Current status and perspective on molecular targets and therapeutic intervention strategy in hepatic ischemia-reperfusion injury

Roles of mitochondrial damage played in HIRI and positive impacts of therapeutic strategies

Admittedly, mitochondrial damage and oxidative stress as mentioned above are closely related and even can influence each other. However, for clarifying the associations more clearly in this section, we will specifically focus on exploring the involvement of mitochondrial-related pathological changes in HIRI initiation and progression. Mitochondria produce ATP through various mitochondrial respiratory chain complexes, which are essential for the proper functioning of hepatic cells. However, during HIRI, mitochondria are the first to be affected by ATP synthesis impairments, leading to their own heterogeneous lesions. This imbalance in ATP production not only disrupts the normal metabolic processes in liver cells but also triggers the generation of high levels of ROS, thereby exacerbating HIRI [13]. Therefore, the factors that affect the function of mitochondria including disturbing ATP synthesis, ROS balance and coenzyme (coenzyme NAD⁺, NADH) dysfunction may be involved in the pathogenesis of HIRI. Methylation-controlled J protein (MCJ), an endogenous negative regulator of mitochondrial respiration, is reported to reduce ATP synthesis by restraining complex I activity and supercomplex formation [14]. On the contrary, the knockdown of MCJ could increase ATP levels to promote the early activation of Kupffer cells (KCs) accompanied by increased production of tumor necrosis factor-alpha (TNF-α), IL-6 and heparinbinding epidermal growth factor (EGF), which contribute to liver regeneration and prevent cell damage during liver regeneration in murine models of HIRI [15]. As we said at the beginning, this abnormality in ATP is often accompanied by an abnormal imbalance of ROS in mitochondria, leading to mitochondrial dysfunction. This dysfunction can manifest as abnormal mitochondrial membrane potential, changes in redox potential, and disruptions in mitochondrial quality control. In a study by Teixeira et al. [16], it was found that preconditioning with polyethylene glycol 35 kDa (PEG35) could mitigate hepatic hypoxia/reoxygenation-induced mitochondrial dysfunction by inhibiting the toxicity of ROS and the exhaustion of ATP, as well as restoring the mitochondrial membrane potential (MMP). Similarly, SS-31, a peptide with strong antioxidant and anti-inflammatory effects, also restrained lipopolysaccharide (LPS)-induced mitochondrial dysfunction by reducing the production of mtROS, increasing the production of ATP in mitochondria and restoring the MMP in Raw 264.7 cells [17].

Considering the inseparable relationship between ATP and mitochondrial respiratory chain, subsequent studies paid more attention to the effect of local respiratory chain complex on mitochondrial damage and HIRI. From a structural point of view, ATP is the product of the fifth complex of mitochondrial respiration and its production is simultaneously affected by different respiratory complexes along the electron transport chain, including respiratory chain complex I (NADH dehydrogenase, specifically at the change of NADH/NAD⁺), complex II (succinic acid-Q oxidoreductase), complex III (UQ-cytochrome C oxidoreductase), complex IV (cytochrome C oxidase) and complex V (ATP synthase) [18]. Coenzyme Q₁₀ (CoQ₁₀) is the only non-protein REDOX carrier in the respiratory chain. It can move rapidly through the membrane to transfer electrons from NADH dehydrogenase in the respiratory chain, promoting MMP maintenance and recovery of superoxide dismutase (SOD) vitality to alleviate hepatic damage in ischemic surgery-induced HIRI mouse model. Meanwhile, the use of a CoQ₁₀-loaded virus-mimicking liposomal system (VMLs), which employs dendritic lipopeptides to mimic the viral envelope, may offer a more effective therapeutic approach compared to using CoQ₁₀ alone for treating HIRI [19]. Additionally, NAD⁺ serves as a vital coenzyme for dehydrogenase in biocatalytic reactions, enhancing the physiological function of CoQ₁₀ and acting as a co-substrate for various enzymes, such as sirtuins [20]. It has been suggested that NAD⁺, short for coenzyme I, protects the kidney from acute stress damage through sirtuin1 (SIRT1, an NAD-consuming deacetylase)/glycogen synthase kinase 3β (GSK-3β) [21]. Specifically, NAD supplementation reduced damage in a SIRT1-dependent manner that was associated with the decrease in GSK-3β levels and activity and an increase in Nrf2 expression. 21 In liver, pretreatment with indirubin-30-oxime (a GSK-3β inhibitor) prevented cyclophilin-D (CypD) phosphorylation located in the mitochondrial matrix to maintain the content and synthesis of ATP by inhibiting GSK-3β activity; on the other hand, NAD⁺ pretreatment promoted the deacetylation of CypD accompanied by decreased mitochondrial efficiency and ROS generation by increasing SIRT3 activity, leading to the decline in the binding of CypD to adenine nucleotide translocator (ANT) and mitochondrial permeability transition (mPT) induction, thus protected against ischemic surgery-induced HIRI [22]. Furthermore, mitochondrial metabolic reprogramming can have a direct impact on glucose metabolism and the mitochondrial respiratory chain, particularly complex I (NADH dehydrogenase) which plays a crucial role in regulating the HIRI process. A separate study showed that HOPE treatment induced mitochondrial reprogramming by elevating ATP levels in liver tissue and reducing mitochondrial succinate accumulation, as well as releasing flavin mononucleotide (FMN) and altering the NADH/NAD ratio. This reprogramming was found to offer protection against mitochondrial injury and HIRI in rat livers donated after circulatory death prior to liver transplantation [23].

In addition, the normal operation of mitochondrial function can maintain the energy balance of cells through the regulation of the dynamic balance between mitochondrial dynamics (fission/fusion) and quality control (biogenesis/mitochondrial autophagy) [24]. Originally, mitochondrial fusion and fission are natural protective mechanisms employed by the body to eliminate damaged, aging and excessive mitochondria, thereby maintaining overall health and normal immune system function. However, in cases of abnormal damage, these processes can become disrupted, leading to mitochondrial fragmentation with abnormal division or prolonged mitochondrial morphology with abnormal fusion. Gradually, more and more evidence has found that HIRI can affect the self-renewal and repair process of the above mitochondria, thus causing devastating damage to the liver. It has been reported that HIRI caused fragmented mitochondria and downregulated the expression of N6-methyladenosine (m6A) demethylase (FTO), a fat mass and obesity-associated protein. But, adeno-associated virus-mediated liver-specific overexpression of FTO (AAV8-TBG-FTO) attenuated HIRI and mitochondrial fragmentation via inhibiting the expression of dynamin-related protein 1 (drp1) that could regulate the mitochondrial fission process in cell survival [25]. Similarly, previous evidence pointed out that augmenter of liver regeneration (ALR) was involved in maintaining physiological operation of mitochondria and preventing mitochondrial fission by inhibiting drp1 phosphorylation to alleviate HIRI-induced apoptosis [26]. With the deepening of the research, it was found that ALR also could interact with transcription factor Yin Yang-1 (YY1) and suppress its nuclear import. This interaction contributed to decreasing ubiquitin-like modifier-activating enzyme 2 (UBA2, YY1 downstream target gene) transcription levels. Consequently, this led to a reduction in drp1 and small ubiquitin-like modification (SUMOylation)-medicated mitochondrial fission in a rat model of hepatic IRI [26]. Currently, the majority of studies focus on probing the impact of mitochondrial division on the HIRI process. Nevertheless, there is a noticeable dearth of research concerning mitochondrial fusion and its associated interventional drugs.

Additionally, emerging evidence indicates that mitochondrial autophagy plays a role in selectively degrading dysfunctional mitochondria, thereby mitigating damage and preventing the accumulation of impaired mitochondria in the liver. Previous studies have shown that lysosomal Ca²⁺ signaling is the vital part of the traditional mucolipin 1 (MTORC1, a master regulator of autophagy)-dependent autophagy pathway, and induces autophagy [27]. Calpastatin (CAST) is an endogenous calpain inhibitor, which can maintain the stability of intracellular calmodulin in the liver [28]. After HIRI stimulation, the aged liver showed a rapidly decreasing CAST resulting in the activation of calmodulin, defective autophagy, cleaving autophagy-related protein 5 (ATG5), mPT onset, mitochondrial depolarization and hepatocyte death. However, CAST overexpression restored autophagy for eliminating abnormal mitochondria and prevented mitochondrial depolarization to protect hepatocytes against HIRI [29]. Similarly, PEG35 preconditioning enhanced autophagy and mitochondrial quality by increasing the levels of autophagy-related proteins (light chain 3 II [LC3II] and protein sequestosome 1/p62 [p62]) and genes involved in mitochondrial biogenesis (peroxisome proliferator-activated receptor gamma, coactivator 1 alpha [ppargc1a], nrf1, mitochondrial fission 1 [fis1], dynamin 1-like gene [dnm1l] and optic atrophy 1 [opa1]) to ameliorate HIRI [16]. However, these studies only observed the simultaneous existence of autophagy and mitochondrial clearance or increase but still did not provide direct evidence of mitophagy, which has specific targets and markers such as phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1), mitofusin 2 (Mfn2) and Parkin, which were different from general autophagy. Other subsequent research has elucidated the direct evidence of mitophagy participating in HIRI pathogenesis in multiple gene-knockout animals. ALR-knockout (KO) mice exhibited more severe hepatocellular necrosis and inflammatory cell infiltration that were associated with decreased mitophagy via inhibiting PINK1 accumulation and PARKIN translocation to mitochondria, which could be reversed by transfecting Mfn2 [30]. Meanwhile, DJ-1 is protein associated with mitochondrial function. In Dj-1 knockout (Dj-1^-/-) mice subjected to hepatic ischemia-reperfusion (I/R) injury, Dj-1 deficiency enhanced mitochondrial accumulation and protein stability of PARKIN, which in turn enhanced mitophagy, contributing to the clearance of damaged mitochondria [31]. Collectively, in various models of apoptosis, there was observed a collective occurrence of mitochondrial fragmentation, disruption of reticular structure, remodeling of mitochondrial ridge, and a notable increase in mitochondrial number. Thus, it is suggested that mitophagy, mitochondrial fusion, and fission may serve as potential mechanisms for HIRI by influencing the quantity and composition of mitochondria (Fig. 1).

Figure 1.

Mitochondrial damage and the crosstalk between Kupffer cells and mitochondria play crucial roles in progression of HIRI. ATP synthesis, ROS balance, membrane potential, quality control in mitochondria, mitophagy, mitochondrial fusion and fission may contribute to the regulation of I/R injury. KCs, Kupffer cells; DNM1L, dynamin 1-like gene; Fis1, mitochondrial fission 1; TNF-α, tumor necrosis factoralpha; IL-6, interleukin-6; EGF, heparin-binding epidermal growth factor; ALR, augmenter of liver regeneration; MCJ, methylation-controlled J protein; ATP, adenosine trisphosphate; NADH, nicotinamide adenine dinucleotide; PEG35, polyethylene glycol 35 kDa; ATG5, cleaving autophagy-related protein 5; ROS, reactive oxygen species; CoQ₁₀, coenzyme Q₁₀; SIRT3, sirtuin3; GSK-3β, glycogen synthase kinase 3β; CAST, calpastatin; HOPE, hypothermic oxygenated perfusion; FMN, flavin mononucleotide; CypD, cyclophilin-D; ANT, adenine nucleotide translocator; Drp1, dynamin related protein 1; YY1, Yin Yang-1; UBA2, ubiquitin-like modifier-activating enzyme 2; FTO, fatmassand- obesity-associated protein; LC3II, light chain 3 II.

The dual role of oxidative stress signaling during HIRI and potential interventions

After reperfusion, the restoration of blood flow led to the release of large amounts of oxidative intermediates, exacerbating the imbalance of oxidative and antioxidant homeostasis in the liver microenvironment. Among these oxidative intermediates, ROS is the most widely known damage agent, secreted by mitochondria and can be eliminated by antioxidant substances. ROS impacts the expression of downstream genes or proteins and regulates different oxidative stress-related signaling pathways, affectingthe process of HIRI [11]. In Wister male rats suffering from I/R, ROS increased significantly as the injury marker, meanwhile the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), malondialdehyde (MDA) and TNF-α also raised in serum, which could be diminished by metformin administration [32]. At the same time, the increase of ROS also involves a variety of immune reactions and the intervention of immune cells, such as mediating the aggregation of neutrophils and macrophages, and the adhesion of leukocytes, thus exacerbating this oxidative stress imbalance and HIRI damage. With the rise of nanomaterials in medical treatment, Zhang et al. [33] designed the Ceria@Apt synthesized by nanoceria and anti-fifth component of complement (C5a) aptamers to inhibit the formation of ROS. The Ceria@Apt alleviated C5a-mediated inflammation via switching valence states to scavenged ROS, restraining neutrophil recruitment and inflammation in the murine model of HIRI [33].

Except for ROS, nitric oxide (NO), as small molecular free radicals, can regulate oxidative stress through regulating various signaling pathways. NO could interact with ROS to derive reactive nitrogen species (RNS) or nitro compounds with high oxidation activity for inducing oxidative and nitrosative stress [34]. Ferreira-Silva et al. [35] found that in addition to ROS and TNF-α, RNS production was also raised in the rat model subjected to hepatic IR by surgery, which could be improved by administration of liposomal quercetin nanoformulation. Another research demonstrated that simvastatin pretreatment could reverse oxidative and nitrosative stress in liver by preventing the increase of NO and nitrotyrosine [36]. Li et al. further interpreted that pretreatment with dietary nitrate significantly decreased oxidative stress by increasing the contents of nitrate, nitrite and NO in liver. Meanwhile, it improved inflammatory responses by decreasing Ly6G⁺neutrophil infiltration and levels of proinflammatory factors (TNF-α, interferon-gamma [IFN-γ], IL-1β, IL-1α, IL-17A, IL-27), while increasing the levels of IL-10 in liver to ameliorate HIRI [37]. Interestingly, investigators also found that NO played a double-edged role in HIRI. Generally, NO is catalyzed through the enzymatic action of nitric oxide synthase (NOS) family including inducible NOS (iNOS), endothelial NOS (eNOS) and neuronal NOS (nNOS) [38]. A zeolitic imidazolate framework-8 (ZIF-8)-based hybrid nanoreactor contains iNOS and platinum nanoparticles (Pt NPs) that act as SOD and catalase (CAT) mimics. This nanoreactor generates NO via the catalysis of iNOS enzyme and eliminates ROS by converting O₂⁻ and H₂O₂ to O₂, accompanied by the inhibition of macrophage activation and neutrophil accumulation [39]. Current evidence suggests that these functional enzymes responsible for the regulation of NO are also involved in the regulation of multiple signaling pathways and thus affect HIRI in multiple ways; for instance, eNOS is also involved in the regulation of mitogen-activated protein kinases (MAPK) family-associated signaling pathway. In the mice with HIRI by surgery, pretreatment of myricitrin, a flavonoid with hepatoprotective property, could raise the NO production and phosphorylation of eNOS expression through activating the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway to ameliorate liver I/R injury [40].

Besides, several regulatory factors that govern cellular redox reactions and contribute to the development of HIRI to varying degrees have also been identified. These factors include nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), nuclear factor kappa B (NF-κB), hypoxia-inducible factor-1alpha (HIF-1α), and tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3). Nrf2 is an emerging cellular regulator that plays a critical role in maintaining redox homeostasis and inhibiting oxidative stress, for example, by regulating downstream target proteins in the HO-1 signaling pathway [41]. Li et al. [42] also pointed out that pretreatment of fisetin promoted cytoplasmic Nrf2 translocate into the nucleus and further elevated HO-1 expression to alleviate cell apoptosis and oxidative stress in IR liver model. Attentively, immune-responsive gene 1 (IRG1), one of the most sensitive genes in proinflammatory conditions, encodes for the enzyme producing itaconate which negatively regulates the inflammatory response. After I/R, IRG1^−/− caused downregulating expression of Nrf2 and its downstream target genes (NADPH quinone oxidoreductase 1 [NQO-1] and HO-1) as well as the upregulation of F4/80-positive macrophages and IL-6 mRNA expression compared with WT mice. However, restoring IRG could reverse oxidative damage by transfecting a plasmid carrying IRG1 [43]. Another study exhibited pretreating veratric acid (VA) also relieved HIRI by Nrf2/HO-1/NQO-1 pathway [44]. Simultaneously, studies have pointed out that the recovery of NADPH oxidase (Nox) 4 activity can produce a similar effect to the activation of Nrf2 signaling pathway. In mice subjected to HIRI, the low doses of sitagliptin could keep the balance of nitrative and halogenative and reduce oxidative stress and inflammatory response via increasing 3-bromotyrosine concentration and upregulating Nrf2 expression, reducing IL-6 and midkine expression and free 3-nitrotyrosine concentration, while partly restoring expression of Nox 4 [45]. These oxidative stress reactions triggered by Nrf2 also interact with many inflammatory reactions or inflammatory transcription factors, thus forming a complex interactive network. Mao et al. [46] found that Nrf2 at T369 was directly phosphorylated due to the overexpression of p21-activated kinase 4 (PAK4, a serine/threonine kinases), causing nuclear export and proteasomal degradation to exacerbate oxidative stress in hepatocyte. Inversely, gastrodin (GSTD), a bioactive compound extracted from a traditional Chinese herb (Gastrodia elata Blume), contributes to the anti-inflammatory and antioxidant effects to protect cells against IR-stressed livers. The pre-administration of GSTD dose-dependently ameliorated surgery-caused HIRI and attenuated oxidative stress and inflammation by inducing HO-1 expression and inhibiting toll-like receptor 4 (TLR4), along with activating Nrf2 and p38 mitogen-activated protein kinases (P38/MAPK) signaling [47]. Notably, the MAPK pathway can trigger the phosphorylation of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK), leading to the activation of the NF-κB inflammatory pathway. Mice subjected to I/R injury by surgery showed the increased NF-κB/p65 phosphorylation and release of TNF-α and IL-6. Nevertheless, kaempferol (KAE), an ingredient isolated and extracted from Penthorum chinense Pursh, reduced transaminase, MDA and raised SOD and GSH in a dosedependent manner and inhibited inflammation via activated Nrf2/HO-1 signaling pathway and restraining NF-κB phosphorylation [48].

In the field of tumor research, it has been well established that fibrocyte growth factor 18 (FGF18), which can regulate the epithelial-mesenchymal transition (EMT) of cell cancer, is involved in the regulation of HIRI to Nrf2. Specifically, injection of FGF18 has been shown to activate Nrf2 by downregulating ubiquitin carboxyl-terminal hydrolase 16 (USP16) expression, leading to increased ubiquitination of kelch-like ECH-associated protein 1 (KEAP1), which ultimately attenuates surgically induced HIRI [49]. Interestingly, vascular endothelial growth factor (VEGF) and Nrf2 are closely related under ROS stimulation, and Nrf2 has functioned as a transcription factor in macrophages, not only maintaining cancer cell growth under oxidative stress but also regulating HIF-1α/VEGF signaling pathways to promote the EMT of cancer cells [50]; however, in the field of HIRI, the regulation of Nrf2 on VEGF is still under study. It is reported that Nrf2 knockdown restricted the expression of VEGF by limiting expression of HIF-1α, failing to palliate oxidative stress in colon tumor cells following hypoxia-mimicking CoCl2 treatment [51]. Whereas, surprisingly, overexpression of HIF-1α could exert protective effects on hepatic IRI by upregulating activation of the A2B adenosine receptor (A2BAR) to prevent adenosine-mediated damage in HIRI with surgery [52]. The finding suggests that HIF-1α is critical to VEGF function played in hypoxia, but its role is currently controversial in HIRI progress. Notably, HIF-1α is also a transcription factor that mediates the expression of the transmembrane protein CEACAM1 (CC1). Recent mechanisms have found that HIF-1α can inhibit hepatic injury in HIRI surgical mice and human donor liver grafts by binding polypyrimidine track-binding protein-1 (Ptbp1) to promote the increase of RNA splicing factor (CEACAM1-short). Specifically, CEACAM1-short exerted a cellular protective effect by inhibiting MAP3K and the p38 pathway (Fig. 2) [53].

Figure 2.

Signaling pathway and targeted therapeutic agents against oxidative stress during HIRI. Oxidative stress could influence HIRI through PI3K/ AKT, Nrf2/HO-1, P38/MAPK, HIF-1α/VEGF, and NF-κB signaling pathways, and regulatory factors including Nrf2, HO-1, NF-κB, HIF-1α and TRAF3 also participate in HIRI development. HIRI, hepatic ischemia-reperfusion injury; A2BAR, A2B adenosine receptor; TLR4, toll-like receptor 4; ROS, reactive oxygen species; ZIF-8, zeolitic imidazolate framework-8; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; NO, nitric oxide; iNOS, inducible nitric oxide synthase; eNOS, endothelial NOS; GSTD, gastrodin; IRG, immune- responsive gene; PAK4, p21-activated kinase 4; KAE, kaempferol; NF-κB, nuclear factor kappa B; P38, p38 mitogen-activated protein kinases; MAPK, mitogen-activated protein kinases; VA, veratric acid; NQO-1, NADPH quinone oxidoreductase 1; Nox 4, NADPH oxidase; VEGF, vascular endothelial growth factor; HIF-1α, hypoxia-inducible factor-1alpha; HO-1, heme oxygenase-1; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-alpha; IL-6, interleukin-6; IL-17A, interleukin-17A; IFN-γ, interferon-gamma; IL-10, interleukin-10; Nrf2, nuclear factor erythroid 2-related factor 2.

The mechanism of discovered abnormal cell death signaling during HIRI and corresponding treatment strategies

Cell death is the initially direct cell-damaging consequence of HIRI, including apoptosis, autophagy, pyroptosis and ferroptosis. Generally, there are two main pathways involved in apoptosis of HIRI process, namely exogenous pathway activated by death receptors and endogenous pathway. The intrinsic apoptosis pathway, also known as mitochondrial death pathway, is activated by different death signals, which eventually cause mitochondrial dysfunction and further induce apoptosis. Under the stimulation of ROS, apoptotic markers B-cell lymphoma-2 (Bcl-2) family and Bcl-2-associated X protein (Bax) can form oligomer complexes, which damage MMP and lead to the release of cytochrome c (Cyt C, proteins encoded by nuclear genes) as well as downstream cascade reaction of cell apoptosis [54]. In the rat model of HIRI following surgery, chlorogenic acid (CGA) pretreatment reduced the levels of MDA and ROS while increasing SOD and GSH levels to relieve oxidative stress. CGA also decreased hepatocyte injury by suppressing the mitochondria-mediated apoptotic pathway accompanied by reduced expression levels of Bax, Cyt-c, cleaved-caspase9, cleaved-caspase3, ENDOG, and apoptosis-inducing factor (AIF) [55]. Saleh and El-Shorbagy [56] demonstrated the same therapeutic effect by chitosan administration on liver I/R injury. The intriguing discovery was that sevoflurane (SEV) preconditioning led to the downregulation of miR-124-3p and the increased expression of TRAF3, which not only decreased the expression of apoptosis-related factors but also promoted the ubiquitination and degradation of cAMP-responsive element-binding protein (CREB) to collectively alleviate HIRI both in vivo and in vitro [57]. It is well known that small mothers against decapentaplegic (SMAD), ERK, JNK, transforming growth factorbeta-activated kinase 1 (TAK1), p38 MAPK, NF-κB or PI3K/AKT are all common participants through direct or indirect involvement in the process of apoptosis. More concretely, some studies have found that SMAD was also involved in the process of apoptosis, which may be related to miR-210 [58]. Notably, in a murine hepatic IRI model by surgery, ghrelin administration delayed the transition from HIRI to liver fibrosis process via the inhibition of SMAD and ERK signaling pathways [59]. Pan et al. [60] also found that the level of miR-210 was increased after hepatic I/R injury but was accompanied by less inflammatory responses and hepatocytes apoptosis in miR-210 KO mice. Mechanistically, miR-210 could restrain SMAD4 expression, impair SMAD4-mediated anti-apoptosis and thus exacerbate cell death and liver damage. Similarly, miRNA can also suppress gene expression through specific binding to genes responsible for regulating apoptosis. In a rat HIRI by surgery, administration of agomir-miR-34a-5p markedly decreased the expression of hepatocyte nuclear factor 4α (HNF4α), cleavage cas-3, Bax, JNK, and P38, thereby ameliorating HIRI [61].

As a matter of fact, among these pathways involved, JNK was highly concerned first as a regulatory target to improve apoptosis in HIRI. Based on the above conclusions, the researchers pay close attention to regulating the processes of HIRI by targeting either the upstream or downstream targets of JNK. In mice and cell line exposed to HIRI, the overexpression of liver-specific Yes-associated protein (YAP) promoted autophagy to thus reduce apoptosis via upregulating the phosphorylation of JNK. However, it is worth noting that the interaction between YAP and transcriptional enhanced associate domain (TEAD) could reverse the activation effect of YAP on JNK, resulting in the upregulation of apoptosis levels [62]. In mice model exposed to hepatic I/R injury by surgery, Guo et al. [63] found that TAK1 was another key upstream target of JNK/p38 proapoptosis signaling pathway, and six-transmembrane epithelial antigen of the prostate 3 (Steap3), an iron regulatory protein, could directly interact with TAK1 and promote TAK1-JNK/p38 signaling activation to accelerate hepatic cell apoptosis. As the study progressed further, tripartite motif-containing (TRIM) 27 was investigated as another key regulator and TAK1-related target to attenuate hepatic I/R injury through reducing inflammation and cell apoptosis. It has been reported that TRIM27 significantly diminished TAK1 activity via promoting the degradation of TAK1 binding protein 2/3 (TAB2/3) repressing K63-linked polyubiquitin, thus contributing to the inhibition of downstream JNK/p38 and NF-κB signaling pathways [64]. Concurrently, Zhang et al. [65] in the same lab further discovered regulator of G-protein signaling 14 (RGS14), a complex scaffolding protein that integrates the G-protein and MAPK signaling pathways, not only inhibited the activation of TAK1 and downstream P38/JNK signaling but also reduced CD11b⁺ cell infiltration in the liver tissues to exert protective effects on HIRI in hepatocyte-specific RGS14 transgenic (RGS14-TG) mice. With deeper investigation, researchers searched out new regulatory targets that apoptosis-regulating kinase 1 (ASK1) could also regulate intracellular signaling pathways, such as JNK and p38MAPK, thereby further inducing cell apoptosis. Researchers have found that the interaction between ring finger protein 5 (RNF5) and ubiquitinated phosphoglycerate mutase family member 5 (PGAM5) could repress ASK1-JNK-p38 signaling pathways via inhibiting ASK1 phosphorylation and increasing PGAM5 degradation to protect against HIRI [66]. Attentively, N-acetylgalactosaminyltransferase-4 (GALNT4) is the first step to catalyze protein O-glycosylation, which interacted with ASK1 and inhibited N-terminal dimerization and activation of ASK1, resulting in repression of downstream JNK/p38 and NF-κB signaling pathways to alleviate HIRI induced by surgery [67].

In addition to the JNK pathway, PI3K/Akt signaling pathway also extensively regulates apoptosis and HIRI. In recent years, numerous studies have aimed to identify key targets involved in the regulation of PI3K/Akt on the one hand, and to seek more effective protective drugs against this pathway on the other hand. β-Arrestin-2 (ARRB2) belongs to a member of regulatory proteins involved in the G protein-coupled receptors (GPCRs) signaling pathway and also takes part in PI3K/Akt-mediated apoptosis; the knockdown of ARRB2 resulted in high expression of caspase 3 and significant inhibition of PI3K/Akt signaling, leading to more severe apoptosis in mice after HIRI [68]. Lipoxin A4 receptor (ALXR), as another key target protein that can bind to GPCRs, is thought to participate in infiltration or apoptosis through AKT signaling. It has been reported that maresin 1 (MaR1) administration repressed inflammatory response, oxidative stress, and hepatocyte apoptosis via enhancing the ALXR/AKT signaling pathway, resulting in alleviation of I/R-mediated liver damage [69]. Moreover, Li et al. [70] found another protein that myristoylated alanine-rich C-kinase substrate (MARCKS), a regulatory molecule in actin dynamics, could not only activate p38/JNK MAPK but also suppress PI3K/AKT signal to exacerbate inflammation and cell apoptosis. Interestingly, MiR-142-3p overexpression could alleviate HIRI by decreasing MARCKS levels. At the same time, natural products have been found to improve apoptosis and HIRI by directly regulating PI3K/AKT activity. Methyl eugenol (ME), a natural enylbenzene, also attenuated inflammation and apoptosis by activating the PI3K/AKT pathway [71]. Additionally, Ding et al. [72] further explained the pro-survival effect of MAPK in endogenous apoptosis. In mice subjected to HIRI by surgery, cordycepin precondition inhibited MAPK phosphorylation and NF-κB expression to reduce Beclin-1 and LC3-induced autophagy, on the one hand, decreased BAX and caspase 9/3-induced apoptosis in a dose-dependent manner by inhibiting activation of the MAPK/NF-κB signaling pathway.

Emerging evidence suggested that the changes in epigenetic modification states such as histone modification and small ubiquitin-like modifier (SUMOylation) can influence apoptosis in HIRI process. Histone deacetylase (HDAC) is mainly responsible for removing acetyl from histone, which is related to inhibition of gene transcription and expression. There are studies finding that carbon monoxide (CO) inhalation pretreatment prominently increased the mRNA expression of SIRT1 and activity of SIRT1 deacetylase by decreasing expression of miR-34a, causing the upregulation of NF-κB p65 and p53 deacetylation to inhibit NF-κB p65/p53-related apoptotic response in mouse with HIRI surgery [73]. Moreover, LP342, as a candidate of the HDAC inhibitors (HDACi), was found to inhibit HDAC I and promote NF-κB p65 acetylation, subsequent depressing of JNK activation and caspase 3 level to alleviate inflammation and apoptosis in male mice with HIRI surgery [74]. Observingly, SUMOylation is a novel form of protein posttranslational modification that inhibits gene transcription of the protein. In mice with HIRI surgery, protein inhibitor of activated STAT1 (PIAS1), a small ubiquitin-like modifier (SUMO) E3 ligase, reduced hepatocyte inflammatory response and apoptosis by promoting nuclear factor of activated T cells 1 (NFATc1) SUMOylation [75]. Meanwhile, NFATc1 downregulation caused by SUMOylation decreased histone deacetylase 1 (HDAC1) and HDAC1-mediated interferon regulatory factor 1 (IRF1) and resulted in inactivating the p38 MAPK signaling, thus alleviating HIRIrelated symptoms [75].

In addition to apoptosis, pyroptosis is another form of programmed cell death that is initiated by inflammasomes and plays a crucial role in eliminating pathogens and reducing infection. This process can be identified by monitoring levels of apoptosis-associated speck-like protein (ASC), gasdermin D (GSDMD), and caspase-1, as well as the presence of IL-1β and IL-18 [76]. Following HIRI surgery, the secretion of ikaros by infiltrating macrophages activated inflammasomes, suppressed SIRT1 expression, and accelerated hepatic pyroptosis and inflammatory response in an adenosine monophosphate-activated protein kinase (AMPK)-dependent manner in mice. Myeloid-specific disruption of SIRT1 activated caspase-1-GSDMD processing, resulting in the increased release of IL-1β and IL-18 to exacerbate HIRI [77]. As a matter of fact, the methods of inhibiting pyroptosis for improving HIRI are usually divided into two types: on the one hand, activating the relevant signaling pathway of a non-dependent pathway of caspase; on the other hand, directly inhibiting caspase function. Caspase 1 and Caspase 11 are proteins associated with cell death and inflammation, which activate inflammatory factors and detect sterile stressors (DAMPs) [78]. Wu et al. [79] pointed out that dexmedetomidine (DEX), a highly selective α2-adrenergic agonist, repressed ASC and GSDMD-N levels and inhibited the activation of the nucleotide-binding oligomerization domain (Nod)-like receptor family pyrin domaincontaining 3 (NLRP3) inflammasome and pyroptosis by increasing miRNAs-494 expression, inhibiting JUND (a proinflammatory transcription factor) and activating the PI3K/AKT/Nrf2 pathway in hypoxia/reoxygenation (H/R) cells. Concurrently, NLRP3 silencing regulated pyroptosis by downregulating caspase 1 activation and IL-1β and IL-18 contents [79]. Kolachala et al. [80] further confirmed that in mice fed a high-fat diet (HFD) and undergoing HIRI, there was a significant increase in the expression of GSDMD and caspase-1. However, the double gene knockout of Caspase 1/11 or the deletion of the Caspase 1 gene improved liver injury by inhibiting inflammation and pyroptosis. Meanwhile, it was verified that suppressing Caspase 1 could suppress pyroptosis and play a protective role in hepatocytes, but not Caspase 11 [80].

Apart from apoptosis and pyroptosis, ferroptosis is a hot research topic but is relatively rare compared to the other two in HIRI study. When induced by a high iron diet, ferroptosis leads to iron overload, lipid peroxidation, and increased expression of Ptgs2 (a marker of ferroptosis), exacerbating HIRI [81]. However, treatment with ferroptosisspecific inhibitors like ferrostatin-1 (Fer-1), α-tocopherol, or iron chelators such as deferoxamine (DFO) can prevent cell death and reduce inflammatory damage in the liver [81]. Proverbially, phospholipids are essential for oxidation in the process of ferroptosis and are regulated by long-chain acyl-coenzyme A synthase 4 (ACSL4). This study further suggested that GP78 upregulated the expression of Ptgs2 and ACSL4, promoting lipid remodeling to accelerate ferroptosis in gp78 overexpressed mice feeding MCD diets after surgery HIRI, which could be reversed by rosiglitazone, an inhibitor of ACSL [82]. Most recently, we found that aqueous extract of Radix Rehmanniae Praeparata regulated intracellular iron metabolism by inhibiting zinc transporter14 (ZIP14)-mediated iron uptake and promoting hepcidinmediated iron efflux processes to ameliorate HIRI [83], suggesting a possibility of improving HIRI by inhibiting ferroptosis (Fig. 3).

Figure 3.

Potential strategies against cell death during HIRI. (A) SMAD, ERK, JNK, TAK1, p38 MAPK, NF-κB, and PI3K/AKT influence HIRI by affecting apoptosis. (B) Several mechanisms influence HIRI progression by affecting pyroptosis. (C) Potential role of ferroptosis in HIRI. Bcl-2, B-cell lymphoma-2; Bax, Bcl-2-associated X protein; MAPK, mitogen-activated protein kinases; JNK, c-Jun N-terminal kinase; YAP, Yes-associated protein; ARRB2, β-Arrestin-2; MaR1, maresin 1; ME, methyl eugenol; CO, carbon monoxide; NF-κB, nuclear factor kappa B; MARCKS, myristoylated alanine-rich C-kinase substrate; PGAM5, phosphoglycerate mutase family member 5; RNF5, ring finger protein 5; ASK1, apoptosis-regulating kinase 1; TAK1, transforming growth factor-beta-activated kinase 1; JNK, c-Jun N-terminal kinase; P38, p38 mitogen-activated protein kinases; MAPK, mitogen-activated protein kinases; PIAS1, protein inhibitor of activated STAT1; NFATc1, nuclear factor of activated T cells 1; GALNT4, N-acetylgalactosaminyltransferase-4; TRIM27, tripartite motif-containing 27; RGS14, regulator of G-protein signaling 14; HNF4α, hepatocyte nuclear factor 4α; CGA, chlorogenic acid; AIF, apoptosis-inducing factor; TRAF3, tumor necrosis factor receptor (TNFR)-associated factor 3; SEV, sevoflurane; CREB, cAMP-responsive element-binding protein; HDAC1, SUMOylation decreased histone deacetylase 1; IRF1, interferon regulatory factor 1; IL-1β, interleukin-1β; IL-18, interleukin-18; Cyt C, cytochrome c; AKT, PI3K, protein kinase B; phosphatidylinositol 3-kinase; NLRP3, nucleotide-binding oligomerization domain (Nod)-like receptor family pyrin domain-containing 3; SIRT1, sirtuin1; GSDMD, gasdermin-D; Nrf2, nuclear factor erythroid 2-related factor 2; ASC, apoptosis-associated speck-like protein; Dex, dexmedetomidine; ZIP14, zinc transporter14; PEG35, polyethylene glycol 35 kDa; Fer-1, ferrostatin-1; DFO, deferoxamine.

The involvement of intracellular DAMPs in HIRI progression and related pharmacological therapy

DAMPs are intracellular molecules with immunoregulatory properties that are released during cell damage, death, or activation. These DAMPs, including HMGB1, IL-33, and ST2 growth stimulator expression factors, can trigger the release of inflammatory mediators, enhance the adhesion and infiltration of inflammatory cells, and modulate immune responses [84]. For instance, pretreatment with the natural product γ-oryzanol (ORY) could restrain the HMGB1/NLRP3/IL-1β signaling pathways, thereby reducing inflammation caused by HIRI [85]. It is important to investigate whether DAMPs contribute to the increase in pro-inflammatory factors that worsen inflammation and tissue damage, or if these pro-inflammatory cytokines stimulate the release of more DAMPs, further amplifying the immune response.

Acknowledgedly, the main and most studied receptor for HMGB1 is TLR4. In a mouse model of surgical ischemia and a hydrogen peroxide-induced hepatocyte model, the pretreatment of pinocembrin, a monomer of flavonoid extracted from Penthorum chinense Pursh, scavenged ROS and attenuated tissue damage in liver accompanied by decreased levels of Bax and MDA and increased Bcl2, SOD and GSH levels via inhibiting the HMGB1/TLR4 signaling pathway [86]. Interestingly, after being fed with inulin, intestinal microbiota in mice can produce large amounts of propionic acid (PA) [87], of which administration also restrained HMGB-1/TLR4 pathway and downregulated the production of inflammatory cytokines and chemokines in macrophages, which alleviated IR-induced liver injury [88]. However, this study only detected the protein expression of HMGB1 and TLR4 at this time, but there was a lack of discussion on the pathway relationship and direct evidence. Subsequently, Kawasoe et al. [89] further verified the effect of the domain 1 of recombinant thrombomodulin (rTMD1) on the HMGB1/TLR4 pathway on protein interaction to facilitate HIRI function. rTMD1 pretreatment diminished the activation of Ly-6G⁺ neutrophils and CD11b⁺ macrophages by blocking the binding of HMGB1 and TLR4 and subsequent reducing HMGB1/TLR4 pathways and the release of pro-inflammatory cytokines and chemokines after reperfusion. It is worth noting that rTMD1 can still improve the damage caused by HMGB1/TLR4 signal, even after the IR insult has started [89]. In addition to HMGB1/TLR4 regulation mode, the level of plasma thrombin, the main effector protease to activate thrombomodulin (TM), is mainly dependent on domain 1 (rTMD1) for its regulatory role [90]. was strikingly increased in clinical patients after hepatectomy. Noguchi et al. [91] further found that as a highly selective inhibitor of thrombin, dabigatran pretreatment could inactivate and degrade the excessive expression of HMGB1 to decrease neutrophilic recruitment by increasing the expression of TM and downregulate the expression of inflammatory cytokines (TNF-α and IL-6) and VCAM-1 caused by HMGB1 infiltration in mice model of HIRI. Subsequently, the researchers found that HMGB1-induced damage in HIRI is also closely related to epigenetic modification. Sosa et al. [92] indicated that human orthotopic liver transplant (OLT) recipients with IRI showed the significant upregulation of disulfide-HMGB1 (dis-HMGB1), histone acetyltransferase GTF3C4 and decrease of histone deacetylase HDAC5, which further elevated hyperacetylation of HMGB1 and translocated into cytoplasmic vesicles as a pro-inflammatory cytokine, ulteriorly activating pro-inflammatory macrophages and promoting TNF-α secretion from macrophages by combining TLR4 to induce cell death.

Once activated by whatever form in HIRI, HMGB1 will inevitably exacerbate inflammatory damage and immune responses by affecting multiple signaling pathways, such as the relatively most studied NF-κB. Nogo-B (Reticulon 4B), a protein mainly localized to the endoplasmic reticulum, was largely expressed on macrophages and facilitated macrophage infiltration in mouse ischemic livers. Nogo-B^KO deficiency inhibited phosphorylation of NF-κB p65 and expression of Nogo-B deletion-induced YAP, which further decreased M1 macrophage polarization in HMGB1-stimulated bone marrow-derived macrophages (BMMs), which could be eliminated with YAP siRNA intervention in vivo and in vitro [93]. This above study indicated that the regulation of immune response by HMGB1 is a worthy research direction in HIRI. With further deeper research, in mice subjected to IR procedure following Annexin A1 N-terminal peptide Acetyl 2-26 (Ac2-26) preconditioning, neutrophil infiltration was reduced, accompanied by fewer Ly6G⁺ cells and MPO activity by suppressing the activation of HMGB1/TLR4/NF-κB signaling pathway [94]. Furthermore, multiple studies have developed that a nanostructured lipid carrier of berberine isolated from traditional medicinal plants (NLC BBR) therapy [95] or lncRNA potassium voltage-gated channel subfamily Q member 1 opposite strand/antisense transcript 1 (KCNQ1OT1) knockdown [96] could palliate HIRI via restraining the HMGB1/TLR4/NF-κB inflammatory signaling. In addition to TLR4, our recent study demonstrated that HMGB1 may also influence TLR3 to aggravate HIRI. Likewise, acteoside (ACT) could prevent HMGB1 bounding with TLR3/4 on LSECs and the subsequent activation of IRF1 nuclear translocation, as well as restrain neutrophils chemotaxis, which eventually exerts protective effect from an immune perspective in mice with HIRI surgery [97]. Besides TLR4/3, TLR9 is another pivotal innate immune receptor. Allyson etal. elaborated that dis-HMGB1 promoted mass generation of monocyte cytokines as well as stimulated the polarization of monocyte-derived macrophage (MDM) from OLT patients by activating TLR4 and TLR9, which further contributed to an enhanced ability of MDM to present antigens and activate T cells and aggravated the degree of liver injury in OLT murine model of IRI [98]. Gratefully, restrained diSHMGB1 with the therapy of HMGB1 neutralizing antibodies or TLR4/9 inhibitors could reverse the above situation.

IL-33 plays a crucial role in DAMPs and can regulate immune responses and contribute to the progression of HIRI. Following HIRI in mice, there is a rapid release of IL-33, and studies have shown that IL-33-deficient mice exhibit reduced I/R injury, as evidenced by decreased neutrophil recruitment, lower levels of ALT, and reduced tissue damage [99]. Furthermore, IL-33 may serve as a potential biomarker for early hepatic I/R injury in human liver transplantation, as its serum levels increase immediately after reperfusion of the liver graft [99]. However, it is worth noting that subsequent studies have pointed out that IL-33 may also play a beneficial protective role, which may be related to the positive regulation of M2 macrophage in HIRI from some perspectives. Notably, after I/R injury, a low dose but long-course administration of IL-33 reduced hepatocyte edema and hepatic vessel congestion and were associated with the increasing polarization of CD45⁺CD11b⁺F4/80^high macrophages to the M2 type [100]. Mechanically, innate lymphoid cells (ILC2s) proliferation stimulated by exogenous IL-33 could promote M2 macrophage polarization via regulating the IL-4/JNK/Stat3 pathway [100]. This suggests that IL-33 is a double-edged sword and its intrinsic mechanism in HIRI needs to be further explored (Fig. 4).

Figure 4.

The mechanism of DAMPs involved in HIRI, especially HMGB1 and IL-33. (A) The mechanism of HMGB1 included in HIRI. (B) IL-33 is a double-edged sword during HIRI progression. NF-κB, nuclear factor kappa B; ROS, reactive oxygen species; NLRP3, nucleotide-binding oligomerization domain (Nod)-like receptor family pyrin domain-containing 3; HMGB1, high mobility group box-1 protein; IRF1, interferon regulatory factor 1; KCNQ1OT1, potassium voltage-gated channel subfamily Q member 1 opposite strand/antisense transcript 1; ACT, acteoside; ORY, γ-oryzanol; NLC BBR, nanostructured lipid carrier of berberine isolated from traditional medicinal plants; rTMD1, recombinant thrombomodulin; Ac2-26, acetyl 2-26; HDAC5, Histone deacetylase 5; TNF-α, tumor necrosis factor-alpha; IL-6, interleukin-6; IL-4, interleukin-4; IL-1β, interleukin-1β; IL-33, interleukin-33; JNK, c-Jun N-terminal kinase; YAP, Yes-associated protein.

Effect of intracellular inflammatory response on HIRI development and providing a promising solution

Although inflammatory targets have been covered in previous chapters, once HIRI happened, apart from the released DAMPs and immune cells mentioned above, hepatic inflammatory signaling is associated with many kinds of intracellular damage and have more complex effects on liver damage and immunologic process than we thought [101,102]. As above research demonstrated, chemokines and chemokine receptors are also elements that trigger or respond to inflammation involved in HIRI. It has been widely regarded as a truism that TLR4 has a powerful recognition function like the eyes of the immune response, and is involved in inflammation and cell damage by TLR4-dependent or independent myeloid differentiation factor 88 (MyD88) pathway. At the same time, this process also involves many transcription factors that participate in TLR4 regulation. Notably, NF-κB is involved in the TLR4-dependent MyD88 pathway or NLRP3 signal pathway, which can cause severe inflammation and cell damage. There is evidence that NF-κB, a key regulator of intranuclear immunity, also has potential to become a downstream regulatory target of receptor tyrosine kinase (AXL) that has significant anti-inflammatory and antiapoptotic effects. Activated AXL decreased hepatic inflammation by promoting the expression level of suppressor of cytokine signaling-1 (SOCS-1) to inhibit TLR4 and its downstream signaling pathways (MyD88/NF-κB) and inflammatory cytokines (TNF-α, IL-1β, and IL-6) expression on one side, as well as alleviated hepatocyte apoptosis and improved HlRI via reducing the proapoptotic proteins BAX and cleaved caspase-3 levels, and increasing the antiapoptotic protein Bcl-2 levels on the other side [103]. In addition, telmisartan, a partial agonist of PPARγ, also can directly decline the expression of pro-inflammatory cytokines and attenuate the downstream inflammatory response by inhibiting TLR4/NF-κB signal pathway to protect liver IR [104]. Moreover, TRAF3, as an adaptor molecule, regulates NF-κB pathways causing inflammatory responses. The ubiquitinated TRAF3 at Lys138 aggravated oxidative stress and activated the NF-κB inflammation pathway induced by H/R in BRL-3A cells, while HOPE therapy effectively relieved IR [105]. On the other hand, morin (MRN), a natural flavanol isolated from plants of the Moraceae family, also counteracted the inflammation by repressing TLR4, NF-κB and NLRP3 expression following the hepatic impairment [106]. It is important to recognize that both ASC and caspase-1 play crucial roles in the NLRP3 inflammasome and collectively impact inflammatory responses. Furthermore, pre-treatment with salidroside, an active compound derived from Rhodiola plants, has been shown to reduce the activation of NF-κB and the NLRP3/ASC/caspase-1 inflammasome, as well as inhibit the TLR4-dependent MyD88 pathway, leading to a dose-dependent decrease in proinflammatory cytokine levels by blocking TLR4 activation. Noteworthy, the hepatoprotective effects of salidroside can be negated by the administration of a TLR4 agonist, LPS [107].

Proverbially, inflammation in the intestinal tract can potentially alter the composition of intestinal microbes and their metabolites. Simultaneously, hepatic inflammation can be triggered by the affected intestinal tract and the intestinal microbes, leading to an intensification of inflammation. Clostridium butyricum (C. butyricum), a probiotic in human gut, restored liver function via decreasing the Firmicutes/Bacteroidetes ratio and increasing the relative abundance of probiotics such as Lactobacillus and Bifidobacterium. This was associated with a decline in the levels of short-chain fatty acids (SCFAs) and butyric acid levels in a rat model of HIRI. Meanwhile, C. butyricum supplement attenuated inflammation accompanied by reduced LPS, TNF-α and IL-6 levels and downregulated TLR4 and NF-κB p65 expressions [108]. Other evidence further explained the mechanism about how intestinal flora regulates inflammation induced by HIRI. The pretreatment of prophylactic antibiotics (Abx) could attenuate HIRI associated with the reduction of CCR2⁺ monocytes recruitment by reshaping the gut microbiota including decreasing the relative abundance of Firmicutes and increasing the relative abundance of Clostridium, increasing the levels of hepatic unconjugated bile acid species (unconjugated BAs, especially UDCA), and activating liver farnesoid X receptor (FXR) [109]. Further, the activation of FXR decreased the expression of TLR4 and suppressed its downstream MAPK/NF-κB signaling pathways and the CCL2-CCR2 axis both in vivo and in vitro, along with the decrease of TLR4, p-ERK1/2, p-P38 and p-JNK1/2 expression [109].

Considering that ischemia and reperfusion usually lead to the impairment of whole hepatic function, hepatocyte regeneration and functional recovery after liver injury have also been a hot research direction. Schmitz et al. [110] found the inhalation of noble gas argon upregulated the pro-inflammatory response by increasing expression of cytokines encompassing IL-1β and IL-6 and reduced hepatic regeneration capacity by declining the mitotic index and the cell cycle marker Ki-67 levels to aggravate liver damage after the mice with HIRI surgery [110]. Observingly, when hepatocytes are damaged, autophagy can restore the volume and function of the liver by eliminating abnormal hepatocytes and enhancing hepatocyte regeneration. Pituitary adenylate cyclase-activating polypeptide was reported to exert hepatoprotective effects by amplifying hepatocellular regeneration in autophagy-dependent manner, which was relied on the improvement of CREB activation and Kruppellike factor 4 (KLF4) expression [111]. On the other hand, in cases of liver dysfunction following I/R injury, autophagy activation may also play a negative regulatory effect under certain circumstances. For instance, autophagy may also decline the proliferation of hepatocytes by enhancing the activation of non-parenchymal hepatocytes. As research pointed out, IRF8, a pivotal upstream intracellular regulatory factor, caused cytokine secretion and autophagy formation. Shi et al. [112] elucidated that IRF8 knockout inhibited inflammatory injury and improved HIRI, whereas, unlike IRF8 knockout mice, the IRF8 overexpression of mice exerted a higher exacerbation of liver injury. Further research showed that IRF8-mediated chemokine release (CXCL1, CXCL9 and CCRL2) significantly aggravated the liver structure damage via accelerating autophagy formation and the NF-κВ pathway in Adeno-Associated Virus 8-IRF8-I/R (AAV8-IRF8-I/R) mice, which could be remedied by hydroxychloroquine (HCQ) or secukinumab pretreatment [112]. Notoriously, the chemokine receptor CXCR4 stands out for its pleiotropic roles, for example, as potential substrates and receptors of dipeptidyl peptidase-4 (DPP-4) and also as a receptor for CXCL12. Sitagliptin acted as DPP-4 inhibitors, of which pretreatment downregulated the expression of chemokine stromal-derived factor 1 (SDF1) and CXCR4 to protect liver in HIRI animals (Fig. 5) [113].

Figure 5.

Pathophysiological effects and consequences of intracellular inflammation in HIRI. Inflammation could influence HIRI through TLR4, JNK/p38, NF-κB and other kinds of signal pathways. Abx, antibiotics; MRN, morin; FXR, farnesoid X receptor; SOCS-1, suppressor of cytokine signaling-1; AXL, receptor tyrosine kinase; TLR4, toll-like receptor 4; NF-κB, nuclear factor kappa B; HOPE, hypothermic oxygenated perfusion; TRAF3, tumor necrosis factor receptor (TNFR)-associated factor 3; HCQ, hydroxychloroquine; PACP, pituitary adenylate cyclase-activating polypeptide; CREB, cAMP-responsive element-binding protein; KLF4, kruppel-like factor 4; IRF8, interferon regulatory factor 8; CCRL2, C-C motif chemokine receptor-like 2; CXCL1, C-X-C Motif Chemokine Ligand 1; CXCL9, C-X-C Motif Chemokine Ligand 9; ASC, apoptosis-associated speck-like protein; NLRP3, nucleotide-binding oligomerization domain (Nod)-like receptor family pyrin domain-containing 3; TNF-α, tumor necrosis factor-alpha; IL-6, interleukin-6; IL-4, interleukin-4; IL-1β, interleukin-1β; TLR4, toll-like receptor 4; JNK, c-Jun N-terminal kinase; P38, p38 mitogen-activated protein kinases; MAPK, mitogen-activated protein kinases.

Regulation of immune cell on HIRI development and establishing protective approaches

The immunity events contributing to hepatocyte damage are fairly complicated and involve the interaction between hepatocytes, neutrophils, macrophages, KC and other immune cells in the pathophysiological process of HIRI. Specifically, there is no consentaneous conclusion on this question about how do immune cells attack hepatocytes to exacerbate injury or ameliorate inflammation during HIRI. As is known to all, neutrophils have chemotactic, phagocytotic and bactericidal properties, and they are attracted to the site of injury by chemotactic substances when HIRI occurs. Chronic plus binge alcohol (Gao-binge)-fed mice underwent hepatic IRI surgery and showed neutrophil infiltration and the increase in mixed lineage kinase-like protein (MLKL) plasma membrane translocation [114]. However, the deletion of Mlkl had non-significant protective effects against IR liver injury in ethanol-fed mouse livers. [114]. Fortunately, dermcidin (DCD) is a peptide derived from sweat glands and its analog is contained on a Cys (C)→Ser (S) substitution at residue 34 (DCD-C34S), all observably preventing IR damage at the beginning of reperfusion. Further, DCD attenuated Gr-1-positive neutrophils infiltration and I/R-elicited inflammation by suppressing the phosphorylation of epidermal growth factor receptor (EGFR) and AKT as well as reducing the expression of a neutrophilic CXC chemokine, CXCL2 and NO production [115]. Besides CXCL2, the adhesion and infiltration of neutrophils are also related to other adhesion molecules. Other evidence demonstrates that endomucin (EMCN) is a negative regulator of leukocyte adhesion. And the overexpression of EMCN inhibited leukocyte adhesion and the infiltration of CD11b⁺Ly6G⁺ neutrophils caused by HIRI via preventing integrin subunit beta 2 (ITGB2, LFA-1) on neutrophils binding to intercellular adhesion molecule-1 (ICAM-1) on liver sinusoidal endothelial cells (LSECs) to abrogate liver injury [60]. Similarly, CD321 also is an essential junctional adhesion molecule (JAM) to advance neutrophil migration into inflammatory tissues [116]. CD321, the essential molecules for trans-endothelial migration of leukocytes, could interact with lymphocyte function-associated antigen 1 (LFA-1) on CD45⁺ neutrophils to dramatically increase infiltrated neutrophil in a murine HIRI model, while anti-CD321 monoclonal antibody (90G4 mAb) treatment could significantly block neutrophil and leukocytes infiltration in the early phase of HIRI [116]. Not only CD321, but carabin, as the negative regulator of calcineurin, also can affect neutrophil migration by interacting with calcineurin and Ras [117]. Carabin-deficient mice (LysMCre: Carabinfl/fl) showed more severe damage and inflammatory symptoms associated with CD11b⁺Ly6G⁺ neutrophils recruitment via the activation of Ras-ERK and calcineurin-NFAT (nuclear factor of activated T cells) pathways, which could be reversed by treatment with anti-P-selectin, E-selectin and avb3 integrin [117].

Furthermore, neutrophils can aggravate HIRI by inducing chemokines to the injured site, but also intensify HIRI through the formation of neutrophil extracellular traps (NETs) [118]. The knockdown of histidine-rich glycoprotein (HRG) led to the increased expression of inflammatory factors (IL-6 and TNF-α), MPO-DNA, nucleosome and Ly6G⁺ neutrophil accumulation, as well as up-regulation of NETs formation, accompanied by the downregulation of FXR and hypoxia inducible factor 2 alpha subunit (HIF2A) in HIRI mice, and gave rise to the susceptibility of the methioninecholine-deficient diet (MCD) mice to HIRI [119]. Another study pointed out that resveratrol, as a natural polyphenol found in kinds of plants, its preintervention repressed endothelin 1 secreted by neutrophils in an autocrine manner to ameliorate oxidative stress and the inflammatory response of neutrophils accompanied by decreased NETs generation via suppressing the ERK/c-Fos signaling pathway [120]. In addition to neutrophils themselves affecting the immune microenvironment and aggravating HIRI, what is noteworthy is that NETs could activate LSECs by promoting the expression of integrins on LSEC to augment infiltration of neutrophil pro-inflammatory immune cells in a murine model of liver IRI. Interestingly, exercise training (ExT) preconditioning as a non-pharmacological therapy before the operation attenuated neutrophils infiltration by intercepting NET formation [121].

Among these immune cells, macrophages are divided into BMDM and liver-derived macrophages called KCs. Notably, Kupffer cells, colonized in the liver and distributed along the LSECs, have caught the researchers’ attention in HIRI process. Under TNF-α stimulation, KCs can secrete neutrophil chemotactic agents and pro-inflammatory factors, conclusively inducing neutrophil immune response [122]. HIRI mice also showed the recruitment of KCs, as well as release of proinflammatory cytokines and ROS derived from the activation of KCs and neutrophils. But a hydrophilic carbohydrate-derived nanoparticle (C-NP) was synthesized as an effective nanoantioxidant against HIRI and suppressed the inflammatory effect of the above immune cells by removing excessive ROS [123]. Proverbially, Rao et al. [124] ulteriorly indicated that the expression of Nrf2 in KCs was significantly increased in HIRI mice. Myeloid-specific Nrf2-KO mice (Nrf2^M-KO) with IR stress-induced liver injury showed more hepatic ROS, inflammation, and hepatocellular apoptosis/necrosis that were associated with the activation of the Ras homolog family member A (RhoA)/Rho-associated coiled-coil containing protein kinase (ROCK) pathway by repressing the target gene tissue inhibitor of metalloproteinase 3 (Timp3). Sulforaphane (SFN) promoted Nrf2 translocation in KCs to enhance Nrf2 interacting with the antioxidant response element (ARE), and declined hepatic injury in ARE-luc mice after IRI [125]. Moreover, researchers have indicated that except for Nrf2, immune regulatory factors IL-18 and a stimulator of interferon genes (STING) also actively participate in macrophages regulation, respectively effecting the activation of macrophages [126] and the release of apoptosis signals by macrophages [127]. Aging mice exhibited more serious HIRI associated with excessive proinflammation in macrophages by intensifying intrahepatic NLRP3 activation in STING-dependent manner [128]. Further research showed that hepatic STING significantly upregulated the expression of caspase 1-GSDMD and inflammatory cytokines IL-1β/IL-18 to cause inflammation and damage but which were prominently restrained by the treatment of knockdown STING in macrophages, VX-765 (caspase 1 inhibitor) or liposome-encapsulated clodronate (the macrophage inhibitor) during HIRI. In vitro, STING strengthened caspase 1-GSDMD processing and inflammatory response by increasing intracellular Ca²⁺to initiate Kupffer cell pyroptosis after H/R, which could be prominently mitigated by BAPTA-AM (a calcium chelator) administration [129].

Moreover, VEGF contains a variety of receptor subtypes that may play their own unique roles in HIRI. Therapy of pro-lymphangiogenic factors like VEGF-D can improve liver repair in mice model of HIRI and attenuate hepatic surgery I/R injury by fortifying the number of hepatic restorative macrophages and the expression of mannose receptor (MR), Fizz1 and IL-10 [130]. In contrast, chemokines and chemokine receptors can be secreted by pro-inflammatory KCs and regulate cell migration further to aggravate injury during HIRI. Wang et al. [131] also reported that after reperfusion, proinflammatory phenotype of the C1QC_KC cluster specifically expressed CXCL12 and responded to the CXCR4 receptor on the T/B cell clusters to upregulate the migration of cells, as well as highly expressed TNFAIP3 interacting protein 3 (TNIP3). Interestingly, TNIP3 secreted by inflammatory cells could ameliorate HIRI by decreasing inflammation via inhibiting NF-κB pathway [131]. In addition, a new subpopulation of KCs that is colony-stimulating factor 3 (CSF3) positive KCs, namely inflammation-associated Kupffer cell subtype (iKC), has been found to exacerbate HIRI injury through the IL-17 signaling pathway, but also activated T cells in adaptive immunity to participate in the immune response of HIRI. At the same time, CSF3⁺KCs can facilitate the antigen presentation of dendritic cells (DCs) through JAG1/Notch2 and CD47/signal regulatory protein gamma (SIRPG) axes, indirectly promote the recruitment of CCR7⁺ CD8⁺ T cells in transplanted steatotic liver microenvironment, and ultimately aggravate inflammatory damage caused by I/R injury [132].

Interestingly, the researchers indicated that KCs and BMDM may co-regulate HIRI processes by regulating similar signaling pathways. Principally, CD47 and signal regulatory protein alpha (SIRPα) are immune test sites, but play a positive dominant role in the immune response of HIRI process. In mice model of IR-induced sterile inflammatory liver injury, adoptive transfer of mesenchymal stem cell (MSC) showed that CD47 interacted with macrophage SIRPα. This interaction inhibited proinflammatory mediators by enhancing downstream SMO activity and increased nuclear expression of Gli1 and Notch1 intracellular domain (NICD) of macrophage, which caused Dvl2 upregulation. Dvl2 upregulation acts as a negative regulator of NLRP3-driven inflammatory response, thereby inhibiting NEK7/NLRP3 activity [133]. Notably, as a Nod-like receptor, NLRP3 is related to the regulation of Notch signal of immune cells mediated by Jagged1 (JAG1) [134]. Jin et al. [135] found that in Notch1-proficient (Notch1^FL/FL) model of liver I/R injury, JAG1-mediated myeloid Notch1 signaling pathway not only reduced macrophage infiltration under IR stress by promoting heat shock transcription factor 1 (HSF1)/Snail activation and inhibiting TXNIP/NLRP3 activation, but also reduced apoptosis of hepatocytes under IR stress. Interestingly, Snail suppressed pro-inflammatory cytokines such as TNF-α, IL-1β and CXCL-1 and ameliorated the inflammatory response and apoptosis through the downregulation of NLRP3 and caspase-1 in the Notch1^FL/FL model. Moreover, adoptive transfer of lentivirus expressing HSF1 transgenic macrophages could improve inflammatory response and hepatocellular apoptosis by enhancing Snail expression and inhibiting NLRP3, thioredoxin-interacting protein (TXNIP), and ASC expression in Notch1 knockout model (Notch1^M-KO) [135].

Along with the deepening of study, the researchers pointed out that the polarization of liver macrophages and its related immune response are closely related to HIRI. HIRI mice exhibited KCs M1 polarization and the increase of TNF-α, IL-1β and IL-6, which was decreased by liraglutide treatment [136]. Another study further demonstrated the intracellular mechanism affecting the direction of hepatic macrophages polarization. Alpha B-crystallin (CRYAB) promoted the differentiation of liver macrophages into M2 phenotype through activating AKT1/mammalian target of rapamycin (mTOR), thereby alleviating the liver IRI in RAW 264.7 cell hypoxia/reoxygenation (H/R) model. It was also found that miR-450b-5p can directly target CRYAB and inhibit the Akt/mTOR phosphorylation and CRYAB level in liver IRI, but miR-450b-5p inhibitors can improve the above situation [137]. Besides, Zhou et al. [138] similarly proved that macrophage migrated to injury location and polarized to M1 state in vitro by activating cathepsin E (Cat E)/AKT/mTOR signaling pathway, which could be inhibited by plasma membrane-bound G protein-coupled bile acid receptor (TGR5) in myeloid cells. While ursodeoxycholic acid administration could relieve HIRI by activating TGR5 and facilitating macrophage M2 polarization. Attentively, Zito et al. [139] pointed out that peripheral blood mononuclear cells (PBMC)-derived M1-like macrophages were a befitting model to research HIRI in vitro. It elaborated that human amnion-derived mesenchymal stromal/stem cells (hAMSCs) pre-conditioning by the IFN-γ pre-treatment could also restrain inflammation induced by M1-differentiated macrophages via reducing IL-1β, IL-12 and phosphotransferase system gene (PTSG) 2 levels, activating hepatocyte growth factor (HGF)-met signaling (Fig. 6) [139].

Figure 6.

The mechanisms by which immune cells affect HIRI. (A) Immune response happened in KCs during HIRI. (B) CSF3⁺KCs promote the recruitment of CCR7⁺ CD8⁺ T cells through JAG1/Notch2 and CD47/SIRPG axes. (C) The neutrophils infiltration and NET formation are closely related to HIRI. (D) The polarization of liver macrophages and its related immune response play an important role in HIRI. HIRI, hepatic ischemia-reperfusion injury; TNF-α, tumor necrosis factor-alpha; IL-6, interleukin-6; IL-12, interleukin-12; IL-1β, interleukin-1β; IL-18, interleukin-18; ASC, apoptosis-associated speck-like protein; GSDMD, gasdermin-D; NLRP3, nucleotide-binding oligomerization domain (Nod)-like receptor family pyrin domain-containing 3; TXNIP, thioredoxin-interacting protein; AKT, protein kinase B; Cat E, cathepsin E; KCs, Kupffer cells; T cell, T lymphocyte; LSEC, liver sinusoidal endothelial cell; STING, stimulator of interferon genes; SFN, sulforaphane; HSF1, heat shock transcription factor 1; SIRPG, signal regulatory protein gamma; ARE, antioxidant response element; Nrf2, nuclear factor erythroid 2-related factor 2; Timp3, target gene tissue inhibitor of metalloproteinase 3; RhoA, Ras homolog family member A; ROCK, Rho-associated coiled-coil containing protein kinase; CXCL12, C-X-C Motif Chemokine Ligand 12; CXCR4, chemokine (C-X-C motif) receptor 4; JAG1, agged1; SIRPα, signal regulatory protein alpha; DCD, dermcidin; EGFR, epidermal growth factor receptor; CXCL2, C-X-C Motif Chemokine Ligand 2; NO, nitric oxide; ERK, extracellular signal-related kinase; ITGB2, LFA-1, integrin subunit beta 2; ICAM-1, intercellular adhesion molecule-1; NET, neutrophil extracellular trap; HRG, histidine-rich glycoprotein; ExT, exercise training; HGF, hepatocyte growth factor; hAMSCs, human amnion-derived mesenchymal stromal/stem cells; PTSG2 phosphotransferase system gene 2; mTOR, mammalian target of rapamycin; TGR5, plasma membrane-bound G protein-coupled bile acid receptor; CRYAB, Alpha B-crystallin; AKT, protein kinase B; VEGF, vascular endothelial growth factor; MR, mannose receptor; IL-10, interleukin-10.