum levels of proinflammatory cytokines, including TNF-α, IL-1β, and IL-6, which correlated with decreased neutrophil accumulation in hepatic tissue[104-106]. In contrast, the effect of IP on the levels of anti-inflammatory IL-10 is controversial. While animal models of RIP showed attenuation of HIRI without IL-10 overexpression, human liver transplantation with donor IP resulted in increased post-reperfusion IL-10 levels, which were correlated with a significant reduction in acute rejection episodes, although there was no effect on HIRI severity[107,108]. IP may also exert its protective effect through the downregulation of CXC chemokine expression and a reduction in neutrophil recruitment upon reperfusion[109].

Oxidative stress and mitochondrial stability

Multiple mechanisms of ROS modulation have been proposed to contribute to the protective effects of IP against HIRI. According to current data, IP reduces ROS production and oxidative stress through the inhibition of the xanthine and xanthine dehydrogenase-xanthine oxidase pathways in the liver[110]. Furthermore, IP reduces mitochondrial ROS production, stabilizes mitochondrial function, inhibits HMGB1 release, downregulates the expression of NF-κB and proinflammatory cytokines, and inhibits Kupffer cell and CD4+ T lymphocyte activation during HIRI[111,112]. A reduction in HIRI-induced T-cell immunoglobulin and mucin domain molecule-4 expression constitutes one of the suggested mechanisms through which the inflammatory response is attenuated during IP[16].

On the molecular level, IP protects the liver through the modulation of adenosine, NO and intracellular kinase pathways. Brief ischemic episodes increase extracellular adenosine (a nucleotide and compound of nucleic acids, ATP and adenosine monophosphate), which activates several protective mechanisms. Through the stimulation of A1 and A2 adenosine receptors, IP enhances the generation of endogenous adenosine, which promotes eNOS expression and NO production, leading to the attenuation of hepatic sinusoidal vasoconstriction, a reduction in inflammatory cell adhesion, the suppression of proinflammatory transcription factor activation and the inhibition of endothelial cells and hepatocyte apoptosis. Activation of adenosine A2 receptor-dependent pathways, modulation of endothelin, and inhibition of caspase are among the proposed biochemical pathways. Moreover, NO may stimulate the production of prostaglandin, a key mediator with vasodilatory, anti-inflammatory, antioxidant and antithrombotic effects. Although the precise downstream targets remain unclear, evidence supports NO as an important pathophysiological factor of hepatic IP. Conversely, while the role of prostaglandins in IP remains controversial, recent evidence suggests that exogenous administration may provide protection against HIRI[113-116].

IP also contributes to the prevention of intracellular calcium overload, as the adenosine generated during IP enhances the efflux of ischemia-related calcium from both hepatocytes and hepatic microcirculatory smooth muscle cells, thereby stabilizing the cell membrane and promoting microvascular vasodilation. Studies suggest that adenosine A2 receptors on sinusoidal endothelial cells play a key role in the liver, with IP or A2 receptor agonists reducing cell death and increasing cyclic adenosine monophosphate levels. Through the activation of A1 receptors, adenosine also diminishes neutrophil infiltration and adhesion to the microvascular endothelium[103,117]. As a result, adenosine downregulates cytokine and adhesion molecule expression, suppresses neutrophil and platelet activity and reduces ROS generation[18].

Key signal pathways (adenosine/NO/kinase)

During IP, adenosine activates several intracellular kinases, including protein kinases A and C, p38 MAPK, and Akt/PKB. Specifically, adenosine can signal through four G-protein-coupled receptors, where its coupling to the Gs and Gq subunits leads to the activation of protein kinases A and C, respectively, and the subsequent elevation of cyclic adenosine monophosphate[118]. PKC represents a family of intracellular enzymes that act as signal transducers. PKC activation increases after reperfusion and serves as an important mediator of IP-initiated extracellular signaling pathways, although the precise downstream mechanisms remain undefined[119].

HSPs are ubiquitous, stress-induced protein families that play important roles as molecular mediators in cell signal transduction, the cell cycle, and apoptosis regulation[120]. In the rat liver, increased expression of the HSPs (HSP72, HSP73, HSP70), and HO-1/HSP32 is associated with reduced ischemic injury and improved survival. Proposed mechanisms include the preservation of mitochondrial integrity, the repair of damaged proteins, protection against oxidative stress, the suppression of proinflammatory cytokines, and the repair of ion channels. However, whether heat shock proteins actively mediate preconditioning or simply serve as markers of ischemic tolerance remains unclear[94,120,121]. HO-1, or HSP32, is a HSP with antioxidant, anti-inflammatory, antiapoptotic, and vasodilatory effects. HO-1 upregulation in hepatocytes and Kupffer cells has been shown to reduce HIRI, suppress cytokine and HMGB1 release and promote immune tolerance in experimental models[122-124]. Pharmacological induction or gene transfer of HO-1 enhances hepatoprotection, while RIP also increases hepatic HO-1 expression and activity, linking its protective role to reduced injury and autophagy induction. However, HO-1 upregulation appears to be delayed, suggesting that it contributes to sustained rather than immediate IP protection[94,125].

NF-κB is an important transcription factor and signaling pathway component that regulates the expression of numerous genes involved in the immune response, inflammation and cell death and proliferation. IP activates NF-κB in the early phase, leading to the transcription of genes, such as cyclin D1, that favor cell survival and promote hepatocyte proliferation[100]. The induction of cyclin D is regulated by MAPK, notably p38 and JNK, among others[126].

A source of recent insight into the mechanisms of IP against HIRI has been the analysis of differential miRNA expression, as these molecules play important regulatory roles in the cell death, proliferation and differentiation processes. Furthermore, the data suggest that miRNA activity may vary among different pathological contexts. A study conducted by Xu et al[127] demonstrated significant downregulation of four miRNAs (mmu-miR-23a, mmu-miR-326, mmu-miR-346_MM1, and mmu-miR-370) following IP after HIRI, indicating their contribution to the protective effects of IP in the liver. Circular RNA (circRNA) is a type of noncoding RNA that forms a covalently closed loop. CircRNAs regulate miRNAs by binding to miRNA response elements, which may competitively suppress their activity[128]. Tian et al[14] reported that circRNA_017753 may play a protective role in IP intervention during HIRI, with three circRNA_017753-miRNA-mRNA regulatory axes connected to the possible target Jade1 protein, a component of the HBO1 acetyltransferase complex and a key regulator of apoptosis.

Regulation of programmed cell death

An important consequence of IP-associated pathophysiological processes is the modulation of programmed cell death mechanisms. The limitation of oxidative stress and energy loss during IP results in a reduction in hepatocyte and sinusoidal endothelial cell apoptosis through the downregulation of caspase-3 activation. The activation of the intracellular Akt-PKB pathway further suppresses apoptotic pathways through regulatory targets such as Bad, caspase-9, and cellular FLICE-inhibitory protein and may act via NF-κB signaling. The PKC and PI3K-Akt signaling pathways, NO-mediated inhibition of caspase activation and preservation of mitochondrial function may also contribute to the antiapoptotic effects of IP[99,129]. A recent study demonstrated that the activation of PKC-δ and PKC-ε isozymes prevents necrosis in early IP by preserving the oxidation state, whereas in late IP, it limits apoptosis through the induction of iNOS and HO-1[119]. These effects reduce the vulnerability of liver cells to necrosis and apoptosis during HIRI. Although direct evidence in the liver remains limited, studies in other ischemia-reperfusion models suggest that interventions that upregulate glutathione peroxidase 4 and maintain iron homeostasis by modulating iron metabolism-related proteins, such as ferritin and transferrin, can attenuate ferroptosis. It is plausible that IP, particularly when combined with antiferroptotic agents, may act through similar mechanisms, a hypothesis that warrants further investigation[90,130]. Similarly, although direct evidence from hepatic models is lacking, IP appears to attenuate pyroptosis by suppressing activation of the NLRP3 inflammasome, thereby reducing the cleavage of caspase-1 and gasdermin D and consequently decreasing the release of proinflammatory cytokines such as IL-1β and IL-18[131]. The anti-HIRI protective mechanisms of IP are summarized in Table 2. Beyond its local hepatic effects, growing evidence suggests that hepatic IP exerts protective effects on remote organs such as the lungs, kidneys, heart, gastrointestinal system and brain.

Table 2 Protective pathways of ischemic preconditioning.

Mechanism	Description
Improvement of hemodynamics	Enhancement of portal vein flow, improvement of micro-, macrovascular and sinusoidal perfusion
Modulation of inflammatory response	Reduction of Kupffer cells activation, attenuation of neutrophil recruitment, reduction in pro-inflammatory cytokine release, downregulation of CXC chemokine expression
Reduction of ROS production	Inhibition of XDH/XOD pathways, reduction of mitochondrial ROS production, inhibition of HMGB1 release, downregulation of NF-κB expression, reduction of TIM4 expression
Modulation of adenosine pathway	Enhancement of the adenosine generation, activation of adenosine A2 receptor-dependent pathways, modulation of endothelin, caspase inhibition, cytokine release downregulation, inhibition of adhesion molecule expression, suppression of neutrophil and platelet activity and reduction of ROS generation, activation of intracellular kinases
NO production	Promotion of eNOS-derived NO production, attenuation of hepatic sinusoidal vasoconstriction, reduction of inflammatory cell adhesion, suppression of pro-inflammatory transcription factor activation, regulation of programmed cell death
NO-associated prostaglandins production	Vasodilation, inhibition of inflammatory response, suppression of oxidative stress, inhibition of platelets aggregation
Regulation of calcium homeostasis	Prevention of intracellular calcium overload through adenosine pathway
Heat shock proteins	Preservation of mitochondrial integrity, repair of damaged proteins, protection against oxidative stress, suppression of proinflammatory cytokines and stabilization of the ion channels
NF-κB activation	Cyclin D1 transcription, promotion of hepatocyte proliferation
Modulation of miRNAs expression	Modulation of Jade1 protein expression, downregulation of mmu-miR-23a, mmu-miR-326, mmu-miR-346_MM1, and mmu-miR-370
Inhibition of programmed cell death pathways	Reduction of hepatocyte and sinusoidal endothelial cell apoptosis, preservation of mitochondrial function, prevention of necrosis

ROS: Reactive oxygen species; XDH: Xanthine dehydrogenase; XOD: Xanthine oxidase; HMGB1: High mobility group box 1; NF-κB: Nuclear factor kappa B; TIM4: T-cell immunoglobulin and mucin domain molecule-4; NO: Nitric oxide; eNOS: Endothelial nitric oxide synthase; miRNA: MicroRNAs.

PROTECTIVE EFFECTS OF HEPATIC IP ON DISTANT ORGANS

Hepatic IP has been shown to attenuate hepatocellular oxidative stress and inflammatory reactions while enhancing microcirculatory and mitochondrial stability and activating cytoprotective signaling cascades. Importantly, the benefits of hepatic IP are not limited to local protection. Increasing evidence indicates that IP also provides remote organ protection, reducing damage to the extrahepatic organs that are vulnerable to the HIRI-associated systemic inflammatory response. These processes are thought to be mediated by complex interactions among cellular and molecular factors, neural pathways, and immunomodulatory mechanisms that extend IP signaling beyond the liver. RIP models have demonstrated that exosomes serve as key mediators, functioning as carriers of bioactive molecules such as hypoxia-inducible factor 1-alpha (HIF-1α) and specific miRNAs that modulate inflammation, apoptosis, and angiogenesis in target organs. Evidence indicates that RIP-derived exosomes, originating primarily from endothelial cells, exert protective effects comparable to those of RIP itself. Nevertheless, the precise mechanisms underlying these effects, as well as the standardization of exosome and miRNA analyses, remain unclear and warrant further investigation[132]. Experimental evidence also suggests that neural pathways play pivotal roles in mediating RIP-induced protection. Vagus nerve simulation transmits protective signals through mediators such as adenosine, bradykinin, and HIF-1α, leading to the activation of pathways involving PKC-ε and mitochondrial preservation. These findings highlight the close interplay between neurogenic and humoral mechanisms in mediating systemic protection during remote hepatic IP[133]. The mechanisms and mediators involved in HIRI-associated remote organ damage are summarized in Table 3, while the protective effects of IP are illustrated in Figure 3.

Figure 3 Remote organ damage during hepatic ischemia-reperfusion injury and protective effect of ischemic preconditioning. HIRI: Hepatic ischemia-reperfusion injury; IP: Ischemic preconditioning; ROS: Reactive oxygen species; XOD: Xanthine oxidase; TNF-α: Tumor necrosis factor α; STAT5: Signal transducer and activator of transcription 5; NO: Nitric oxide; SIgA: Secretory immunoglobulin A; IL-1β: Interleukin 1β; LDH: Lactate dehydrogenase; CK-MB: Creatine kinase-MB; Akt: Protein kinase B.

Table 3 Hepatic ischemia-reperfusion injury-associated remote organ damage.

Organ	Underlying mechanisms	Mediators involved	Relative manifestations
Lungs	Systemic inflammatory response. Neutrophil and lymphocytes infiltration. Oxidative stress	TNF-α. ICAM-1. E-selectin. PAF. IL-6. IL-18. Substance P neutrophil-chemoattractant protein. ROS. WISP1	ALI. ARDS
Myocardium	Systemic inflammatory response. Oxidative stress. Hemodynamic instability	Cytokines. ROS. Adhesion molecules	Stress-induced cardiomyopathy postreperfusion syndrome. Arrhythmias
Kidney	Hemodynamic instability. Systemic inflammatory response. Oxidative stress. Renal endothelial and mitochondrial injury	ROS. PINK1. LC3. HIF-1. TNF-α. IL-6. IL-8. ET-1. IL-18. ICAM-1	AKI
Gastrointestinal system	Systemic inflammatory response. Oxidative stress. Postoperative liver failure	LPS. PRRs. TLR4. MAPK signaling pathway. NF-κB. Adhesion molecules	Mesenteric congestion increased intestinal permeability. Venous ischemia. Mucosal barrier disruption. Bacterial translocation
Pancreas	Oxidative stress increased apoptosis	lCAM-1. ROS	Pancreatic dysfunction. Acute pancreatitis
Brain	Increased BBB permeability. Infiltration of immune cells. Neuroinflammation. Neurodegeneration. Apoptosis	ROS. iNOS. TNF-α. IL-1β. IFN-γ. IL-6. Caspase-3. Bcl-2/Bax proteins ratio. NF-κB. Protein kinase 3	Cognitive impairment. Neuron injury

TNF-α: Tumor necrosis factor α; ICAM-1: Intercellular adhesion molecule 1; PAF: Platelet-activating factor; IL-6: Interleukin 6; IL-8: Interleukin 8; ROS: Reactive oxygen species; WISP1: WNT1-inducible signaling pathway protein 1; ALI: Acute lung injury; ARDS: Acute respiratory distress syndrome; PINK1: PTEN-induced kinase 1; LC3: Light chain 3; HIF-1: Hypoxia-inducible factor 1; ET-1: Endothelin-1; IL-18: Interleukin 18; AKI: Acute kidney injury; LPS: Lipopolysaccharide; PRRs: Pattern recognizing receptors; TLR4: Toll-like receptor 4; MAPK: Mitogen-activated protein kinase; NF-κB: Nuclear factor kappa B; BBB: Blood-brain barrier; iNOS: Inducible nitric oxide synthase; IL-1β: Interleukin 1β; IFN-γ: Interferon-γ.

Lung injury

HIRI initiates a profound systemic inflammatory response, which can extend beyond the liver and adversely affect distant organs. Among the extrahepatic manifestations, pulmonary complications are particularly significant, presenting clinically as acute lung injury or, in a post-transplantation context, as acute respiratory distress syndrome[134,135]. Specifically, postoperative acute lung injury secondary to HIRI is a common complication frequently associated with adverse outcomes, including increased morbidity and mortality[136]. TNF-α released from reperfused Kupffer cells into the systemic circulation interacts with pulmonary capillaries, induces the expression of ICAM-1 and E-selectin adhesion molecules and promotes the infiltration of neutrophils and lymphocytes into lung tissue, causing lung injury. In addition to TNF-α, other proinflammatory mediators, including platelet-activating factor, IL-6, IL-18, substance P and neutrophil chemoattractant protein, further contribute to this damage[12]. In addition, the ROS generated during HIRI circulate systemically and contribute to pulmonary endothelial injury, disrupting barrier integrity and increasing vascular permeability[134,137]. A study conducted by Tong et al[138] revealed that WNT1-inducible signaling pathway protein 1 is responsible not only for hepatic injury during HIRI but also for the development of lung injury following hepatic reperfusion.

Hepatic IP has been demonstrated to attenuate the systemic inflammatory response, decrease ROS production and inhibit neutrophil infiltration, thereby conferring protection on pulmonary tissue as well. In an experimental model, hepatic IP limited xanthine accumulation and xanthine oxidase pathway activity during ischemia, leading to a reduction in ROS generation and an amelioration of HIRI and subsequent lung inflammation and microvascular damage[139]. Furthermore, IP attenuated hepatic TNF-α release from Kupffer cells, a process modulated by NO production, which reduced both liver injury and lung injury after HIRI[9]. The suppression of P-selectin upregulation constitutes one of the important mechanisms for preventing lung neutrophil infiltration and microvascular dysfunction induced by HIRI[110].

Myocardial injury

During reperfusion of ischemic hepatic tissue, proinflammatory cytokines and ROS are released into the systemic circulation via hepatic venous outflow, reaching the heart first and predisposing it to early injury[140]. Specifically, ROS induce myocardial injury through both direct mechanisms, such as necrosis and membrane disruption, and indirect effects mediated by cellular signaling pathways[141,142]. A recent experimental study in animals subjected to HIRI revealed cardiomyocyte necrosis accompanied by hyperemia, hemorrhage, and edema[138]. HIRI-associated myocardial injury has also been linked to the generation of ROS, the release of inflammatory cytokines and eicosanoids, and the activation of adhesion molecules[143]. Clinically, severe cardiac dysfunction is an uncommon but recognized complication following liver transplantation. It may manifest as stress-induced cardiomyopathy, arrhythmias or postreperfusion syndrome, driven by systemic inflammation and hemodynamic instability[144].

The cardioprotective effects of IP have been mostly studied in experimental RIP models, in which animals undergo hepatic IP before myocardial ischemia-reperfusion injury is induced. Cardiac enzyme (lactate dehydrogenase and creatine kinase-MB) levels, myocardial infarct size and rate-pressure product were significantly reduced in the RIP group[145]. The cardioprotective effects of hepatic IP appear to be mediated by glycogen synthase kinase-3β and signal transducer and activator of transcription 5-dependent signaling pathways and by circulating protective factors, such as erythropoietin and miR-144[20,146].

Kidney injury

HIRI during liver surgery or transplantation is a major cause of postoperative acute liver failure. Acute kidney injury frequently complicates acute liver failure, with an incidence of 40%-85%, depending on the diagnostic standards and causation[147]. The underlying pathophysiology is multifactorial, involving hemodynamic changes, systemic inflammatory reactions, oxidative stress, renal endothelial and mitochondrial injury and various molecular mechanisms[148,149]. Many molecular mediators have been implicated in acute kidney injury secondary to HIRI. ROS, PTEN-induced kinase 1 and microtubule-associated protein 1 light chain 3 mediators of mitophagy and autophagy, HIF-1, TNF-α, IL-6, IL-8, endothelin-1, IL-18, cytokines and ICAM-1 adhesion molecules are among the key factors implicated as mediators of renal injury[149].

An experimental study revealed that hepatic IP activated the Akt signaling pathway, elevated NO levels and attenuated the inflammatory response, which are key contributors to renal injury. Furthermore, while high levels of ROS are well known to cause reperfusion injury, moderate levels appear to be essential for IP in cardiac, hepatic, and renal cells. Specifically, ROS activate cytoprotective kinases (extracellular signal-regulated kinase 1/2, MAPK, and Akt) and upregulate protective genes. In the kidney, ROS derived from moderate hepatic IP may trigger pathways that provide renal cytoprotection against later oxidative injury[150].

Intestinal injury

Clamping of the hepatoduodenal ligament during liver surgery or transplantation obstructs portal vein inflow, leading to mesenteric congestion, increased intestinal permeability, and venous ischemia, which in turn promotes bacterial translocation into the portal circulation[151]. Circulating bacterial components, such as lipopolysaccharide, activate various pattern recognizing receptors, mainly Toll-like receptor 4, where lipopolysaccharide binding may trigger MAPK signaling pathway and NF-κB activation leading to the upregulation of inflammatory cytokines, chemokines, and adhesion molecules, thereby contributing to liver and intestinal injury[152]. Other proposed mechanisms include remote oxidative injury and postoperative liver failure. Free ROS induces enterocyte apoptosis and necrosis, disrupts epithelial integrity, and affects the adhesion molecule expression, leading to mucosal barrier disruption[12].

Study conducted by Ren et al[153] demonstrated that hepatic IP preserved intestinal mucosal ultrastructure, lowered serum endotoxin levels and bacterial translocation following liver transplantation, increased fecal secretory immunoglobulin A, and decreased serum TNF-α. On the microbial level, hepatic IP promoted restoration of intestinal microbiota, which may further support hepatic function through a positive feedback mechanism.

Pancreatic injury

Pancreatic dysfunction and acute pancreatitis are recognized as complications that can occur after major hepatectomy and liver transplantation. Proposed risk factors include the duration of the surgery, hepatitis B virus infection as an indication for transplantation, the administration of immunosuppressive drugs, posttransplant biliary complications and portal congestion during vascular control[154]. Mechanistically, ICAM-1 upregulation, ROS-mediated oxidative damage and increased apoptosis constitute a possible molecular basis for HIRI-associated pancreatic injury[155,156]. In an experimental model of pancreatitis induced by ischemia-reperfusion injury, hepatic IP led to a reduction in plasma lipase and IL-1β levels, accompanied by improved histological findings regarding pancreatic damage[157].

Brain injury

The reperfusion phase following prolonged hepatic ischemia generates ROS, iNOS, and proinflammatory cytokines such as TNF-α, IL-1β, interferon γ and IL-6, which circulate systemically and compromise blood-brain barrier permeability, allowing the infiltration of immune cells and neurotoxic molecules[158,159]. Specifically, neutrophils and T lymphocytes can enter the central nervous system (CNS) through transcytosis or paracellular pathways. Once activated, these immune cells also secrete proinflammatory cytokines and chemokines, which further disrupt the integrity of the blood-brain barrier and exacerbate CNS injury. In addition, astrocytes and microglia are further stimulated by inflammatory mediators and infiltrating immune cells, which, together with peripheral inflammatory signaling pathways, may lead to neuroinflammation, neurodegeneration and the development of cognitive impairment[158]. Moreover, the activation of apoptosis-related caspase-3 and changes in the Bcl-2/Bax protein ratio during HIRI may significantly affect neuronal injury and apoptosis[160]. Together with the activation of NF-κB and downregulation of protein kinase 3, these molecular and histological alterations may have a negative effect on cognitive function following HIRI[161].

The beneficial effect of hepatic IP on the CNS was demonstrated by Yang et al[23] in an experimental model of hepatic RIP against cerebral ischemia-reperfusion injury. In detail, hepatic RIP attenuated cerebral ischemia-reperfusion injury by reducing the infarct volume and lowering serum lactate dehydrogenase and creatine kinase-MB levels, limiting neurological deficits and apoptosis. This significant neuroprotective effect is mediated through activation of the Akt-dependent signaling pathway.

CHALLENGES IN METHOD APPLICATION AND FUTURE DIRECTIONS

Hepatic IP represents a promising and effective approach against HIRI; however, a consensus on the most effective protocol in terms of ischemia duration, number of cycles and reperfusion time is still lacking. Typical experimental designs involve three to four cycles of short ischemia (5-10 minutes) followed by brief reperfusion (5-15 minutes) before a prolonged ischemic insult. This heterogeneity makes results difficult to compare, leading to limited translation into clinical practice[162]. In addition, although highly promising, most existing evidence on hepatic IP is at the preclinical level, while clinical investigations in humans have demonstrated inconclusive results[163]. Traditionally, most studies on IP have focused on its ability to modulate apoptosis, the inflammatory response and oxidative stress. However, a growing body of evidence highlights the importance of diverse mechanisms involved in HIRI, including the regulatory roles of miRNAs as well as alternative forms of programmed cell death such as ferroptosis, pyroptosis, and necroptosis.

Future studies should aim to standardize hepatic IP protocols in order to optimize timing, cycle duration, and reproducibility across experimental and clinical settings. In addition, further investigations are warranted to elucidate the interactions of IP with other HIRI-associated mechanisms, including those involving miRNAs and nonapoptotic cell death pathways; such knowledge may support the development of combined therapeutic strategies, such as IP in combination with pharmacological inhibitors of ferroptosis or pyroptosis. Furthermore, combining IP with pharmacological agents targeting oxidative stress, inflammation and mitochondrial dysfunction holds significant promise for augmenting its protective efficacy. Combined protective interventions, such as IP coupled with anesthetic conditioning and antioxidant or prostaglandin-based therapies, could increase hepatic tolerance against HIRI and extend protection to remote organs affected by reperfusion injury. It will also be important to further explore whether IP can attenuate systemic complications and remote organ injury, thereby extending its benefits beyond the liver. Ultimately, well-designed clinical trials are essential to validate these findings and to demonstrate the role of IP as a reliable protective method in hepatic surgery and transplantation. To create consensus guidelines, clearly defined and approved criteria for both primary and secondary outcomes should be established. These may reasonably include clinical parameters such as length of stay in the intensive care unit, overall hospital stay duration, peak serum concentrations of bilirubin and transaminases, prothrombin time, and a quantitative apoptosis index[163]. Further randomized clinical trials with rigorous methodological standards and adequate technical standardization are needed to increase the reproducibility of the method.

CONCLUSION

HIRI remains a major clinical challenge in liver surgery and transplantation, particularly in the context of advanced surgical techniques, oncological treatments involving major hepatectomies, and the increasing use of donors under expanded criteria and donors after cardiac death grafts, all of which are associated with a high risk of postoperative complications. The onset of HIRI is attributed to multifactorial pathogenic processes, involving oxidative stress, mitochondrial dysfunction, calcium overload, and inflammatory and molecular signaling. The limited understanding of the underlying mechanisms is strongly associated with the complexity of developing effective therapeutic strategies. IP has emerged as a promising protective intervention, capable of attenuating hepatic damage through the activation of cellular defense mechanisms, attenuation of the systemic inflammatory response, moderation of ROS production and modulation of programmed cell death pathways. Beyond its local hepatoprotective effects, IP also appears to exert systemic benefits, potentially reducing injury to distant organs, including the lungs, kidneys, heart, gastrointestinal system and brain. Evidence from experimental studies suggests that molecular and immunomodulatory pathways triggered by hepatic IP are responsible for its global protective effects. These findings highlight IP as a valuable strategy for improving outcomes in liver transplantation and other high-risk surgical settings. However, further experimental and clinical research is needed to clarify the optimal protocol and optimize its clinical application.