Cell-specific roles of autophagy in liver fibrosis: implications for targeted pharmacotherapy

Abstract

Background

Liver fibrosis represents a pivotal stage in the progression from chronic liver disease to cirrhosis, characterized by intricate pathological alterations in both parenchymal and non-parenchymal cells. Autophagy, a fundamental mechanism for maintaining intracellular homeostasis, plays a critical role in these processes. This study aims to elucidate the cell type-specific roles of autophagy in liver fibrosis and propose targeted intervention strategies.

Methods

We systematically reviewed the functional roles of autophagy in hepatocytes, macrophages, hepatic stellate cells (HSCs), and liver sinusoidal endothelial cells (LSECs) by analyzing current experimental and mechanistic evidence. We further conceptualized therapeutic approaches based on selective modulation of autophagic activity in different cell populations.

Results

In hepatocytes, autophagy exerts protective effects by reducing lipid accumulation and preventing apoptosis caused by lipid metabolism disorders. In macrophages, autophagy regulates polarization and suppresses inflammatory cytokine release, thereby attenuating inflammation and slowing fibrosis progression. Conversely, autophagy in HSCs and LSECs exhibits dual effects: it may accelerate fibrosis by promoting cell activation and extracellular matrix remodeling or mitigate fibrosis by inhibiting these processes. These findings underscore the cell context-dependent role of autophagy during fibrogenesis.

Conclusions

Selective enhancement or inhibition of autophagy in distinct liver cell types offers a promising strategy for regulating fibrosis. This approach provides new insights and a theoretical basis for developing targeted therapies for liver fibrosis.

1. Introduction

Liver fibrosis is a chronic liver injury caused by various factors, such as excessive alcohol consumption, viral infections (including hepatitis B and C), non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), and autoimmune hepatitis. If these factors persist or treatment is delayed, it can result in excessive accumulation of extracellular matrix (ECM). This accumulation disrupts the liver’s structure, gradually causing a loss of function that can ultimately lead to irreversible cirrhosis [1]. This process is accompanied by severe complications, including portal hypertension and liver failure, which pose significant threats to patient health [2]. Globally, the incidence and mortality rates of liver fibrosis are at a high level [3]. A meta-analysis of NAFLD patients found that the risk of liver-related mortality significantly increases as the stages of liver fibrosis progress, exhibiting an exponential growth trend [4]. Additionally, multiple independent studies have emphasized the degree of fibrosis as a major determinant of poor prognosis and increased mortality in NASH patients [5–7]. Therefore, it is crucial to deepen our understanding of its pathogenesis and to develop effective intervention strategies.

Liver fibrosis is a response of the liver to long-term injuries and involves interactions among different cells and molecules [8]. The activation of hepatic stellate cells (HSCs) drives the development and progression of liver fibrosis. Under normal physiological conditions, HSCs exist in a quiescent state in the space of Disse, responsible for the storage and metabolism of vitamin A and lipid droplets (LDs). However, under pathological conditions, such as exposure to drugs, alcohol, or inflammatory factors, HSCs transform into activated myofibroblast-like cells, gaining the ability to proliferate, migrate, contract, and secrete ECM, which results in liver fibrosis due to excessive ECM accumulation [9,10]. Liver fibrosis is not entirely irreversible. Appropriate interventions can promote its regression, leading to the senescence, apoptosis, or return of HSCs to a quiescent state. This process reduces ECM synthesis and deposition, facilitating liver tissue repair and regeneration [11–13]. In this process, autophagy plays an important regulatory role. Autophagy has two main roles: it promotes the senescence and apoptosis of HSCs by increasing oxidative stress, which negatively regulates their activation and inhibits excessive ECM synthesis [14]. Additionally, autophagy regulates HSC energy metabolism by hydrolyzing LDs to release fatty acids, influencing HSC activation [15]. Additionally, autophagy plays a significant role not only in HSCs but also in various other cells within the liver microenvironment, such as liver macrophages, hepatocytes, and liver sinusoidal endothelial cells (LSECs). Specifically, autophagy regulates liver macrophage polarization, which affects the inflammatory response [16]. In hepatocytes, it alleviates lipid accumulation and reduces apoptosis [17]. For LSECs, moderate autophagy protects against oxidative stress, supports normal function, and inhibits HSC activation [18]. However, excessive autophagy can compromise LSEC structure, leading to functional impairment and affecting the fenestration and basement membrane characteristics. This diminishes their ability to regulate vascular tension and immune responses, as well as maintain the quiescent state of HSCs, which is detrimental to liver microenvironment homeostasis [19–21]. In conclusion, autophagy has a complex and extensive role in liver fibrosis, directly regulating the activation and function of key cells while also influencing the overall homeostasis of the liver microenvironment. Given this complex regulatory role, therapeutic strategies targeting autophagy in liver fibrosis have naturally become a research hotspot. This article systematically reviews the mechanisms of autophagy in hepatocytes and non-parenchymal cells during liver fibrosis and explores potential therapeutic strategies-such as small molecule compounds, gene editing technologies, and natural compounds-to modulate autophagic activity, providing new insights for liver fibrosis prevention and treatment.

2. The biological basis of autophagy

Autophagy is a conserved cellular process that maintains cellular homeostasis and facilitates adaptation to stress by recycling and degrading damaged or superfluous components. This is achieved through the formation of double-membraned autophagosomes, which encapsulate cytoplasmic cargo and deliver it to lysosomes for degradation [22]. Based on the delivery route of cargo to lysosomes, autophagy is primarily classified into macroautophagy, microautophagy, and chaperone-mediated autophagy. Among these, macroautophagy (hereafter referred to as autophagy) is the most extensively studied. Functionally, autophagy is categorized as non-selective or selective. Non-selective autophagy is triggered by stresses such as nutrient starvation to carry out bulk degradation of cytoplasmic components, thereby providing emergency nutrients. In contrast, selective autophagy precisely targets specific cargoes for elimination, including damaged mitochondria (mitophagy), the regulation of iron homeostasis through ferritin degradation (ferritinophagy), and the breakdown of lipid droplets (lipophagy). This specificity is orchestrated by autophagy receptors (e.g. p62/SQSTM1, NBR1, NDP52), which recognize the cargo and tether it to the autophagosomal membrane by binding to lipidated LC3/GABARAP proteins [23].

The core mechanism of autophagy is governed by autophagy-related (ATG) proteins acting in a coordinated sequence [22] (Figure 1). Increasing the levels of the ULK complex, which includes UNC-51-like kinase 1 (ULK1), ULK2, FIP200, ATG13, and ATG101, is essential for starting the autophagy process. The initiation of autophagy is strictly regulated by the mammalian target of rapamycin complex 1 (mTORC1) and AMP-activated protein kinase (AMPK) signaling pathways. ATG9 is the only transmembrane protein among many autophagy-related proteins, providing membrane material for the formation of the autophagosome membrane [24]. Next, VPS34 complex I, which includes VPS34, beclin-1, ATG14, and VPS15, catalyzes the production of phosphatidylinositol-3-phosphate (PI3P) on the autophagic membrane. This step is crucial for recruiting additional autophagy-related proteins. To further expand the isolation membrane, the PI3K complex, which typically includes VPS34, interacts with the ATG5-ATG12 complex and the LC3 system. This interaction promotes the binding of ATG5-ATG12 and converts LC3 from its form LC3-I to LC3-II through a positive feedback loop. LC3-II is inserted into the autophagosome membrane and acts as a marker for autophagosome formation. In selective autophagy, specific autophagy receptors (like p62/SQSTM1) recognize and bind to target substrates, which associate with LC3-II to mediate their encapsulation into the autophagosome [25]. As the isolation membrane expands and closes, the autophagosome is completely formed and eventually fuses with the lysosome to create the autolysosome. Inside the autolysosome, the autophagosome’s contents are degraded by acidic hydrolases into small molecules, such as amino acids, fatty acids, and nucleotides, which are then recycled by the cell.

Figure 1.

Autophagy, also referred to as macroautophagy, is crucial for maintaining cellular balance. (A) Activation of autophagy occurs when AMPK inhibits the mTORC1 complex. This reduction in mTORC1’s inhibitory effect on ULK1 promotes the production of autophagic vesicles. (B) The processes of nucleation, elongation, and maturation of autophagosomes involve ATG9 supplying membrane material, while the VPS34 complex catalyzes the production of PI3P and recruits additional autophagy-related proteins. The PI3K complex binds to both the ATG5-ATG12 complex and the LC3 system, facilitating the formation of LC3-II, which then integrates into the autophagosome membrane. Ultimately, as the autophagosome elongates, it gradually closes to create a complete bilayer membrane structure. (C) The generation of autophagosomes: mature autophagosomes fuse with lysosomes, resulting in the formation of autolysosomes. (D) Degradation: the contents of autophagosomes are degraded by acidic hydrolytic enzymes (such as proteases, lipases, nucleases, etc.) in lysosomes.

Autophagy is regulated by various internal and external signals, primarily influenced by nutrient and energy status. During starvation or energy deficiency, AMPK is activated and mTORC1 is inhibited, which promotes autophagy; in contrast, when nutrients are abundant, this process is inhibited [26]. Additionally, specific stress conditions, such as oxidative stress and endoplasmic reticulum stress, can activate distinct signaling pathways that regulate autophagy [27]. Various molecules and signaling pathways, including p53, Bcl-2 family proteins, and calcium ion signaling, also regulate autophagy by influencing the activity and localization of key components like the ULK complex, VPS34 complex, and ATG proteins [28,29]. In summary, autophagy regulation is complex. A deeper understanding of its mechanisms not only clarifies cellular physiological and pathological processes but also provides a theoretical foundation for developing therapeutic strategies for diseases related to autophagy.

3. Autophagy of HSCs and liver fibrosis

HSCs are crucial in liver fibrosis. Continuous damage to liver parenchymal cells and immune cell stimulation activate HSCs, leading them to transform into myofibroblasts. These cells express α-smooth muscle actin (α-SMA), vimentin, and continuously produce ECM and type I collagen (COL1). Activated HSCs release various pro-fibrotic cytokines, including TGF-β1 and TNF-α, which further promote the fibrotic process [30]. Therefore, HSCs are both the main executors of liver fibrosis and important regulators.

3.1. Autophagy in HSCs promotes their activation

The activation of HSCs is a crucial step in the development of liver fibrosis. The loss of LDs is a key characteristic of HSCs activation [8]. As a result, researchers have investigated how autophagy regulates this process. Hernandez-Gea et al. [31] and Thoen et al. [32] reported that autophagy enhances the breakdown of lipid droplets in inactive HSCs, facilitating their activation, as confirmed by both in vivo and in vitro studies. In models of liver fibrosis, the breakdown of lipid droplets through autophagy further supports the activation of HSCs [31]. Conversely, inhibiting HSC autophagy leads to the accumulation of lipid droplets, thereby preventing HSCs activation [31,33]. Furthermore, additional studies indicate that HSC autophagy directly contributes to the degradation of LDs. It may be linked to abnormalities in retinoic acid signaling and the activation of the TGF-β signaling pathway [34]. Collectively, these factors drive the activation of HSCs and exacerbate the progression of liver fibrosis. Autophagy speeds up the breakdown of lipid droplets, releasing free fatty acids, ATP, and triglycerides. This process supplies the energy needed for HSC activation and further accelerates the progression of liver fibrosis [31] (Figure 2A). Importantly, this pro-fibrotic role of HSC autophagy is supported by direct evidence from human patients. The foundational study by Hernández-Gea et al. demonstrated that primary HSCs isolated from human livers with hepatitis B virus–induced fibrosis exhibited a comprehensive autophagic response. This was characterized not only by a marked increase in the autophagy marker LC3-II and a greater number of autophagic vacuoles observed by electron microscopy but also by a concurrent decrease in the levels of p62/SQSTM1. This combination of increased LC3-II, elevated autophagic vacuoles, and reduced p62 provides direct biochemical and ultrastructural evidence of enhanced autophagic flux in activated HSCs in human liver disease [31].

Figure 2.

The autophagy of HSCs plays two opposing roles in the development of liver fibrosis. (A) The autophagy of HSC breaks down stored lipid droplets, resulting in the production of free fatty acids. These free fatty acids enter the mitochondria for β-oxidation, which produces ATP. This ATP provides the energy required for HSCs activation, thereby promoting liver fibrosis. (B) The autophagy of HSCs can inhibit their activation by inducing senescence, apoptosis, and ferroptosis in activated HSCs. This process slows the progression of liver fibrosis.

3.2. Targeted inhibition of HSC autophagy

In light of this evidence that autophagy fuels HSCs activation, therapeutic efforts have focused on inhibiting autophagy as a key strategy in antifibrotic therapy. Consequently, significant research is directed toward exploring methods to suppress HSC autophagy, such as modulating critical signaling pathways, developing novel therapeutics, and utilizing nano-delivery systems, to slow the progression of liver fibrosis.

Research indicates that docosahexaenoic acid (DHA) counteracts liver fibrosis via a specific disruption of the RAB25-mediated lipid droplet recognition. This disruption inhibits autophagy in HSCs, resulting in lipid droplet regeneration and a consequent restoration of the quiescent phenotype [33]. In addition, several signaling pathways, including prostaglandin E2 (PGE2)-EP4/Erk, TGF-β1-ERK/JNK, and Akt/mTOR, regulate autophagy activity in HSCs and are potential targets for liver fibrosis treatment. For example, the specific antagonist E7046 can inhibit the binding of the pro-inflammatory cytokine PGE2 secreted by liver M2 macrophages to the surface receptor EP4 on HSCs, thereby blocking the activation of the Erk pathway, inhibiting the expression of autophagy-related proteins Beclin-1 and LC3-II, and reducing autophagy and activation of HSCs [35]. TGF-β1 promotes autophagy and activates HSCs via the ERK and JNK pathways. However, this process can be reversed by the ERK inhibitor PD98059 and the JNK inhibitor SP600125 [36]. Ginsenosides Rh2 and Rg3 reduce autophagy and HSC activation by activating the Akt/mTOR signaling pathway [37,38]. The α1-adrenergic receptor antagonist doxazosin inhibits autophagy in HSCs by activating the PI3K/Akt/mTOR signaling pathway [39]. The unfolded protein response (UPR) caused by endoplasmic reticulum stress promotes autophagy in HSCs via the IRE1-p38 MAPK pathway. DNIRE1 mutants and the p38 inhibitor SB203580 effectively inhibit this process [40].

In drug development, chemical agents like ursodeoxycholic acid, Pianzihuang, and taurine show promise in inhibiting autophagy in HSCs; however, their specific mechanisms still need further research [41–43]. At the same time, salvianolic acid B disrupts lysosomal homeostasis and degradation functions, successfully blocking the later stages of autophagy in HSCs [44]. The autophagy inhibitor chloroquine (CQ) inhibited autophagy in primary HSCs in vitro, as evidenced by the accumulation of p62 and LC3-II, leading to reduced HSC activation [45].

Researchers have developed various nano-delivery systems—such as HCQ@VA-Lip-Exo, HCQ@ROL-LNPs, and HA@PRB/COL NPs—to deliver autophagy inhibitors (CQ or PRB) to HSCs, thereby preventing autophagosome-lysosome fusion [46–48]. Additionally, the Lip/siATG7/CK system achieves autophagy inhibition by specifically knocking down the key autophagy gene ATG7 in HSCs [49] (Table 1).

Table 1.

Modulation of HSC autophagy as a therapeutic strategy for alleviating liver fibrosis.

Therapeutic agent/approach	Therapeutic strategy	Impact on autophagy	Mechanism of alleviating liver fibrosis	References
Docosahexaenoic acid	Autophagy inhibition	Inhibition of the binding of Rab25 and PI3KCIII	Activation of HSC↓, LDs↑	[33]
CQ, BafA1	Autophagy inhibition	Inhibition of the fusion of autophagosomes and lysosomes	Activation of HSC↓	[34]
DN IRE1 mutant cells, p38 inhibitor SB203580	Autophagy inhibition	Inhibit endoplasmic reticulum stress and the activity of p38 MAPK	Activation of HSC↓	[40]
E7046	Autophagy inhibition	Blocking the binding of PGE2 to its receptor EP4	Activation of HSC↓	[35]
Ursodeoxycholic acid, Pian Zai Huang, Taurine	Autophagy inhibition	/	Activation of HSC↓	[41–43]
PD98059, SP600125	Autophagy inhibition	Inhibition of ERK and JNK signaling pathways	Activation of HSC↓	[36]
Ginsenoside Rh2 and Rg3	Autophagy inhibition	Activation of the Akt/mTOR signaling pathway	Activation of HSC↓	[37,38]
Doxazosin	Autophagy inhibition	Activation of the PI3K/Akt/mTOR signaling pathway	Apoptosis of HSC↑	[39]
SAB	Autophagy inhibition	Increase lysosomal pH value	Activation of HSC↓	[44]
HCQ@VA-Lip-Exo, HCQ@ROL-LNPs, HA@PRB/COL NPs	Autophagy inhibition	Prevent the fusion of autophagosomes and lysosomes	Activation of HSC↓	[46–48]
Lip/siATG7/CK	Autophagy inhibition	Silences the expression of the key autophagy gene ATG7	Activation of HSC↓	[49]
CQ	Autophagy inhibition	increasing p62 and LC3 accumulation	Activation of HSC↓	[45]
m6A modification, DHA, ELAVL1/HuR (ferritinophagy)	Autophagy enhancement	Enhance the stability of Beclin-1 mRNA	HSC ferroptosis↑, Activation of HSC↓	[55,63–65]
Sorafenib, FBXW7 (ferritinophagy)	Autophagy enhancement	Enhance the stability of ATG16L1 mRNA	HSC ferroptosis↑, Activation of HSC↓	[66]
Artemisinin (ferritinophagy)	Autophagy enhancement	/	HSC ferroptosis↑, Activation of HSC↓	[67]
Curcumin (ferritinophagy)	Autophagy enhancement	/	HSC ferroptosis↑, Activation of HSC↓	[68]
SchB (ferritinophagy)	Autophagy enhancement	/	HSCs senescence↑	[53]
Oroxylin A (ferritinophagy)	Autophagy enhancement	Activate the cGAS-STING pathway	HSCs senescence↑	[54]
Valinomycin (mitophagy)	Autophagy enhancement	Enhance the phosphorylation of Parkin and E3 ligase activity	HSCs apoptosis↑	[57]
SPD	Autophagy enhancement	Enhancing the expression of MAP1S to promote autophagosome formation	Activation of HSCs↓, NRF2↑, Antioxidant capacity↑	[56,59]
DHI	Autophagy enhancement	Inhibited the interaction between YAP and TEAD2, promoting the conversion of LC3-I to LC3-II	COL1A1↓	[69]

BafA1: bafiloamycin; CQ: chloroquine; PI3KCIII: phosphatidylinositol 3-kinase III; RAB25: Rab-GTP enzyme; E7046: specific antagonist of prostaglandin E2 receptor EP4; DN IRE1cells: the overt negative mutant IRE1 cells; SAB: salvianolic acid B; HCQ@VA-Lip-Exo: HCQ: autophagy inhibitor hydroxychloroquine; VA: vitamin A; Exo: exosome; Lip: liposome; HCQ@ROL-LNPs: HCQ: autophagy inhibitor hydroxychloroquine; LNPs: liposome nanoparticles; ROL: retinol; HA@PRB/COL N Ps: HA: hyaluronic acid; PRB: autophagy inhibitor probucol; COL: collagenase type I; NPs: nanoparticle; Lip/siATG7/CK: Lip: liposomes; siATG7: siRNA targeting autophagy-related gene 7; CK: KDEL-grafted chondroitin sulfate; m6A: N6-methyladenosine; DHA: dihydroartemisinin; ELAVL1/HuR: RNA binding protein; SchB: Schisandra protein B; NCOA4: nuclear receptor coactivator 4; FTH1: ferritin heavy chain 1; Parkin: E3 ubiquitin ligase; mtDNA: mitochondrial DNA; TOM20: mitochondrial protein TOM20; SPD: spermidine; MAP1S: microtubule-associated protein 1S; NRF2: nuclear factor erythroid 2-related factor 2; DHI: dihydrotanshinone I; YAP: yes-associated protein; TEAD2: TEA family member 2; COL1A1: type I collagen.

After examining how autophagy in HSCs contributes to liver fibrosis, we discovered that HSCs exhibit significantly enhanced autophagic activity in the early stages of liver injury. As a key marker of cell activation, autophagy helps clear organelles and LDs, supplying the energy and materials needed for HSCs to proliferate and activate. However, further research revealed that autophagy in HSCs can inhibit liver fibrosis under certain conditions. This inhibitory effect arises mainly because autophagy in HSCs can degrade pro-fibrotic mediators, such as type I collagen and multivesicular bodies (MVBs), effectively reducing the generation and release of fibrosis-related factors [14,50,51]. The autophagic activity of HSCs is dynamic and changes in response to various external stimuli. For instance, TGF-β1 stimulates HSCs to undergo autophagy and activation via the PI3K/AKT signaling pathway [36]. In contrast, platelet-derived growth factor (PDGF) activates its downstream signaling molecule SHP2, which then inhibits HSCs’ autophagic activity by activating the mTOR signaling pathway. This leads to the release of more extracellular vesicles (EVs), further promoting HSC activation [14]. This differential response likely arises from various stimuli activating distinct signaling pathways. Therefore, HSC autophagy plays a complex and variable role in liver fibrosis, potentially promoting or inhibiting fibrosis depending on the circumstances. Next, we will explore how HSCs autophagy exerts anti-fibrotic effects through various mechanisms, highlighting its significant role in liver fibrosis in detail how HSCs autophagy exerts anti-fibrotic effects through various mechanisms, further revealing its important role in the process of liver fibrosis.

3.3. Autophagy of HSC inhibits liver fibrosis

Recent studies have shown that autophagy plays an anti-fibrotic role in HSCs during liver fibrosis. This role involves several mechanisms, such as promoting cellular senescence, inducing cell death, and affecting related signaling pathways. Specifically, selective autophagy in HSCs, particularly the degradation of ferritin, is mediated by nuclear receptor coactivator 4 (NCOA4), which helps recognize and bind ferritin heavy chain 1 (FTH1), guiding it into autophagosomes for degradation [52]. This process releases iron ions and causes iron overload in HSCs, which worsens oxidative stress. As a result, HSCs enter a state of senescence, slowing the progression of liver fibrosis [53,54]. Furthermore, by triggering ferroptosis, autophagy in HSCs suppresses their activation and alleviates liver fibrosis [55].

Additionally, autophagy helps reduce fibrosis by regulating pro-fibrotic mediator levels. For instance, in LX-2 cells, overexpressing phospholipase D1 (PLD1) triggers autophagy in HSCs, which in turn decreases the accumulation of Col-I [50]. Autophagy breaks down MVBs in HSCs, lowering the levels of pro-fibrotic exosomes and helping to prevent liver fibrosis [14]. Simultaneously, autophagy in HSCs decreases ECM production, increases LDs numbers, and inhibits HSCs activation, which collectively slows the progression of liver fibrosis [56].

Mitophagy in HSCs is crucial for combating fibrosis. Mitophagy reduces liver fibrosis by promoting the apoptosis of HSC [57]. Conversely, inhibiting mitophagy in HSCs leads to an increase in mitochondria and ATP production, which provides energy for HSCs activation and worsens liver fibrosis [58] (Figure 2B).

At the signaling pathway level, HSC autophagy enhances the stability and activity of nuclear factor erythroid 2-related factor 2 (NRF2) by decreasing the expression of Kelch-like ECH-associated protein 1 (KEAP1). This process improves the cell’s antioxidant defense and positively impacts fibrosis [59–61]. However, the exact mechanisms underlying these processes require further investigation for better understanding.

3.4. Targeted enhancement of HSC autophagy

Given the crucial role of HSC autophagy in liver fibrosis, strategically enhancing specific autophagic pathways has emerged as a promising approach not only to inhibit but to actively reverse established fibrosis. Compelling evidence for this regenerative potential comes from interventional studies in experimental models where treatment was initiated after fibrosis was fully developed. One effective approach is to promote ferritinophagy specifically in HSCs. This action induces ferroptosis and helps inhibit fibrosis. For example, Sorafenib, a ferroptosis inducer, is particularly effective against HSCs and inhibits liver fibrosis by specifically inducing ferroptosis in these cells [62]. This mechanism involves N6-methyladenosine (m6A) modification. The m6A reader protein YTHDF1 recognizes m6A binding sites, enhancing the stability of Beclin-1 mRNA and initiating ferritinophagy [55]. Similarly, dihydroartemisinin also promotes this process by upregulating m6A modification [63,64]. The RNA-binding protein ELAVL1/HuR activates HSCs ferritinophagy by binding to the AU-rich elements (AREs) in the 3′ untranslated region (3′UTR) of Beclin1 mRNA [65]. In contrast, the RNA-binding protein ZFP36/TTP promotes the decay of ATG16L1 mRNA by binding to AREs, inhibiting HSCs ferritinophagy. However, treatment with Sorafenib or the ubiquitin ligase FBXW7 can target a specific amino acid sequence, ‘SFSGLPS’, in the ZFP36 protein. This targeting induces ubiquitination and degradation of ZFP36, reducing its expression and promoting ferritinophagy [66]. Furthermore, the active component Oroxylin A from Scutellaria baicalensis activates HSCs’ ferritinophagy through the cGAS-STING signaling pathway, inducing HSCs’ senescence [54]. In addition, several natural compounds, including artemisinin [67], schisandrin B [53], and curcumin [68] have been identified as inducers of HSCs’ ferritinophagy, although their specific molecular mechanisms are yet to be thoroughly investigated. These compounds promote ferritinophagy through various pathways, leading to either ferroptosis or senescence in HSCs, which ultimately helps inhibit liver fibrosis. Some natural compounds inhibit liver fibrosis by inducing autophagy in HSCs. Spermidine enhances the formation of autophagosomes by promoting the expression of MAP1S, thereby facilitating autophagy in HSCs [56,59]. This process involves MAP1S sequestering key proteins such as LC3, p62, and KEAP1 into autophagosomes, reducing KEAP1 levels by degrading the MAP1S-KEAP1-P62 complex, stabilizing NRF2, and enhancing the cell’s antioxidant stress capacity [59]. Dihydrotanshinone I can block YAP nuclear translocation in the Hippo pathway, inhibiting the YAP/TEAD2 interaction, promoting the conversion of LC3-I to LC3-II, activating HSC autophagy, and suppressing Col1a1 expression [69]. Additionally, targeting mitophagy in HSCs is also a method to alleviate liver fibrosis. For example, the mitophagy inducer valinomycin can reduce the expression of the apoptosis-related protein BCL-B, promote the phosphorylation of Parkin, enhance its activity as an E3 ubiquitin ligase, increase mitophagy in HSCs, promote HSC apoptosis, and alleviate liver fibrosis [57] (Table 1). Thereby, strategies such as promoting ferritinophagy in HSCs and enhancing mitophagy provide new targets for the treatment of liver fibrosis.

4. Autophagy of hepatocyte and liver fibrosis

4.1. The key role of hepatocyte autophagy in inhibiting liver fibrosis

Hepatocytes are the primary cell type in the liver, making up ∼80% of its volume and performing most liver functions [70]. These cells have a high autophagic capacity because they contain many lysosomes and lysosomal enzymes [71]. Emerging evidence underscores a pivotal role for hepatocyte autophagy in retarding liver fibrosis progression, partly mediated through the regulation of lipid metabolism. A key mechanism involves the activation of peroxisome proliferator-activated receptor alpha (PPARα), a master transcriptional regulator of energy and lipid homeostasis. PPARα activation upregulates rate-limiting enzymes such as carnitine palmitoyltransferase 1, facilitating mitochondrial import and β-oxidation of long-chain fatty acids. This enhances lipid turnover, ameliorates hepatic steatosis, and thereby attenuates fibrogenesis [72,73]. Beyond transcriptional regulation, selective autophagic degradation of lipid droplets—known as lipophagy—offers a direct pathway for lipid clearance [74]. The efficacy of this process depends on specific molecular machinery, as illustrated by VPS4A, which has been identified as a dedicated lipid droplet autophagy receptor in both murine and human livers. This makes VPS4A a promising therapeutic target for lipophagy-deficient conditions such as NAFLD [75]. Enhancing lipophagy in hepatocytes helps protect the liver from lipid-mediated toxicity, thereby presenting a viable therapeutic strategy for NAFLD [76]. Moreover, by promoting lysosomal exocytosis of lipids, hepatocyte lipophagy prevents the harmful intracellular accumulation of free fatty acids, thereby mitigating hepatocyte damage and inflammation, which in turn suppresses liver fibrosis [77].

Hepatocyte mitophagy plays a vital role in preventing liver fibrosis (Figure 3). The clearance of damaged mitochondria through mitophagy prevents NLRP3 inflammasome activation and reduces IL-1β release, thus blocking the subsequent activation of HSCs and recruitment of immune cells, which are key events in the inhibition of liver fibrosis [78,79]. Furthermore, mitophagy protects against mitochondria-dependent hepatocyte apoptosis and liver fibrosis by facilitating the timely removal of impaired organelles. A key mechanism underlying this protection is the suppression of mitochondrial ROS production, as demonstrated in the context of a high-fat diet, where mitophagy attenuates hepatic inflammation, injury, and subsequent fibrogenesis [80,81].

Figure 3.

Mitophagy in hepatocytes inhibits liver fibrosis by removing damaged mitochondria. (A) By clearing damaged mitochondria, mitophagy suppresses ROS-induced NLRP3 inflammasome activation, thereby inhibiting hepatocyte pyroptosis and the release of pro-inflammatory cytokines such as IL-1β and IL-18. This attenuation of the inflammatory response ultimately alleviates liver fibrosis. (B) Mitophagy delays the apoptosis process of hepatocytes by inhibiting the release of cytochrome C from mitochondria, which plays a role in alleviating liver fibrosis.

The protective role of mitophagy also extends to modulating the immune response. In acute liver injury, it prevents the leakage of mitochondrial DNA and subsequent activation of the cGAS-STING signaling pathway in macrophages, thereby suppressing a potent inflammatory cascade [82]. Moreover, mitophagy modulates macrophage-mediated inflammation by reducing the release of pro-inflammatory factors such as TNFα, disrupting another critical pathway that promotes fibrogenesis [83].

Hepatocyte autophagy mitigates liver fibrosis by suppressing the release of pro-inflammatory factors such as IL-1β and IL-18. For example, hepatitis B virus infection causes high mobility group box 1 (HMGB1) to move from the nucleus to the cytoplasm in hepatocytes, increasing its secretion into the extracellular space, which activates macrophage inflammasomes and worsens liver fibrosis. Activating hepatocyte autophagy reduces HMGB1 secretion and inhibits NLRP3 inflammasome activation in macrophages, resulting in lower pro-inflammatory factor release [84]. Hepatocyte autophagy also counteracts IFNγ-induced liver injury by inhibiting STAT1 signaling hyperactivation and preventing NOS2 overexpression, which helps maintain cellular energy homeostasis, avoid ATP depletion, and protect against necrotic cell death [85]. Furthermore, through the maintenance of energy homeostasis, hepatocyte autophagy suppresses IL-1β/TNF-induced lysosomal permeabilization and cathepsin-dependent necrosis. It also degrades damage-associated molecular patterns in multivesicular bodies, thereby reducing the secretion of proinflammatory exosomes [86].

Collectively, the anti-fibrotic role of hepatocyte autophagy is not merely cell-autonomous but is significantly amplified through its orchestration of intercellular communication. By maintaining intracellular metabolic and mitochondrial quality control, hepatocytes attenuate the release of multiple pro-fibrotic signals. Specifically, hepatocyte mitophagy prevents the leakage of mitochondrial DAMPs (e.g. mtDNA), thereby inhibiting the cGAS-STING-driven pro-inflammatory activation of macrophages [82]. Concurrently, autophagic degradation of DAMPs like HMGB1 in hepatocytes directly curtails their paracrine potential to trigger NLRP3 inflammasome activation in macrophages, breaking a key inflammatory circuit that drives HSC activation [84]. This multi-faceted crosstalk positions the hepatocyte as a central signaling hub, whereby its autophagic activity governs the inflammatory tone of the hepatic milieu and directly influences the activation state of both immune (macrophages) and fibrogenic (HSCs) effector cells.

4.2. Targeted enhancement of hepatocyte autophagy

Researchers are actively exploring strategies to enhance hepatocyte autophagy, which plays a crucial role in inhibiting liver fibrosis. These strategies include protein and peptide regulation, nucleic acid-based interventions, and drugs or natural compounds (Table 2). In the area of protein and peptide regulation, studies have shown that the vertebrate-specific autophagy regulator RUBCNL/Pacer promotes the fusion of autophagosomes and lysosomes by binding to the PI3K-III and HOPS complexes, thus accelerating the maturation of hepatocyte autophagosomes [87,88]. Furthermore, insights from human fibrotic liver tissues have informed the development of targeted therapeutic peptides. Analysis of clinical samples revealed that upregulation of TRIB3 impairs autophagic flux by disrupting SQSTM1-LC3 binding. This mechanism-based understanding led to the design of the cell fusion peptide Pep2-A2, which specifically blocks the TRIB3-SQSTM1 interaction. Subsequent animal studies confirmed that Pep2-A2 restores functional autophagy and exerts robust anti-fibrotic effects [51]. In parallel, nucleic acid-based strategies also demonstrate strong therapeutic potential, supported by clinical evidence. For instance, the miR-125a/VDR axis has been implicated in human cirrhosis, where miR-125a updrive leads to suppressed VDR expression and impaired autophagic flux. Accordingly, intervention with miR-125a-antagomir to restore VDR signaling has been shown to enhance autophagic flux and ameliorate fibrosis in preclinical models [89].

Table 2.

Targeted enhancement hepatocyte autophagy to alleviate liver fibrosis.

Hepatocyte autophagy activator	Impact on autophagy	Mechanism of alleviating liver fibrosis	References
EMPA	Activate the AMPK-TFEB signaling pathway	Lipid metabolism↑	[90]
RUBCNL/Pacer	Regulation of autophagosome maturation	Lipid metabolism↑	[87]
mTORC1	Phosphorylating perilipin 3	Lipid-mediated toxicity↓	[76]
SLC7A11	Activation of the AMPK signaling pathway	Activation of HSC↓	[78]
TXNIP/VDUP1	Inhibition of MTORC1 activity and nuclear translocation of TFEB	Fatty acid oxidation↑	[72]
α-DBI	Promoted the lipidation of LC3	Inflammation, fatty degeneration↓	[96]
Pep2-A2	Promote the binding of LC3 and P62	Activation of HSCs↓	[51]
Spermidine	Depleting HDAC4 increases the acetylation and stability of MAP1S	Oxidative stress↓	[92]
EGCG	Activate the AMPK-mTOR pathway	HMGB1,NLRP3↓	[94]
Gemigliptin	Enhance the expression of ULK1	Inflammation, apoptosis↓	[95]
Sirtuin 3 (mitophagy)	Upregulate Bnip3 expression	Hepatocyte mitochondria-dependent apoptosis↓	[80]
Miz1 (mitophagy)	Upregulate Parkin expression	TNFα↓	[83]
SELENOM (mitophagy)	Upregulate Parkin expression	Mitochondrial oxidative stress and apoptosis↓	[81]
Liraglutide (mitophagy)	Inhibit lipid deposition and reduce mitochondrial dysfunction	NLRP3,ROS, apoptosis↓	[79]
WY14643 (lipophagy)	Increase the interaction between JMJD3 and PPARα	Lipid degradation↑	[73]

EMPA: empagliflozin; AMPK: AMP-activated protein kinase; TFEB: transcription factor EB; mTORC1: mechanistic target of rapamycin complex 1; SLC7A11: solute carrier family 7 member 1; TXNIP/VDUP1: sulfiredoxin interaction protein; α-DBI: diazepam combined with inhibitor antibodies; Pep2-A2: cell fusion peptide; HDAC4: histone deacetylase 4; MAP1S: microtubule-associated protein 1S; EGCG: green tea polyphenol epigallocatechin-3-gallate; HMGB1: high-mobility group box 1; NLRP3: NACHT, LRR, and PYD domains-containing protein 3; Bnip3: Bcl-2 nineteen-kilodalton interacting protein 3; Miz1: Myc-interacting zinc-finger protein 1; Parkin: Parkin RBR E3 ubiquitin ligase; TNFα: tumor necrosis factorα; SELENOM: selenoprotein M; ROS: reactive oxygen species; PPARα: peroxisome proliferator-activated receptorα; WY14643: PPARα agonist pirinixic acid; JMJD3: histone demethylase Jumonji-D3.

Researchers have identified several promising candidate drugs and natural compounds. For example, the sodium-glucose co-transporter 2 inhibitor empagliflozin lowers blood glucose levels and reduces O-GlcNAc glycosylation. This action activates AMPK and phosphorylates the transcription factor EB (TFEB), promoting the expression of autophagy-related genes and stimulating hepatocyte autophagy [90,91]. Spermidine enhances the acetylation and stability of microtubule-associated protein 1S (MAP1S) in hepatocytes by depleting histone deacetylase 4, thereby reducing its binding to MAP1S [92]. Because MAP1S interacts with LC3 and positively regulates autophagic flux, its increased acetylation and stability subsequently activate the autophagic process in hepatocytes [93]. Green tea polyphenol epigallocatechin-3-gallate induces hepatocyte autophagy by activating the AMPK-mTOR pathway [84,94]. Sitagliptin promotes hepatocyte autophagy by boosting ULK1 expression [95]. Injecting the monoclonal antibody α-DBI, which neutralizes the autophagy inhibitor ACBP/DBI, promotes the conversion of LC3-I to LC3-II. This process activates autophagy in hepatocytes [96,97]. Additionally, rapamycin, a known autophagy activator, initiates hepatocyte autophagy by inhibiting mTORC1 activity [72,98].

Research has also focused specifically on autophagic regulation of hepatocyte organelles, such as mitophagy and lipophagy. For lipophagy, mTORC1 specifically activates this process by phosphorylating perilipin 3 (Plin3) [76]. Similarly, the PPARα agonist WY14643 enhances the histone demethylase Jumonji-D3′s ability to recruit PPARα, promoting the transcription of autophagy-related genes and activating hepatic lipophagy [73]. For mitophagy, solute carrier family 7 member 11 (SLC7A11) augments this process by reducing reactive oxygen species levels and enhancing α-ketoglutarate-dependent prolyl hydroxylase activity, leading to AMPK signaling pathway activation [78]. The long-acting glucagon-like peptide-1 analog liraglutide enhances mitophagy by reducing lipid accumulation in hepatocytes, inhibiting NLRP3 inflammasome formation, activating pyroptosis, reducing mitochondrial dysfunction, and decreasing ROS production [79]. Sirtuin 3 activates the ERK-CREB signaling pathway to upregulate Bnip3 expression, thereby promoting hepatocyte mitophagy [80]. Selenoprotein M (SELENOM) induces mitophagy through the AMPKα1-MFN2 signaling pathway, which upregulates Parkin expression and promotes Parkin-mediated mitophagy [81]. Furthermore, hepatocyte Myc-interacting zinc-finger protein 1 (Miz1) is a critical suppressor of NASH progression. It functions by maintaining Parkin-mediated mitophagy. Miz1 directly binds to peroxiredoxin 6 (PRDX6) and sequesters it in the cytoplasm, thereby preventing PRDX6 from translocating to mitochondria and inhibiting Parkin. This mechanism alleviates the suppression on mitophagy and ensures the timely clearance of damaged mitochondria [83]. In summary, hepatocyte autophagy is an important therapeutic target for liver fibrosis, regulated by various factors; thus, activating autophagy or inhibiting its suppressive pathways presents new strategies for treatment.

5. Hepatic macrophage autophagy and liver fibrosis

5.1. Autophagy regulates macrophage polarization to inhibit liver fibrosis

Liver macrophages show considerable diversity during liver fibrosis, and their polarization states—M1 (pro-inflammatory) and M2 (anti-inflammatory)—are vital for the progression of fibrosis. M1 macrophages are activated by lipopolysaccharides (LPS) and IFN-γ, which leads to the release of pro-inflammatory factors like TNF, IL-1β, and IL-12. These factors worsen the inflammatory response. In contrast, M2 macrophages are stimulated by IL-4 and IL-13, releasing anti-inflammatory factors such as IL-10 and TGF-β, which aid in the repair of liver injury [99,100]. Research shows that during liver fibrosis, the imbalance in M1 and M2 macrophage polarization—marked by an increase in M1 macrophages and a decrease in M2 macrophages—is a critical factor contributing to heightened inflammatory responses and worsened fibrosis [101]. Therefore, maintaining the balance between these two macrophage phenotypes is crucial for managing liver inflammation and the progression of fibrosis.

Autophagy helps maintain cellular homeostasis and is crucial for regulating macrophage polarization [102]. Research indicates that PINK1/Parkin-mediated mitophagy in liver macrophages inhibits inflammatory cytokine expression. This process partially reverses M1 macrophage polarization and weakens their pro-inflammatory response [103]. Additionally, macrophage autophagy suppresses the transcription of pro-inflammatory genes (e.g. TNF-α, IL-1β, and IL-6) by inhibiting JUN/c-JUN N-terminal kinases and MAPK8/9 activation, thereby curbing M1 macrophage polarization [104,105]. Conversely, macrophage autophagy activates the STAT3 and STAT6 signaling pathways, which promote M2 macrophage polarization. This activation leads to the release of anti-inflammatory factors (e.g. IL-10, TGF-β) and supports liver tissue repair, ultimately alleviating inflammation and fibrosis [16,104]. In summary, autophagy significantly inhibits liver fibrosis by balancing macrophage polarization, specifically by suppressing M1 polarization and promoting M2 polarization (Figure 4).

Figure 4.

Hepatic macrophage autophagy prevents liver fibrosis by influencing how macrophages polarize. Hepatic macrophages polarize in response to inflammatory factors, transforming into two types: pro-inflammatory M1 and anti-inflammatory M2. M1 macrophages release pro-inflammatory factors that worsen inflammation and contribute to liver fibrosis. In contrast, M2 macrophages secrete anti-inflammatory factors that aid tissue repair and help reduce liver fibrosis. Macrophage autophagy suppresses M1 polarization by inhibiting the JUN signaling pathway and encourages M2 polarization by activating the STAT3 and STAT6 signaling pathways, which together help reduce liver fibrosis.

5.2. Autophagy regulates the secretion of inflammatory cytokines to inhibit liver fibrosis

In the complex process of liver fibrosis, macrophages activate the NLRP3 inflammasome by releasing pro-inflammatory cytokines, synthesizing chemokines, and producing TGF-β1. This activation promotes the activation of HSC and worsens liver fibrosis progression [106]. Macrophage autophagy serves as a crucial internal balance mechanism that alleviates the production of inflammatory factors and improves the liver’s inflammatory and fibrotic state. Autophagy in macrophages can reduce the production of intracellular ROS, inhibit the activation of the NLRP3 inflammasome, decrease the secretion of inflammatory factors such as IL-1β, and relieve liver inflammation and fibrosis [107–109]. Conversely, when macrophage autophagy is suppressed, pro-inflammatory cytokine secretion increases, which worsens liver fibrosis progression. For example, knocking out the ATG5 gene inhibits macrophage autophagy, leading to the accumulation of ROS in mitochondria. This accumulation activates the ROS-NF-κB-IL1α/β signaling pathway and increases the secretion of IL-1α and IL-1β, which promotes the progression of liver fibrosis [110,111]. Increased levels of hypoxia-inducible factor-1α (HIF-1α) inhibit macrophage autophagy, which in turn promotes liver fibrosis by raising IL-1β production [112]. Another study found that liver fibrosis from Schistosoma japonicum infection is linked to suppressed macrophage autophagy. The infection induces IL-7, which binds to its receptor on macrophages. This binding inhibits autophagic activity, hindering the clearance of damaged liver cells and egg granulomas, thereby worsening liver fibrosis [113]. Besides decreasing pro-inflammatory factors, macrophage autophagy also alleviates liver injury by regulating anti-inflammatory factor levels. During liver injury, macrophage autophagy activation increases levels of the anti-inflammatory cytokine IL-10 [114]. This factor has anti-inflammatory and immunoregulatory functions, which can alleviate the inflammatory response and fibrosis in the liver [115]. However, when macrophage autophagy is suppressed, IL-10 secretion drops, which further worsens liver injury [116].

5.3. Targeted enhancement of liver macrophage autophagy

Given the critical role of liver macrophage autophagy in regulating polarization and inflammatory cytokine secretion, enhancing this autophagic function could be a strategy to intervene in liver fibrosis (Table 3). Studies indicate that various drugs and compounds can enhance liver macrophage autophagy through different mechanisms, which inhibits the progression of liver fibrosis. For example, the lipid-lowering drug ezetimibe enhances liver macrophage autophagy by activating the AMPK-TFEB signaling pathway, thereby reducing the release of inflammatory cytokines from macrophages [108]. Recombinant IL-1 receptor antagonists reduce the production of pro-inflammatory cytokines and enhance macrophage autophagy by blocking IL-1A/B signaling. This action helps regulate the liver’s inflammatory response [111,117]. Anti-CD127 blocking antibodies prevent IL-7 from binding to its receptor on macrophages. This restoration of macrophage autophagic function helps reduce collagen deposition in the liver [113]. Alpha-lactose blocks the interaction between galectin and its receptors, which downregulates Gal-3 expression. This results in an increase in M1 macrophages, a decrease in M2 macrophages, and promotes liver macrophage autophagy via the Akt/mTOR signaling pathway [118]. Rapamycin inhibits mTORC1 activity, which relieves the suppression of ULK1 on cellular autophagy. This induction of macrophage autophagy inhibits M1 macrophage polarization while promoting M2 macrophage polarization [16]. In addition to drug therapy, compounds such as spermidine, the cannabinoid receptor 2 agonist JWH-133, and monoacylglycerol lipase inhibitors have shown potential in activating macrophage autophagy and treating liver fibrosis. SPM enhances liver macrophage autophagy by accelerating the formation of autophagosomes through increased expression of ATG5. This process inhibits M1 macrophage polarization and promotes M2 macrophage polarization [116]. JWH-133 stimulates the expression of the stress-induced protein heme oxygenase-1 (HO-1) in macrophages, which activates liver macrophage autophagy and helps alleviate liver steatosis [119]. MAGL inhibitors improve macrophage autophagy by encouraging the fusion of autophagosomes with lysosomes and reducing the production of inflammatory factors in macrophages [120]. Additionally, nano-iron oxide particles enhance macrophage autophagy and decrease IL-10, an anti-inflammatory cytokine, by activating the Notch1/HES1 signaling pathway [114]. STING, a regulator of DNA-mediated innate immunity, boosts macrophage activity and autophagic flow while inhibiting inflammasome activation when its signaling pathway is engaged [121]. Toyocamycin reduces the expression of X-box binding protein 1 (XBP1), which restores BNIP3-mediated mitochondrial autophagy in macrophages. This process hinders the cGAS/STING/NLRP3 signaling pathway’s activation and prevents the production of TGF-β1, ultimately inhibiting HSC activation [122].

Table 3.

Targeted enhancement macrophage autophagy to alleviate liver fibrosis.

Macrophage autophagy activator	Impact on autophagy	Mechanism of alleviating liver fibrosis	References
Ezetimibe	Activate the AMPK-TFEB signaling pathway	NLRP3 inflammasome IL-1B↓	[108]
Recombinant IL1RN	/	IL1A/B↓	[111,117]
Anti-IL-7 neutralizing antibody	Activation AMPK signaling pathway	IL-7↓	[113]
α-lactose	Activate the Akt/mTOR signaling pathway	Galectin 3↓	[118]
Rapamycin	Inhibits mTORC1 activity, enhances the expression of ULK1	M1 polarization↓, M2 polarization↑	[16]
SPM	Promotes ATG5 expression and accelerates autophagosome formation	M1 polarization↓, M2 polarization↑	[116]
JWH-133	HO-1	Liver steatosis↓	[119]
MAGL inhibitors	Encouraging the fusion of autophagosomes with lysosomes	Inflammatory factors↓	[120]
Nano-iron oxide particles (γ-Fe2O3)	Activation Notch1/HES1 signaling pathway	IL-10↑	[114]
DMXAA	STING signaling activation	HBV replication, macrophage inflammasome activation↓	[121]
Toyocamycin	BNIP3-mediated mitochondrial autophagy	cGAS/STING/NLRP3↓	[122]

IL1RN: interleukin 1 receptor antagonist; ULK1:Unc-51 like kinase 1; SPM: spermidine; JWH-133: cannabinoid receptor 2 receptor agonist; HO-1: oxygenase-1; MAGL: monoacylglycerol lipase; Notch1: neurogenic locus homolog 1; HES1: hairy and enhancer of split 1; STING: stimulator of interferon genes; DMXAA: STING agonist dimethylxanthenone-acetic acid; HBV: hepatitis B virus; BNIP3: Bcl-2 nineteen-kilodalton interacting protein 3; cGAS: cyclic GMP-AMP synthase.

6. Autophagy of liver sinusoidal endothelial cell (LSEC) and liver fibrosis

6.1. The effect of hepatic LSEC autophagy on liver fibrosis

LSECs are located between the hepatic sinusoidal lumen and hepatocytes. Normal LSECs have a unique fenestrated structure and loose intercellular connections, lacking a basement membrane, which gives them high permeability. These characteristics enable LSECs to effectively exchange substances, supporting liver function and the stability of the blood microenvironment [123]. However, when LSECs experience ongoing damage and inflammation, sinusoidal capillarization occurs. The loss of the fenestrated structure, along with the formation of a basement membrane-like structure, reduces permeability. This reduction affects blood flow and substance exchange in the hepatic sinusoids, leading to hepatocyte injury and death. Additionally, it activates inflammatory responses and promotes ECM deposition, all of which contribute to the progression of liver fibrosis [124]. In addition to their exchange function, LSECs also keep intrahepatic cells (including KCs and HSCs) in a quiescent state by regulating signaling pathways, vascular tone, and immune modulation, thus preventing liver fibrosis. However, in the context of both acute and chronic liver injury, LSECs undergo dedifferentiation and lose their normal function, which leads to the activation of KC and HSC and ultimately exacerbate liver inflammation and fibrosis [125,126].

The involvement of LSEC autophagy in liver fibrosis is complex (Figure 5). On one hand, impaired autophagy in LSECs exacerbates fibrotic progression. For instance, specific knockout of ATG7 disrupts autophagic flux, reduces nitric oxide (NO) production, and increases oxidative stress, thereby activating HSCs and aggravating liver fibrosis [18]. Similarly, knocking out the ATG5 gene inhibits LSEC autophagic function, intensifies the inflammatory response in endothelial cells, and promotes endothelial-mesenchymal transition and apoptosis, thereby advancing liver inflammation and fibrosis [127]. In diabetic liver fibrogenesis, elevated IL-33 activates the ERK/MAPK pathway, inducing autophagy deficiency in LSECs, which in turn drives endothelial-mesenchymal transition and HSC activation [128]. On the other hand, overactivation of selective autophagy in LSECs—particularly Caveolin-1 (Cav-1)-related autophagy—also contributes to fibrogenesis. As a critical structural and functional component of LSEC caveolae, Cav-1 is essential for maintaining fenestrae and endothelial homeostasis. The autophagic degradation of Cav-1 disrupts this foundation, downregulates NO-dependent signaling (NO/eNOS/SGC/cGMP/PKG/VASP), and induces F-actin remodeling, collectively leading to LSEC defenestration, as observed in CCl₄-induced early liver fibrosis [19,20]. Additionally, LSEC autophagy also facilitates the autophagic degradation of MVBs derived from LSECs, resulting in a decrease in the number of EVs secreted by MVBs, including a reduction in the secretion of the anti-fibrotic microRNA miR-342-5p, which further promotes the activation of HSC and the occurrence of liver fibrosis [129]. In summary, LSEC autophagy exerts dual—and often opposing—roles in liver fibrosis, largely by governing paracrine crosstalk with HSCs. Whether through the maintenance of endothelial NO homeostasis, the preservation of Cav-1 and fenestrae, or the regulated secretion of anti-fibrotic EVs, LSEC autophagy serves as a critical signaling hub that directly influences the activation state of HSCs. Thus, fine-tuning LSEC autophagic activity is essential to restore intercellular homeostasis and mitigate fibrosis.

Figure 5.

The autophagy of LSEC plays a dual role in liver fibrosis. (A) Moderate autophagy helps reduce inflammation and oxidative damage in LSECs, preventing their apoptosis and thereby decreasing fibrosis. (B) Excessive autophagy promotes fibrosis through two distinct mechanisms. First, Cav-1-related autophagy leads to the loss of Cav-1, inducing sinusoidal capillarization. Second, it facilitates the autophagic degradation of MVBs, which reduces the secretion of EVs containing the anti-fibrotic miRNA, miR-342-5p. The diminished level of this protective miRNA further promotes the activation of HSC, collectively driving liver fibrosis.

6.2. Intervention on liver fibrosis targeting autophagy of hepatic LSECs

Because LSECs play a dual role in autophagy during liver fibrosis, new research strategies are focusing on interventions that target this process (Table 4). Autophagy inducers like rapamycin can stimulate autophagy in LSECs and reduce liver fibrosis by lowering oxidative stress [18,128]. In addition, by blocking the IL-33/ST2 binding, the αIL33 antibody inhibits the ERK/MAPK pathway, thereby relieving autophagy suppression in LSECs and thus attenuating fibrosis [128].

Table 4.

Modulation of LSEC as a therapeutic strategy for alleviating liver fibrosis.

Therapeutic agent/approach	Therapeutic strategy	Impact on autophagy	Mechanism of alleviating liver fibrosis	References
Rapamycin	Autophagy enhancement	Suppressing the mTORC1 signaling pathway	Oxidative stress↓	[18]
αIL33, PD98059	Autophagy enhancement	Inhibits the ERK/MAPK pathway	LSECs dysfunction, capillarization, endothelial inflammation↓	[128]
3-MA	Autophagy inhibition	Suppressing the formation of autophagosomes	Autophagic degradation of Cav-1↓	[20,130]
BafA1, ATG5-siRNA, overexpressed Cav-1	Autophagy inhibition	Inhibiting fusion between autophagosomes and lysosomes	Autophagic degradation of Cav-1↓	[20,131]
Spironolactone	Autophagy inhibition	Suppressing the AMPK-ULK1 signaling pathway	LSECs defenestration↓	[19]
Spironolactone, si-ATG5	Autophagy inhibition	Inhibits the fusion of autophagosomes with lysosomes	Activation of HSC↓	[129]

αIL33: IL33 neutralizing antibody; PD98059: ERK/MAPK inhibitor; Cav-1: caveolin-1.

To evaluate how autophagy in LSECs affects liver fibrosis, researchers have used various autophagy inhibitors. For example, 3-methyladenine (3-MA) inhibits class III PI3K (especially Vps34), suppressing the formation of autophagosomes and blocking the early stages of autophagy [130]; while bafilomycin A1 (BafA1) inhibits the degradation of autophagic contents by blocking lysosomal acidification, thus inhibiting the later stages of autophagy [131]. The application of both inhibitors suppresses autophagy in LSECs, thereby maintaining their fenestrated structure and improving the NO-dependent pathway, consequently alleviating liver fibrosis [20]. Additionally, the aldosterone antagonist spironolactone shows potential in inhibiting LSEC autophagy. Spironolactone operates through two mechanisms. First, it suppresses AMPK-ULK1-mediated, Cav1-related selective autophagy in LSECs, thereby alleviating liver fibrosis [19]. Second, it downregulates the expression of V-ATPase (especially ATP6V0a2), which uses energy from ATP hydrolysis to pump protons (H⁺) into lysosomes, maintaining their acidic environment. Spironolactone weakens the acidic environment of lysosomes, inhibits the fusion of autophagosomes with lysosomes, and reduces the autophagic degradation of MVBs. This results in an increased number of EVs and the secretion of miR-342-5p, while also suppressing the activation of HSCs and the formation of liver fibrosis [129]. In summary, strategies targeting the autophagy of liver endothelial cells offer a promising new approach for treating liver fibrosis. By finely regulating the autophagic activity of LSECs, it is possible to alleviate oxidative stress and inhibit the progression of liver fibrosis through the modulation of NO-dependent pathways and EV secretion.

7. Summary and outlook

Recent advancements have significantly deepened our understanding of the role of autophagy in liver fibrosis, refining our insights into hepatic homeostasis, the regulatory mechanisms of fibrosis, and providing a theoretical foundation for novel therapeutic strategies. This review systematically elaborates on the cell-specific roles of autophagy in hepatocytes, HSCs, liver macrophages, and LSECs. Overall, the induction of autophagy in hepatocytes and macrophages generally exerts protective effects, slowing fibrosis progression. However, the role of autophagy in HSCs and LSECs is more complex and dualistic: it can promote fibrosis by providing energy and degrading specific substrates, yet it can also inhibit fibrosis by inducing cellular senescence, ferroptosis, or modulating signaling pathways. This paradoxical role underscores the paramount importance of achieving cell-type-specific modulation of autophagy.

Translating these mechanistic insights into effective anti-fibrotic therapies remains challenging. A promising strategy lies in the development of precision nanomedicine. For instance, retinol or hyaluronic acid-based nanoparticles (e.g. HCQ@ROL-LNPs, HA@PRB/COL NPs) can deliver hydroxychloroquine specifically to HSCs [46–48], or the Lip/siATG7/CK system can be used to specifically knock down the key autophagy gene ATG7 in HSCs [49]. These approaches successfully concentrate the therapeutic effect on activated HSCs, enhancing anti-fibrotic efficacy while minimizing off-target toxicity.

Despite these promising preclinical advances, several limitations must be acknowledged. The current evidence base relies heavily on animal models, which cannot fully capture the complexity, heterogeneity, and chronicity of human liver disease. Moreover, the pharmacological tools available often lack specificity. For instance, while rapamycin inhibits mTORC1 to activate autophagy, prolonged exposure can lead to mTORC2 degradation, adversely affecting translation, cell growth, and proliferation [132,133]. Similarly, lysosomal function inhibitors like chloroquine, hydroxychloroquine, and BafA1 not only block autophagy but also disrupt broader endolysosomal trafficking, impairing endosome degradation and vesicle transport [134]. Perhaps most importantly, direct clinical evidence supporting the efficacy of autophagy-targeting therapies in human liver fibrosis remains scarce, underscoring a significant translational gap.

To bridge this gap, future research should prioritize several key directions. First, the efficacy of targeted nanotherapies must be rigorously compared against conventional inhibitors in established fibrosis models, focusing on endpoints such as collagen deposition and HSC activation markers. Second, the therapeutic potential of inducing selective autophagy pathways, notably ferritinophagy, to trigger HSC ferroptosis and reverse fibrosis warrants further investigation using specific pharmacological inducers alongside ferroptosis inhibitors. Third, there is a pressing need to identify and validate circulating biomarkers that reflect hepatic autophagic activity and fibrosis stage. Promising candidates include EV-associated miRNAs (e.g. HSC-derived miR-29a or hepatocyte-derived miR-125a) and proteins that accumulate during autophagic dysfunction (e.g. 14-3-3ζ and importin α4) [89,135,136]. Validating these biomarkers is crucial for enabling non-invasive patient stratification and treatment monitoring in future clinical trials.

Finally, successful clinical translation will require careful consideration of several critical aspects. The therapeutic timeframe is crucial, as aHSCs at advanced disease stages may become unresponsive, limiting the efficacy of interventions. Furthermore, the choice of which autophagic step to inhibit is highly context-dependent. For instance, in viral hepatitis, blocking autophagy initiation (e.g. with 3-MA) may be preferable to preventing autophagosome-lysosome fusion (e.g. with BafA1). This is because viruses like HBV can exploit accumulated autophagosomes for replication, and some viral proteins (e.g. HBsAg) actively inhibit fusion by suppressing SNAP29, meaning additional fusion blockade might exacerbate viral replication [137,138].

In conclusion, future success hinges on shifting from broad to precise autophagy modulation—controlling it in the right cells and context. By integrating targeted technologies with a nuanced understanding of disease, we can steer the liver away from scarring and toward repair, ultimately aiming to reverse fibrosis in patients.

Funding Statement

This study was supported by Zhejiang Provincial Natural Science Foundation of China [No. LY23H030002], Zhejiang Provincial Medical and Health Science and Technology Project [No. 2023KY412].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.