Qiu-Cheng Li, Qi Li,2,3, Pan-Hong Jia, Shao-Ning Li, Xiao-Man Xiong, Xiang-Dong Zhou,2,3?

1. Department of Respiratory Medicine, the First Affiliated Hospital of Hainan Medical College, Haikou 570102, China

2. Key Laboratory of Hainan Trauma and Disaster Rescue, Haikou 571199, China

3. Research Unit of Island Emergency Medicine, Chinese Academy of Medical Sciences 2019RU013, Hainan Medical University, Haikou 571199,China

Keywords:Autophagy Acute lung injury Infectious factors Non-infectious factors

ABSTRACT Autophagy is a highly conserved catabolic process, involving the degradation of cellular components, including damaged organelles, denatured proteins and various pathogens. It is a defense and stress mechanism for maintaining cell and tissue homeostasis. More and more evidences show that autophagy is closely related to many diseases. Acute lung injury is caused by intrapulmonary or extrapulmonary factors, with hypoxia and low lung compliance as the main manifestations. At present, its pathogenesis is not clear. It is known that the imbalance of inflammatory response and oxidative stress mechanism are the important mechanisms of its occurrence. The inflammatory response caused by acute lung injury has been reported to involve a series of changes in autophagy expression. Autophagy may be protective or harmful in acute lung injury. This article will summarize the respective roles of autophagy in acute lung injury caused by infectious factors (such as pathogen infection, lipopolysaccharide, sepsis) and non-infectious factors (acute pancreatitis, chlorine poisoning), so as to provide new ideas and strategies for better prevention of acute lung injury.?Corresponding author:ZHOU Xiang-dong, Ph.D., Chief Physician.

Acute lung injury (ALI) is a group of syndromes caused by various factors based on the increase in alveolar and capillary permeability, leading to alveolar edema with a large number of inflammatory cell infiltration, and then acute respiratory distress syndrome. The fatality rate can be as high as 50%[1].The etiology of ALI is complex, including infectious factors (such as bacteria,viruses) and non-infectious factors (such as acute pancreatitis,inhalation of harmful gases). The current pathogenesis of ALI is not clear, so it is urgent to explore the pathogenesis of ALI. The current main treatment options include glucocorticoid anti-inflammatory,mechanical ventilation to improve oxygen saturation and so on.Autophagy plays different physiological and pathological roles in animal cells[2]. Studies have shown that regulating autophagy can reduce ALI, suggesting that autophagy may become a new target for ALI treatment[3]. However, the role of autophagy in ALI caused by different etiologies is not the same.

1. Autophagy and its related mechanisms

Autophagy is the process of cell catabolism, through the lysosomemediated removal of cytoplasmic defects, damaged organelles,denatured proteins and pathogens, etc., which is conducive to the normal functioning of cells and the maintenance of homeostasis. It is also the body's defense. Stress regulation mechanism[4]. Autophagy can be divided into three types: macroautophagy, microautophagy and molecular chaperone-mediated autophagy according to different lysosome entry pathways[3]. Macroautophagy is also called autophagy and is most closely related to ALI.Autophagy mainly includes 5 parts[5]: (1) initiation of the autophagy process (induction of phagocytic cell membrane); (2) phagocytic vesicle formation; (3)autophagosome extension and maturation; (4) phagocytic-lysosome The fusion of the complex; (5) Degradation of phagosomes. The above process is mediated by autophagy-related genes(ATG)to form autophagy-related protein families (ATGs). At present, more than 30 autophagy-related genes have been found in yeast genetics, and 11 related genes have been identified in mammals[6]. Mercer et al.[7]explained the change process of autophagy in detail from a molecular perspective.The ATGs that are obviously related to the regulation of autophagy are mainly divided into four categories[8,9]: ①Atg1/unc-51-like kinase initiation complex (ULK1), including ULK1,ATG1(yeast) and Atg101, passed in mammals ULK1 acts on the target of rapamycin (mTOR) to regulate autophagy initiation; ②Class III phosphatidylinositol 3-kinase nucleation complex, including Atg18/Atg2 complex, PI3KC3, Vps34, Beclin-1 and Atg6, which are involved The formation of phagocytic vesicle core; ③Microtubuleassociated protein light chain 3 and two ubiquitin complexes (LC3/ATG8, Atg5-Atg16L1-ATG12), participate in autophagosome extension; ④Transmembrane-related proteins (ATG9, Atg18, WIPI-1 and VMP1) regulate the maturation of autophagosomes. The formed autophagosome is fused with the lysosome and combined to produce an autophagolysoso-me complex. The surrounding substances are decomposed by proteolytic enzymes, and the active ingredients are reabsorbed and utilized again. The activation of autophagy depends on the dissociation of mTOR and ULK1 complex. The autophagy regulatory pathway includes the following three pathways: ① The mTOR pathway inhibits Atg1 from recruiting Atg13 and Atg17 through mTOR to inhibit autophagy. mTOR is the main regulatory target for autophagy induction. Rapamycin is a commonly used mTOR inhibitor. Inhibiting the activation of mTOR sites can regulate the intensity of autophagy[10,11]; ②Atg6/Beclin-1 mediated pathway, which forms a complex with class III PI3K Vps34, which is a 3-methyladenine drug inhibitor Target, Beclin-1 is an important interface between autophagy and apoptosis pathways. Anti-apoptotic proteins Bcl-2 and Bcl-XL combined with Beclin-1 can inhibit autophagy[12]; ③Two ubiquitin-like pathways In the binding process,Atg7 and Atg10 mediate the binding of Atg12 to Atg5/19, and then interact with Atg16/20. The second key conjugation reaction involves Atg8 or microtubule-associated protein light chain 3 (LC3)[13].

2. The role of autophagy in ALI caused by infectious factors

2.1 ALI caused by Pseudomonas aeruginosa infection

Pseudomonas aeruginosa (PA) is an opportunistically pathogenic gram-negative bacillus and an extracellular pathogen. Annexin A2(AnxA2), a member of the Annexin family, is expressed in a variety of cells (such as endothelial cells, monocytes, and macrophages).AnxA2 is used in non-small cell lung cancer, chronic obstructive pulmonary disease (COPD) and chronic inflammatory diseases It plays multiple roles in lung diseases and participates in the functional expression of multiple cells at the same time; inhibits the activity of AnxA2, and the phagocytic ability of peritoneal macrophages is reduced, suggesting that AnxA2 is involved in the endocytosis of macrophages and EGFR-mediated signal transduction[14,15]. Using wild-type mice to compare with AnxA2-/-mice found that the survival rate of AnxA2-/-mice after PA infection was reduced, the inflammatory response was more obvious, and the lung parenchyma damage was more serious[16]. IFN-γ is associated with Pseudomonas aeruginosa infection. Pretreatment of cells with IFN-γ can enhance autophagy and reduce the survival rate of bacteria. However, when the expression of Beclin-1 is knocked out,the bacterial clearance rate is reduced, suggesting the inhibition of autophagy. Phagocytosis can attenuate the killing function of lung macrophages(AMs)[17]. The autophagy induced by Pseudomonas aeruginosa is mediated by the AnxA2-Akt1-mTOR-ULK1/2 and Beclin-1-ATG7-ATG5 signaling pathways[17,18]. Recent studies have shown that PA activates Toll-like receptor 2 (TLR2)through Src kinase Lyn-mediated heterophagocytic degradation of AMs,and Wnt5A-Rac1-Disheveled pathway has also been used to induce heterogeneous phagocytosis of AMs[19,20]. It can be seen from the above that when PA infection causes ALI, up-regulation of autophagy facilitates bacterial clearance, reduces lung inflammation,and has a protective effect on the body. AnxA2 gene expression,Beclin-1-ATG7-ATG signal pathway activation,TLR2 activation,are involved PA infection initiates the process of autophagy.

2.2 ALI caused by H1N1 virus infection

Studies have reported that H1N1 virus infection can promote the expression of circ-GATAD2A(circular RNA GATA Zinc Finger Domain Containing2A). The formation of circ-GATAD2A promotes virus replication by inhibiting autophagy; knocking out the Vps34 gene, inhibiting class III phosphatidylinositol The formation of 3-kinase nucleation complex can also promote virus replication and aggravate lung injury[21]. After epithelial cells are infected with influenza A virus,the Atg8/LC3-II autophagy markers in human lung epithelial cells are significantly increased. Influenza virus infection inhibits the fusion of autophagosomes with acidified LAMP1 lysosomes, thereby preventing the autophagy substrates.Degradation;Viral M2 protein targets Atg6/Beclin-1, interferes with the binding of Atg6/Beclin-1 and PI-3 kinase complex, and inhibits the fusion of autophagosomes and lysosomes through the interaction with LC3.After the degradation of autophagosomes, the down-regulation of autophagy leads to the death of virus-infected lung tissue cells and the increase of virus release [22]. Nowadays, therapeutic drugs such as oseltamivir, etc.The literature proves that oseltamivir can induce autophagy of H1N1 virus to achieve the purpose of eliminating the virus[23]. The above suggests that the mechanism of ALI caused by H1N1 infection is related to the weakening of autophagy. Upregulation of autophagy expression can improve the body's ability to clear viruses, reduce virus release, and achieve the effect of treating H1N1 causing ALI.

2.3 ALI caused by H5N1 virus infection

H5N1 induces JNK phosphorylation through the TAK1-MKK4 pathway, and further phosphorylates Bcl-2 to promote the dissociation of the Bcl-2-Beclin1 complex. The activation of the JNK signaling pathway can up-regulate the level of autophagy and promote virus replication[24]. Studies have found that H5N1 virus can also regulate autophagy by activating the Akt-TSC2-mTOR signaling pathway. The expression of autophagy marker LC3-II in human lung A549 cells increases when the virus is infected.When 3-MA and Atg5 siRNA are used to inhibit autophagy, it can increase The viability of A549 cells reduces lung injury[25]. Avian influenza virus can cause autophagy death of lung epithelial cells,which may induce autophagy death through kinase AKT, tumor suppressor protein TSC2, and mTOR target. Specific autophagy inhibitors can alleviate autophagy caused by H5N1 Sexual death and acute lung injury in mice[25,26].The above studies show that the level of autophagy is positively correlated with virus replication.Excessive autophagy is closely related to ALI. Down-regulating autophagy expression can inhibit virus replication and reduce lung damage. However,some studies have shown that specific methods of inducing autophagy can inhibit the replication of influenza A virus. For example, Tu elongation factor (TUFM) can be used as a host restriction factor to inhibit the replication of avian influenza virus in human cells. It interacts with NLRX1 to enhance autophagy.Autophagy prevents the replication of avian influenza virus in human cells [27].

2.4 ALI caused by new coronavirus (COVID-19) infection

The new type of coronavirus belongs to βCoV, and the formation of ALI caused by the new type of coronavirus is the main cause of its death. Studies have confirmed that βCoV induces the formation of ATG5-dependent autophagosomes in infected cells through NSP6 (nonstructural protein 6), and replicates through the doublemembrane vesicles (DMV) of autophagosomes[28,29]. Analysis of the genome sequence of COVID-19 revealed that NSP6 gene mutation, as a multi-pathway transmembrane protein, has multiple phenylalanine residues on its surface, which destroys autophagy by binding to the endoplasmic reticulum(ER) membrane The degradation of the virus by the body, and the expression of the NSP6 gene has an inducing effect on the formation of autophagosomes[30].During viral infection, unfolded protein(UPR)can accumulate and be activated in the endoplasmic reticulum. As a source of viral protein,it participates in the formation of double-membrane vesicles and assists in virus replication. In addition,UPR and autophagy are related to each other, and induction of UPR can promote Autophagy,so COVID-19 infection may induce autophagy through UPR[31,32].Autophagy inhibitors prevent the fusion of autophagosomes with lysosomes, inhibit autophagy flux and increase the accumulation of autophagosomes. Autophagosomes can induce apoptosis of virusinfected cells and disrupt the virus replication cycle. It exists during virus infection In the case of autophagy inhibitors, the interference of multiple processes can inhibit the replication of the virus; combined with the mechanism of action of chloroquine(CQ)as an antiviral drug, it indicates that these autophagy inhibitors may interrupt the early steps of the virus life cycle, that is, the virus Fusion with lysosomes, thereby reducing virus replication and protecting cells from virus-induced cell death[33]. In summary, the new coronavirus up-regulates autophagy to provide the basis for its replication,accelerate cell death, and aggravate lung tissue cell damage.Inhibition of autophagy may be one of the treatment options for the new coronavirus. More research is needed to clarify these autophagy The exact mechanism of action of the inhibitor and COVID-19 and the impact at different stages of the virus life cycle.

2.5 ALI caused by LPS

LPS is a pathogen-associated molecular pattern (PAMP) that can recognize bacterial invasion and activate the innate immune system.LPS stimulation can regulate the autophagy of lung epithelial cells, lung endothelial cells and AMs. In Atg4b-deficient mice,ATF3 activity is weakened after LPS stimulation, and the lungs are more sensitive to LPS-mediated damage;LPS can increase pulmonary microvascular endothelial cells.Permeability, aggravating inflammation and exudation, is one of the reasons for the clinical manifestations of ALI. After using siATG5, siATG7 or chloroquine to inhibit autophagy, it can significantly increase the permeability of pulmonary microvascular endothelial cells and aggravate LPSinduced lung injury in mice. Autophagy has a protective effect in LPS-induced lung injury[34,35]. Studies have reported that TLR4 or MYD88 gene knockout mice can effectively reduce LPS-induced MTOR activation and enhance autophagy-related marker LC3B, and studies have shown that MTOR positively regulates the activation of cell NF-κB, suggesting that LPS can activate TLR4/ MYD88-MTOR-NF-κB signaling pathway inhibits autophagy and regulates the release of inflammatory factors [36]. At the same time, AMPK is an inhibitor of mTOR and an autophagy activator. AMPK inhibits the activation of mTOR through phosphorylation of TSC2 (mTOR inhibitor), thereby preventing the inhibitory phosphorylation of ULK1 and promoting autophagy expression. LPS induces AMPK inactivation, leading to mTOR Increased activation and reduced ULK1 activity inhibited autophagy [37].

Calcium/calmodulin-dependent protein kinases (CaMK) are a family of serine/threonine kina ses that are sensitive to intracellular calcium ion concentration. CaMKKβ can be used as the upstream kinase of AMP kinase and up-regulate intracellular calcium ion concentration through Bcl-2 Thereby regulating autophagy;activated CaMKIα can phosphorylate AMPK to form a CaMKIα-AMPK-ATG7 complex. Autophagy mediated by this signaling pathway significantly attenuates LPS-induced lung neutrophil inflammation[38]. Lipoxins (LXs) are endogenous lipids synthesized by immune cells (such as macrophages and neutrophils), which have anti-inflammat-ory and pro-degradation effects. The increase of LXs in the lung microenvironment promotes neutrophils Apoptosis, while enhancing the phagocytosis/clearance of apoptotic neutrophils by macrophages, has dual effects of anti-inflammatory and promoting decomposition; research has found that BML-111 (Lipoprotein A4 receptor agonist) stimulates the autophagy of AMs through MAPK And inhibit apoptosis, reduce ALI-related inflammation and tissue damage[39]. In summary, in ALI caused by LPS, autophagy,as a defense and stress mechanism, is beneficial to maintain the permeability of pulmonary microvascular endothelial cells, reduce inflammation, improve symptoms of pulmonary edema, and prolong survival time. A moderate increase in autophagy can be a potential therapeutic target for LPS-induced ALI.

However, in some studies using 3-MA to inhibit autophagy,the LPS-induced lung inflammation in mice was significantly inhibited; LPS stimulated autophagy to promote actin cytoskeleton rearrangement and VE-cadherin cleavage, thereby destroying the lung blood barrier function Inhibition of autophagy can prevent the decomposition of VE-cadherin, reduce barrier dysfunction,and improve pulmonary vascular damage after the action of LPS;studies have shown that mice that knock out the Atg5 gene can reduce the release of LPS to the contents of neutrophil granules Induce and reduce the damage of inflammatory factors to the lung endothelial cell barrier and the inflammatory response, thereby reducing ALI[40]. Studies have shown that by inhibiting p38 MAPK activation and TLR4/NF-κB signal activation, it can down-regulate autophagy and reduce the level of inflammatory factors to improve lung function[41,42]. The reason is that LPS can promote excessive autophagy of neutrophils, stimulate the release of granular contents,and increase pulmonary microvascular permeability. Treatment with 3-MA can inhibit the activation of TLR4/NF-κB and p38 MAPK and reduce ALI, indicating excessive Autophagy is also one of the mechanisms by which LPS causes ALI.

2.6 ALI due to sepsis

In a mouse model of sepsis induced by cecal ligation and puncture (CLP), the levels of LC3-II, ATG5, and ATG7 in the lungs of sepsis mice were down-regulated, indicating that sepsis may inhibit autophagy; Paamycin or activated protein C(APC)stimulates autophagy to reduce inflammation and reduce lung injury,suggesting that autophagy can be upregulated as a potential target for the treatment of sepsis[43]. Studies have found that low physiological doses of carbon monoxide(CO)can enhance the Beclin-1-dependent autophagy process in lung epithelial cells, thereby improving the survival rate of septic mice, and knock-out Beclin-1 mice CO affect pus The protective effect of toxemia is weakened[44]. Some studies have found that the level of LC3-II in the lungs of septic mice is significantly increased; compared with wild-type mice, mice with high expression of the LC3 gene have accelerated the fusion of autophagosomes and lysosomes. After the survival time is prolonged,the study concluded that the role of autophagy in sepsis may be related to the autophagy flux: the preservation of autophagy flux has a cytoprotective effect on sepsis, and the autophagy flux is impaired.Phage accumulation may be one of the causes of lung injury in the late stage of sepsis[45]. In summary, autophagy plays a protective role in ALI caused by sepsis, and proper regulation of autophagy can effectively reduce septic lung injury.

2.7 Inflammasome and autophagy

Inflammasomes are a type of macromolecules composed of intracellular pattern recognition receptors (PRRs), namely NOD-like receptors(NLRs), including risk-related molecular patterns (DAMPs)and pathogen-related molecular patterns (PAMPs). Complex[46]. It can activate NLRP3 under the stimulation of microbial infections such as bacteria, viruses, fungi, and small toxic particles such as silica and asbestos[47]. After NLRP3 is activated, the assembled NLRP3 inflammasome can split inactive caspase-1 into active caspase-1, and activated caspase-1 can cleave pro-IL-1β and pro-IL-18 into Active IL-1β and IL-18 are released outside the cell[48].IL-1β and IL-18 can stimulate the production of inflammatory factors, regulate the accumulation of macrophages and neutrophils,and trigger the "cascade effect" of inflammation to damage alveolar epithelial cells, leading to increased alveolar permeability and increasing the incidence of pulmonary edema.

Pathogen infection and related products can cause the activation of inflammasomes in the body, and uncontrolled inflammation is closely related to acute lung injury. LPS and sepsis cause the high expression of caspase-1, IL-1β and IL-18 in the plasma of ALI,suggesting that LPS/sepsis can cause the caspase-1-dependent inflammasome pathway to be activated, and IL-18 in circulation The increase of LPS is related to disease severity and mortality; at the same time, LPS can up-regulate the expression of IL-1RI on the surface of macrophages through the NF-κB signaling pathway,leading to pyroptosis of alveolar macrophages and aggravating lung inflammation[49,50]. NLRP3 inflammasome can stimulate the release of inflammatory factors on the one hand, and on the other hand can activate macrophages to aggravate lung injury.In LPS-induced ALI, a large number of abnormal mitochondria accumulate in macrophages of autophagy-deficient mice, activate NLRP3 inflammasome,and increase the mortality of mice; Atg7 gene knockout mice and autophagy protein Atg16l1 deficient mice and wild-type Compared with mice, NLRP3 inflammasomes in their bodies were activated,and the expression of IL-1β and IL-18 increased, suggesting that autophagy can regulate the activity of inflammasomes and reduce the release of inflammatory factors[51]. Damaged mitochondrial DNA is one of the main factors for activating inflammasomes. lc3b-/-or Beclinl+/-macrophages tend to accumulate abnormal mitochondria and produce a large amount of ROS. After the action of LPS, the mitochondria are prone to more serious structural disorders. And dysfunction, it stimulates the activation of inflammasomes and the release of inflammatory mediators, suggesting that autophagy inhibits its activation by eliminating the components that induce inflammasome activation[52]. It can be seen that pathogen infection can cause the activation of inflammasomes, promote the release of inflammatory factors and even trigger the "inflammatory waterfall effect". Enhancing autophagy can clear the activated inflammasomes and reduce the damage of inflammatory response to lung tissue cells.

3. The role of autophagy in ALI caused by noninfectious factors

3.1 ALI caused by Cl2

Cl2 exposure to lung epithelial cells can lead to mitochondrial dysfunction and ROS accumulation, which may be the main cause of lung injury[53]. Studies have shown that Cl2can promote the formation of mitochondrial superoxide, which in turn affects mitochondrial oxygen consumption, membrane potential and glycolysis, and inhibits the activity of complexes in the mitochondrial transport chain; after Cl2interferes with cells, the level of autophagy marker LC3-BII increases High, the level of p62 protein decreases,and the decrease of p62 level is considered to be a manifestation of autophagy. After using an autophagy activator (trehalose) to interfere with Cl2exposed cells, it is found that their mitochondrial respiratory function is improved; instead, 3-MA treatment is used to expose Cl2The latter cells may further develop biological dysfunction[54]. These results indicate that Cl2exposure leads to an increase in autophagy,and autophagy has a protective effect in Cl2induced lung injury.Properly enhancing autophagy has a protective effect on Cl2induced ALI. The mechanism may be to prevent mitochondrial damage by inducing autophagy. , Reduce inflammation and improve Cl2toxicity.

3.2 ALI caused by acute pancreatitis

Acute pancreatitis(AP)activates the TLR4/NF-κB signaling pathway, induces the release of TNF-α, IL-1β, IL-6 and other inflammatory factors, further recruits inflammatory cells and promotes the occurrence of inflammation. The systemic inflammatory response is acute The pathological features of pancreatitis are also the main cause of ALI[55]. Diakopoulos et al.[56]found through Atg5 gene-deficient mice that normal autophagy plays an important role in maintaining pancreatic homeostasis;abnormal autophagy is the key point that causes AP pathological damage. During AP, autophagy flux is impaired. The aggregation of autophagosomes activates trypsinogen, suggesting that AP is closely related to impaired autophagic flux[57]. A large number of inflammatory mediators and cytokines produced during AP act on Toll-like receptors, activate the NF-κB signaling pathway, and cascade to produce a large number of inflammatory factors, leading to local or systemic explosive inflammatory response; autophagy is impaired, and it is inflammatory The mediator clearance ability was significantly weakened, and further activated the inflammasome to aggravate the inflammatory response and damage tissue cells; the activation of NF-κB and the production of TNF-α in mice with Beclin-1 knockout were increased compared with wild-type mice[58].At the same time, p62 cannot be degraded in time when autophagy is damaged, and p62 can further activate NF-κB, release a large number of inflammatory factors, and aggravate tissue damage. In summary, acute pancreatitis is closely related to abnormal autophagy,and the inflammatory response is regulated by activating the TLR4/NF-κB signaling pathway to aggravate lung tissue damage, but the causal relationship between impaired autophagy and AP is not yet clear, suggesting that it may be regulated Abnormal autophagy, and then inhibit the activation of NF-κB pathway to achieve the purpose of treating AP-induced ALI.

4. Outlook

At present, significant progress has been made regarding the role of autophagy in ALI, and autophagy plays different roles in different modes of ALI, but its specific molecular mechanisms still need to be further explored. The onset of ALI is rapid and the condition is dangerous. At present, there is no definite treatment method for ALI.The treatment of inhibiting or activating autophagy has a positive effect on many factors causing ALI. At the same time, autophagy has a potential therapeutic target for the new coronavirus. To provide new ideas for effective treatment of the new coronavirus. At present, most researches on autophagy are based on cell or animal experiments, and it takes a long time to explore the transformation of the above-mentioned treatment methods into clinical applications.Future research needs to explore specific markers of autophagy expression in the human body to provide a basis for realizing the regulation of autophagy expression to achieve the purpose of treating diseases.

Author’s contribution

The first author: Li Qiucheng: collect relevant documents and write articles; Corresponding author: Zhou Xiangdong: article project conception and review; other authors participate in document collection and analysis.