Microautophagy, chaperone-mediated autophagy (CMA) and macroautophagy are the three forms of autophagy in mammalian cells. In spite of the differences among the three types of autophagy, the basic functions and features are merging with cargo at the lysosome for degradation and recycling (12,13). Microautophagy involves lysosomes directly engulfing cytoplasmic components, mainly smaller organelles within the cell. Microautophagy can be either selective or non-selective, is capable of degrading entire organelles and can be activated by signals such as environmental stress. The main function of microautophagy is to remove dysfunctional intracellular proteins and organelles (13-15). CMA utilizes heat shock proteins and transporter proteins on the lysosomal membrane to degrade proteins expressing targeting motifs. Molecular chaperones such as heat shock protein family A (Hsp70) member 8/HCS70 recognize the targeting motif and transfer the protein to the lysosomal membrane. At the lysosomal membrane, the target binds to lysosomal-associated membrane protein (LAMP)-2A on the lysosome and is subsequently transferred to the lysosomal lumen for degradation (13,16). Macroautophagy is the most common form of autophagy. Macroautophagy primarily targets larger organelles, removing intracellular waste and damaged organelles. The central process of macroautophagy involves the formation of autophagosomes by the encapsulation of lipid membrane structures around the substrates designated for degradation. Autophagosomes fuse with lysosomes to form autolysosomes after the phagocytosis of cytoplasmic material. Thus, the substrate undergoes degradation (13,17).

Autophagy is a dynamic and complex process (if not specified, the form of autophagy discussed in this paper refers to macroautophagy). The macroautophagy process can be summarized in four steps: Initiation, autophagosome formation, autophagolysosome formation and autophagolysosomal degradation (18). i) Autophagy initiation: This stage comprises the generation and expansion of phagocytic vesicles. This step involves two protein complexes: The vacuolar protein sorting 34 (VPS34) complex [VPS34, BECLIN1, autophagy-related 14 (ATG14) and VPS15] and the Unc51-like kinase 1 (ULK1) complex (19). ii) Autophagosome formation: During this process, phagocytic vesicle expansion occurs, and two ubiquitin-like binding systems regulate the expansion and completion of autophagosomes: The ATG12-ATG5-ATG16L system and the ATG8/microtubule-associated protein 1A/1B-light chain 3 (LC3) system (18). Ubiquitin-like system ATG12-ATG5-ATG16L: ATG16L interacts with ATG5-ATG12 to form ATG5-ATG12-ATG16L, which adheres to autophagosomes and participates in the extension of autophagic precursor membranes (20,21). ATG8/LC3 ubiquitin-like system: LC3 is ATG8's mammalian homologue, and ATG4 cleaves it into cytoplasmic LC3-I (the cytosolic form of LC3). During the formation of autophagosomes, cytosolic LC3-I participates in ubiquitin-like reactions through interactions with ATG7 and ATG3 and then couples with phosphatidylethanolamine (PE) to generate lipidated LC3-II (the conjugate form of LC3-I with PE). As an autophagosome structural protein, LC3-II is attached to the membrane of autophagosomes. Subsequently, ATG4 excises LC3-II from the outer membrane of autophagic lysosomes during the autophagic degradation process, and the generated product, LC3-I, can be recycled. In autophagic lysosomes, LC3-II on the inner membrane, along with encapsulated content, is degraded by lysosomes (22). Unlike ATG5-ATG12-ATG16L, LC3B-II is distributed on both the outer and inner surfaces of autophagosomes. LC3B-II is essential for the extension and completion of autophagic membranes. After autophagosome membrane closure, the ATG16-ATG5-ATG12 complex detaches from the vesicle, but part of LC3B-II still covalently binds to the membrane. Thus, LC3B-II is currently the most widely recognized molecular marker (21,23). iii) Autophagy lysosome formation: After autophagosome formation, LC3-II on the outer membrane is removed from the PE by ATG4 and liberated back into the cytoplasm (24). LAMP, the lysosomal membrane protein, and RAB7, a small GTPase, are crucial in the fusion processes of autophagosomes and lysosomes. This process is characterized by the formation of isolated membrane structures containing cytoplasmic constituents (25). Furthermore, acetylation plays a significant role in autophagic lysosome biogenesis (26). iv) Autophagolysosomal degradation: The inner membrane of the autophagosome is degraded by lysosomal enzymes during autophagic lysosome formation so that the contents of the two are well mixed. As the contents continue to degrade, raw materials necessary for cellular life activities, such as amino acids, are continuously produced, which are conveyed to the cytosol to be re-utilized by the cell, whereas residues that cannot be recycled may be expelled from the cell or retained in the cytosol (27).

Several forms of cellular stress can activate autophagy [including, but not limited to, DNA damage, protein aggregates, intracellular pathogens, reactive oxygen species (ROS), hypoxia, damaged organelles and nutrient or growth factor deficiencies]. This process involves the promotion or inhibition of multiple signaling pathways to coordinate the various stages of autophagy (28). Autophagy is regulated by three major nutrient-sensing pathways: The mammalian target of rapamycin complex 1 (mTORC1) pathway, the adenosine monophosphate-activated protein kinase (AMPK) pathway and the oxidized nicotinamide adenine dinucleotide-dependent histone deacetylase sirtuin 1 (SIRT1) pathway (29).

The mammalian target of rapamycin 1 (mTOR1), a serine/threonine protein kinase, is a member of the PI3K-associated kinase family and is sensitive to rapamycin (30). mTORC1 phosphorylates ULK1 and ATG13 to inactivate the autophagy regulatory complex composed of ULK1, ATG13, FAK family interacting protein of 200 kDa (FIP200) and ATG101, which affects autophagy vesicle formation. Under nutrient-rich conditions, mTORC1 inhibits autophagy-promoting kinase activity of the ULK1 complex by mediating site-specific phosphorylation of ULK1 and ATG13. However, there are adaptive changes in how the organism is regulated during starvation and cellular stress, such as mTORC1 inhibition and ULK1 dissociation from mTORC1. Consequently, site-specific phosphorylation of ULK1 and ATG13 is deregulated (19). Meanwhile, the autophosphorylation of threonine located at site 180 activates the ULK1 complex, and in this state, ULK1 phosphorylates ATG13, FIP200 and ATG101 in the ULK1 complex. After that, the active ULK1 complexes are transferred to the isolation membrane of the endoplasmic reticulum, thereby initiating autophagy (31,32). Furthermore, AMPK affects mTORC1 as well. In the presence of sufficient glucose, active mTORC1 inhibits ULK1 activation in two mechanisms: Phosphorylating the ULK1-specific site (serine 757) and disrupting the ULK1 and AMPK interaction. After ULK1 activation is inhibited, autophagy initiation is also impaired. In the presence of glucose insufficiency, AMPK activity inhibits the phosphorylation of mTORC1, then ULK1 interacts with AMPK and is phosphorylated, resulting in ULK1 activation and autophagy initiation (31). In the class III phosphatidylinositol 3-kinase complex I, the phosphorylation of ATG14, autophagy and beclin 1 regulator 1 (AMBRA1) and nuclear receptor binding factor 2 (NRBF2) inhibits the nucleation step of autophagy. However, mTORC1 regulates autophagy by phosphorylating ATG14, AMBRA1 and NRBF2 (25). A study showed that mTORC1 regulates the elongation process in autophagosome formation and influences LC3 binding to the autophagosome membrane through targeting WD repeat domain, phosphoinositide interacting 2 (WIPI2) and P300 acetyltransferases, respectively. For instance, activating P300 by inhibiting its intramolecular autoinhibition promotes the acetylation of LC3, depriving it of the ability to be lapidated (33). By binding to ATG16L, WIPI2 promotes the binding of ATG12-ATG5-ATG16L complexes to phagophores, therefore enhancing lipidation of LC3 by PE (34). In addition to directly inhibiting autophagy, mTORC1 can also indirectly inhibit it by regulating the transcription of genes associated with lysosomal biogenesis. For instance, recent research on transcription factor EB (TFEB) has found that TFEB regulates the expression of genes involved in lysosomal biogenesis and autophagy, including those associated with autophagosome formation, fusion of autophagosomes with lysosomes, and lysosomal biogenesis (35). In addition, overexpression of TFEB increased the expression of UV radiation resistance associated, WIPI, microtubule-associated protein 1 light chain 3B, sequestosome 1, VPS11, VPS19 and ATG9B, which are involved in various steps of autophagy (36). Furthermore, mTORC2 can also play multiple roles as an important regulator in autophagy regulation, including the indirect inhibition of autophagy through the activation of mTORC1 (37). In summary, mTOR inhibits autophagy induction. Therefore, the development of novel mTOR inhibitors that can precisely regulate autophagy provides new avenues for the treatment of clinically difficult diseases (Fig. 1).

In the cell, AMPK acts as an energy sensor, actively upregulating catabolism and inhibiting anabolism (38). By specifically phosphorylating different autophagy-associated protein complexes or different components of the same protein, AMPK can affect different phases of autophagy and thus promote autophagy (39). AMPK antagonizes mTORC1 to regulate the activity of the ULK complex. Two distinct mechanisms induce autophagy when AMPK is activated: Inhibition of mTOR and direct phosphorylation of ULK1 (40). Most regulatory factors can affect mTORC1 by interfering with tuberous sclerosis complex (TSC) and ras homolog enriched in brain (RHEB). RHEB binds to GTP to form RHEB-GTP, which then binds and activates mTORC1. The TSC complex, however, has GTPase activity that hydrolyzes GTP to inactivate RHEB-GDP. Therefore, the TSC complex can inhibit mTORC1 activity by modulating RHEB (41). AMPK is phosphorylated and activated under low-energy conditions or starvation situations, and it inhibits mTOR activity in two ways. i) AMPK directly phosphorylates TSC2, promoting GTPase-activating protein (GAP) activity in the TSC complex. GTP dephosphorylation of RHEB-GTP results in its conversion to the inactive RHEB-GDP, which interrupts RHEB-mediated mTORC1 activation. ii) AMPK inhibits the mTOR signaling pathway by directly phosphorylating regulatory associated protein of mTOR complex 1 (RAPTOR), stopping RAPTOR from binding to mTOR or its substrates (35,37). The inactivation of mTOR leads to decreased ULK1 phosphorylation and increased ULK1 binding to AMPK, thereby exhibiting a unique role in autophagy regulation. AMPK promotes the activity of ULK1, which induces autophagy during glucose deprivation. AMPK interacts with the serine/proline-rich region of ULK1 and directly phosphorylates ULK1 at several sites, resulting in changes in the conformation of ULK1. The conformational changes in ULK1 enhance interactions between ULK1 and the other components of the ULK1 complex, increasing its activity and stability. mTORC1 regulates the interactions between AMPK and ULK1 through various mechanisms. In the nutrient-rich environment, ULK1 is phosphorylated by mTORC1, inhibiting its interaction with AMPK (42,43). In addition to this, mTORC1 can also phosphorylate ATG13, which decreases ULK1 complex activity. In contrast, under starvation conditions, mTORC1 activity is inhibited, leading to the rapid dephosphorylation of ULK1 and ATG13, which activates ULK1 kinase and initiates autophagy. Accompanying this change is that AMPK and ULK1 interact more effectively, increasing ULK1 activity and promoting ULK1-ATG13-FIP200 complex formation (30). Thus, AMPK and mTORC1 coordinately regulate ULK1 to induce autophagy in response to cellular nutrient levels. AMPK, ULK1 and mTORC1 form a signaling triad that constitutes a transient feedback mechanism to maintain the dynamic homeostasis of autophagy (44). AMPK also affects autophagy by regulating the activity of the PI3KC-3/VPS34 complex and the transcriptional regulation of autophagy (45) (Fig. 1).

SIRT1, an NAD⁺-dependent multifunctional enzyme, acts as a regulator of autophagy (46). In mammals, there are seven different SIRT isoforms. Although all SIRTs possess NAD⁺-dependent catalytic structural domains, their localizations and functions differ due to the different lengths and sequences between their C- and N-terminals. Among mammalian SIRTs, SIRT1 shares close homology with the yeast Sir2 and is one of the most thoroughly studied SIRTs (47,48). SIRT1 is distributed in the nucleus and cytoplasm, where it primarily functions by deacetylating various histones and non-histone proteins and thus participates in the regulation of a variety of cellular processes, such as cell proliferation, differentiation, autophagy and cell survival (49). Under conditions of hunger or nutrient deprivation, the body experiences energy deprivation, leading to elevated intracellular NAD⁺ levels that activate SIRT1 (50-52). Thus, SIRT1 is able to sense the energy status of the cell and adapt to energy deprivation by regulating autophagy (50,52). The mechanism by which SIRT1 regulates autophagy is as follows. i) SIRT1 directly deacetylates autophagy-related proteins to promote autophagy (52,53). ii) SIRT1 deacetylates and activates transcription factors to enhance autophagic activity (54). iii) SIRT1 inhibits mTOR, hence facilitating the autophagic process (55). iv) SIRT1 activates autophagy to degrade damaged or excess organelles and proteins (56). Afterwards, the cell recycles metabolites such as amino acids and fatty acids for use in times of hunger or nutrient deprivation, thereby maintaining cell homeostasis and energy balance (56). Thus, SIRT1 activates autophagy during hunger or nutrient deprivation through multiple mechanisms, which not only helps cells to remove damage and maintain metabolic homeostasis, but also optimizes energy utilization and enhances cellular viability. SIRT1 regulates autophagy through its deacetylase activity and mediates it by associating with the various steps of autophagy, including initiation, elongation, maturation, fusion and degradation (49,57,58). SIRT1 acts by regulating TSC2 stability during autophagy initiation (59). TSC2 inactivates the mTORC1 signaling pathway by inhibiting the RHEB, which results in the activation of mTORC1. RHEB requires GTP to activate mTORC1; however, the GAP structural domain of TSC2 is capable of stimulating GTP hydrolysis (58,60). Increased TSC2 stabilization increases GTP hydrolysis, which accelerates the inactivation of RHEB. These changes downregulate mTOR signaling and initiate autophagy (58,60). Under hypoxic conditions, SIRT1 mediates autophagy initiation through regulating BNIP3. SIRT1 deacetylates forkhead box (FOX)O3, enabling it to bind to the BNIP3 promoter and induce BNIP3 expression (61,62). BNIP3 can act on the Bcl-2-Beclin1 complex to dissociate Beclin1 from it, thereby increasing the concentration of Beclin1, which further triggers autophagy (63). The deacetylase function of SIRT1 also regulates the elongation and maturation phases. Under nutrient-rich conditions, EP300, an E1A-binding protein P300 acetyltransferase, inhibits ATG16-ATG5-ATG12 complex formation by acetylating ATG5, ATG7 and ATG12, which prevents autophagosome elongation (64). By contrast, in times of nutrient deprivation or starvation, SIRT1 deacetylates ATG5, ATG7 and ATG12 directly, which contributes to the formation of the ATG16-ATG5-ATG12 complex, thereby enhancing autophagic vesicle elongation (65). The SIRT1 protein also regulates the translocation of the LC3-I protein from the nucleus to the cytoplasm, which contributes to autophagy. Under nutrient starvation, nuclear LC3-I, after deacetylation by SIRT1, interacts with tumor protein p53 inducible nuclear protein 2 to move LC3-I from the nucleus to the cytoplasm. Deacetylated cytoplasmic LC3-I binds to PE by interacting with ATG7 and ultimately forms LC3-II (62,66). During the fusion and degradation of autophagosomes with lysosomes, RAB7, as a Ras-related GTP-binding protein, functions to facilitate autolysosome formation by facilitating autophagosome translocation to lysosomes (67). Under nutrient starvation, SIRT1 deacetylates FOXO1 and increases RAB7 expression (68). The interaction between SIRT1 and autophagy is bidirectional. On the one hand, SIRT1 can regulate autophagic activity; on the other hand, autophagy also controls the level of SIRT1 by affecting lysosomal degradation (69). Early autophagosome formation is fundamental and critical for the initiation of the overall autophagic response (70). Therefore, the mechanism by which SIRT1 acts in the early stages of autophagy is even more important for the regulation of autophagy (Fig. 2).

During the process of growth and development, autophagy is an essential catabolic process for basic biological activities (71). According to current research findings, the functions of autophagy include the following: i) Immune Response: Autophagy removes intracellular pathogens, such as viruses and bacteria, through a process called heterophagy. It also regulates inflammation and antigen presentation, contributing to the normal function of the immune system (72). ii) Nutrient recycling: In times of nutrient deprivation or starvation, the autophagic process recycles cellular components and provides nutrients to cells. In degrading and recycling cytoplasmic material, autophagy provides cells with vital components, such as amino acids, for survival and energy production (56). iii) Energy homeostasis: During periods of metabolic stress, such as fasting or exercise, autophagy is upregulated to provide a source of energy by breaking down cellular components. As a result, cells adapt to changing nutrient supplies and maintain a balance of energy (56). iv) Development and differentiation: Autophagy participates in various differentiation and developmental processes, including cellular differentiation, tissue remodeling and embryonic development. It helps to remove unnecessary or excessive cellular components, shape tissues and organs, and promote normal cell development and differentiation (73,74). v) Cellular quality control: Autophagy sustains cellular homeostasis by cleaning out damaged or dysfunctional cellular components (e.g., misfolded proteins, damaged organelles and excess or aggregated proteins). By eliminating these components, autophagy contributes to preventing the accumulation of toxic substances and maintaining cellular homeostasis (75).

Numerous different triggers can contribute to the ongoing aggravation of progressive fibrosis disease. However, regardless of the initiating event, all fibrotic diseases are characterized by the activation of ECM-producing myofibroblasts, which are the primary mediators of fibrotic tissue remodeling (76,78). When tissues are injured, myofibroblasts from various sources remodel the extracellular environment to initiate healing responses, which restore tissue integrity and promote the replacement of parenchymal cells (79). Typically, the deposition of ECM proteins in the initial stages of tissue healing contributes to the tissue repair process. In mild injuries, the fibrotic matrix is taken up during the tissue repair process (80). However, sustained injury can lead to dysregulation of this process, resulting in excessive deposition of ECM proteins, myofibroblast activity and the gradual development of a chronic inflammatory environment infiltrated by macrophages and immune cells (81). In such a microenvironment, cells are exposed to a significant release of cytokines and growth factors by myofibroblasts, such as TGF-β and WNT1, which are significant players in the fibrotic process (82). TGF-β and WNT1 bind to their stem cell surface receptors and initiate downstream signaling, ultimately leading to nuclear translocation of SMAD2/3 and CREB binding protein/β-catenin transcriptional regulators. This leads to the upregulation of target gene expression, further enhancing myofibroblast differentiation and the production and secretion of ECM proteins, such as collagen, laminin and fibronectin (83,84). As excess ECM deposition proceeds, the matrix undergoes structural changes and hardens (76). Cells sense ECM tension through mechanotransduction of cell surface integrin receptors, which activate the Hippo signaling pathway and its major downstream effectors Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) (85). In addition, activated YAP and TAZ translocate to the nucleus and promote the upregulation of pro-growth genes, such as connective tissue growth factor and platelet-derived growth factor, which promote myofibroblast proliferation and activation through the PI3K/AKT/mTOR pathway (85).

In clinical practice, biomarkers of fibrotic diseases are crucial for early diagnosis, disease progression tracking, prognostic evaluation and treatment efficacy assessment (86). The following are generic fibrosis markers in fibrotic diseases. i) Collagen: The main component of fibrosis, as well as its degradation products in serum, can be used as markers (87,88). ii) Fibronectin: An important component of the ECM, upregulated during fibrosis (89). iii) Matrix metalloproteinases and their inhibitors: They reflect matrix remodeling processes, balancing the disintegration and accumulation of the ECM, and serve a crucial role in regulating fibrosis (90-92). iv) MicroRNAs: MicroRNAs regulate the expression of fibrosis-related genes in a variety of fibrotic diseases and have potential diagnostic and therapeutic value (93-95).

Biomarkers of fibrotic disease exhibit considerable variability across different tissues and organs. Liver fibrosis markers include the following: i) The aspartate aminotransferase (AST) to alanine aminotransferase (ALT) ratio, which is often elevated in liver fibrosis (96); ii) fibrosis indices: AST to platelet ratio index (based on AST level and platelet count) and FIB-4 (based on age, AST, ALT and platelet count) (97); iii) FibroTest: A score is calculated in combination with various serum markers (e.g., α2-microglobulin, apolipoprotein-A1, transferrin, total bilirubin, γ-glutamyltransferase, etc.) and is used to assess the degree of liver fibrosis (98); iv) hyaluronic acid: Serum levels of hyaluronic acid are significantly elevated during liver fibrosis (99). Pulmonary fibrosis markers include the following: i) Krebs von den Lungen-6 (KL-6): KL-6, released after damage to alveolar epithelial cells, is commonly used in the monitoring of idiopathic pulmonary fibrosis (100-102); and ii) surfactant protein D, which reflects lung tissue damage and the inflammatory state (102-104). Renal fibrosis markers include i) Neutrophil gelatinase-associated lipocalin, which is elevated in acute kidney injury and renal fibrosis (105); and ii) urinary collagen degradation products, which reflect collagen metabolism and fibrotic activity (106,107). Cardiac fibrosis markers include i) Galectin-3, which is involved in the fibrotic process and it is an important marker of cardiac fibrosis (108-110); ii) ST2 protein, which reflects cardiac stress and fibrotic activity with significant prognostic value (109,110); and iii) collagen degradation products, reflecting cardiac collagen metabolism (111).

The mechanism of fibrosis varies among organs, so the selection of appropriate markers needs to be tailored to the specific type of disease.

Macrophages are immune cells found in almost all tissues. Macrophages participate in non-specific immunoregulation by phagocytosis of bacteria and other pathogens, while also transmitting signals to lymphocytes to participate in specific immunoregulation, thus contributing to immunity, repair and homeostasis of the body (112). Macrophages may be categorized into two subpopulations, M1 and M2, which secrete pro-inflammatory and anti-inflammatory factors, respectively (113). Continued research has indicated that macrophages play a critical role in regulating organ fibrosis (114). When an organ or tissue is infected or injured, macrophages polarize into the pro-inflammatory M1 phenotype, secreting proinflammatory cytokines to remove antigens and necrotic cells (115). Pro-inflammatory macrophages are categorized as M1-type macrophages, while anti-inflammatory macrophages are categorized as M2-type macrophages (115). During the organ or tissue repair phase, M2 macrophages secrete anti-inflammatory cytokines that suppress inflammation as well as contribute to tissue repair and remodeling (116). In a pathological situation, persistent pro-inflammatory macrophages produce pro-inflammatory factors continuously, resulting in chronic inflammation, which ultimately accelerates the progression of organ fibrosis significantly (117). It has been confirmed that autophagy regulates macrophage polarization (118). By inhibiting M1 proinflammatory macrophage polarization, macrophage autophagy ameliorates organ fibrosis and attenuates chronic inflammation (9).

After chronic or severe organ damage, fibrous connective tissue accumulates and parenchymal cells decrease, resulting in fibrosis. Continuous progression may result in severe damage to organ structure and function, or even death, posing a serious threat to human health and life (119). Pathologically, organ fibrosis is characterized by an imbalance in ECM homeostasis, resulting in excessive accumulation of collagen, fibronectin and other ECM components (120). Thus, fibrosis can also be seen as the result of abnormal tissue repair.

Pulmonary fibrosis (PF) is a common pathological feature and the ultimate outcome of numerous lung diseases. The main feature of PF is the excessive accumulation of ECM in the lungs, leading to the thickening of the alveolar walls, which ultimately causes the destruction of the alveolar structure and respiratory failure (121,122). Type II alveolar epithelial cell dysfunction or disorder is considered to be the initiating factor for PF. In addition, the contribution of macrophages in the evolution and progression of fibrotic diseases should not be overlooked (121,123). Depending on their location, macrophages in the lung are classified as alveolar macrophages (AMs) or interstitial macrophages (124). Normally, AMs reside in the alveolar cavity and are the most important component of the alveoli. AMs play a crucial role in preventing inflammation and fibrosis in the lungs.

In silicosis, autophagy inhibits inflammation and AM apoptosis and plays a protective role in the progression of silicosis. Du et al (125) found that crystalline silica (CS) triggered autophagy activity in AMs, thereby protecting AMs from CS-induced apoptosis. In AMs, diosgenin promoted autophagy and therefore attenuated CS-induced pulmonary fibrosis. Mechanistically, diosgenin can exert antifibrotic effects by enhancing macrophage autophagy activity. Diosgenin leads to mitochondrial dysfunction through silica inhalation and activates beclin1, which increases the expression of two key proteins of mitochondrial autophagy, PTEN-induced putative kinase 1 (PINK1) and parkin RBR E3 ubiquitin protein ligase (PARKIN), in AMs in mice. Diosgenin-mediated AMs mitochondrial autophagy eliminated damaged mitochondria and further ameliorated silicosis fibrosis. In ATG5 knockout mice, the protective effect of diosgenin was lost, and macrophages in these mice lacked autophagy function (126,127). In addition, microRNA-205-5p promotes macrophage autophagy by inhibiting S-phase kinase-associated protein 2-mediated ubiquitination of Beclin1, thereby suppressing lung fibrosis in silicosis mice (128). These experimental results suggest that tissue-resident macrophage autophagy can inhibit PF during silicosis progression. In addition, the lung has some macrophages of monocyte origin. The origin of these macrophages is different from that of tissue-resident macrophages. A study by Jessop et al (129) showed that in mouse monocyte-derived macrophages, exposure to CS can enhance autophagic activity. ATG5 gene knockout in mice resulted in impaired macrophage autophagy derived from monocytes, and more fibrosis was observed in ATG5 knockout mice exposed to silica compared to littermate control mice (129). These experimental results show that, similar to tissue-resident macrophages, monocyte-derived macrophages are also able to protect against CS-induced PF. In conclusion, macrophage autophagy has been shown to inhibit chronic inflammation, therefore inhibiting PF. However, there are different views among scientists, e.g., it has been suggested that autophagy may exacerbate lung injury and PF under certain circumstances, such as when autophagy levels are excessive or uncontrolled (130).

Renal fibrosis is the ultimate pathway leading to end-stage renal failure in almost all chronic progressive kidney diseases. In the kidney, macrophages play a crucial role in the generation and evolution of renal fibrosis (131). A study confirmed that macrophage autophagy suppresses the pro-inflammatory response of macrophages. Loss of macrophage autophagy leads to abnormal macrophage polarization, increased M1 and decreased M2, which causes worsening inflammation (132). Therefore, macrophage autophagy plays a crucial role in both macrophage polarization and suppressing inflammation. Liu et al (133) experimentally demonstrated that ubiquitin-specific peptidase 19 (USP19) can regulate NLR family pyrin domain containing 3 (NLRP3) function by affecting autophagy and thereby significantly influencing inflammation and macrophage polarization. In terms of mechanisms, USP19 increases autophagic flux and reduces mitochondrial ROS production, thus inhibiting inflammation and promoting macrophage polarization in M2 (133) (Fig. 3). In a study using two mouse models of experimental renal fibrosis, Bhatia et al (134) found that macrophage mitochondrial autophagy regulates the PINK1/mitofusin 2/PARKIN pathway, which could protect mice's kidneys from fibrosis (Fig. 3). Based on a renal fibrosis model, Zhang et al (135) found that fibrosis and macrophage infiltration were positively correlated with lymphangiogenesis. The activation of the VEGF-C/VEGFR3 signaling pathway inhibited macrophage autophagy, consequently facilitating macrophage M1 polarization, which then increased the transdifferentiation of M1 macrophages into lymphatic endothelial cells (LECs). By contrast, rapamycin-induced macrophage autophagy decreased M1 macrophage polarization and transdifferentiation to LECs. Thus, macrophage autophagy reduces LEC production, thereby inhibiting renal fibrosis (135). In summary, when the kidney is severely injured or repetitively injured, a large number of macrophages infiltrate into the damaged area and persist at the infiltration site, leading to chronic inflammation and renal fibrosis. Macrophage autophagy inhibits macrophage polarization to M1, thereby suppressing inflammation and renal fibrosis.

In liver disease, macrophage autophagy similarly influences the progression of liver fibrosis. Lodder et al (136) demonstrated that the autophagic pathway in macrophages exerts a protective effect against hepatic fibrosis by limiting the release of inflammatory cytokines such as IL-1A and IL-1B from macrophages, which are key mediators of liver fibrosis. By contrast, rapamycin-induced autophagy activation limits the production of IL-1A and IL-1B in macrophages. These results reveal macrophage autophagy as a paracrine pathway that regulates IL-1-dependent activation of hepatic myofibroblasts.

In conclusion, macrophage autophagy protects against organ fibrosis.

The endoplasmic reticulum, a complex and closed intracellular tubular endomembrane system interwoven into a three-dimensional network structure, is the central organelle responsible for the production of secreted and transmembrane proteins. The endoplasmic reticulum has the function of folding, assembling and modifying proteins (137). The biological functions of the endoplasmic reticulum are tightly regulated. Cellular homeostasis is disrupted when cells encounter various strong stimuli, such as nutritional deficiencies and oxidative stress stimuli, initiating a series of self-protective mechanisms, such as ERS (138). It has been indicated that ERS is activated when the folding capacity of the endoplasmic reticulum breaks down, resulting in accumulation of misfolded and unfolded proteins and disrupted protein homeostasis (139). In mammalian cells, three endoplasmic reticulum transmembrane proteins act as ERS sensors: Activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1)α and protein kinase RNA-like endoplasmic reticulum kinase (PERK) (140,141) (Fig. 4). Under conditions of protein homeostasis, BIP (a molecular chaperone located in the endoplasmic reticulum membrane that facilitates degradation and refolding of unfolded and misfolded proteins accumulated in the endoplasmic reticulum to restore homeostasis) binds to these sensors, rendering them inactive (140,142). During ERS, BIP dissociates from the sensor (at this time, the affinity of BIP for unfolded and misfolded proteins increases), triggering the unfolded protein response (UPR), which is one of the adaptive responses to stress (141,143). Through activation of PERK, IRE1 and ATF6, the UPR promotes protein expression and restores the normal protein structure of misfolded or unfolded proteins. When the ERS-activated UPR is insufficient or does not completely eliminate the accumulated misfolded and unfolded proteins, as a complementary form, the ubiquitin-proteasome system collaborates with the UPR to degrade the misfolded and unfolded proteins, thus restoring the normal morphology of the endoplasmic reticulum (144). If the stimulus to the cell is persistent or excessively severe so that the synergistic action of the UPR and the ubiquitin-proteasome still does not fully restore the endoplasmic reticulum to its normal state, autophagy appears to be the last resort for restoring endoplasmic reticulum homeostasis. Persistence of ERS activates autophagy (145). Autophagy and ERS exhibit tight reciprocal regulation, both of which are crucial for maintaining cellular homeostasis and responding to environmental stimuli (146,147). The specific relationship includes the following four aspects: i) ERS induces autophagy: ERS is triggered when unfolded or misfolded proteins accumulate in the endoplasmic reticulum. In response to this stress, cells initiate the UPR, and by activating sensors such as IRE1, PERK and ATF6, these UPR signaling pathways can facilitate the initiation of autophagy (146,148). ii) Autophagy to alleviate ERS: Autophagy can selectively degrade damaged endoplasmic reticulum regions, remove accumulated unfolded proteins and restore endoplasmic reticulum function (149). iii) Feedback regulation and homeostasis: A feedback regulatory mechanism exists between ERS and autophagy to ensure that cells balance survival and apoptotic signaling in response to stress (150,151). iv) Disease association: Dysregulated autophagy and ERS response are closely associated with the development of multiple diseases, e.g., PF (152), inflammatory bowel disease (147) and cardiovascular disease (153).

An association between ERS-induced autophagy and fibrotic disease has been identified. During fibrosis, autophagy regulates ERS to influence disease progression primarily through the following mechanisms: i) Reducing ERS load and modulating UPR: Autophagy reduces the endoplasmic reticulum burden and relieves ERS by breaking down aberrant proteins and damaged endoplasmic reticulum and activating the UPR, preventing further fibrosis development (154). ii) Modulation of inflammatory response: ERS is often accompanied by an inflammatory response, and autophagy can reduce tissue damage and the fibrosis process by removing inflammatory signaling molecules and inhibiting the excessive release of inflammatory mediators (147,155). iii) Maintaining cell survival and function: By regulating autophagy, cells can more effectively respond to protein folding stress, avoid apoptosis or necrosis, and protect the tissue structure (156,157). iv) Involvement in ECM metabolism: Autophagy indirectly affects fibrosis progression by regulating collagen degradation (154). Shu et al (158) demonstrated that ERS in renal proximal tubular cells can induce renal fibrosis. Furthermore, the PERK-mediated UPR signaling pathway links ERS to autophagy activation. Autophagy activation contributes, at least in part, to fibrosis associated with ERS (158). Xiong et al (159) found that cardiac Ca²⁺ concentrations increased and endoplasmic reticulum markers were upregulated during tris(2-chloroethyl) phosphate (TCEP)-induced cardiac fibrosis. However, Ca²⁺ overload and subsequent cardiac fibrosis were attenuated with the use of CDN1163, an inhibitor of the sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase, which also suppressed the upregulation of endoplasmic reticulum markers. Furthermore, CDN1163 supplementation inhibited TCEP-induced excessive autophagy in the heart. Thus, by inhibiting sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase expression, TCEP can lead to Ca²⁺ overload, which triggers ERS and excessive autophagy, ultimately leading to cardiac fibrosis (159). Ren et al (160) found that milk fat globule-egf factor 8 (MFG-E8) gene defects exacerbated pancreatic fibrosis. By contrast, pancreatic fibrosis was attenuated in cerulein-induced chronic pancreatitis mice treated with injections of exogenous MFG-E8. Meanwhile, ERS and CMA levels were reduced in cerulein-induced chronic pancreatitis mice after the administration of exogenous MFG-E8. Further experiments revealed that MFG-E8 inhibited the ERS-induced CMA pathway by targeting the specific CMA activator QX77, which reversed the effects of MFG-E8. This suggests that MFG-E8 inhibits pancreatic fibrosis by suppressing the ERS-induced CMA pathway. Recombinant MFG-E8 is likely to be developed as a novel medication for pancreatic fibrosis in chronic pancreatitis (160). Zheng et al (161) found that tunicamycin-induced ERS was associated with family with sequence similarity 172, member A (FAM172A)-mediated calcium flux. The autophagic process was observed in both normal fibrous tissue and epidural scar tissue cell lines after tunicamycin treatment. Further experimentation revealed that overexpression of FAM172A inhibited the autophagic process in normal fibrous tissue and epidural scar tissue. However, when the expression of FAM172A was inhibited, the autophagic process was increased in both cell lines. In a mouse model, FAM172A inhibited epidural fibrosis. Thus, ERS-associated calcium currents mediate the downregulation of FAM172A expression, which promotes autophagy in fibroblasts, a critical pathogenic factor in epidural fibrosis (161).

In summary, ERS-mediated autophagy exhibits a promoting effect on fibrosis development.

EndMT, a special type of epithelial-mesenchymal transition (EMT), refers to the process in which endothelial cells lose their original characteristics and transform into mesenchymal cells under the action of multiple stimuli. During the transformation process, endothelial cells gradually lose their morphology and function and acquire mesenchymal cell phenotypic characteristics such as proliferation, migration and collagen synthesis (162). In the process of acquiring the mesenchymal phenotype, endothelial cells (ECs) lose endothelial-specific markers such as vascular endothelial cadherin, platelet and endothelial cell adhesion molecule 1 and vonWillebrand factor. Instead, they start to express mesenchymal markers, such as vimentin, α-smooth muscle actin and type I collagen (163,164). At the same time, the cell morphology changes from compact cobblestone-like to elongated spindle-like. Cells acquire an impaired antiplatelet-generating capacity and loss of cell-to-cell connectivity and polarity, but enhanced mesenchymal cell properties, such as invasion and migration. Partial EndMT is a process in which ECs remain in an intermediate stage of transdifferentiation for an extended period rather than permanently acquiring a mesenchymal phenotype (165). This is the stage at which cells gain a mesenchymal phenotype, while retaining endothelial markers and exhibiting progenitor-like characteristics. This intermediate stage of phenotypic transformation is unstable and can be reversed under certain conditions, making it a potential target for therapy (166,167).

EndMT has been shown to promote the formation of heart valves and septa during embryonic development and has also been indicated to contribute to certain pathological conditions, such as cardiac and renal fibrosis and PF, tumor progression, and wound healing processes (168). ECs, however, are not fully understood regarding their regulatory role in cellular metabolism to drive EndMT. Currently, it has been found that autophagy regulates the EndMT, which contributes to the development of fibrotic diseases (Fig. 5). Zhang et al (169) showed that advanced glycosylation end-products (AGEs)/AGE receptor (RAGE) regulates autophagy in heart failure, which results in EndMT-induced cardiac fibrosis. RAGE knockdown inhibited autophagy-regulated EndMT, attenuated cardiac fibrosis and improved cardiac function. Therefore, mechanistic studies focusing on the AGEs/RAGE-autophagy-EndMT axis may provide novel therapeutic targets for the treatment of heart failure (169). Pan et al (170) found that early perivascular fibrosis induced by doxorubicin was associated with the EndMT program. Disturbed autophagy leads to ROS accumulation, and ROS can trigger NF-κB-Snail activation, which could be the basis for doxorubicin involvement in EndMT induction. In an animal model of disseminated intravascular coagulation, irisin attenuated perivascular fibrosis and EndMT. Irisin can improve autophagy disorders, eliminate ROS and reverse EndMT by modulating uncoupling protein 2, irisin's proven target. ROS accumulation and autophagy disorders are identified as the factors contributing to EndMT in CMEC, which is involved in the initiation and development of perivascular fibrosis in disseminated intravascular coagulation (170) (Fig. 5). Zhou et al (171) analyzed the late proliferative endometrium of both normal patients and those with severe intrauterine adhesions (IUA), confirming that the endometrial tissue in patients with IUA exhibited autophagy deficiencies, which promoted EMT and fibrosis. Further analysis of the sequencing results of autophagy-related gene expression revealed that the most significant differential gene, iodothyronine deiodinase 2, triggers autophagy defects in endometrial epithelial cells through the MAPK/ERK-mTOR pathway, leading to EMT. Meanwhile, in vivo experiments also confirmed that targeting autophagy could inhibit EMT and alleviate fibrosis (171). Singh et al (172) found that the deletion of ATG7, an essential autophagy gene in ECs, resulted in disrupted autophagic flux, an increase in mesenchymal markers, loss of ECs and significant changes in the structure of the ECs. In in vitro experiments, deletion of ATG7 led to upregulation of key pro-fibrotic genes and TGF-β signaling. In in vivo experiments, mice with specific knockout of ATG7 in the ECs exhibited a reduction in endothelial-specific markers. Higher sensitivity to collagen accumulation and bleomycin-induced PF was also observed. The study provided novel evidence indicating that the loss of in vivo endothelial autophagy exacerbates the fibrotic response in mice. This occurs through a regulatory effect of autophagy to restrain TGF-β-dependent EndMT. These findings suggest that it is possible that autophagy regulates the crosstalk between EndMT and organ fibrosis. The autophagy gene ATG7 has been demonstrated to regulate organ fibrosis by modulating shifts in EndMT (172).

Autophagy is able to change the secretory phenotype of tissue cells. For example, autophagy leads to a secretory phenotype that facilitates the production of profibrotic cytokines, which results in the secretion of the corresponding pro-fibrotic cytokines, leading to fibrotic disease progression. Livingston et al (173) found that autophagy was sustained at high levels in renal tubular cells after ischemic acute kidney injury, leading to the expression and secretion of fibroblast growth factor (FGF)2. FGF2 is a key paracrine factor produced by renal tubular cells of the secretory phenotype that promotes fibroblast activation and interstitial fibrosis during maladaptive renal repair (173). Zhou et al (154) demonstrated that ERS in fibroblasts triggers the upregulation of autophagy, which promotes the phenotypic transformation of fibroblasts and the synthesis and secretion of collagen. The study also found that autophagy regulates the secretion of inflammatory factors, which contribute to the development of fibrotic diseases. In a study by Nam et al (174), after unilateral ureteral obstruction, autophagy induced in distal renal tubular epithelial cells was demonstrated to confer protection against renal tubular interstitial fibrosis by modulating the TGF-β/SMAD4 signaling pathway and the NLRP3 inflammatory vesicle/caspase-1/IL-1β signaling pathway.