Autophagy regulation is an effective strategy to improve the prognosis of chemically induced acute liver injury based on experimental studies

Abstract Acute liver injury (ALI) induced by chemicals in current experimental studies is characterized by inflammation, oxidative stress and necrosis, which can greatly influence the long‐term outcome and lead to liver failure. In liver cells, different autophagy forms envelop cytoplasm components, including proteins, endoplasmic reticulum (ER), mitochondria and lipids, and they effectively participate in breaking down the cargo enclosed inside lysosomes to replenish cellular energy and contents. In general, autophagy serves as a cell survival mechanism in stressful microenvironments, but it also serves as a destructive mechanism that results in cell death in vitro and in vivo. In experimental animals, multiple chemicals are used to mimic ALI in patients to clarify the potential pathological mechanisms and develop effective strategies in the clinic. In this review, we summarize related publications about autophagy modulation to attenuate chemically induced ALI in vitro and in vivo. We also analysed the underlying mechanisms of autophagy regulators and genetic modifications to clarify how to control autophagy to protect against chemically induced ALI in animal models. We anticipate that selectively controlling the dual effects of hepatic autophagy will help to protect against ALI in various animals, but the detailed mechanisms and effects should be determined further in future studies. In this way, we are more confident that modulating autophagy in liver regeneration can improve the prognosis of ALI.


| INTRODUC TI ON
Although most liver cells are quiescent under normal conditions, liver regeneration is initiated after partial hepatectomy to compensate for liver function. The liver is also a digestive organ that is always exposed to orally ingested antigens and harmful products from intestinal bacteria since liver tissue is surrounded by systemic blood circulation from portal blood. In response to liver injury, liver-specific apoptosis and autophagy occur simultaneously but act independently through different pathways, influencing each other and participating in the initiation of liver injury and liver regeneration. Acute liver injury (ALI) is characterized by inflammation, oxidative stress and necrosis, which can greatly influence the long-term outcome and lead to liver failure. Autophagy, a form of cell death marked by partial chromatin condensation is considered to be up- (b) although mutagenic microenvironments continuously stimulate gene mutations in mammals, autophagy effectively maintains the genomic integrity of cells or tissues 4 ; (c) autophagy is also able to supply more energy and maintain cell or tissue homeostasis in mammals by degrading cytosolic components in lysosomes 5 ; (d) autophagy significantly degrades unfolded protein aggregates and inhibits endoplasmic reticulum (ER) stress to maintain ER function 6 ; and (e) autophagy promotes cell growth and proliferation in local injured tissues. 7 As a result, activation of apoptotic pathways further promotes the inactivation of autophagy. BCL2-interacting protein 3 (BNIP3) can significantly increase the apoptosis rate by sequestering B-cell lymphoma protein-2 (Bcl-2) family proteins and reducing the binding between Bcl-2 and Beclin-1. 8 Activation of apoptosis further cleaves and inactivates Beclin-1, resulting in suppression of autophagy in a caspase-dependent manner. 9 Moreover, autophagy improves the survival rate of quiescent hepatocytes by continually recycling nucleic acids, complex carbohydrates, lipids and proteins in liver tissue.
It is worth noting that excessive accumulation of autophagic factors will ultimately result in cell death after disruption of the adaptive mechanism under extremely harmful conditions. 10 The final products of autophagy contain small sealed membrane vesicles similar to apoptosis, but autophagic cell death significantly increased the contents of autophagosomes and autolysosomes in vivo. 11,12 It is generally anticipated that impairment of lysosomal activities would result in autophagosome deposition, cellular dysfunction and activation of caspase-dependent cell death. 13 On the other hand, purified Beclin-1-C is generated in a caspase-dependent manner and effectively promotes the generation of mitochondrial cytochrome c to stimulate apoptosis. 14 Remarkably, autophagy is a homeostatic response that helps to clear damaged cells and hepatotoxic factors in vivo, but it also results in a stressful microenvironment and tissue injury ( Figure 1).
As liver cell apoptosis is a prominent pathological process during severe liver injury, interventions on autophagy in liver tissue help to regenerate liver tissue and inhibit cellular apoptosis. Pharmacological autophagy modulators or genetic modifications may provide protective effects in liver tissue via their hepatoprotective and anti-inflammatory effects. In this review, we summarize related publications about autophagy modulation for attenuating ALI in vitro and in vivo.
We also analysed the underlying mechanisms of autophagy regulators and genetic modifications to clarify how to control autophagy to protect against chemically induced ALI in animal models. We conclude that strategies targeting the regulation of autophagic flux in liver tissue will further improve the prognosis of patients with ALI.

| CURRENT FORMS OF AUTOPHAGY IN LIVER TISSUE
Macroautophagy, chaperone-mediated autophagy (CMA) and microautophagy are the three main types of autophagy in mammals ( Figure 2). The complete process of macroautophagy includes six steps: initiation, nucleation, elongation, closure, maturation and degradation. [15][16][17] Recognition of ER stress activates mechanistic target of rapamycin kinase (mTOR) and AMP-activated protein kinase (AMPK) for activation of unc-51-like kinase (ULK) protein and subsequent activation of phosphatidylinositol-3-phosphate (PI3P) in the ER membrane. Although the generation of the omegasome is not indispensable for autophagosome formation, double FYVEcontaining protein 1 (DFCP1) is an effector protein for omegasome formation and subsequent autophagosome generation. 18 After synthesis of PI3P from the nascent phagophore, the mammalian effector protein WD repeat domain phosphoinositide-interacting protein (WIPI) recognizes this protein. 19 The PI3P-binding protein complex contains WIPI, and autophagy-related (Atg)2 accumulates on the isolation membrane and contributes to expansion. 18 PI3P effectively recruits Atg12-Atg5-Atg16L and promotes the conversion of microtubule-associated protein light chain 3 (LC3)I into LC3II. After that, cytoplasmic materials are sequestered by the phagophore (a preautophagosomal membrane structure), which thereafter expands and encloses its cargo to form an autophagosome (a double-membrane vesicle). 16,20 Then, these autophagosomes fuse into lysosomes and generate autolysosomes for degradation of the enclosed cargo by acid hydrolases and recycling into biologically active monomers to maintain cellular metabolic homeostasis. 20 CMA is a form of autophagy that uniquely and selectively degrades abundant substrate proteins by delivering them into the lysosome one by one. 21 Heat shock cognate (HSC)70 recognizes a specific cytosolic protein that contains a KFERQ-like pentapeptide and subsequently interacts with lysosomal-associated membrane protein 2 (LAMP2A) to degrade cellular contents. 16 Under stress conditions, microautophagy effectively maintains organelles and membrane homeostasis by degrading cytoplasmic contents in lysosomes through invagination or deformation of the lysosomal membrane. 22 The liver is also a large and special organ that has abundant mitochondria and is an F I G U R E 1 Autophagy is a survival mechanism or a cell death mechanism in liver injury    important site for the metabolization of glucose and fat storage.
In consideration of this, other selective autophagy processes, such as mitophagy and lipophagy, were also found to exist in cultured hepatocytes and liver tissue. Mitophagy is a kind of autophagy that effectively removes damaged mitochondria to reduce mitochondria-

| AUTOPHAGY AND CHEMI C ALLY INDUCED ALI IN ANIMAL MODEL S
To mimic ALI in patients, multiple chemicals, such as acetaminophen Autophagy regulation is reported to exert protective effects in these models via various mechanisms (Table 1).

| Modulation of autophagy reduces APAPinduced liver injury
The liver is the largest organ for the metabolization of drugs and detoxification of toxins, and frequent exposure to toxic drugs leads to a high prevalence of liver failure. Drug-induced ALI commonly targets mitochondria and then induces cell death; thus, modulation of mitophagy will be a major target for improving cell survival. In the clinic, Glycycoumarin, which is purified from licorice, is able to alleviate APAP-induced oxidative stress and liver injury via activation of protective autophagy and the JNK signalling pathway but not via activation of the nuclear factor erythroid 2 like 2 (NRF2) signalling pathway. 38 However, another study showed that pre-treatment with alpha-mangostin partly inhibited the activation of autophagic cell death via down-regulation of p-mTOR, p-AKT and the LC3II/LC3I ratio, subsequently decreasing the release of inflammatory factors, including tumour necrosis factor (TNF)-α and IL-1β, and inhibiting the activation of apoptotic pathways in APAP-induced ALI models. 39 Related studies indicate that autophagy activation may play a dual effect in APAP-induced ALI models since autophagy can serve as a survival mechanism or a cell death mechanism in vivo. Galanos  in ALF mice. 46 It is worth noting that although autophagy is an important process that helps to clear damaged cellular contents to preserve liver function in D-GalN/LPS-treated animals, it also aggravates liver injury after activation of multiple cell death pathways.

| Modulation of autophagy reduces ConAinduced ALI
It is widely accepted that ConA-treated animal models generally serve as an experimental model of acute immune hepatitis (AIH).
ConA is reported to induce immune hepatitis by promoting hepato-

| Modulation of autophagy reduces CCl 4induced ALI
CCl 4 is reported to induce high levels of oxidative stress, inflammation, necroptosis and apoptosis in liver tissue by up-regulating hypoxia-inducible transcription factor-1α (HIF-1α) expression and activating the TLR4/NF-κB pathway. 66 In CCl 4 -induced ALF models, the expression of KLF6 is activated to enhance autophagy and liver regeneration through transcriptional induction of Atg7 and Beclin-1 in a p53-dependent manner. 33 Another study indicated that the caspase-9 inhibitor z-LEHD-FMK aggravated CCl 4 -induced ALI in HepG2 cells, AML12 cells and mouse models via down-regulation of cytoprotective autophagy, while up-regulation of HIF-1α resulted in oxidative stress and TLR4/NF-κB-mediated inflammation. 66 Pretreatment with genipin has been proven to induce the conversion of LC3 and inhibit p62 accumulation in vivo, subsequently attenuating hepatic tissue haemorrhage and necrosis. 74 According to these studies, we predict that genetic modifications will repair liver injury by regulating autophagy.

| CON CLUS IONS
The formation of autophagosomes and packaging of cytoplasm in liver cells participate in breaking down enclosed cargo inside lysosomes to replenish new energy sources and other components.
Autophagy generally serves as a cell survival mechanism in stressful microenvironments, but it also serves as a destructive mech- After clarification of how to selectively control the dual effects of hepatic autophagy in various animals, we are more confident in the ability to improve the prognosis of ALI by modulating autophagy.

ACK N OWLED G M ENTS
This work was supported by the National Natural Science Foundation of China (no. 81700553) 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.

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