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New progress in understanding roles of nitric oxide during hepatic ischemia-reperfusion injury

Hepatic ischemia-reperfusion injury (HIRI) is a major clinical cause of morbidity and mortality in liver surgery and transplantation. Many studies have found that nitric oxide (NO) plays an important role in the HIRI and its increase or decrease can affect the progression and outcome of HIRI. However, the role of NO in HIRI is controversial and complicated. NO derived by endothelial NO synthase (eNOS) shows a protective role in HIRI, while excessive NO derived by inducible NO synthase (iNOS) accelerates inflammation and increases oxidative stress, further aggravating HIRI. Nevertheless, the overexpression of eNOS may exacerbate HIRI and iNOS-derived NO in some cases reduces HIRI. Here we review the new progress in the understanding of the roles of NO during HIRI: (1) NO possesses different roles in HIRI by increasing NO bioavailability, down-regulating leukotriene C4 synthase, inhibiting the activation of the nuclear factorκB (NFκB) pathway, enhancing cell autophagy, and reducing inflammatory cytokines and reactive oxygen species (ROS). And NO has both protective and deleterious effects by regulating apoptotic factors; (2) eNOS promotes NO production and suppresses its own overexpression, exerting a hepatoprotective effect reversely. Its activation is regulated by the PI3K/Akt and KLF2/AMPK pathways; and (3) iNOS derived NO mainly has deteriorating effects on HIRI, while it may have a protective function under some conditions. Their expression should reach a balance to reduce the adverse side and make NO protective in the treatment of HIRI. Thus, it can be inferred that NO modulating drugs may be a new direction in the treatment of HIRI or may be used as an adjunct to mitigate HIRI for the purpose of protecting the liver.


INTRODUCTION
Hepatic ischemia-reperfusion injury (HIRI) is a major complication often seen in liver surgery and organ transplantation. It manifests as cellular damage during the ischemic phase and worsens during reperfusion. Depending on the different conditions of ischemia, HIRI can be divided into warm ischemia-reperfusion (WIR) injury and cold ischemia-reperfusion injury (IRI), which have similar pathophysiology but different clinical injury sites [1].
With a high incidence of cases, liver cancer has increased by 114.0% and ended up with 1007800 cases in 2016 [2]. Many liver cancer patients are supposed to be treated by liver transplantation or hepatectomy, where HIRI occurs during the operation [3]. Although HIRI is receiving increasing attention to improve the success rate of surgery and improve prognosis, very few of them are known.
The pathophysiological process of hepatic IRI involves the interaction of many different cell types and numerous signaling pathways such as anaerobic metabolism, acidosis, oxidative stress, and intracellular calcium overload. Among the interactions, the imbalance in the ratio of endothelin (ET) to nitric oxide (NO) is one of the mechanisms involved in HIRI. Normally, their function is to regulate blood flow to the hepatic sinusoids. In contrast, in the first few hours after reperfusion, as ET rises, plasma expression of NO decreases, leading to an increase in the ET/NO ratio and the possible appearance of HIRI [4,5].
Many pieces of evidence show that NO plays an important role in ischemia-reperfusion (I/R) [6,7]. However, as a vasodilator, the role of NO has been controversially discussed by scientists [8,9]. In past studies, NO was regarded as a negative factor because of its cytotoxic effect [10]. Nevertheless, a recent study indicated that NO can induce either a positive or negative effect during the early phase of HIRI and have a protective effect during late HIRI [11]. Therefore, it is important to further explore the protective mechanism of NO in HIRI.
NO is a small molecule free radical that can easily penetrate cell membranes. It is also an important effector and messenger molecule of biological information, which has undergone many extensive types of research in the past few years. There are two sources of NO in the human body-enzymatic production and non-enzymatic production. Non-enzymatic production mainly comes from chemical degradation and inorganic nitrogen transformation on the body surface or ingested. For enzymatic production, NO is oxidized from L-arginine by NO synthase (NOS) [12].
There are Ca 2+ -independent and Ca 2+ -dependent NOS in the human body. Ca 2+ -dependent NOS can be subdivided into neuronal (nNOS) and endothelial (eNOS). eNOS is an enzyme continuously expressed in vascular endothelial cells and exerts biological functions through producing NO. In contrast to eNOS, Ca 2+ -independent inducible NOS (iNOS) is activated by some exterior factors including viruses, bacteria, pro-inflammatory interferon, and cytokines [13]. iNOS produces a large amount of NO in hepatocytes, cholangiocytes, and Kupffer cells (KCs), helping macrophages to mount an immune response [14].

Increase of NO bioavailability involved in its protective effect in HIRI
Hide et al [16] found that NO bioavailability was reduced during reperfusion by detecting the levels of cyclic guanosine monophosphate, a second messenger of NO. They concluded that the decreased NO bioavailability can be explained by the reduction of eNOS activity leading to less synthesis of NO and increased NO clearance by reacting with ROS and forming peroxynitrite, which may later react with cell components such as proteins, lipids, and DNA, further damaging the cell. Therefore, increasing NO bioavailability can protect the liver from further damage during HIRI. It is reported that obestatin enhances NO bioavailability by up-regulating eNOS expression [17]. Also, simvastatin maintains NO bioavailability by preventing kruppel-like factor 2 (KLF2) down-regulation [16].

NO can down-regulate LTC4S by inhibiting the nuclear factorκB pathway
Many studies reveal that cysteinyl leukotrienes are directly associated with hepatic IRI. Leukotriene C4 synthase (LTC4S) is one of the enzymes that are responsible for LTC4 synthesis, showing a strong relationship with the NO [18,19]. In I/R rats, the gene expression level of LTC4S is much higher. However, this is reversed by V-PYRRO/NO, which acts as an NO donor. Hong et al [20] also found that another NO donor, sodium nitroprusside, could down-regulate the mRNA expression of LTC4S by inhibiting nuclear factor-kappa B (NF-κB) activation in an NF-κB α inhibitor-independent manner by detecting the protein levels of NF-κB p65 and p50 in the nuclear extracts using Western Blot.

NO can regulate some apoptotic signal transduction pathways and factors
NO has a significant role in regulating some apoptotic signal transduction pathways which can be potentially activated to induce or inhibit the hepatic cell apoptosis process caused by HIRI during the hepatic operation or other hepatic diseases. The signal pathways or apoptosis-related genes including caspases [21][22][23][24][25], Bcl-2 gene family [26][27][28][29][30], mitogen-activated protein kinase (MAPK) [31], and NF-kB [32]. Studies have shown that the caspase family is strongly related to hepatic cell apoptosis[33]. Zhao et al [21] found that steatosis-induced decline in adenosine monophosphate-activated protein kinase (AMPK)-catalyzed phosphorylation permits caspase-6 activation, leading to hepatocyte death. And Gao et al [22] indicated that caspase-3A is involved in cadmium (Cd)-induced cell apoptosis in common carp, which showed 71.8% sequence similarity and 59.3% sequence identity to human caspase-3. Zhang et al [23] found that Cd treatment increased the level of iNOS and NO. The overexpression of NO leads to chicken hepatic cell apoptosis by inducing the mitochondrial apoptotic pathway. In two other studies, mouse liver cell apoptosis can be inhibited by reducing NO content, down-regulating Bax protein expression, and increasing Bcl-2 protein expression [24,25]. Besides, an imbalanced Bax/Bcl-2 ratio is caused by decreasing levels of NO and iNOS and increasing Bcl-2 expression through the NF-kB pathway. And this imbalanced ratio may show a protective role in the damaged liver [30]. Jiang et al [34] also found that 7-mer peptide can increase the level of Bcl-2 and decrease the level of Bax expression to reduce apoptosis and protect against IRI.

NO protects against I/R-induced liver injury by enhancing autophagic flux
NO has an important role in protecting against I/R-induced liver injury by enhancing autophagic flux. During severe environments such as IRI, the cell will undergo an autophagic process, which is an adaptive response to reduce the injury. Studies have found that the protective mechanism of NO during HIRI is associated with autophagic flux. Shin et al [35] demonstrated that NO could enhance light chain-3 lipidation and autophagosome-lysosome fusion during hepatic I/R. Also, eNOS-induced NO enhances autophagy via p38 MAPK activation during liver I/R. Simvastatin, which is used to protect the donor  liver, can activate autophagy and increase NO release during hepatic transplantation. This also indicates the possible connection between NO and autophagy [36].

NO decreases inflammatory cytokines and reduces ROS by inhibiting the mitochondrial respiratory chain
During reperfusion, the surge of inflammatory factors, cytokine liberation, neutrophil infiltration and ROS generation occurred, which led to hepatic injury. An increased level of NO can reduce cytokines such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-1 which stimulate infiltration and endothelial injury. Also, continuous NO production can reduce ROS and proinflammatory cytokine generation as well as neutrophil infiltration [37]. Inversely, NO deficiency can induce TNF-α expression as a result of ROS surging. Ragy et al [38] proved this by adding Nomega-nitro-L-arginine methyl ester (L-NAME) in IRI model rats treated with oxytocin. In this group, not only did the parameter damage increase but also the inflammatory factor such as TNF-α level increased compared with the control group.

Activated eNOS produces NO to protect HIRI
eNOS performs various biological functions by promoting the production of NO, which is important for maintaining vascular tone and cardiovascular hemostasis, and inhibiting platelet activation and aggregation. It has been confessed that eNOS shows a hepatoprotective effect in HIRI by improving the production of NO (Figure 1). There are two main regulation pathways for eNOS activation, one dependent on intracellular concentration of Ca 2+ and the other independent. The increasing intracellular Ca 2+ level can enhance the affinity of calmodulin binding to eNOS and activate enzymes to produce NO [39]. For the Ca 2+ -independent regulation pathway, phosphorylation of the Ser1177 residue or dephosphorylation of the Thr495 residue activates it to produce NO[40].

Calcium-dependent eNOS activation
At the early stage of HIRI, the ischemia will lead to a shortage of oxygen and nutrients, which can decrease ATP availability. Without energy, ATP-dependent ion channels or transporters cannot work. The incompetence of the Na + /K + pump leads to depolarization of the cell membrane, resulting in the influx of Ca 2+ [41]. Besides, anaerobic glycolysis induces an increase in H + , which activates intracellular proteases to increase cellular permeability. Furthermore, Na + /Ca 2+ exchange is activated due to a high concentration of H + , leading to a further influx of Ca 2+ . Consequently, eNOS is activated due to the increase of intracellular concentration to produce NO, carrying anti-HIRI activities at the initial stage.

Calcium-independent eNOS activation
Phosphoinositide 3-kinase/Akt pathway induced eNOS activation: The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is a cell survival pathway that regulates cell proliferation and apoptosis, as well as an endogenous negative feedback regulator that functions in anti-inflammation and antiapoptosis effects in IR.
PI3K can activate Akt to act on the phosphorylation of eNOS. It has been proven that telluric acid has a hepatoprotective effect by elevating the expression of eNOS, which is accompanied with elevated expression of p-PI3K and p-Akt proteins. Besides, the activation of PI3K/Akt also inhibits NF-κB and activates nuclear erythroid-related factor-2, reducing pro-inflammation cytokine expression and inducing anti-oxidative effects [42]. Moreover, through the PI3K/Akt pathway, apelin preconditioning can increase the expression of eNOS and counteract the pathological effects of the angiotensin II/angiotensin II type 1 receptor system in HIRI [43]. Thus, the activation of the PI3K/Akt pathway leads to the phosphorylation of eNOS and continuous catalysation of NO production, which is essential to counteract HIRI.

KLF2 induced eNOS activation:
There may exist other ways of influencing the eNOS activity during IR. It has been proven that WIR injury can decrease the expression of KLF2 in endothelial cells. Also, this reduction is accompanied by a decrease in phosphorylated eNOS (p-eNOS), one of the KLF2 targets. And the IR damage can be mitigated by pretreatment with simvastatin through a KLF2-dependent mechanism, upregulating the mRNA expression of KLF2 and eNOS as well as the protein expression of KLF2 and p-eNOS [16,44]. Hu et al [45] also demonstrated that hypothermic machine perfusion inhibited NF-κB signaling and activated eNOS/NO signaling through KLF2 expression, thereby alleviating the inflammatory response and oxidative stress injury. It has demonstrated that KLF2 activators can be candidate therapeutic agents for HIRI. AMPK induced eNOS activation: AMPK plays a key role in the regulation of cellular energy homeostasis. The activation of this kinase is a response to the stimulus. Mahfoudh et al [46] reported that repeated administration of trimetazidine protected against WIR injury by decreasing liver damage and oxidative stress. The underlying mechanism involves the activation of the AMPK/eNOS signaling pathway. In addition, similar mechanisms have been identified in the protective effect of Institut Georges Lopez 1 solution on cold-stored fatty liver grafts. The effect is mainly exerted through the activation of the AMPK pathway, which targets eNOS to produce NO, offsetting aggravated microcirculatory changes, and improving vascular resistance and function during IR[47,48].
Other pathways: SEW2871, a selective sphingosine-1-phosphate receptor 1 (S1PR1) agonist, can restore the expression of eNOS and vascular endothelial (VE) cadherin in sinusoidal endothelial cells during HIRI in vivo and does not influence the expression of p-Akt. Thus, there may be a regulation pathway between S1PR1 and eNOS [49]. And the expression of VE-cadherin is important for vascular integrity, which is the basis for eNOS expression [49].
Moreover, ischemia preconditioning (IPC) can protect HIRI through p38 MAPK activation, which induces eNOS-derived NO expression to enhance cell autophagy in HIRI [35]. However, pretreatment with 3,7-dimethyl-1-propargylxanthine, an adenosine A2 receptor (A2AR) antagonist, can repeal the protective effect induced by IPC. Therefore, it can be inferred that there may be a relationship between the A2AR and eNOS [50].
The study of Kebschull et al[51] showed that low-dose erythropoietin (EPO) treatment significantly increased hepatic NO bioavailability by up-regulating eNOS expression. EPO-mediated eNOS phosphorylation is promoted by EPOR-mediated activation of the Janus kinase 2/PI3K/Akt pathway and common β receptor (βcR)-dependent activation of AMPK. In addition to this, activation of the βcR2vascular endothelial growth factor receptor-2 complex is also involved in the regulation, but its downstream signaling is currently unclear.

ROLE OF INOS IN HIRI
As mentioned above, iNOS-derived NO may have different functions in HIRI [15]. Although in most cases iNOS is considered to be harmful to the HIRI, it does not affect or even protects the HIRI in some conditions. In a study of models with liver ischemia and partial liver resection, iNOS mRNA expression was not found to be significantly altered compared to the sham group. While during 6 to 8 h after hepatectomy, iNOS expression and NO production were promoted by cytokines, thereby improving liver microcirculation and preventing cell apoptosis [52]. The protective effect of iNOS has only been demonstrated in a few specific experiments and lacks widespread validation. Due to differences in experimental subjects, measurement criteria, and experimental time constraints, iNOS-derived NO exhibits a more complex and unclear role than eNOS.

iNOS aggravates HIRI
Hide et al [14] found a surge of NO in WIR in aged livers, which was mainly induced by iNOS production. The surge of NO derived from iNOS can increase the expression of reactive nitrogen species (RNS) and inflammatory cytokines, resulting in cytotoxic damage in hepatocytes. Besides, the damage from iNOS is also confirmed in other studies. As intrahepatic macrophages, KCs are activated in early IRI, producing excessive amounts of iNOS-derived NO and leading to massive production of proinflammatory factors, cytokines, and ROS, which are key links to impaired microcirculation in the liver and deteriorate HIRI [5,53].
At the late phase of HIRI, the function of iNOS will be at a prominent stage. Excess NO derived from iNOS has cytotoxic effects that induce inflammation and excessive oxidation, and performs many deleterious functions in HIRI. Increased iNOS expression is associated with increased TNF-α and NF-κB, which leads to increased expression of pro-inflammatory genes, inflammatory mediators, and regulatory enzymes [54]. They are both important to trigger inflammation reactions and may have deleterious effects on IRI. Besides, in studying the role of iNOS/NO in the interferon regulatory factor-1 (IRF1) signaling pathway of primary human hepatocytes, Du et al [55] found the existence of a positivefeedback loop between iNOS and IRF1. The IRF1 and p53 can upregulate the p53 up-regulated modulator of apoptosis (PUMA), which is a modulator of apoptosis, resulting in hepatocyte death and further damage to hepatic IRI.

REGULATING INOS AND ENOS EXPRESSION TO PROTECT HIRI
The extent and intensity of eNOS and iNOS in HIRI are both higher than those in the normal state, while excess NO will produce peroxynitrite to aggravate IR damage. These can be reduced by using high doses of tadalafil and pentoxifylline to mitigate the deterioration of nitrosative stress and endothelial cell injury [53].
Iwasaki et al [6] demonstrated that L-NAME, an NOS inhibitor, attenuated liver damage in IRI of cholestatic livers by inhibiting the NO production. Comparing the expression of iNOS and eNOS with L-NAME treatment, they found that this kind of protection was mainly mediated by the inhibitory effects of iNOS. It also prevented the increase of asymmetric dimethylarginine, which is an exogenous inhibitor of eNOS, to protect against IRI at the early stage.
Bone marrow mesenchymal stem cell (BMMSC) transplantation can regulate NOS synthesis by increasing eNOS expression as well as inhibiting iNOS expression and excessive NO production to protect HIRI and reduce hepatocyte apoptosis. Its regulations are closely related to the inhibition of NOS-induced macrophage activation, the suppression of large amounts of iNOS and NO synthesized by macrophages, and the amelioration of endothelial damage. And the combined use of BMMSCs and normothermic machine perfusion can increase the balance of ET/NO ratio [5].
Besides, the eNOS traffic inducer (NOSTRIN) can significantly inhibit NO release by decreasing the enzymatic activity of eNOS. Pretreatment with N-acetylcysteine or thymoquinone can up-regulate eNOS along with NO production and down-regulate iNOS and NOSTRIN expression to attenuate HIRI injury, showing the protective effect of increasing eNOS and NO levels and inhibiting iNOS expression against IRI in rat liver [56].

Inhibiting iNOS to protect HIRI
After reperfusion, the expression of inflammatory factors such as macrophage inflammatory protein-2 and iNOS increase with the activation of NF-κB, leading to a series of inflammation reactions. Alphalipoic acid can reduce the formation of excess NO during reperfusion by decreasing the expression of iNOS mRNA and reduce cellular damage from NO-forming NOS superoxide and peroxide anions [57]. Beyond that, in a study of vildagliptin function in lung injury after hepatic IRI, significant inhibition of iNOS mRNA expression and NO was observed by the involvement of the hypoxia-inducible factor (HIF)-α/hepatocyte growth factor/iNOS pathway. The evaluated HIF-α can increase iNOS expression in various models. Therefore, targeting HIF-α expression can reduce tissue damage [58]. Furthermore, hepatic IR-induced iNOS protein expression can be diminished by eupatilin, which also suppresses the Toll-like receptor 2/NF-κB pathway to ameliorate inflammation response [59]. In addition, neuralsphingomyelinase (N-SMase) can produce ceramide, which is a mediator of iNOS expression. Inhibition of N-SMase leads to a decrease in iNOS levels, along with a decrease in protein nitrification and nitrite/nitrate levels in WIR [60].

Inhibiting overexpression of and eNOS
Some studies have demonstrated the hepatoprotective effect of genetic eNOS overexpression in smallfor-size liver transplantation and illustrated the importance of promoting eNOS expression for hepatoprotection [61]. However, there is insufficient evidence for a protective effect of eNOS overexpression, and evidence that eNOS overexpression is detrimental to HIRI [62]. The dual effect of eNOS in HIRI remains controversial.
The fact is that the expression of eNOS will be deteriorated by oxidative stress and endothelial damage during the progression of ischemia, while the function of iNOS will be stimulated by oxidative stress during reperfusion and aggravate the liver injury. The imbalance of eNOS and iNOS can also March

THERAPEUTIC PERSPECTIVES
NOS drugs as well as drugs for the regulation of NOS enzymes may be the way forward for liver protection. However, more in-depth studies are still needed. Not only do drugs need to be stable, but they also need to avoid the harm that NO and NOS can cause to reduce side effects. Besides, despite a number of experimental studies demonstrating the beneficial effects of NO-releasing compounds and some drugs that promote NO release in ameliorating hepatic IRI, the results of trials and evaluations in the clinical setting are still lacking. Perhaps more randomised controlled clinical trials should be strengthened in the future to obtain more therapeutic results. In a nutshell, increasing or decreasing NO availability in the hepatic tissue may both be ways to prevent and treat HIRI and identifying ways to balance the expression of eNOS and iNOS is important to protect IR and can be a promising direction for clinical research.

CONCLUSION
In general, NO along with eNOS and iNOS can play complex roles in HIRI. NO can down-regulate LTC4S by inhibiting the NFκB pathway, inhibit apoptotic related genes such as Bax and Bcl-2, enhance autophagic flux, decrease inflammatory cytokines, and reduce ROS by inhibiting the mitochondrial respiratory chain. Furthermore, NO induced by different NOS results in a duality of action in HIRI. NO derived by eNOS prefers to protect endothelial cells and attenuate liver injury in HIRI. However, iNOS promotes the production of NO in response to stimuli, thus exacerbating liver damage. But their role is not set in stone. Overexpression of eNOS also worsens HIRI, whereas iNOS has also been reported to have a protective effect against HIRI. Actually, these views remain controversial, and the underlying mechanisms are urgently needed to be clarified.

FOOTNOTES
Author contributions: Zhang YP and Liu XR are co-first authors and contributed equally to this work, and they wrote and revised the paper; Yang MW participated in data collection and revised the paper by making point-by-point responses to the reviewers; Yang SL and Hong FF are co-corresponding authors who contributed equally to this work, and they are responsible for improving grammars and languages, conceptualizing the idea, and obtaining funding.

Conflict-of-interest statement:
There is no conflict of interest associated with any of the authors or other coauthors in this manuscript.

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