Protective role of heme oxygenase-1 in fatty liver ischemia–reperfusion injury

Kupffer cells are liver-resident macrophages located in the sinusoidal lumen, mainly at sinusoidal branch points. They constitute liver sinusoidal cells along with other kinds of cells, such as endothelial cells, stellate cells and dendritic cells. All of these non-parenchymal cells interact in the process of hepatic IR damage. Previously, it was thought that Kupffer cells were immobilized or ‘fixed’. Now, studies have shown that they move along the sinusoid to the damaged areas of the liver [44]. Kupffer cells contribute to liver damage during ischemia and reperfusion. During the early stage of reperfusion, Kupffer cells alter their morphology and produce large amounts of ROS induced by ischemia followed by reperfusion [45]. In addition, hepatocyte apoptosis occurs in the phases of IR, resulting in the release of endogenous damage-associated molecular patterns (DAMPs), such as high mobility group box-1 (HMGB1) and denatured nuclear DNA. DAMPs activate Kupffer cells by binding to Toll-like receptor (TLR), which in turn generates an inflammatory reaction, producing a great deal of pro-inflammatory cytokines, such as TNF-α and interleukin-1 (IL-1) [46]. These pro-inflammatory cytokines play an essential role in aggravated hepatic IR damage. The release of cytokines through oxygen-free radicals and activated Kupffer cells facilitates the removal of protein–polysaccharide complexes from the surface of vascular endothelial cells and increases the exposure of adhesion molecules on the surface of endothelial cells. This phenomenon promotes the adhesion of neutrophils and platelets to the sinus endothelial cells, thereby exacerbating endothelial cell injury and ultimately causing serious damage to microcirculation and aggravating the degree of tissue ischemia.

Kupffer cells are considered to play an important role in liver IR damage. The initial stages of reperfusion make dramatic morphological changes to activated Kupffer cells and compel them to extend into the central sinusoid [47]. Activated Kupffer cells are major contributors to the release of not only large amounts of cytokines but also intracellular ROS [48]. Evidence suggests that liver parenchymal cell damage occurs in the hypoxic phase of IR injury. Pharmacological preconditioning for protection against hepatic IR injury by reducing Kupffer cell activation has been reported. Indeed, Mosher et al. [49] reported that gadolinium chloride relieves liver cell injury caused by liver IR by inhibiting Kupffer cell activity. Kupffer cells are a kind of macrophage located in the hepatic sinusoid and are the first cells to come into contact with the exogenous immune reactive substance. Previous reports have shown that a high-fat diet increases the number of activated Kupffer cells and is associated with the severity of inflammation [50]. In the early stage of liver IR, the release of DAMPs induced by ischemic injury and the binding of TLRs on the surface of Kupffer cells leads to the activation of Kupffer cells [51]. Activated Kupffer cells further enhance the inflammatory response by releasing a large amount of inflammatory cytokines [52].

A study examining that, whether or not HO-1 up-regulation exerts a direct protective effect on active Kupffer cells which aggravate reperfusion injury identified by cobalt protoporphyrin (inducer of HO-1) and zinc protoporphyrin (antagonist of HO-1), respectively. Found that down-regulated the expression of HO-1 by zinc protoporphyrin, the reperfusion injury was aggravated by Kupffer cells activation [53]. The production of inflammatory cytokines and the CD14 expression were decreased in the cobalt protoporphyrin-pretreated group. There was an interesting experiment that has proved the importance of Kupffer cells in IR injury. Kupffer cells and circulating monocytes were ablated by liposomal clodronate in CD11b diphtheria toxin receptor mice, which subsequently suffered liver ischemia. The depletion of Kupffer cells reduces the expression of HO-1 and increases the sensitivity to liver IR injury, but the ablation of circulating monocytes kept the IR injury from becoming more serious. Indicated that Kupffer cells are the main cells expressing HO-1 in the liver and exert anti-inflammatory effects against inflammation-induced oxidative damage, such as IR injury [54].

CO is a product generated by HO-1 degrading heme. It is a signaling molecule and plays a critical role in anti-inflammatory activities, anti-apoptosis activities and vasodilation [55]. The pretreatment of liver donors with CO improves their hepatic IR damage by increasing the number of Kupffer cells and the anti-inflammatory HSP70 pathway expression [56]. Furthermore, HO-1 overexpression in animal liver transplants is mainly achieved through less infiltrating macrophages and inhibiting the expression of inducible nitric oxide synthase [57, 58].

In general, hepatic IR injury is characterized by the recruitment of neutrophils and infiltration into the portal area after ischemia [59]. Acute inflammatory reactions include two consecutive stages: in the Kupffer cell-dominant stage (0–6 h of reperfusion), ROS increase liver injury, and Kupffer cell activation and lymphocyte infiltration induce the secretion of cytokines that further exacerbate the inflammatory response; in the second stage (6–24 h of reperfusion), the neutrophil activation was completely achieved and expressed various types of mediators, including ROS, protease, CXCL-1 and CXCL-2, aggravated liver injury [60]. The retinoic acid receptor-related orphan receptor-γt (RORγt)/IL-17A axis plays an important role in regulating the sub-acute neutrophil-mediated inflammatory responses. The recruitment of lymphocyte and IR injury were found to be diminished in IL-1R1- and IL-17A- knockout mice [61].

Studies on liver injury have identified a neutrophil elastase inhibitor with therapeutic potential that can promote the secretion of high-mobility group box 1 and then reduce the IL-6 expression [62]. In addition, the MMP-9-deficient model of hepatic IR showed an improvement in liver damage that was associated with neutrophil translocation through the hepatic sinusoids [63]. The migration of neutrophils across the endothelial cells and extracellular matrix barriers is a complicated process in hepatic IR injury. CD11b⁺/CD18⁺ neutrophils are very important for the adhesion on the hepatocyte surface and vascular endothelial cells [64]. In addition, CD44 plays an important role in neutrophil infiltration induced by IR injury in mouse liver. Indeed, pretreatment with CD44 antibody reduced the neutrophil infiltration and ameliorated the sinus congestion and hepatocyte necrosis [65]. Although preclinical data have shown good results, a clinical trial of anti-adhesion therapy for IR injury showed no significant improvement [66].

Recently, several investigators have focused on the following three foci concerning the role of neutrophils and lymphocytes in hepatic IR injury: (1) the rapid progress of liver IR injury is not consistent with the time of the T cell response; (2) most CD4⁺ T cells are mainly natural killer T (NKT) cells recruited after liver reperfusion; (3) NKT cells rapidly produce cytokines after stimulation. Shimamura found that the proportion of NKT cells rapidly increases after portal vein clamping-induced IR injury in the liver [67]. The proportion or amount of NKT cells in the total hepatocytes peaked at 10–20 h after reperfusion. The hepatic injury amplitude was reduced by 50% at 6 h after perfusion injury in NKT cell-deficient mice. Interestingly, NKT cells produce interferon-gamma within 2 h after reperfusion [68]. This is due to the fact that NKT depletion in this model reduces biochemical and histological damage, and the adoptive transfer of NKT cells to lymphocyte-deficient mice restores the level of damage to wild-type animals. Experiments have also shown that the aspartate aminotransferase (ALT) in mice treated with synthetic adenosine 2A receptor (A2AR) agonists was reduced by 58%. A2AR agonists can directly reduce the production of IFN-γ by activated NKT cells. All of these results indicated that IR injury and pro-inflammatory cytokine/chemokine transcription levels were significantly reduced following systemic treatment with A2AR agonists [69].

Some investigators have shown that CD4⁺ T cells induce neutrophil recruitment in liver reperfusion injury [68, 70, 71]. However, the mechanism underlying direct tissue injury after liver blood flow recovery is not clear. Using a partial ischemia model, Kuboki et al. found that NKT cells and not natural killer cell (NK cells) play the dominant role in hepatic IR injury through the activation of CD1D-dependent T cell receptor [72]. In contrast, the antibody depletion of NKT cells alone or with NK cells significantly ameliorated liver injury after 8 h of reperfusion. The loss of regulatory T cells has no effect on IR injury. Although the exact mechanism underlying tissue damage is unclear, the release of IFN-γ from the recruited NKT cells can stimulate other proinflammatory cytokines, which may exacerbate liver damage and affect the neutrophil function.

The expression of activated caspase-3 and caspase-9 is high in NAFLD patient specimens. Furthermore, the degree of liver cell apoptosis is notably increased and closely associated with the ponderance of the disease [73]. Hepatocyte apoptosis was found to be positively correlated with liver fibrosis [73]. The present study from Syn WK’s team found that there are three indivisible risk factors associated with apoptosis of NAFLD hepatocytes: dyslipidemia in the liver; cellular stress resulting from changes in oxidation, metabolism and cytokines; and mitochondrial dysfunction [74]. The expression of caspase-3 in plasma and the content of soluble Fas are increased in fatty liver patients. These two biomarkers have a good correlation with liver histopathology [75].

The progress of classical NAFLD is explained by the “two-strike theory”. The first attack is caused by metabolic changes that induce lipid accumulation and steatosis in the liver; the second attack is due to mitochondrial dysfunction caused by metabolism, oxidation, and cytokine secretion [76]. Antioxidants, such as vitamin E and betaine, reduce the level of transaminase via reducing cellular oxidative stress and inhibiting hepatic parenchymal cells apoptosis [77]. Cytokines such as TNF-α, IFN-γ and IL-6 activate the downstream pathway leading to hepatocyte apoptosis [74, 78].

Mitochondrial dysfunction is an extremely important factor influencing the progress of NAFLD hepatocyte apoptosis [77]. It destroys the balance of lipid metabolism in liver cells, mediates oxidative stress and increases the level of ROS. The overproduction of ROS may destroy mitochondrial proteins, phospholipids and even mitochondrial DNA [79, 80]. The mitochondrial DNA depletion of hepatocytes results in a reduction in the expression of mitochondrial DNA-encoded polypeptides and increased mitochondrial dysfunction [81].

Apoptosis and necrosis are common reactions in the liver when suffering injuries induced by ischemia, radiation and poisonous substance [30‐32]. The liver, especially the hepatic parenchymal cells, is sensitive to damage caused by IR injury [33, 34]. In IR injury, the occurrence of oxidative damage in the enzyme complexes and the decrease in the anti-apoptotic protein level cause apoptosis of hepatic parenchymal cells [35]. The inhibition of the caspase family was found to notably attenuate hepatic injury induced by IR, indicating that apoptosis plays a key role in IR damage [36]. Hepatocyte apoptosis leads to elevated AST and ALT levels and further reduction of the liver function [37].

A liver IR injury rodent model was generated by 1-h portal vein occlusion and 24 h of reperfusion. The hepatocyte apoptosis was markedly increased in the liver IR injury group compared with the sham operation group and became more aggravated over time (55-fold increase after 4 h of reperfusion and 200-fold increase after 24 h of reperfusion) [82]. The interaction between various types of hepatocytes, like parenchymal cells, Kupffer cells and neutrophils, promotes hepatic IR injury [53].

Liver IR damage induces hepatocyte apoptosis and produces a series of changes, including oxygen-free radical production, calcium overload and changes in the permeability of mitochondrial membranes and the expression of cytokines and apoptotic genes [45, 83, 84]. Ca²⁺ overload, anaerobic metabolism, acidosis and oxidative stress trigger hepatocyte apoptosis during hepatic IR injury. Intracellular Ca²⁺ overload activates Ca²⁺-dependent enzymes, eventually leading to cell apoptosis [85]. Oxidative stress causes mitochondrial dysfunction and lipid peroxidation and induces apoptosis by stimulating the production of active molecules, like ROS [86].

Molecular hydrogen, a new type of antioxidant, ameliorated hepatocyte apoptosis in hepatic IR injury by inhibiting the level of oxygen radicals [87]. The process of aerobic and anaerobic metabolism inhibits redox reaction in hepatocytes, resulting in the depletion of intracellular ATP. This shortage of energy leads to mitochondrial damage and microcirculation failure. Enhanced anaerobic glycolysis reduces the intracellular pH and ultimately leads to apoptosis associated with acidosis [88]. A number of cytokines are involved in the regulation of apoptosis, including Bax (pro-apoptotic factor) and Bcl-2 (anti-apoptotic factor) [89]. In steatosis hepatocytes suffering from hypoxia/reoxygenation injury, the Bax expression was found to be significantly up-regulated, while the expression of Bcl-2 was dramatically down-regulated [90].

The overexpression of HO-1 exerts strong cytoprotective functions in many liver IR injury models. The expression of HO-1 in normal or fatty liver transplantation or after transplantation can be increased by drugs or gene engineering, which maintains the tissue structure and organ function and prolongs the survival time of the graft [57]. The overexpression of HO-1 reduces the hepatic apoptotic IR damage by reducing the C/EBP homologous protein levels and expression of NF-κB mediated genes, such as MCP-1 and IL-6, while increasing the IκBa expression (repressor of NF-κB) [91]. Adenovirus-mediated HO-1 overexpression was reported to increase the expression of anti-apoptotic molecule BAG-1 and reduce the number of apoptotic cells in mice [92]. Interestingly, the expression of Bcl-2 and BAG-1 (anti-apoptotic genes) was increased in grafts with a good liver function, while the Caspase-3 expression was significantly decreased in such grafts. Obviously, the cytoprotective effect of HO-1 is directly related to the increase in the expression of anti-apoptotic genes [93]. CO, the degradation product of HO-1 can reduce the apoptosis induced by IR damage through synergistic action of miR-34 A/SIRT1 signaling pathway [94].

5-ALA is an important intermediate product of heme synthesis and is the basic substance of aerobic energy metabolism. It acts as a precursor of photosensitizers for a photodynamic diagnosis (PDD) and photodynamic therapy (PDT) to identify and kill tumor cells [95]. In the human body, 5-ALA is synthesized to protoporphyrin IX (PpIX) in the mitochondria, and divalent iron ion is inserted into PpIX to form heme. Hayashi et al. showed that the usage of SFC as a form of iron enhanced the specific effect of 5-ALA in tumor PDT [96].

Several studies found that the coalition of 5-ALA and SFC mediated the overexpression of HO-1 [97, 98]. Pretreatment with the coalition of 5-ALA and SFC resulted in the increased expression of HO-1 in mouse kidney, and CO produced by the degradation activity of HO-1 showed a protective effect against IR damage in kidney [97]. Previous findings obtained by the authors have shown that application of the precursor of heme anabolism (5-ALA) along with the byproduct of heme catabolism (ferrous iron) in the macrophage cell line RAW264.7 up-regulated the HO-1 gene expression to produce an anti-inflammation effect [99]. Our laboratory’s data indicated that the treatment of coalition of 5-ALA and SFC ameliorated the IR damage in steatosis livers. The protective effects were mediated by the high concentration of CO, and ROS were reduced by inhibiting the TNFα/NF-κB pathway [99].

Astaxanthin (ASTX) is a xanthophyll carotenoid, that can be produced by a variety of seaweed and microorganisms [100]. ASTX has many antioxidant properties because it has a unique and remarkable molecular structure of hydroxyl and keto moieties on each ionone ring [101]. The antioxidant properties of ASTX have been previously observed in the plasma, eye and liver of rats fed microalgae biomass, which contained ASTX, dispersed in olive oil. Furthermore, the levels of antioxidant enzymes were up-regulated when ASTX was administered to ethanol-induced gastric ulcer rats [102]. In addition, an in vitro study showed the beneficial effect of ASTX in tubular epithelial cells stimulated by high glucose, which induced inflammation and oxidative stress [103].

Some reports have shown that ASTX dissolved in olive oil had no negative effects and in fact exerted protective effects against oxidative stress [104]. Recently, Ni et al. reported that ASTX was more useful for preventing and treating steatotic liver than vitamin E in mice [105]. Furthermore, several reports have shown that ASTX attenuates hepatic [106], retinal [107], and renal [108] IR injury in rodent models. Our previous study demonstrated the protective effect of ASTX in steatotic liver affected by IR injury. The histopathological features of IR injury, including necrosis, apoptosis infiltration of macrophages, lipid peroxidation products, liver enzyme leakage and inflammatory cytokine expression, were corrected by ASTX. The inhibition of ROS production and inflammatory cytokine expression, induction of HO-1/Nrf-2 in Kupffer cells and inactivation of MAPK may be involved in the inhibitory mechanism of ASTX against IR injury in the steatotic liver. The decrease in the expression of cleaved caspase-3/9 and Bax and the up-regulation of Bcl-2 in hepatocytes may also play a role [90].

Molecular hydrogen has a novel medical application. Similarly, nitric oxide and CO, which exert cytoprotective effects against cellular stress, have also drawn attention [109]. Molecular hydrogen exerts cytoprotective effects in the nervous, cardiovascular and digestive systems [110‐112]. According to our unpublished data, hepatic protection by molecular hydrogen was observed in a mouse model of fatty liver IR, and hypoxia/reoxygenation-induced damage was observed in fatty hepatocytes in an in vitro model. Hydrogen saline exerted marked hepatic protection by preventing hepatocyte death and inhibiting macrophage recruitment compared with mice treated with normal saline. Furthermore, its application inhibited hypoxia/reoxygenation-induced damage in fatty hepatocytes. During ischemia/hypoxia and subsequent reperfusion/reoxygenation, a large number of harmful substances, such as pro-inflammatory cytokines, which produced by activated Kupffer cells then resulting in hepatocyte apoptosis.

Recently, some researchers have found that molecular hydrogen exerts potent pharmacological effects by reducing oxidative stress, inflammation and apoptosis [113, 114]. Molecular hydrogen has emerged in the form of hydrogen-rich water or inhaled hydrogen gas and is recommended as an effective treatment for cardiac arrest in the clinical setting. There are many ways to administer molecular hydrogen, with oral treatment being the most convenient, although some hydrogen gas will escape into the stomach via this route. The administration of hydrogen saline to the target tissue allows for more efficient molecular hydrogen delivery [115]. A recent report derived from rat models has highlighted the novel, promising therapeutic application of imaging-guided hydrogen bubble delivery to prevent myocardial IR injury [116].

Growing evidence indicates that the protective effect of molecular hydrogen is not only interrelated with the elimination of oxygen-free radicals but also associated with various intercellular signals [117, 118]. Kawamura et al. reported that hydrogen gas ameliorated lung injury by promoting the expression of Nrf-2, which contributes to HO-1 expression [119]. Cai et al. [120] revealed that molecular hydrogen therapy relived TNF-α-induced rat osteoblast inflammatory injury via the down-regulation of the NF-κB pathway. Furthermore, there is also evidence that molecular hydrogen reduces lung injury induced by transplantation by increasing the expression of HO-1 [119]. In our research, hydrogen saline exposure was found to significantly increase the expression of HO-1 according to in vivo data. The protective mechanism underlying hypoxia/reoxygenation-mediated hepatic injury was clarified in an in vitro study. Our results demonstrated that HO-1 positively regulates the expression of Sirt1, while HO-1 and Sirt1 attenuate the Kupffer cell activation. Sirt1 inhibits the activity of the p53 pathway by directly inducing the production of the apoptosis regulator Bcl-2 and inhibiting the transcription of Bax and the activation of cleaved caspase-3.