NAFLD is a common chronic liver disease characterized by excessive accumulation of hepatic fat in the absence of chronic viral infection and alcohol abuse (1, 2). NAFLD is a metabolic disease involving abnormal liver fat metabolism, which has recently been renamed as metabolism-associated fatty liver disease (MAFLD) (3), as represented by NAFLD in the following article. Non-alcoholic fatty liver (NAFL) continues to progress, leading to hepatocyte swelling, liver damage, inflammation, and varying degrees of fibrosis. This promotes the occurrence of non-alcoholic steatohepatitis (NASH), which can eventually progress to cirrhosis over time (4). The incidence and prevalence of NAFLD have been reported to be rapidly increasing worldwide, reaching approximately 30%. It is projected that the prevalence of NASH will experience a significant rise of 56% by the year 2030 (5). The traditional treatment of NAFLD focuses on addressing pathological changes caused by excessive fatty accumulation through exercise, dietary adjustments, lifestyle improvements, and reduction of liver fat accumulation (6, 7). While these methods alleviate NAFLD to some extent, they are not sufficient to prevent the progression of NAFLD. Therefore, there is an urgent need for new treatment methods for NAFLD in clinical practice.

The pathogenesis of NAFLD is mainly characterized by the accumulation of triglycerides (TGs) in hepatocytes, and the long-term accumulation of liver lipids promotes the occurrence of steatosis and lipotoxicity (8, 9). However, lipotoxicity induces mitochondrial dysfunction, triggering endoplasmic reticulum stress (ERS) and lipid peroxidation (10). This process further promotes the activation of KCs, leading to inflammation and the release of inflammatory cytokines and transforming growth factors, and activates HSCs, thus facilitating the progression of NAFLD (11, 12).

Autophagy is an important process for maintaining cellular homeostasis, and forms autophagosomes by degrading unnecessary organelles, proteins and other components in cells, and forms autolysosomes by combining with lysosomes to eliminate phagocytized goods and maintain intracellular homeostasis (13, 14). The term “autophagy” was initially coined by Christian de Duve in 1967, coined from the Greek terms “auto” meaning self, and “phagy” meaning to consume (15). In recent years, researchers have conducted in-depth studies on autophagy and believe that autophagy is a widely existing necessary physiological regulatory process in mammalian cells (16). Significantly, autophagosomes and autolysosomes are two essential components in the formation of autophagy (17). It is generally believed that the fusion between autophagosomes and lysosomes is necessary for the formation of autolysosomes. However, distinct mechanisms regulate the formation of autophagosomes as well as their subsequent fusion with lysosomes to form autolysosomes.

An increasing number of studies have shown a close relationship between the development of NAFLD and impaired autophagy function (18). Restoring autophagy can promote the breakdown of fat, improve hepatic steatosis, and reduce liver cell damage (19). In addition, autophagy can maintain the homeostasis and function of non-parenchymal cell types, thereby inhibiting liver inflammation and fibrosis and slowing down the progression of NAFLD. This makes it one of the important targets for treating NAFLD (20, 21). However, the current literature on the relationship between drugs and autophagy in NAFLD needs to be better organized. Therefore, this review will discuss the important role of autophagy in NAFLD specifically focusing on the two processes of autophagosome formation and autolysosome formation, to provide theoretical support for the clinical treatment of NAFLD.

In mammals, autophagy mainly includes macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA) (22). Microautophagy is selective organelle degradation that occurs through the interaction of proteins with lysosomes, vacuolar membranes, or late endosomal surface proteins (23). CMA is a highly selective form of autophagy that often involves binding to the molecular chaperone heat-shock cognate protein in order to transport substrate proteins to the lysosomal surface for internalization and rapid degradation within the lysosomal lumen (24). This review concentrates on macroautophagy (hereinafter referred to as autophagy) and its role in NAFLD.

Autophagy is governed by a complex series of regulatory mechanisms, including initiation, formation of autophagosomes, formation of autolysosomes, degradation, and termination of recycling (25). Meanwhile, autophagy is primarily initiated by autophagy-related (ATGs) proteins, which are encoded and modified by highly conserved ATG genes. These ATG proteins, in conjunction with autophagy regulatory factors, play a crucial role in regulating the autophagic process (26, 27). Moreover, the AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) are two pivotal components involved in the regulation of autophagy (28).

When autophagy is initiated, AMPK is regulated by upstream kinases such as liver kinase B1 (LKB1) and calcium/calmodulin-dependent protein kinase kinase 2 or β (CaMKK2/β), while simultaneously inhibiting the expression of mammalian target of rapamycin 1 (mTOR1) (29, 30). Subsequently, AMPK phosphorylates the downstream UNC-51-like kinase 1 (ULK1) complex, which serves as the initiation kinase complex of autophagy and primarily consists of ATG13, FIP200, ATG101, ULK1 or ULK2 in mammals (31, 32). Activation of ULK1 promotes phosphorylation of the downstream III phosphoinositide 3-kinase (PI3K) complex I, which is composed of autophagy and Beclin 1 regulator 1 (AMBRA1), Beclin-1, class III PI3K vacuolar protein sorting 34 (Vps34), p115 and ATG14 (17). Subsequently, downstream proteins of the class III PI3K complex, including double-FYVE-containing protein 1 (DFCP1) and WD repeat domain phosphoinositide-interacting protein (WIPI), are recruited to the cell wall for the production phosphatidylinositol 3-phosphate (PI3P), which contributes to phagosome extension (33, 34). Simultaneously, PI3P recruits ATG2 and transmembrane protein ATG9 as lipid transporters within the autophagosome intermembrane and interfollicular regions to facilitate phagocytosis (35) ( Figure 1 ).

Subsequently, the engagement system of two ubiquitin-like proteins promotes phagosome extension and formation of autophagosomes (36). Ubiquitin-like protein ATG12 is bound to ATG5 by the combination of E1 activase ATG7 and E2 binding enzyme ATG10 and further bound to ATG16L1 to form the ATG12-ATG5-ATG16L1 ubiquitin-like complex (37, 38). Furthermore, the homolog of ATG8, microtubule-associated protein 1A/1B-light chain 3B (LC3), undergoes hydrolysis by ATG4 protein to generate LC3I. Subsequently, the ubiquitin-like binding of E1 activase enzyme ATG7, the E2 binding enzyme ATG3, and the E3 ligase ATG12-ATG5-ATG16L1 activates LC3I and facilitates its interaction with phosphatidylethanolamine (PE) to form lipidized ATG8/LC3-II (39–42). Subsequently, P62/sequestosome1 (SQSTM1) functions as an autophagy adaptor protein that mediates the interaction between ubiquitin-like proteins and LC3II, facilitating expansion of phagocytic cells and promoting closure of the isolation membrane to facilitate formation of autophagosomes (43) ( Figure 1 ).

Finally, autophagosomes form autolysosomes by transporting their cargo into lysosomes and fusing with them (44). In the process of autophagosome-lysosome fusion, the ADP-ribosylation factor-like GTPase AR18 and Ras-associated protein 7 (Rab7) play a pivotal role in localizing autophagosomes and lysosomes, as well as mediating fusion through recruitment of specific adapter proteins and motor proteins (45, 46). Additionally, tethering factors such as homotypic fusion and vacuole protein sorting (HOPS) are crucial for autolysosomal formation (47). They are recruited through interaction with pleckstrin homology and RUN domain containing M1 (PLEKHM1) and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes to facilitate lipid and content mixing, promoting fusion between autophagosomes and lysosomes (47). The pacer autophagy enhancer is targeted by STX17 and phosphatidylinositol 3-phosphate (PtdIns3P) within the SNARE complex localized in autophagy vacuoles. This targeting facilitates the recruitment of the VP34-Beclin 1-UVRAG complex to the autophagy vacuoles (48). Meanwhile, the PtdIns3P protein on autophagosomes facilitates fusion with lysosomes by recruiting the HOPS complex tethering factor (48, 49). Notably, transcription factor EB (TFEB) and transcription factor enhancer 3 (TFE3) are also involved in the formation of autolysosomes (50). Ultimately, lysosomal proteases release degraded products into extracellular environment to promote recycling of these materials (51) ( Figure 1 ).

Autophagy degradation can be classified into two categories: non-selective and selective. Non-selective autophagy refers to the random phagocytosis and degradation of cytoplasmic substances such as proteins and organelles. However, selective autophagy is a cellular response mechanism that specifically targets cargoes for degradation in lysosomes upon exposure of cells to various stresses, such as DNA damage (52, 53). Furthermore, studies have found that the progression of NAFLD is not only related to non-selective autophagy (hereinafter referred to as autophagy) but also closely associated with selective forms of autophagy, including mitophagy, lipophagy, and endoplasmic reticulum (ER) autophagy (54).

The liver utilizes non-selective autophagy to eliminate, degrade, and recycle senescent or dysfunctional cellular components, organelles, and proteins in response to nutrient deficiency or stressful conditions (55). This ensures the maintenance of cellular energy balance. In a state of starvation, autophagy degrades LDs in hepatocytes to release fatty acids to the mitochondria for β-oxidation, thereby regulating lipid storage and energy balance in hepatocytes (55). On the contrary, dysregulation of autophagy weakens lipolysis and fat phagocytosis in hepatocytes, leading to mitochondrial dysfunction and increased production of reactive oxygen species (ROS) in the liver (56). This activation of KCs induces the proliferation and activation of HSCs, thus promoting the development of NAFLD. However, improving specific selective autophagy can alleviate the progression of NAFLD (57).

For instance, lipophagy is the process through which cells selectively recognize lipids and, activated by autophagy-related molecules, contribute to the degradation of LDs in hepatocytes (58). Under normal conditions, the liver mediates the decomposition of triacylglycerols (TAGs) stored in LDs and induces mitochondrial β-oxidation through two major pathways: lipid catabolism and lipophagy (59). Thus, adipose triglyceride lipase (ATGL) is a cytosolic lipase involved in lipolysis, which releases free fatty acids (FFAs) that can serve as substrates for mitochondrial β-oxidation or as potent signaling molecules for various cellular processes (60). The ATGL and patatin-like phospholipase domain-containing protein 8 (PNPLA8) act as selective autophagy receptors for lipid engulfment, facilitating lipolysis and β-oxidation of FFAs (61). During the process of lipophagy, lipid droplet-associated proteins perilipin 2 (PLIN2) and perilipin 3 (PLIN3) are degraded through CMA by a synergistic effect of Hsc70 and lysosome-associated membrane protein 2A (LAMP-2A) receptors (62). Similarly, the interactions of ATG14 with ULK1 and LC3 also induce lipophagy, leading to the release of FFAs and mitochondrial β-oxidation (63). The regulation of TFEB can enhance lysosome biosynthesis, autophagosome formation, and their fusion with lysosomes and promote autophagy flux, a crucial regulatory factor in lipolysis (64). The studies have shown that overexpression of TFE3 can regulate coordinated lysosomal expression and regulation (CLEAR) elements, leading to a significant reduction in hepatocyte steatosis (65). Another study found that important proteins and pathways involved in maintaining lipid phagocytosis and lipid droplet homeostasis include the Rab GTPase in LDs, Rab10, and Rab7 (primarily activated during autophagy), as well as the mTORC1-perilipin-3 pathway (66).

In addition, mitophagy can degrade functionally impaired mitochondria and misfolded proteins in the liver, regulating cell death to maintain a stable state of liver lipid metabolism and inhibit the production of ROS. This process is important in the treatment of NAFLD (67, 68). Simultaneously, mitophagy also triggers the activation of inflammatory vesicles (56). Normally, PINK1 facilitates the translocation of Parkin from cytoplasmic lysates to depolarized mitochondria. Parkin ubiquitinates outer membrane proteins and recruits P62 to damaged mitochondria, resulting in changes in autophagosome membrane structure and initiating selective mitophagy (69, 70). During hypoxia, HIF1-α promotes the expression of BNIP3 (BCL2/adenovirus E1B interacting protein 3) and its relocation to the mitochondrial envelope. Subsequently, BNIP3 interacts with Bcl-2 to facilitate mitochondrial remodeling, while NIX directly interacts with LC3 through the LIR domain, recruiting autophagosomes to mitochondria and inducing mitophagy (71–73). Studies have demonstrated that macrophage-stimulating protein 1 (Mst1) mediates mitochondrial autophagy by regulating Parkin expression through the AMPK pathway, which further promotes NAFLD (74). However, the early pathogenesis of NAFLD involves the loss of mitophagy due to the deletion of a key regulator, Parkin, which accelerates the onset of crucial disease features in NAFLD (68). Another study demonstrated that increased expression and mitochondrial localization of PINK1/Parkin promoted PINK1-mediated mitophagy for clearing damaged mitochondria (75). This reduction in oxidative stress inhibited NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) inflammasome activation and slowed hepatic steatosis progression in NAFLD patients (75).

Interestingly, ERS activates the unfolded protein response (UPR) and induces ER autophagy, which is an essential factor in restoring the degradation of substances in the ER to normal levels (76). In the normal liver, ERS facilitates autophagy-mediated lipid degradation in hepatocytes to maintain the protective mechanism of physiological hepatocyte function (77). The ER autophagy receptor is a crucial factor that affects the level of ER autophagy. Notably, current investigations have identified several ER-phagy receptors, namely FAM134, SEC62, RTN3L, CCPG1, ATL, TEX264, CALCOCO1, C53, and p63 (78). Under ERS induction, the disruption of the ER structure is recognized by the ER receptor and leads to binding with LC3/GABARAP/ATG8, thereby promoting ER autophagy (79). However, during the process of ERS-induced autophagy, UPR mainly regulates autophagy through inositol requiring enzyme 1 (IRE1α), double-stranded RNA-activated protein kinase (PKR)–like ER kinase (PERK), activating transcription factor 6 (ATF6), and Ca²⁺ regulation. By upregulating ER autophagy receptors, UPR promotes autophagy, and C/EBP homologous protein (CHOP) plays a critical regulatory role in this process (80, 81). Specifically, IRE1α promotes autophagy by modulating Beclin1 through the tumor necrosis factor receptor-associated factor 2 (TRAF2) and c-Jun N-terminal kinase (JNK) pathways. Additionally, the PERK pathway induces autophagy by suppressing mTORC1 activity (82, 83). ATF6 activates death-associated protein kinase 1 downstream through Beclin1, thereby triggering autophagy (84). Additionally, Ca²⁺ in the ER can also participate in autophagy via protein kinase C (PKC) and calcium Ca²⁺/calcium calmodulin-dependent kinase II (CaMKII). Animal studies have shown that autophagy induces autophagy by downregulating mTOR signaling, ERS-related genes (PERK, IRE1, ATF6, BIP, and CHOP), and autophagy-related genes. This reduces hepatic steatosis and lipid accumulation (85). Another study demonstrated that exposure of human liver cells to low concentrations of per- and polyfluoroalkyl substances induces ERS through activation of the UPR signaling pathway, and modulates NAFLD (86).

Many clinical drugs for treating NAFLD have been shown to activate autophagy and affect the progression of NAFLD through different signaling pathways, including the classic hypoglycemic drug metformin (87), the well-established lipid-lowering agent simvastatin (88), and the mTOR target inhibitor rapamycin (89). Emerging therapeutic drugs, such as glucagon-like peptide-1 (GLP-1), sodium glucose co-transporter 2 (SGLT2) inhibitors, and immune-related GTPase M (IRGM), have been studied extensively (90). Additionally, studies have demonstrated that herbal extracts containing active ingredients such as resveratrol, ginsenoside, and curcumin exhibit hepatoprotective effects by inducing autophagy in the liver (91). More detailed information will be provided in the subsequent sections.

In the pathological progression of NAFLD, various cell types including parenchymal and non-parenchymal hepatocytes, KCs, HSCs, liver sinusoidal endothelial cells (LSECs), and cholangiocytes are implicated (92) ( Figure 2 ).

As previously described, we discussed the biological processes and degradation of autophagy, as well as its function in cellular metabolism. Through a literature review, we demonstrated the close association between autophagy and the progression of NAFLD in both liver parenchyma and non-parenchymal cells ( Figure 3 ). However, there is a lack of research on the treatment of NAFLD by regulating autophagy through drug-targeting signaling pathways for LSECs and cholangiocytes. Therefore, we will start by focusing on the formation of autophagosomes and autolysosomes in three specific cell types, namely hepatocytes, KCs, and HSCs. This will allow us to explore how drugs targeting autophagy through signaling pathways can be used to treat the progression of NAFLD, thereby deepening our understanding of the relationship between autophagy and NAFLD.

Defects in autophagy are implicated in the pathogenesis of numerous diseases, including tumors, neurodegenerative disorders, and metabolic conditions (16). Moreover, dysfunctional autophagy is intricately associated with the pathological progression of NAFLD (194).

The upstream proteins activate AMPK in autophagy to facilitate phosphorylation of the ULK-1 complex and suppress activation of mTOR (29, 30). Subsequently, phosphorylation of downstream PI3KC3 complex I induces recruitment of ATG9, a downstream autophagy factor, onto the cell membrane and initiates autophagosome nucleation. Through interaction between DFCP1 and WIPI proteins, PI3P is generated, which facilitates cell nucleation and autophagosome formation (33, 34). Concurrently, the induction of WIPI and ATG16L1 triggers the formation of two downstream ubiquitin complexes: ATG12-ATG5-ATG16L1 and LC3-II (33, 35, 37, 38). These complexes are crucial in facilitating autophagosome elongation. Subsequently, the ubiquitination of LC3II through its association with p62/SQSTM1 as an autophagosomal substrate facilitates autophagosome maturation (43). In conjunction with the HOPS complex, VPS34 complex II and Rab7 further regulate this process, enabling fusion between cargo-containing autophagosome sand lysosomes (45, 47, 48). Ultimately, lysosomes digest this fused product, leading to degradation, reactivating mTORC1, inhibiting autophagy, and terminating the autophagic process (51). The degradation modes within autophagy encompass both non-selective and selective processes. Non-selective autophagy is a cellular process that eliminates senescent or dysfunctional components like organelles and proteins due to nutrient deficiency or stress conditions while degrading them for recycling. This process helps maintain cellular energy balance (54). On the other hand, selective autophagy is a cellular response mechanism that targets explicit cargoes for degradation within lysosomes when cells are exposed to various stresses like DNA damage (52, 53). However, during the development of NAFLD pathology, selective autophagy primarily plays a role in degrading LDs through mitophagy and ER-phagy processes (57).

The pathogenesis of NAFLD is intricate and primarily associated with the interplay of steatosis-induced inflammation, lipotoxicity, oxidative stress, and the ER (8). During disease progression, excessive accumulation of fat in hepatocytes, increased levels of FFAs and TG stimulate hepatocyte steatosis, induce mitochondrial dysfunction, and ERS. This promotes excessive production of ROS, impairs autophagy, promotes the capillarization of LSECs, and stimulates the activation of inflammasomes in KCs. Simultaneously, it also enhances the secretion of fibrotic factors that stimulate the activation of adjacent HSCs and facilitate the progression of NAFLD to liver fibrosis (126). However, autophagy has a dual role in HSCs, promoting the growth of quiescent HSCs, while inhibiting the growth of activated HSCs. This prevents fibrosis changes in NAFLD and its involvement in all stages of NAFLD progression (115). In recent years, there have been reports discussing drugs that target autophagy for the treatment of NAFLD, including herbal extracts, emerging GLP-1 activators, and SGLT2 inhibitors (91). These drugs have demonstrated their ability to induce or restore the process of autophagy, ameliorate hepatocyte steatosis, reduce inflammation caused by KCs, inhibit HSC activation, decrease the occurrence of liver fibrosis, and delay the progression of NAFLD (115).

Three types of autophagy have been identified, namely microautophagy, macroautophagy, and CMA. However, numerous unresolved questions remain regarding the intricate interplay among these processes and the specific triggering mechanisms involved in autophagy. Unresolved inquiries persist concerning identification of novel receptors involved in selective autophagy for autophagy-mediated degradation. Despite extensive knowledge of liver autophagy, the specific mechanism of action of drugs targeting autophagy in the pathogenesis of NAFLD remains to be determined. Certain drugs have not transitioned from animal experiments to clinical practice due to a lack of theoretical support from human clinical research. Similarly, the current research stage in NAFLD pathology lacks sufficient investigation into drug targeting of autophagy through signaling pathways, specifically in hepatic sinusoidal endothelial cells and cholangiocytes. In the future, by regulating the role of autophagy through signaling pathways, we will be able to identify better therapeutic targets to treat NAFLD more effectively.

The activation of autophagy can inhibit hepatocyte steatosis, suppress the production of inflammatory factors in KCs, prevent the activation of HSCs, and maintain LSECs. In contrast, impaired autophagy is involved in different stages of NAFLD. However, despite ongoing clinical studies and the use of some drugs to improve autophagy in NAFLD patients, the incidence of this disease continues to rise. The maintenance of autophagy in innate liver cells is an urgent problem that needs to be addressed in the medical field. This study aims to provide novel perspectives and insights for the clinical management of NAFLD by summarizing the physiological process of autophagy, elucidating the association between autophagy and NAFLD, and exploring the role and relevant pathways of intracellular therapeutic agents.

QS: Writing – original draft, Writing – review & editing, Conceptualization, Project administration, Visualization. MY: Writing – review & editing, Project administration, Visualization. SW: Investigation, Writing – review & editing. XC: Writing – original draft, Investigation, Project administration. SC: Writing – original draft, Project administration. RZ: Investigation, Writing – original draft. ZX: Writing – review & editing, Visualization. YL: Supervision, Writing – review & editing.