Regulation of autophagy protects against liver injury in liver surgery‐induced ischaemia/reperfusion

Abstract Transient ischaemia and reperfusion in liver tissue induce hepatic ischaemia/reperfusion (I/R) tissue injury and a profound inflammatory response in vivo. Hepatic I/R can be classified into warm I/R and cold I/R and is characterized by three main types of cell death, apoptosis, necrosis and autophagy, in rodents or patients following I/R. Warm I/R is observed in patients or animal models undergoing liver resection, haemorrhagic shock, trauma, cardiac arrest or hepatic sinusoidal obstruction syndrome when vascular occlusion inhibits normal blood perfusion in liver tissue. Cold I/R is a condition that affects only patients who have undergone liver transplantation (LT) and is caused by donated liver graft preservation in a hypothermic environment prior to entering a warm reperfusion phase. Under stress conditions, autophagy plays a critical role in promoting cell survival and maintaining liver homeostasis by generating new adenosine triphosphate (ATP) and organelle components after the degradation of macromolecules and organelles in liver tissue. This role of autophagy may contribute to the protection of hepatic I/R‐induced liver injury; however, a considerable amount of evidence has shown that autophagy inhibition also protects against hepatic I/R injury by inhibiting autophagic cell death under specific circumstances. In this review, we comprehensively discuss current strategies and underlying mechanisms of autophagy regulation that alleviates I/R injury after liver resection and LT. Directed autophagy regulation can maintain liver homeostasis and improve liver function in individuals undergoing warm or cold I/R. In this way, autophagy regulation can contribute to improving the prognosis of patients undergoing liver resection or LT.


| INTRODUC TI ON
Ischaemia/reperfusion (I/R) initiates a process with transient ischaemia and reperfusion that maintains liver tissue in a microenvironment characterized by anoxia and reoxygenation, which subsequently leads to tissue injury and a profound inflammatory response. 1 Hepatic I/R injury in individuals or animals undergoing liver resection or liver transplantation (LT) may result in liver failure with high mortality. Both partial hepatectomy (PH) and LT are effective strategies for treating various liver diseases; however, LT is the only effective strategy for rescuing patients with end-stage liver disease or irreversible liver malignant tumours. 2 Notably, PH and orthotopic LT cause severe complications because they can initiate cell death and hepatic I/R injury. A large number of liver cells die, inducing hypohepatia or even liver failure. In addition, hepatic I/R plays a vital role in the pathophysiology of ischaemic-type biliary lesions since a large number of cholangiocytes undergo apoptosis and necrosis. 3 In general, I/R can be classified into two types: warm I/R or cold I/R (Figure 1). Warm I/R is observed in patients or animal models undergoing liver resection, haemorrhagic shock, trauma, cardiac arrest or hepatic sinusoidal obstruction syndrome when vascular occlusion inhibits normal blood perfusion in liver tissue. 4 Cold I/R is a condition affecting only patients who have undergone LT because the donated liver graft is preserved in a hypothermic environment prior to entering a warm reperfusion phase. 5 Warm ischaemia induces nutrient depletion, adenosine triphosphate (ATP) depletion and anaerobic glycolysis and promotes the generation of an acidic milieu in the cytoplasm, which subsequently suppresses a myriad of protective enzymes. Although a harsh acidic environment can protect against injury in the liver parenchyma during an acute ischaemic event, severe liver acidosis eventually results in the death of primary hepatocytes after the activation of apoptosis-or necrosisrelated pathways. 6 On the other hand, cold ischaemia mainly induces cell death in the sinusoidal endothelium and nonparenchymal cells in liver tissue. 7,8 Although hypothermia has been shown to reduce energy metabolism and preserve the function of liver grafts, a prolonged ischaemia period and hypothermia lead to cell swelling and liver injury after damaging Na/K ATPase membrane pumps, leading to the accumulation of sodium and chloride. 9, 10 Hypothermia has also been shown to induce a strong adaptive immune response in resected liver grafts characterized by recruitment of T cells into the ischaemic graft. 11 Similarly, liver reperfusion-induced inflammation initiates an innate immune-dominant response in conventional T F I G U R E 1 Warm I/R or cold I/R initiates three types of cell death: apoptosis, necrosis and autophagy. Hepatic I/R also induces ROS accumulation, the immune response and inflammation at injury sites, which in turn aggravates the death of hepatocytes, sinusoidal endothelial cells and nonparenchymal cells lymphocytes, which subsequently leads to injury of both parenchymal and nonparenchymal cells in situ and in liver transplants. 12,13 Moreover, other inflammatory cells are recruited in the response to hepatic I/R after restoration of blood flow and pH neutralization. 8 In the early stage of reperfusion, resident hepatic macrophages are activated to induce reactive oxygen species (ROS) generation and oxidative stress, while neutrophils are recruited to release inflammatory factors and induce tissue damage at the late stage of reperfusion. 14 Liver I/R also upregulates the release of a cascade of inflammatory factors, including tumour necrosis factorα (TNFα), interleukin (IL)-1, IL-2, IL-6 and high mobility group box 1 (HMGB1). 7 Mitochondrial ROS, immune responses and inflammatory factor activation lead to further activation of cell death-related pathways.
Apoptosis, necrosis and autophagy are three main types of cell death in rodents and patients with warm or cold I/R injury. The initiation of apoptosis, necrosis and autophagy may be induced by similar effectors and is usually regulated by similar signalling pathways. 15 Hepatic I/R activates phagocytosis and degradation of apoptotic bodies after cells undergo cell shrinkage, nucleus condensation, chromatin margination and fragmentation of both the nucleus and cytoplasmic structures. This process is determined as apoptosis in cells with normal in appearance. Physiologically, apoptosis is critical for the elimination of damaged or senescent cells and tissue remodelling in injury environments. 16 Apoptosis is initiated either by extrinsic stimuli through cell surface death receptors, including TNFα, Fas and TNF-related apoptosis-inducing ligand (TRAIL) receptors, or by intrinsic stimuli via a mitochondrial pathway. 17 Thus, activation of caspases leads to cell structure destruction and apoptosis. 18 Compared to apoptosis, pathological necrosis is a disordered and passive cell death in response to acute injury that involves cell bursting but is not generally triggered in normal development.
Necrosis progression is typically not associated with the activation of caspases, and the cellular nucleus becomes distended and remains largely intact. 16,19 The morphologic feature of necrosis is elimination of organelles, formation of large plasma membrane blebs, bleb rupture and subsequent loss of the plasma membrane permeability barrier. 20 Rupture of the plasma membrane promotes the release of cellular contents into the extracellular environment and induces a significant inflammatory response. 21 Under physiological conditions, a relatively low level of autophagy plays a homeostatic role in maintaining cell structures and functions by recycling long-lived proteins and whole organelles. 22 Under stress conditions, autophagy plays a critical role in promoting cell survival and maintaining liver homeostasis by generating new ATP and organelles after degradation of macromolecules and organelles in liver tissue. Activation of autophagy inhibits the progression of caspase-dependent apoptosis, and the activation of caspase-dependent apoptosis inhibits the autophagic process. However, under some conditions, autophagy proceeds to autophagic cell death, which promotes the progression of apoptosis or necrosis in mammals. 23,24 Recent studies have highlighted therapies targeting the suppression of I/R injury in liver tissue that rely on complex mechanisms; however, there is a particular emphasis on therapies that control autophagy in liver tissue to reduce inflammation and organ failure.
In this review, we comprehensively discuss current strategies and the underlying mechanisms for alleviating I/R injury in liver resection and LT via autophagy regulation. We hope that more investigators will seek to control autophagy regulation specifically to maintain liver homeostasis and improve liver function in individuals undergoing warm or cold I/R. In this way, autophagy regulation will contribute to improving the prognosis of patients undergoing liver resection or LT.

| AUTOPHAGY IN LIVER TISSUE
Three main autophagy-related pathways participate in the degradation of cytosolic contents in lysosomes. Macroautophagy facilitates the degradation of entire organelles or parts thereof after the development of a double-membrane autophagosome and the subsequent generation of autolysosomes that degrade cytoplasmic material. 25 Macroautophagy is selectively initiated by organelle injury, and it is upregulated nonspecifically through bioenergetic instability. 26 Microautophagy, which is mediated by acidic organelles such as late endosomes, is the least studied type of autophagy. 27 It is initiated by amino acid starvation. 28 Microautophagy decomposes small autophagic substrates by packaging a cytoplasmic portion into the lumen of a lysosome after the lysosomal membrane is rearranged and develops a protruding arm-like structure. 29 Chaperone- showing that reduced autophagic flux is able to impair the adaptive capacity of cells or tissues, enabling them to withstand oxygen damage, including that caused by ischaemia or hypoxia. 33 Komatsu et al. demonstrated that autophagy-related protein (ATG)7 deficiency resulted in enlarged livers in up to 30% of the body of mice and abnormal structures in mitochondria and peroxisomes of hepatocytes. 34 Disabled autophagy in hepatocytes may induce severe hepatic injury since the limited half-life of primary hepatocytes leads to the accumulation of detrimental cellular byproducts. 35 Moreover, autophagy upregulation is initiated to inhibit ROS-induced hepatocellular necrosis after the expression levels of microtubule-associated protein 1 light chain (LC)3-II is upregulated, increasing the number of autophagosomes in liver tissue under partial warm ischaemia without reperfusion. However, inhibition of autophagy by chloroquine treatment aggravates mitochondrial oxidative stress and mitochondrial dysfunction. 36 Zhu et al showed that rapamycin (an autophagy activator) effectively downregulated endoplasmic reticulum (ER) stress, inhibited the mechanistic target of rapamycin (mTOR) pathway and enhanced autophagy to protect against liver injury induced by I/R. 37 However, others have debated whether autophagy activation initiates or aggravates hepatic I/R injury by enhancing liver inflammation. Rapamycin was reported to inhibit liver regeneration and liver weight reconstitution, accompanied by upregulation of inflammatory factors, including TNFα and IL-1Ra, but downregulation of hepatocyte growth factor (HGF) and angiogenesis-related factors, including vascular endothelial growth factor receptor 2 (VEGFR2) and angiopoietin, in mice after PH. 38 Currently, expert opinion suggests that macroautophagy can be further classified into nonselective autophagy and selective macroautophagy, which targets special organelles or specific compounds for degradation. It is widely accepted that macroautophagy is able to degrade various organelles or substrates, such as mitochondria, the ER, peroxisomes, ribosomes, lipid droplets, iron-based compounds, glycogen, protein aggregates and cytoplasmic pathogens. 25 Specific names have been ascribed to autophagy of specific compounds, including mitophagy (mitochondria), reticulophagy (ER), pexophagy (peroxisomes), ribophagy (ribosomes), lipophagy (lipids) and ferritinophagy (iron-based compounds). 39 Reticulophagy effectively maintains the structure and function of the ER via the interaction of diverse receptors with LC3 to generate autophagosomes under various conditions, including starvation, nonalcoholic fatty liver disease (NAFLD), viral infection and fibrosis. 39 Lipophagy is another important macroautophagic form that is involved in lipid homeostasis and metabolism in liver diseases such as alcoholic liver and nonalcoholic liver diseases, including fibrosis, cirrhosis and hepatocellular carcinoma. 39 Notably, mitophagy plays a critical role in removing worn-out mitochondria, which have a half-life of 10 to 25 d in healthy liver. 40 Mitophagy is another important mechanism in which selective abnormally cleaved proteins or damaged mitochondria are eliminated to inhibit the accumulation of mitochondrial-derived ROS and mutated mitochondrial DNA ( Figure 2). 41 Hepatic I/R triggers mitophagy via two distinct pathways: phosphatidylinositol-3-kinase (PI3K)-dependent and PI3K-independent signalling pathways. 42 Kim et al 43

| AUTOPHAGY REG UL ATION AT TEN UATE S WARM HEPATI C I/R INJ URY
Many previous studies have shown that autophagy upregulation or downregulation exerts protective effects in the attenuation of warm hepatic I/R injury according to the specific circumstances (Table 1).
Starvation or ischaemic preconditioning, chemical exposure, plant extract treatment, MSC and/or MSC derivative application, and F I G U R E 2 Autophagy and mitophagy contribute to the degradation of organelles to maintain liver function and histology and repair liver injury in hepatic I/R

TA B L E 1 Autophagy upregulation or downregulation exerts protective effects in attenuating warm hepatic I/R injury
gene modification contribute to maintaining liver homeostasis and liver function in warm hepatic I/R models and patients via autophagy regulation and related mechanisms.

| Starvation or ischaemic preconditioning
I/R initiates liver injury through transient ischaemia and reperfusion; intriguingly, preconditioning tissue with starvation or ischaemia effectively protects against liver injury. Short-term starvation through calorie restriction or fasting has been shown to exert beneficial effects on multiple organs and preserve organ function under stress conditions. Short-term starvation protects against liver I/R injury, as

| Plant extracts
A large amount of evidence has shown that activation of au- However, the PI3K inhibitors wortmannin and LY294002 effectively reduced liver damage and the mortality rate of recipient rats via suppression of autophagy. 77 It has also been reported

| AUTOPHAGY REG UL ATI ON IN LIVER DONATION -AF TER-C ARDIAC DE ATH (D CD) AND AG ED OR S TE ATOTI C LIVER G R AF TS US ED FOR LT
Insufficient liver graft donation is a major disadvantage in LT in the clinic; therefore, liver grafts donated after death or donated by elderly patients or steatotic liver patients can partially remedy the shortage in the clinic. However, these grafts are more sensitive to warm ischaemia, cold storage and reperfusion, which remarkably increase graft dysfunction and liver failure. 83

| Autophagy regulation in DCD liver with hepatic I/R injury
During hepatic I/R, animals showed higher levels of ALT and AST and accelerated autophagy, accompanied by upregulated glycogen synthase kinase-3β (GSK-3β) and AMPK expression in transplanted DCD liver tissue. 84  and LC3B-II.

| Autophagy regulation in aged liver grafts with hepatic I/R injury
Cell and tissue ageing is a normal phenomenon in elderly people, and it is closely related to the high incidence and severity of diseases in this population. Ageing has been associated with elevated levels of ALT, AST, lipofuscin accumulation, steatosis, fibrosis and defective liver regeneration after PH. In addition, ageing is often associated with reduced autophagic flux and lower liver regeneration capacity. In elderly patients, ageing liver tissue shows slow hepatic blood flow and decreased quantities of mitochondria and endoplasmic reticula, which results in poorer regeneration after PH and LT. 86 In comparison to 8-to 12-week-old mice, 12-to 13-month-old mice presented more severe liver damage and higher liver inflammatory responses when exposed to 90 min of warm ischaemia and 8 h of reperfusion. 86 Older mice showed decreased liver regeneration, as

| Autophagy regulation in steatotic liver grafts with hepatic I/R injury
Steatotic livers are more sensitive to liver resection or LT-induced hepatic I/R, which results in an increased risk of postoperative complications. 92,93 Along with mitochondrial dysfunction, ER stress and inflammation after hepatic I/R, lipid accumulation and large cell volume, which obstructs the adjacent sinusoid space, result in reduced delivery of oxygen and nutrients, which contributes to hepatic I/R injury in steatotic livers. 94 The impairment of autophagy in steatotic liver aggravated liver injury induced by cold I/R in steatotic rat livers, while pretreatment with the autophagic stimulator simvastatin effectively attenuated I/R-induced liver injury. Simvastatin pretreatment effectively protects against microcirculation deterioration and endothelial dysfunction in moderately steatotic livers during cold storage and warm reperfusion via an NO-mediated mechanism. 95 On the other hand, the combined action of melatonin and trimetazidine significantly improved the liver function of steatotic liver grafts preserved in Institut Georges-Lopez (IGL)-1 solution via AMPK activation, autophagy activation and ER stress inhibition. 96 With relevance to the prognosis of patients after LT and those with steatotic livers, hypothermic reconditioning (HR), which is implemented by insufflation of gaseous oxygen, notably improved graft function by attenuating mitochondrial dysfunction and restoring hepatocellular autophagy in rats. 97 Domart et al 98 demonstrated that IP before prolonged ischaemia decreased hepatocyte necrosis and liver dysfunction by activating autophagy and maintaining the ATP level in steatotic human livers. Moreover, IP inhibited hepatocellular necrosis and reduced the incidence of liver rejection via upregulation of autophagy in recipients who accepted steatotic grafts compared to recipients who accepted non-IP steatotic grafts. 99

| CON CLUS ION
In general, activation of autophagy is recognized as a mechanism that confers protection against hepatic I/R injury, while excessive autophagy is acknowledged for its role in accelerating the apoptosis of hepatocytes and/or liver nonparenchymal cells. It is widely accepted that stimulation of autophagy generally promotes liver regeneration and inhibits liver dysfunction, although the pathogenesis of warm and cold liver I/R injury is consistent with the complex interplay between autophagy, necrosis and apoptosis. Autophagy initiation is a potential mechanism to improve hepatocyte survival in patients after warm or cold I/R because it degrades intracellular components and eliminates organelle and protein waste. However, the results of studies on autophagy regulation during warm and/ or cold liver I/R remain discordant. A fraction of studies documented downregulation of autophagy in protecting against warm or cold liver I/R injury via inhibition of inflammation and cellular apoptosis and necrosis. The dual role of autophagy suggests that upregulation or downregulation of autophagy may be an effective treatment strategy for the inhibition of liver damage. Autophagy regulation with gene modification in animal models provides a specific strategy to modify the expression of autophagic proteins and therefore is as an effective treatment to preserve liver function in hepatic I/R models. We suggest that future studies focus on clarifying the autophagy regulation mechanism in liver grafts from specific populations eligible for LT, which will contribute to increasing the LT rate and decreasing the low graft function rate in patients with end-stage liver diseases.

ACK N OWLED G EM ENTS
This work was supported by the National Natural Science Foundation of China (no. 81700553 and no. 82000636) and Zhejiang Basic Public Welfare Research Program(no. LGF20H030008).

CO N FLI C T O F I NTE R E S T
The authors declare no competing financial interests. Validation (equal). Lanjuan Li: Conceptualization (lead).

DATA AVA I L A B I L I T Y S TAT E M E N T
Not applicable.