Autophagy, Oxidative Stress, and Alcoholic Liver Disease: A Systematic Review and Potential Clinical Applications

Ethanol consumption triggers oxidative stress by generating reactive oxygen species (ROS) through its metabolites. This process leads to steatosis and liver inflammation, which are critical for the development of alcoholic liver disease (ALD). Autophagy is a regulated dynamic process that sequesters damaged and excess cytoplasmic organelles for lysosomal degradation and may counteract the harmful effects of ROS-induced oxidative stress. These effects include hepatotoxicity, mitochondrial damage, steatosis, endoplasmic reticulum stress, inflammation, and iron overload. In liver diseases, particularly ALD, macroautophagy has been implicated as a protective mechanism in hepatocytes, although it does not appear to play the same role in stellate cells. Beyond the liver, autophagy may also mitigate the harmful effects of alcohol on other organs, thereby providing an additional layer of protection against ALD. This protective potential is further supported by studies showing that drugs that interact with autophagy, such as rapamycin, can prevent ALD development in animal models. This systematic review presents a comprehensive analysis of the literature, focusing on the role of autophagy in oxidative stress regulation, its involvement in organ–organ crosstalk relevant to ALD, and the potential of autophagy-targeting therapeutic strategies.


Introduction
Alcohol consumption causes multi-organ damage and is linked to a wide variety of diseases. In 2016, ethanol intake caused an estimated 3 million alcohol-related deaths and resulted in 132.6 million alcohol-related disability-adjusted life years worldwide [1]. Alcohol intake causes a wide spectrum of damage in the liver, ranging from steatosis to alcoholic hepatitis, cirrhosis, and hepatocellular carcinoma [2][3][4]. One-quarter of cirrhosis deaths and one-fifth of liver cancer deaths worldwide in 2019 were associated with alcohol toxicity [4]. Some of the mechanisms underlying this toxicity are: (1) ethanol metabolism due to acetaldehyde accumulation, (2) an increase in the nicotinamide adenine dinucleotide (NAD)H/NAD + ratio, and/or (3) ROS generation [5]. Oxidative stress may be involved in functional and structural changes in mitochondria, leading to impaired oxidative phosphorylation, increased mitochondrial DNA (mtDNA) damage, and changes in mitochondrial protein profiles [6][7][8]. An increase in ROS levels leads to lipid peroxidation, as lipids accumulate in the liver during ethanol metabolism, which can escalate organ damage [9,10]. AMPK enhances mitophagy in hepatocytes, and the AMPK-NFE2L2-UQCRC2 axis regulates liver mitophagy.
Acute ethanol exposure induces NOX4 and CYP2E1 overexpression and significantly increases autophagy. Antioxidants efficiently block CYP2E1-and NOX4mediated autophagy induction.
Autophagy is inhibited in ethanol-treated HepG2 cells. ALR-expressing HepG2 cells have increased survival rates, improved mitochondrial membrane potential, and increased ATP levels after ethanol treatment. This protection is associated  AMPK enhances mitophagy in hepatocytes, and the AMPK-NFE2L2-UQCRC2 axis regulates liver mitophagy.
Autophagy is inhibited in ethanol-treated HepG2 cells. ALR-expressing HepG2 cells have increased survival rates, improved mitochondrial membrane potential, and increased ATP levels after ethanol treatment. This protection is associated with the upregulation of autophagy markers and downregulation of p62 and mTOR phosphorylation.

First Author, Year Models and Methods Summary of Effects
You et al., 2018 [35] Mouse hepatocytes treated with 80 mM ethanol for 6 h.
SNX10 deficiency upregulates LAMP2A expression and CMA activation via Nrf2 and AMPK signaling in vitro, significantly ameliorating ethanol-induced liver damage and hepatic steatosis.
Mahli et al., 2015 [36] Primary human hepatocytes and HepG2 cells treated with 50 mM alcohol over 16-24 h. Alcohol and steatosis increase CYP2E1 levels and activity, lipid peroxidation, oxidative stress, pro-inflammatory gene expression, and autophagy via the CYP2E1 and JNK pathways. Autophagy improves the effects of alcohol on lipid accumulation and inflammatory gene expression in liver cells. Thomes et al., 2013 [37] Hep G2 cells treated with 50 mM ethanol over 24 h.
Ethanol treatment increases LC3-II expression and decreases its degradation in a dose-dependent manner depending on ADH and CYP2E1 expression. Blocking ethanol oxidation and ROS production prevents the enhancement of LC3-II expression. Direct exposure to acetaldehyde enhances LC3-II content.
Ethanol-induced autophagy requires ethanol metabolism, ROS generation, and mTOR signaling inhibition in vitro. It is selective for cells with damaged mitochondria and accumulated lipid droplets (but not long-lived proteins) and protects cells from ethanol's toxic effects. Increasing autophagy reduces acute ethanol hepatotoxicity and steatosis.
Macroautophagy is activated under acute conditions. Hepatic steatosis and liver damage are exacerbated by autophagy inhibition and alleviated by autophagy activation.  Chronic alcohol consumption increases DNA-PKcs in the liver, leading to liver damage and mitochondrial dysfunction through p53 activation and defective mitophagy. Chronic alcohol intake causes liver damage, disturbed fat metabolism, increased inflammation and oxidative stress, and decreased autophagy. Expressing the ALDH2 gene reduced these effects. Lysosomal inhibitors had the same effects as alcohol on p62 accumulation.
Inhibiting autophagy increased liver damage and fat accumulation in mice with normal or increased CYP2E1 levels but not in mice lacking CYP2E1. Autophagy did not affect CYP2E1 activity or induction by alcohol. Mice with normal or increased CYP2E1 levels had decreased autophagy-related gene expression and increased p62 levels.
Alcohol-treated mice experienced fat accumulation, increased autophagy, decreased mitochondrial function, and increased CypD levels. Their mitochondria were more sensitive to damage than those of mice lacking CypD. CypD deficiency impaired autophagy but did not prevent fat accumulation caused by alcohol.
Mice lacking the HFE gene had liver damage, fibrosis, and increased cell death. Iron overload in these mice caused stress responses and impaired autophagy-related gene expression and activity.
Macroautophagy was activated during chronic alcohol consumption. Inhibiting autophagy worsened liver damage and fat accumulation, while activating autophagy improved these conditions.

Summary of Effects
Wu et al., 2012 [40] Male SV129 mice gavaged with a total of 3 g/kg body weight ethanol over 4 days.
Alcohol treatment caused liver damage, increased CYP2E1 levels, and oxidative stress in mice with normal or increased CYP2E1 levels but not in mice lacking CYP2E1. Alcohol impaired autophagy in mice with increased CYP2E1 levels. Inhibiting autophagy worsened alcohol-induced liver damage, fat accumulation, and oxidative stress in these mice. Thomes et al., 2012 [49] Lieber-DeCarli model with GFP-LC3 tg mice for 4-6 weeks.
Chronic alcohol-fed mice had reduced proteasome activity and increased autophagy markers in liver cells.
Inhibiting the proteasome further increased autophagy markers.

First Author, Year Models and Methods Summary of Effect
Liang et al., 2019 [60] Lieber-DeCarli model with female C57BL/6 mice and intraperitoneal LPS injection (1 mg/kg) on the final day.
Chronic alcohol feeding increased liver damage and inflammation in mice lacking the Atg7 gene in immune cells and increased inflammatory gene expression in normal mice. Mice lacking Atg7 experienced impaired mitochondrial function, increased oxidative stress, and increased inflammation. Silencing p62 or deleting Atg7 caused the accumulation of IRF1 and increased inflammatory gene expression.
Mice lacking the Atg5 gene in immune cells had similar fat accumulation compared to normal mice when fed alcohol but had increased liver damage, inflammation, and cell death. Blocking the IL-1 receptor reduced alcohol-induced inflammation. Mice lacking the CB2 gene in immune cells experienced worsened alcohol-induced inflammation and fat accumulation. Activating the CB2 receptor reduced alcohol-induced liver inflammation and fat accumulation in normal mice but not in mice lacking the ATG5 gene in immune cells. Macrophage autophagy mediated the protective effects of the CB2 receptor.
An increase in the UPR, as indicated by XBP1 mRNA splicing, triggered autophagy. The Nrf2-mediated antioxidant response was activated during ER stress.
Blocking the IRE1α pathway in HSCs reduced their activation and autophagy activity, reducing fibrosis through a p38 MAPK-dependent mechanism.
Induction of autophagy through the activation of Nrf2 and MAPK and increased HO-1 levels.
Reduced liver damage and histopathological changes via increased antioxidant activity.
Reduced alcohol-induced hepatotoxicity, oxidative stress, and regulation of AST, ALT, oxidation-related enzyme, inflammatory cytokine, and caspase levels.
Induction of autophagy through the blunted activation of the JNK/MAPK pathway.
CBD prevented ethanol-induced autophagy reduction and reduced oxidative stress and acute alcohol-induced liver steatosis in mice.
CBZ alleviated hepatic steatosis and liver damage and improved insulin sensitivity.
Mouse model of acute ethanol intake with carvacrol pretreatment (10 mL/kg).
Induction of autophagy, likely through the inactivation of p38, and inhibition of cytochrome p450.
Carvacrol reduced the TG content and ethanol-induced liver histopathological changes.
Induction of autophagy through the activation of the AMPK, MAPK, and PI3K/Akt/GSK3β pathways, and inhibition of CYP2E1.
CMZ suppressed chronic ethanol-induced oxidative stress and pro-inflammatory cytokine production, attenuated acute ethanol-induced fatty liver.
Acute alcohol intake rat model ± intraperitoneal cilostazol (10 mg/kg/d for 4 days; primary rat hepatocytes were examined.
Autophagy induction via AMPK pathway activation.
Cilostazol protected hepatocytes from apoptosis in vivo and in vitro.
KD alleviated alcoholic liver damage by reducing oxidative stress and lipid accumulation. Melatonin [86] Pineal gland hormone.
Improved mitochondrial oxidation of NADH and decreased mitochondrial ability to oxidize FAD.
Prevented lysosomal destruction of liver tissue by limiting the increased activity of lysosomal enzymes and the resulting oxidative stress.
Acute ethanol intake mouse model. Murine hepatocytes were exposed to ethanol ± NAC.
Reduction of autophagy via mTOR activation and reversed ROS levels.
NAC reduced TG and TBARS contents and ROS stress and reversed ethanol-induced mTOR inhibition.
Induction of autophagy via AMPK/mTOR pathway activation.
PLT reduced ethanol-induced liver cell damage by inhibiting hepatocyte apoptosis through autophagy promotion.
Induction of autophagy through activating the AMPK/p62/Nrf2/mTOR signaling pathways and reduced oxidative stress.
PCP ameliorated ethanol-induced liver function damage and fat accumulation.
Induction of autophagy via inhibition of mTOR signaling.
Rapamycin reduced ethanol-induced steatosis. Lieber-DeCarli mouse model plus acute ethanol binge, HepG2 cells exposed to oleic acid and alcohol.
Resveratrol increased the number of autophagosomes, reduced hepatic lipid accumulation, and protected against alcoholic liver steatosis.
HepG2 and HL7702 cells exposed to ethanol or acetaldehyde and silibinin.
Induction of autophagy via PINK1 and Parkin activation.
Induction of autophagy via Nrf2 activation.
Sulforaphane prevented binge ethanol-induced oxidative stress and steatosis in CYP2E1 KI mice and lipid accumulation in HepG2 (E47) cells.
Tangeretin dose-dependently normalized serum ALT and AST levels, liver weight, and serum and liver triacylglycerol contents; restored mitochondrial respiratory function; and suppressed steatosis. Chronic ethanol intake mouse model ± TMP, LO2 cells exposed to ethanol (100 mM) and/or TMP (40 µM for 24 h).
Reduced necroptosis and leakage of damage-associated molecular patterns and promoted the clearance of impaired mitochondria.
Chronic plus binge ethanol intake mouse model ± torin 1.
Induction of autophagy via inhibition of mTORC1 and mTORC2 and increased hepatic TFEB levels.
Torin 1 reduced steatosis and liver damage induced by ethanol.
UDCA attenuated and prevented the progression of alcoholic hepatic cholestasis.
VL-17A cells exposed to

Ethanol Metabolism
Hepatocytes remove ethanol from the bloodstream via non-oxidative and oxidative metabolism. Ethanol is metabolized oxidatively via two major pathways: alcohol dehydrogenase (ADH) and cytochrome P450 enzymes (in this case, CYP2E1) [108]. These pathways produce ethanol metabolites, such as acetaldehyde and ROS, and deplete the stores of some antioxidants such as glutathione (GSH) [109,110]. The metabolites produced induce oxidative stress [including endoplasmic reticulum (ER) stress and mitochondrial damage], autophagy, and inflammation, which add to the hepatic inflammation caused by the action of bacterial lipopolysaccharide (LPS) [111][112][113][114].
The main alcohol metabolism pathway is initiated by ADH [113], an NAD + -requiring enzyme that is strongly expressed in hepatocytes and oxidizes ethanol to acetaldehyde in the cytosol [108], decreasing the NAD + /NADH ratio [115]. In normal liver, acetaldehyde is quickly metabolized to acetate by aldehyde dehydrogenase (ALDH). In chronic alcohol users, the ADH/ALDH pathway becomes saturated and generates reactive aldehydes and lipid hydroperoxides that can bind to DNA and proteins, producing adducts that further induce hepatocyte damage and inflammation [116,117]. The decrease in the NAD + /NADH ratio also promotes fat accumulation in the liver by reducing fatty acid oxidation (through peroxisome proliferator-activated receptor-α target genes) and enhancing fatty acid synthesis (by increasing the expression levels of lipogenic genes such as SREBP1c) [118][119][120], reducing sirtuin-1 (SIRT1)-related autophagy by decreasing transcription factor EB (TFEB) deacetylation [121], and increasing oxidative stress, as the re-oxidation of NADH to NAD + in mitochondria requires ROS production [122].
The second major pathway centers around CYP2E1, a nicotinamide adenine dinucleotide phosphate (NADPH)-requiring enzyme, and gains relevance over the ADH pathway with chronic ethanol intake [113]. CYP2E1 metabolizes ethanol to acetaldehyde by converting NADPH and oxygen to NADP + and water, resulting in the generation of ROS such as H 2 O 2 , hydroxyl (HO •− ), and carbon-centered HO •− [123,124]. Through this process, CYP2E1 facilitates DNA and protein adduct formation, activates stress proteins, induces ER stress, and affects lysosomal function and autophagy, leading to mitochondrial damage, hepatocellular death, and hepatic carcinogenesis via oxidative DNA damage [113,125].
Nicotinamide N-methyltransferase (NNMT) is another important enzyme involved in liver metabolism and alcoholic liver disease [126,127]. NNMT is highly expressed in the human liver and plays a crucial role in maintaining NAD+ homeostasis [128]. It catalyzes the methylation of nicotinamide and similar compounds using the methyl donor S-adenosyl methionine (SAM-e) to produce S-adenosyl-L-homocysteine (SAH) and 1-methylnicotinamide. NNMT can affect the autophagic pathway [129,130] and may counteract oxidative stress in liver vessels due to its putative protective role in the endothelium [131]. Increased NNMT in the liver is associated with a better metabolic profile, including reduced serum triglyceride and free fatty acid levels [132]. In the liver, 1-methylnicotinamide produced by the NNMT degradation of nicotinamide increases sirtuin 1 (SIRT1) by inhibiting its degradation [132].
Thus, ROS generation through alcohol metabolism, with a reduction in the NAD + /NADH ratio and the activation of CYP2E1, is a major hallmark of alcohol-related oxidative stress. One of the best indicators of ROS overproduction is an increase in hepatic CYP2E1 levels. ROS-induced oxidative stress is closely related to protein modification, lipid peroxidation, mitochondrial damage, inflammation, iron overload, and antioxidant responses.

Protein Modifications
ROS induce reversible protein modifications, mainly at the level of the sulfur-containing residues cysteine and methionine, indicating a redox-based signal. Oxidative stress can cause changes in protein structure, localization, physical interactions, and post-translational modifications through the oxidative modification of reactive cysteines [133]. These changes can affect the immune response against neoantigens [134]. In liver sinusoidal endothelial cells, oxidative stress can also alter the proper functioning of fenestrae through spectrin oxidation [135]. For instance, oxidative stress reduces disulfide bond formation and causes the accumulation of unfolded proteins, triggering additional ER stress [136]. Cells counteract these protein modifications via three main signaling pathways: inositol-requiring transmembrane kinase/endoribonuclease 1 (IRE1), activating transcription factor 6, and protein kinase RNA-like ER kinase. Collectively, they form the unfolded protein response (UPR), which occurs mainly via the ubiquitin-proteasome pathway [15].

Lipid Peroxidation and DNA Alteration
ROS can react with lipid species to promote lipid peroxidation, inducing apoptosis and ferroptosis [9,140], among other changes. Lipid peroxidation products can bind to DNA and enhance carcinogenesis by producing etheno-DNA adducts and mutations in oncogenes and onco-suppressor genes [10,141,142]. These adducts have been identified in the livers of patients with ALD [143]. In addition, ROS can directly modify DNA, thereby affecting cell viability. HO •− can directly attack the DNA backbone, mainly guanine, due to its low redox potential. The main products of its oxidation are 8-hydroxyguanine and 8-hydroxydeoxyguanosine, which are mutagenic and carcinogenic [144].

Mitochondrial Damage
ALD is characterized by structural and functional abnormalities in hepatic mitochondria, including enlargement [145], mtDNA damage [6], reduced adenosine triphosphate (ATP) levels [146], mitochondrial protein synthesis [147], increased ROS production [7,8], and alterations in mitochondrial membrane permeability and mitochondrial permeability transition, resulting in intrinsic and extrinsic apoptosis [124]. In intrinsic apoptosis, cellular death occurs via the release of cytochrome C and other pro-apoptotic factors that interact with apoptotic protease activating factor 1 and caspase-9 [148]. In the extrinsic pathway, cell death is triggered by the ROS-induced release of apoptosis signal-regulating kinase 1, a member of the mitogen-activated protein kinase (MAPK) family, resulting in the cleavage of pro-caspase-3 to active caspase-3 [149,150]. Mitochondrial permeability transition has been found to activate caspase-3 in hepatocytes in a p38 MAPK-dependent manner [151].
ROS also harm mtDNA integrity by affecting mtDNA-coded proteins and RNA transcription, which in turn regulates the mitochondrial respiratory chain. A vicious cycle is established in which mitochondria with oxidized mtDNA and limited repair mechanisms [152] become dysfunctional and produce abundant ROS, leading to further mitochondrial impairment. This loop can ultimately result in severe nuclear DNA damage and cell death [153]. Mitophagy degrades mitochondria with damaged DNA [154] and enhances longevity in rodent models [155]. However, the autophagy rate declines with age and chronic alcohol intake [53], promoting the accumulation of mtDNA mutations and a decline in mitochondrial function [156].

Inflammation
ROS also play a key role in the development of ethanol-induced inflammation. The depletion of mitochondrial GSH by CYP2E1 activation [157,158] is one factor that links inflammation with oxidative stress as it impairs hepatocyte tolerance to tumor necrosis factor-α (TNF-α) [115]. TNF-α exacerbates oxidative damage and inflammation and stimulates MAPK activation [159], resulting in ROS accumulation through superoxide (O 2 •− ) generation. This process causes oxidative damage and eventual TNF-α production, perpetuating the cycle [150,160]. The overexpression of CYP2E1 due to ethanol-induced oxidative stress also induces inflammation via the Notch1 pathway [161], and ROS mediate interleukin (IL)-1β and IL-18 signaling via inflammasome NLRP3 activation [162,163].
Nicotinamide adenine dinucleotide phosphate oxidases (NOXs) are also important sources of inflammation-related ROS generation, as they generate O 2 •− from oxygen using NAD(P)H. In mice, chronic alcohol consumption increased NOX4 expression in the mitochondrial fraction and NOX4 inhibition ameliorated alcohol-induced liver damage [164]. Upon ethanol administration, NOX-derived ROS are key mediators of nuclear factor-κB activation and subsequent TNF-α production [165], sensitizing Kupffer cells to LPS, thereby contributing to ALD [166].

Iron Overload
Metals such as zinc and iron are involved in ROS-induced oxidative stress caused by ethanol intake. Hepatic iron overload has been observed in approximately 50% of patients with ALD [167]. It causes cellular damage and ferroptosis, which may contribute to ROSassociated alcohol toxicity through Fenton reactions [168] and cause lipid peroxidation and subsequent cell membrane damage and rupture, thereby promoting the autophagydependent release of damage-associated molecular patterns [169].

Protective Mechanisms and the Antioxidant Response
Several mechanisms, including the involvement of antioxidants, counteract the harmful effects of ethanol-induced oxidative stress. The antioxidant enzymes superoxide dismutase (SOD), catalases, and GSH peroxidases, in concert with other proteins, are responsible for ROS removal and restoring the reduced protein and lipid pool [170]. Ethanol inhibits the expression of antioxidant enzymes (e.g., SOD1) and depletes the levels of non-enzyme antioxidants (e.g., GSH), thereby reducing the cellular ability to modulate oxidative stress [171,172]. ROS weaken this antioxidant response by S-glutathionylation, which reduces the expression of downstream antioxidants, such as SOD2, catalases, and sestrin 3 via the phosphoinositide 3 kinase (PI3K)/AKT pathway [173]. This process also increases mitochondrial generation of H 2 O 2 , facilitating further oxidative damage [174].

Autophagy and ALD
The three main types of autophagy which have different means of cargo delivery to the lysosome are macroautophagy, microautophagy, and chaperone-mediated autophagy [15,175]. In macroautophagy, cytosolic materials are sequestered by autophagosomes and then fused with lysosomes for degradation. In microautophagy, cytoplasmic cargo is engulfed directly into the lysosomes [176]. Chaperone-mediated autophagy involves the direct shuttling of specific proteins across the lysosomal membrane for lumen degradation [177].
Autophagosomes migrate to the lysosomes and fuse with them to form autolysosomes for cargo degradation. The cytoskeleton and proteins such as STX17, Rab7, lysosomal-associated membrane protein (LAMP)1/2, [20], synaptosomal-associated protein 29 (SNAP29), and vesicleassociated membrane protein 8 (VAMP8) [199,200] seem to be involved in this migration. The deacetylation of STX17 by the inactivation of the cyclic adenosine monophosphate response element-binding protein promotes the formation of the STX17-SNAP29-VAMP8 SNARE complex, leading to autolysosome formation [199,200]. LAMP2 appears to be key to proper STX17 function [201]. Another key process for autophagy regulation is lysosomal biogenesis, which appears to be regulated by TFEB [202].

Mitophagy
Mitophagy is a selective form of macroautophagy that mediates mitochondrial degradation in the autolysosomes. In type-1 mitophagy, small (0.2-0.3-µm) pre-autophagosomal structures grow into cup-shaped phagophores that envelop and sequester mitochondria into mitophagosomes, often in coordination with mitochondrial fission. In type 2 mitophagy, cup-shaped phagophores are not formed; rather, LC3 aggregates sequester individual mitochondria into mitophagosomes. In both types of mitophagy, mitophagosomes form, acidify, fuse with lysosomes, and degrade their contents [154,156]. While type 1 is primarily related to physiological mechanisms, such as nutrient deprivation, type 2 is related to (and activated by) sensors of mitochondrial damage, such as those caused by oxidative stress [203]. The main oxidative-stress-dependent pathway is that of phosphatase and the tensin homolog-induced putative kinase 1 (PINK1)/Parkin/p62 [203][204][205].
PINK1 is a serine/threonine kinase that translocates to the outer mitochondrial membrane, where it is stabilized by a low mitochondrial transmembrane potential and senses mitochondrial depolarization [206]. It recruits and phosphorylates Parkin (a ubiquitin E3 ligase) [207], which ubiquitylates several proteins on the outer mitochondrial membrane, including voltage-dependent anion channels. p62 recognizes ubiquitylated proteins [204] which trigger their degradation through the lysosome pathway via autophagy via LC3-II interaction [208,209].
The nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant response to acute alcohol exposure in hepatocytes is controversial; it may induce p62-dependent autophagy, which may have a protective role by increasing chaperone-mediated autophagy [35]. However, some researchers have found that this activation may worsen the negative effects of alcohol ingestion by activating ferroptosis [33] (Figure 2). Alcohol exposure also activated type 2 mitophagy in hepatocytes through the PINK1-Parkin-LC3 pathway and mitochondrial depolarization [12,42] (Tables 1 and 3, Figure 2).

Acute Alcohol Intake and Autophagy in Other Cell Types
No study included in our systematic review evaluated autophagy in Kupffer cells (KCs) after acute ethanol intake or exposure. A model in which HSCs were acutely exposed to ethanol revealed increased autophagy, oxidative stress, and cellular activation, with the former regulating the latter two processes via the Nrf2-Keap1-antioxidant response element pathway [62]. The inhibition of autophagy reversed HSC activation and suppressed oxidative stress [62] (Table 5).
Antioxidants 2023, 12, x FOR PEER REVIEW 18 of 31 Figure 2. Effects of ethanol consumption on liver autophagy and relationships to oxidative stress. The symbol "↑" represents an increase in expression or activation while the symbol "↓" represents a decrease in expression or inactivation.
The nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant response to acute alcohol exposure in hepatocytes is controversial; it may induce p62-dependent autophagy, which may have a protective role by increasing chaperone-mediated autophagy [35]. However, some researchers have found that this activation may worsen the negative effects of alcohol ingestion by activating ferroptosis [33] (Figure 2). Alcohol exposure also activated type 2 mitophagy in hepatocytes through the PINK1-Parkin-LC3 pathway and mitochondrial depolarization [12,42] (Tables 1 and 3, Figure 2).

Acute Alcohol Intake and Autophagy in Other Cell Types
No study included in our systematic review evaluated autophagy in Kupffer cells (KCs) after acute ethanol intake or exposure. A model in which HSCs were acutely exposed to ethanol revealed increased autophagy, oxidative stress, and cellular activation, with the former regulating the latter two processes via the Nrf2-Keap1-antioxidant response element pathway [62]. The inhibition of autophagy reversed HSC activation and suppressed oxidative stress [62] (Table 5).

Chronic Ethanol Intake and Autophagy in Hepatocytes
However, the effect of chronic ethanol intake on autophagy in hepatocytes remains unclear. Some studies have shown that chronic ethanol intake or exposure activates autophagy in these cells [47,52,58] and has a protective role similar to acute intake [47]. In these studies, CYP2E1 expression or the reduction of proteasome activity appeared to induce autophagy (e.g., through an increase in LC3 level) [41,49]. Babuta et al. [52] observed decreased LAMP1/2 and lysosomal marker expression, despite increased autophagy [with Figure 2. Effects of ethanol consumption on liver autophagy and relationships to oxidative stress. The symbol "↑" represents an increase in expression or activation while the symbol "↓" represents a decrease in expression or inactivation.

Chronic Ethanol Intake and Autophagy in Hepatocytes
However, the effect of chronic ethanol intake on autophagy in hepatocytes remains unclear. Some studies have shown that chronic ethanol intake or exposure activates autophagy in these cells [47,52,58] and has a protective role similar to acute intake [47]. In these studies, CYP2E1 expression or the reduction of proteasome activity appeared to induce autophagy (e.g., through an increase in LC3 level) [41,49]. Babuta et al. [52] observed decreased LAMP1/2 and lysosomal marker expression, despite increased autophagy [with reduced mTOR and Ras homolog enriched in brain (Rheb) and increased LC3-II expression] in mouse hepatocytes after chronic ethanol intake.
The inhibition of chronic intake exposure-induced autophagy in hepatocytes and the development of steatosis, inflammation, and oxidative stress can be attenuated by ALDH2 expression [39], CYP2E1 inhibition [40], and LAMP2A overexpression [50,51]. LAMP2A plays a protective role by activating the Nrf2 and AMPK pathways by increasing chaperonemediated autophagy [35]. LAMP2 suppression, which is probably mTORC1-dependent, has been observed in patients with severe alcoholic hepatitis [50] (Tables 2 and 4, Figure 2). Mitophagy in hepatocytes is suppressed after chronic ethanol exposure, probably due to the upregulation of the DNA-dependent protein kinase catalytic subunit and p53 activation [53]. In this context, AMPK has been shown to enhance mitophagy via the Nrf2-ubiquinolcytochrome C reductase core protein 2 pathway [31].
Mice lacking proteins required for LC3, Atg5, and Atg7 lipidation show different vulnerabilities to acute and chronic alcohol exposure. For instance, Atg5-knockout (KO) mice are more susceptible to liver damage with acute alcohol treatment, whereas Atg7-KO mice are more sensitive to liver damage after chronic plus binge ethanol intake [43]. Atg5 inhibition appears to improve chronic ethanol consumption-induced liver damage [43]. Similarly, CYP2E1 activation is associated with increased autophagy after acute ethanol intake [43] but reduced autophagy after chronic ethanol intake ( Figure 2). The mechanisms involved in these differences are not adequately understood [43].

Chronic Ethanol Intake and Autophagy in Other Cell Types
In KCs exposed chronically to alcohol, autophagy seems to have a protective role associated with decreased Myeloid differentiation factor 2/Toll-like receptor 4 expression [56] and the mediation of the anti-inflammatory and anti-steatogenic effects of the cannabinoid 2 receptor [63]. The inhibition of autophagy in macrophages after chronic ethanol treatment appeared to increase ethanol-induced liver damage, inflammation, ROS generation, interferon regulatory factor 1 accumulation, and the induction of hepatic C-C motif chemokine ligand 5 and C-X-C motif chemokine ligand 10 expression [60,61] (Table 5).
HSCs from ethanol-fed mice had an increased UPR, which triggered autophagy and induced an Nrf2-mediated antioxidant response under ER stress conditions [146]. The IRE1α pathway blockade significantly reduced autophagy activation in a p38 MAPKdependent manner, thereby reducing the fibrogenic response [64] (Table 5).

Alcohol-Induced Organ-Organ Crosstalk and Autophagy
Information on the autophagic pathways involved in organ-organ crosstalk in ALD is scarcely available, and the studies that have been performed have focused mainly on the crosstalk between adipose and liver tissue. Rodriguez et al. [210] showed that the ablation of the mTOR (Raptor) pathway in adipocytes (but not hepatocytes) contributed to acute alcohol treatment-induced liver damage with increased inflammation, suggesting the implication of adipose tissue in the development of alcoholic steatohepatitis. Consistently, Li et al. [153] reported exacerbated alcohol-induced hepatic steatosis in adipocyte-specific Raptor-KO mice. Adipocyte-specific Atg5-KO mice had increased circulating levels of fibroblast growth factor 21 (FGF21) and adiponectin and were resistant to chronic alcohol treatment-induced adipose tissue atrophy and liver damage [211]. Although this area of research, particularly in the context of ALD, is relatively new, current evidence suggests that autophagy modulates the crosstalk between adipose tissue and the liver by controlling the synthesis of FGF21 and adiponectin. Such autophagy-mediated crosstalk has been detected in other liver diseases, such as non-alcoholic fatty liver disease, in which autophagy inhibition in white adipose tissue after four months of a high-fat diet ameliorated liver pathology in mice [212].

Autophagy-Targeting Treatments for ALD
Effective therapies for ALD are lacking, and given its important role in this disease, autophagy is a potential therapeutic target. The roles of several pharmacological agents in the prevention or amelioration of ALD through autophagic modulation have been examined ( Table 6). Most of these studies have been performed using mouse models, although other animal models (i.e., rat and zebrafish) have also been used. The mTOR pathway is one of the most relevant potential therapeutic targets in this context. The activation of mTOR and AMPK signaling is involved in ethanol-induced autophagy under oxidative stress [14,213]. Rapamycin (also known as sirolimus) is a lipophilic macrolide antibiotic that was first isolated from Streptomyces hygroscopicus and has been shown to inhibit mTORC1, thereby reversing alcohol-induced mTOR activation and attenuating related liver damage [15,40,51]. Torin 1 is a selective ATP-competitive small molecule that inhibits the mTOR pathway through direct inhibition of the mTORC1 and mTORC2 complexes [55,105], and it may also ameliorate ALD. Upstream of mTOR, the pathway can be modulated by the inhibition of AMPK. The activation of AMPK inhibits mTOR-dependent signaling through different molecules, such as calcitriol (the active form of vitamin D) [69] and palmatine (a protoberberine alkaloid found in several plants) [87]. Although no clinical trials have been performed to study the effects of these drugs on ALD, studies are being undertaken to examine their effects on Sjögren syndrome (ClinicalTrials.gov identifier: NCT05605665) and Alzheimer's disease (ClinicalTrials.gov identifier: NCT04629495). The possibility of rapamycin delivery via nanoparticles, which may reduce side effects and have been shown to ameliorate metabolic fatty liver disease in a mouse model [214], adds to the interest in testing the effect of this drug on ALD.
Ethanol intake activates FOXO3a, which transcriptionally regulates several autophagy genes [46]. The SIRT1/FOXO pathway is involved in the alleviation of chronic alcoholic liver damage by preventing fat accumulation and reducing ROS production, inflammation, and cell death [215]. Modulators of autophagy through this pathway, such as resveratrol, quercetin, and salvianolic acid A [46,[90][91][92][93][94][96][97][98], have shown promise for the treatment of ALD in animal models. We are not aware of a clinical trial examining the effects of these products, but potential interest due to the promising experimental results and favorable side effect profiles, particularly with resveratrol, is attenuated by the lack of efficacy against non-alcoholic fatty liver disease [216].
Alcohol-induced autophagy can also be suppressed by antioxidants such as N-acetylcysteine (NAC) [38,40]. NAC and the CYP2E1 inhibitor chlormethiazole [40,72,73] appear to attenuate the toxic effects of ethanol in the liver [40]. NAC has been tested in a clinical trial conducted in patients with alcoholic hepatitis; although infections were less frequent and 1-month mortality was reduced in the prednisolone-NAC group relative to the prednisolone-only group, other side effects and the 6-month survival rate were similar across the two groups [217]. Another clinical trial examining the efficacy of NAC against alcoholic hepatitis is ongoing (ClinicalTrials.gov identifier: NCT03069300), given that the body of evidence provides a rationale for the use of this drug in patients with ALD and/or alcohol use disorders [218].
Other products, such as zinc and carbamazepine, appear to be involved in autophagy activation after ethanol intake or exposure; thus, they could be tested as potential treatments for ALD. Interestingly, zinc exposure stimulates autophagy, with an additive effect of co-stimulation with ethanol for 24 h [107]. Carbamazepine, a mood-stabilizing drug, induces autophagy by reducing the level of intracellular inositol [47]. The administration of Nrf2 activators, such as sulforaphane [102] from vegetables of the genus Brassica and glycycoumarin from Glycyrrhiza uralensis [81], is also of potential interest in the treatment of this disease. Data from animal models suggest that these agents improve alcohol-induced liver steatosis and oxidative stress and promote autophagy ( Table 6). The activation of the Nrf2 pathway appears to protect against alcohol-induced liver fibrosis and hepatotoxicity, whereas Nrf2 knockdown is associated with increased alcohol-induced hepatocyte damage [81,219,220].

Discussion
Acute ethanol consumption increases autophagy activation in the liver, whereas chronic alcohol intake or exposure decreases autophagy activation. The mechanisms underlying this difference have not yet been fully elucidated. Increased autophagy in hepatocytes and KCs seems to have a protective role against the pathogenesis of alcohol-related liver damage and ALD, and the inhibition of autophagy makes hepatocytes susceptible to hepatotoxicity, steatosis, and oxidative stress. Autophagy leads to HSC activation and oxidative stress in these cells.
Most studies in this field have examined the classical components of autophagic pathways (e.g., p62, LC3, and Beclin 1) in liver cells; expanding the scope of research to examine other components is desirable. For instance, autolysosome formation, a key step in autophagy, has not been analyzed in detail in the context of ALD. In addition, autophagy in different organs and tissues, especially in cells of the immune system, needs to be assessed because of the involvement of these cells and tissues in inflammation and oxidative stress, which play key roles in ALD development. Selective autophagy pathways such as mitophagy and lipophagy should also be analyzed further. Autophagy-related organ-organ crosstalk in ALD has received little research attention despite the growing interest in and potential of this field. Regarding clinical applications, autophagy-targeting therapies have not been tested successfully in clinical trials, although data from animal and in vitro models suggest potential roles for several drugs, including those involved in the mTOR pathway and antioxidants that modulate autophagy. Although a large number of drugs could potentially be useful in ALD treatment, based on basic research, the potential difficulties of carrying out large clinical trials, particularly those not funded by drug companies, could prove difficult to navigate, in spite of the fact that autophagy targeting may have protective effects against several liver diseases [47]. We hope to see expansion in this area in the near future.
It is important to acknowledge that the variations observed in the definition of acute or chronic ethanol intake or exposure, as well as the diverse experimental protocols employed across the included studies, have posed challenges in clearly elucidating the underlying mechanisms associated with acute and chronic ethanol exposure and have prevented us from employing a meta-analysis or other numerical approaches to demonstrate the extent of variability (e.g., classic coefficient of variation or robust coefficient of variation estimators [221].

Conclusions
In conclusion, autophagy activation in hepatocytes and KCs appears to have a protective role against alcohol-induced liver damage and oxidative stress. However, further research is needed to elucidate these mechanisms and develop potential clinical applications.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.