Macrophages represent a highly heterogeneous class of immune cells that serve a pivotal role in the body's immune system. Macrophages can polarize into different phenotypes depending on stimuli from various tissue microenvironments. The primary phenotypes are inflammatory or classically activated macrophages (M1) and alternatively activated macrophages (M2) that promote healing. In response to inflammatory stimuli, the actions of LPS or interferon, whether alone or synergistically, promote macrophage polarization towards the M1 phenotype, thereby increasing the release of pro-inflammatory factors [such as TNF-α, nitric oxide (NO), IL-1β and IL-6], which are essential for pathogen elimination and defense (19). During the repair phase of late inflammation, M1 macrophages polarize toward M2 macrophages, secreting anti-inflammatory factors (including IL-4, IL-10, IL-13 and TGF-β) to inhibit the inflammatory response and facilitate tissue repair and remodeling (Fig. 1). Dang et al (20) noted that macrophage polarization within the lungs has a notable influence on the onset of, and recovery from, septic lung injuries. An imbalance in the shift between M1 and M2 macrophages, which leads to ongoing production of pro-inflammatory mediators, can result in tissue harm. It has previously been reported that, in cases of SALI, M1 macrophages exacerbate lung damage due to unchecked inflammatory responses (21). Additionally, Li et al (22) demonstrated that in Traditional Chinese Medicine (TCM), the compound Lianhua Qingdian effectively alleviates SALI by enhancing the infiltration of M2 macrophages. This mechanism may involve promoting the transformation of macrophages from the M1 to the M2 phenotype by activating the peroxisome proliferator-activated receptor γ signaling pathway, inhibiting the NF-κB signaling pathway and alleviating inflammatory responses. However, it is important to note that these results were obtained from a mouse model of LPS-induced ALI and there is still an overall lack of supporting clinical data in humans.

M2 macrophages possess anti-inflammatory and tissue repair properties, thus serving a key role in the recovery process of patients with ARDS. Yang et al (23) found that, in LPS-induced ARDS mouse models, the M2/M1 alveolar macrophage ratio notably decreased as the severity of lung injury increased. Conversely, the intratracheal administration of exogenous M2 alveolar macrophage-derived EVs markedly improved LPS-induced ARDS yet reduced the levels of inflammatory factors in bronchoalveolar lavage fluid. Similarly, in patients with ARDS who underwent continuous mechanical ventilation or succumbed to disease within 28 days, there was found to be a persistent M1 phenotype and insufficient M2 transformation (24). It may be inferred that the anti-inflammatory and pro-repair effects mediated by M2 macrophages are key for alveolar epithelial cell reconstruction and pulmonary function recovery in patients with ARDS during the recovery phase.

Chemokines are a class of small molecular weight cytokines produced by leukocytes in response to external stimuli. During the inflammatory response, chemokines facilitate the migration of circulating leukocytes to injured tissues by recruiting and activating immune cells, thereby forming a concentration gradient, which is further established by the binding of chemokines to glycosaminoglycans in the extracellular matrix and endothelium (26). Neutrophils tend to be the first immune cells to reach the site of infection, where they serve a key role in engulfing pathogens and clearing cellular debris through phagocytosis. Subsequently, monocytes migrate to the infection, where they transform into macrophages, continuing the fight against pathogens while releasing various cytokines, including TNF-α, IL-1β, IL-6, prostaglandins and leukotrienes. The chemokine family is generally divided into four categories: Two primary subfamilies (CXC and CC) and two secondary subfamilies (CX3C and C). The distinct chemokine groups interact with specific receptors found on various cell types and are coordinated with the adhesion molecules present, thus contributing to the inflammatory response (27).

Chemokine receptors are part of the heptameric transmembrane guanosine triphosphate-binding G protein-coupled receptor superfamily, which trigger an intracellular signaling cascade through the associated trimeric G proteins. This mechanism promotes the adhesion of target cells to the endothelial lining and directs their migration toward the infection site (28). Among these, monocyte chemoattractant protein 1 (MCP-1) (29), also known as CC chemokine ligand 2, is classified within the CC subfamily of chemokines. In the early stages of inflammation, MCP-1 attaches to the CC motif chemokine receptor 2 receptor, stimulating the accumulation of monocytes and their transformation into macrophages (30). MCP-1 serves a key role in directing the appropriate immune response associated with infection and inflammation (31). A different category of CXC chemokines, such as macrophage inflammatory protein 2 [also referred to as C-X-C motif chemokine ligand (CXCL) 2] and CXCL8 (often called IL-8), are able to promote the infiltration of innate immune cells and work in conjunction with lipid signaling. Additionally, prostaglandin E2 (PGE2) and leukotriene B4, produced by ω-6 polyunsaturated fatty acid, serve as pro-inflammatory lipid mediators that activate inflammatory vesicles and initiate the endoplasmic reticulum (ER) stress response (32). These processes serve a key role in the onset, progression and resolution of inflammation.

The mitochondrial apoptotic pathway is endogenous. When triggered by factors such as DNA damage, metabolic stress, ER stress or growth factor deprivation, the integrity of the mitochondrial membrane is disrupted (37,38). Increased mitochondrial membrane permeability leads to the release of cytochrome c (Cyt c) from the mitochondria into the cytoplasm. Cyt c interacts with apoptosis-activating factor 1 to create an ATP-dependent apoptotic complex. This complex activates caspase-9, which subsequently triggers the activation of downstream caspases-3 and −7, consequently initiating a cascade of caspase reactions that leads to apoptosis (Fig. 2). Furthermore, once released from the mitochondria, Cyt c can bind to inositol triphosphate receptors located in the ER. This binding results in an elevation of local calcium levels, which then enhances the release of Cyt c and triggers the initiation of apoptosis (39). Conversely, protein tyrosine phosphatase, which is located between the inner and outer membranes of the mitochondria, can facilitate the creation of the mitochondrial permeability transition pore (MPTP), permitting the movement of molecules of ≤1.5 kDa in size. Abnormal MPTP opening has been found to impair mitochondrial function and promote apoptosis (40).

Mitochondria serve as the primary source of cellular energy and are also the principal site for the production of intracellular ROS. During apoptosis, mitochondrial damage and ROS production exacerbate each other, resulting in a vicious cycle (41). ROS can compromise both the integrity and functionality of the mitochondrial membrane, resulting in the release of apoptotic factors, which in turn, further enhances ROS production, aggravating mitochondrial impairment (42). Wang et al (43) induced oxidative stress in Hepa1-6 cells through the application of fluorine (F), which led to increased levels of intracellular ROS and propane-1,2-diol and mitochondrial injury. The oxidative stress induced by F resulted in marked elevations in intracellular ROS, malondialdehyde (MDA) and NO concentrations. Furthermore, there was a notable upregulation in the protein expression of Cyt c, caspase-9 and caspase-3, along with a considerable increase in apoptotic cell count and extensive mitochondrial vacuolation, as evidenced by transmission electron microscopy. This demonstrated that ROS mediates mitochondrial damage, thereby exacerbating apoptotic injury.

The p53 protein functions as a transcription factor, triggering the expression of various target genes and serving a key role in maintaining genomic stability, regulating the cell cycle, facilitating DNA repair and promoting apoptosis. Under standard physiological circumstances, p53 is targeted for degradation by murine double minute 2 homolog (MDM2) and murine double minute X (MDMX), which ubiquitinate it, thereby keeping its intracellular levels low through proteasomal degradation. By contrast, when cells face stressors such as DNA damage, hypoxia or infection, the process of p53 ubiquitination is suppressed, causing a rapid increase in its cellular concentrations (47). Various sensor proteins, such as the ataxia-telangiectasia-mutated protein, ataxia-telangiectasia and Rad3-related protein, checkpoint kinase 1, checkpoint kinase 2, DNA-dependent protein kinase and the p14ARF protein, become activated and p53 stability is enhanced through post-translational modifications, including acetylation, methylation and phosphorylation. Stabilized p53 proteins form tetramers in the nucleus that bind to p53 response elements on target DNA, thereby regulating gene transcription (48). On one side, p53 is involved in apoptosis mediated by mitochondria through activating the transcription of pro-apoptotic proteins such as Bax, Puma and Bid. Conversely, p53 can trigger apoptosis without relying on transcription. The p53 protein, in a manner that does not depend on its transcriptional functions, translocates to the mitochondria, where it competes with Mcl-1 for binding to Bak through protein interactions, leading to the release of Bak from Mcl-1 and initiates Bak oligomerization (49). Furthermore, p53 interacts with Bcl-XL, prompting the detachment of Bax from the Bax/Bcl-XL complex, subsequently enhancing oligomerization. The oligomerization of Bax and Bak modifies the permeability of the outer mitochondrial membrane, allowing the release of Cyt c into the cytoplasm, which then activates caspase proteases, ultimately prompting apoptosis (50).

In addition, the activation of p53 can lead to the disruption of the EC cytoskeleton, increased vascular permeability and the induction of apoptosis in alveolar type II epithelial cells, severely affecting the synthesis and secretion of pulmonary surfactant (PS). This results in increased alveolar surface tension, alveolar collapse and ventilator-associated lung injury. Previous research has found that MDM2 promotes the degradation of p53 protein through the ubiquitination pathway, maintaining p53 at low levels. When MDM2 function is lost, uncontrolled activation of p53 leads to apoptosis, barrier disruption and amplification of inflammation, ultimately exacerbating lung injury (51).

Oxidative stress influences apoptosis through various pathways. An important component of this process is the action of ROS, which function as ‘redox messengers’ and are crucial for redox regulation and cellular signaling. Generally, ROS are understood to encompass free radicals derived from oxygen (O₂), including superoxide anion (O₂⁻), hydroxyl radical (HO⁻), peroxyl radical and alkoxyl radical, along with non-free radical O₂ derivatives such as hydrogen peroxide (H₂O₂). However, ROS, once produced in excess, may lead to oxidative modification of cellular macromolecules, which in turn may cause notable damage to cellular proteins and DNA. Mokrá (54) suggested that oxidative stress is not only an important causative factor in ALI but may also contribute to extensive damage to lung tissues and worsening inflammatory responses through activation of apoptotic pathways.

Mitochondria serve as the primary location for the production of ROS, with 1–2% of the O₂ consumed by mitochondria being utilized for the generation of ROS, particularly during the electron transfer process to O₂ within the electron transport chain. In the presence of inflammation and tissue injury, a marked increase in the release of mitochondrial ROS (mtROS) occurs. This increase may result in mitochondrial membrane depolarization that relies on the pore-forming protein gasdermin D (GSDMD). Consequently, there is a reduction in the mitochondrial membrane potential, prompting GSDMD to associate with the mitochondrial membrane, thereby forming a pore. This process ultimately enhances the permeability of the mitochondrial membrane, facilitates the release of apoptotic factors and triggers the activation of mitochondrial apoptotic pathways. In addition, it has been found that ROS can also inhibit the degradation and transport of mitochondrial proteins, which is the main cause of mitochondrial dysfunction (55). In addition, oxidative stress can trigger the activation of transcription factors such as NF-κB and p53 (56), influencing the expression and functionality of proteins associated with apoptosis (such as Bcl-2). Previous research has demonstrated that, in cells treated with H₂O₂, there is a marked reduction in Bcl-2 protein levels (57), thereby modulating the apoptotic process.

Under normal physiological conditions, apoptotic cells are cleared through phagocytosis, primarily by macrophages, thereby preventing the initiation of an inflammatory response. Effective clearance of apoptotic cells is key for managing inflammatory diseases, preventing secondary necrosis and restoring normal tissue function. Phagocytes are guided by chemokine ‘find-me’ signals in order to migrate towards apoptotic cells (67). The current four identified signals that facilitate locating cells are chemokine C-X3-C motif ligand 1 (CX3CL1; fractalkine), nucleotide triphosphates [including ATP and uridine triphosphate (UTP)], sphingosine-1 phosphate (S1P) and lysophosphatidylcholine (LysoPC). Among these, the release of ATP, UTP and CX3CL1 occurs during the early stages of apoptosis, while LysoPC and S1P are lipid chemokines produced in the later stages of apoptosis (67).

Phagocytes near apoptotic cells recognize specific ligands on their surface, referred to as ‘eat-me’ signals, including cell-surface calreticulin, ICAM-1 and complement component 1q, which activate signal transduction pathways and facilitate the phagocytosis of apoptotic cells (68). Following phagocytosis, apoptotic cells undergo cytosolic burial, maturing gradually and secreting anti-inflammatory and pro-tissue healing factors. However, when the clearing process is hindered, apoptosis can advance to necrotic apoptosis, which is marked by the rupture of the cell membrane and a marked release of intracellular DAMPs, thereby intensifying inflammation and causing tissue injury.

In normal lung tissue, the process of cytosolic burial helps to avert tissue injury and regulate the inflammatory response, largely facilitated by alveolar macrophages. Compared with other macrophage types, alveolar macrophages exhibit a longer lifespan and an enhanced self-renewal capacity. Under steady-state conditions, even during acute lung inflammation, the efficient cellular clearance of alveolar macrophages results in a minimal presence of apoptotic cells in the airways. When the function of alveolar macrophages is compromised and when apoptotic cells are generated in large quantities and cannot be rapidly cleared through cytosolic burial, a sustained release of DAMPs is prompted, perpetuating the inflammatory response (69). In certain patients with bacterial pneumonia, failure to resolve inflammation promptly or the occurrence of an inflammatory storm, results in impaired lung function and the subsequent development of ALI or ARDS, which further prolongs the inflammatory response in the lungs (70). A previous study has indicated that an increased quantity of uncleared apoptotic cells is found in the airways of patients with ARDS, respiratory failure or chronic lung inflammation, all of which are conditions frequently marked by impairments in the cellular clearance capabilities of alveolar macrophages (71). Therefore, the timely and effective clearance of apoptotic cells is key in modulating the inflammatory response and protecting lung function.

Alveolar type II epithelial cells are capable of secreting the alveolar surface-active substance pulmonary surfactant (PS). The functions of PS include reducing alveolar surface tension, maintaining the relative stability of the alveolar structure, facilitating the absorption of alveolar fluid, maintaining fluid balance in the lungs, preventing pulmonary edema and atelectasis and enhancing the lung's defense function (81,82). PS is a complex formulation that consists primarily of 90% phospholipids and 10% proteins (83). It contains four surfactant proteins (SPs) that are linked to surface-active agents, namely SP-A, SP-B, SP-C and SP-D. Among these, SP-A and SP-D are hydrophilic proteins that are part of the collectin family and are key in innate immune defense. Conversely, SP-B and SP-C are highly hydrophobic apolipoproteins that are key in the biophysical functionalities of surfactants (84). A previous study found that SP-A knockout mice exhibited a higher severity of lung injury and mortality in a severe acute respiratory syndrome coronavirus (COVID) 2-induced mouse ALI model, indicating that SP-A serves a key role in pathogen defense (85).

Under physiological conditions, 80–90% of PS is distributed in large aggregates (LAs) with high surface activity. However, during infection, the upregulation of inflammatory mediators and activation of apoptosis-inducing signals leads to the disruption or inhibition of PS. The content of PS in LAs is markedly reduced, shifting to small aggregates with markedly low surface activity, which are mainly products of PS degradation. Furthermore, activated immune cells generate ROS, which may disrupt surfactant functionality by diminishing the synthesis of SP and phospholipids (86). Additionally, high levels of NO free radicals and TNF-α produced during inflammation can directly impede the production of SP-A, SP-B and SP-C (87). As a result, the synthesis and activity of alveolar surface-active substances are reduced, leading to an increase in alveolar surface tension. On the other hand, the increased alveolar surface tension fails to maintain the normal alveolar structure, leading to alveolar collapse. During mechanical ventilation, atelectatic alveoli may be damaged by the force generated by the cyclic opening and closing of the ventilator, ultimately exacerbating respiratory failure. Dargaville et al (88), through a 2-year follow-up study of preterm infants with ARDS treated with minimally invasive surfactant application, found that infants treated with surfactant had a lower incidence of adverse respiratory outcomes within a period of 2 years. This finding, to some extent, suggests that alveolar surfactant can improve lung injury and maintain normal alveolar function.

A healthy pulmonary endothelium largely suppresses inflammation, maintains blood flow and prevents thrombosis. Some studies have shown that various stimuli, including hypoxia, cytokines, chemokines, thrombin, LPS, DAMPs and apoptosis, can lead to the activation and damage of ECs (94,95). On the one hand, ECs produce endothelium-derived diastolic factors (EDRFs) such as NO and prostacyclin as well as endothelium-derived contractile factors such as endothelin, epoxyeicosatrienoic acid and thromboxane A₂ to regulate vascular tone (96). Activated ECs exhibit impaired synthesis or release of EDRFs, resulting in sustained vasoconstriction, reduced vessel diameter, slowed blood flow and impaired pulmonary microcirculation. On the other hand, activated ECs recruit activated neutrophils and form neutrophil extracellular traps with activated platelets, shifting to a pro-coagulant phenotype. Anticoagulant molecules such as thrombomodulin (TM) are other important markers of endothelial injury. Under normal conditions, TM is expressed on the surface of ECs, where it exerts anticoagulant effects by activating the protein C system through binding to thrombin (97). During endothelial injury, TM is shed from the cell surface into circulation, forming soluble TM (sTM), exposing collagen fibers and other subendothelial matrix proteins. The expression of platelet adhesion molecules is upregulated and platelets are activated and rapidly adhere to form a microthrombus, initiating the hemostatic process. Previous research has shown that patients with ARDS who exhibit high levels of sTM have a 3.5-fold increased risk of 60-day mortality, which can serve as a biomarker for early prognostic assessment, and perhaps provide a reference for clinical risk stratification and treatment decision-making (98).

Coagulation factors interact with tissue factor (TF), which is present on AECs and macrophages, initiating an exogenous coagulation cascade. Within the intact vessel wall, TF remains hidden. Following damage to the vascular endothelium, TF becomes accessible to blood and can bind directly to coagulation factor VII to create the TF-VIIa complex, which is capable of activating clotting factors IX and X in the bloodstream, resulting in the formation of active enzymes (coagulation factors IXa and Xa) that subsequently convert plasminogen to thrombin. Thrombin then cleaves fibrinogen into fibrin, which polymerizes to create a fibrin network that forms a thrombus, incorporating aggregated platelets (99). Naderpour et al (100) studied patients with COVID-19 during the pandemic and found that the combined use of tissue plasminogen activator and heparin in patients with severe respiratory failure improved oxygenation and reduced overall mortality. Thus, impaired pulmonary microcirculation further exacerbates tissue ischemia and hypoxia, promoting EC injury, pro-inflammatory factor release and activation of apoptotic pathways, thereby exacerbating lung tissue injury.

Free radicals serve a key role in intracellular signal transduction and various physiological processes at optimal concentrations. However, excess of these free radicals can lead to oxidative damage to proteins. The body's antioxidant defense system comprises both endogenous antioxidants and exogenous free radical scavenging mechanisms. Endogenous antioxidants primarily eliminate excess free radicals and are categorized into enzymatic and non-enzymatic ROS scavengers. Enzymatic antioxidants, including superoxide dismutase (SOD) (109), catalase (CAT) (110) and glutathione peroxidase (GPX) (111), facilitate the conversion of O₂ radicals into H₂O₂ and O₂, which are further catalyzed into H₂O and O₂. Non-enzymatic ROS scavengers include coenzyme Q10 (also known as ubiquinone) (112,113) and acetylated phospholipids (plasmalogens) (114), among others. Coenzyme Q10 is integral to the mitochondrial electron transport chain, functioning as a component of the mitochondrial respiratory chain while scavenging lipid peroxidation free radicals (113,115). Exogenous free radical scavengers, primarily sourced from dietary intake, encompass hydrophilic agents, such as vitamin C (116) and glutathione, as well as lipophilic agents, including vitamin E (117), flavonoids (118,119) and carotenoids (120), which effectively scavenge O₂⁻ and HO⁻.

Mitochondria, as the primary site of ROS production, are often subjected to high levels of ROS, leading to oxidative damage of mitochondrial DNA. This damage subsequently activates the mitochondrial-associated apoptotic pathway, thus exacerbating lung injury. Antioxidants serve a key role in mitigating inflammation-induced oxidative stress by scavenging free radicals and maintaining the body's redox balance. This action effectively inhibits the mitochondrial apoptotic pathway, thereby protecting lung tissue from the combined detrimental effects of inflammation and apoptosis. Numerous antioxidant drugs have been employed in clinical treatments, including recombinant SOD, vitamin C, vitamin E, α-lipoic acid (121), bioflavonoids, selenium (122), glutathione, coenzyme Q10 (123) and various TCM herbal medicines such as Curcuma longa (124), Astragalus (125) and Rhodiola rosea (126). Curcumin (124), through its β-diketone group and Astragalus (125), through active components such as polysaccharides, saponins and flavonoids, directly scavenge ROS while simultaneously activating the endogenous antioxidant enzyme system, which includes SOD, CAT and GPX. This dual action results in a multi-level, multi-target antioxidant protective effect. Similarly, study has demonstrated that Rhodiola injection not only exhibits direct ROS scavenging capabilities, but also protects mitochondrial function, regulates the AMP-activated protein kinase/mTOR autophagy signaling pathway and maintains the balance between cell apoptosis and survival (126). This creates a comprehensive protective network ranging from oxidative stress prevention to cellular damage repair.

However, there is currently very few related clinical trial available (127). These therapeutic agents face important challenges in clinical application. Previous research has indicated that vitamin E supplementation does not reduce all-cause mortality in patients and high doses of vitamin E may even increase overall mortality (128). A similar issue is observed with vitamin C, whereby excessively high levels in the body can induce oxidative stress-related DNA damage, similar to the effects of lower levels. Potential limitations of antioxidant therapy may arise from the need to preserve an equilibrium between oxidation and reduction. Elevated levels of ROS can activate endogenous antioxidant defenses to protect damaged tissues. Notably, scavenging ROS may increase the risk of infection and disrupt ROS-dependent signaling pathways. Consequently, the timing, concentration and dosage of antioxidants, along with their bioavailability and effective targeted delivery to specific organs, are important factors that are often challenging to regulate in clinical practice.

Oxidative stress is a key mechanism in SALI. Traditional antioxidants, such as vitamin C or vitamin E, have limited clinical efficacy due to their lack of targeting specificity and dose-dependent effects. To overcome this bottleneck, progress has been made in recent years with novel targeted antioxidants and biologics. For instance, developments have been made in mitochondrial-targeted antioxidants such as MitoQ, which has been shown to protect cells from apoptosis and inhibit H₂O₂-induced growth factor receptor signaling. This is primarily achieved through its unique triphenylphosphonium carrier system, which precisely delivers the compound to the interior of mitochondria, directly scavenging mtROS and effectively blocking the mtROS-Ca²⁺/calmodulin-dependent kinase II-mediated apoptotic signaling pathway. In an investigation using MitoQ to intervene in LPS-induced ALI in mice, it was found that, compared with the model group, MitoQ treatment significantly reduced the levels of oxidative markers in mouse serum (a 50% decrease in MDA content; P<0.05), while also improving lung injury in mice (145).

Edaravone, approved in Japan as a free radical scavenger for ischemia-reperfusion injury, primarily works by inhibiting lipid peroxidation and preventing the abnormal opening of the mitochondrial MPTP, thereby reducing apoptosis in alveolar epithelial cells. In a randomized clinical trial, edaravone was used to treat critically ill patients with COVID-19 admitted to the ICU. Results showed that the edaravone treatment group experienced a reduction in mechanical ventilation dependency and a shortened ICU treatment duration (146).

Numerous studies have shown that specific biological substances can alleviate pulmonary injury by mitigating inflammatory oxidative stress, offering new avenues for the clinical management of SALI. For example, the nebulized formulation of recombinant human SOD can specifically neutralize O₂⁻ within lung tissue, directly targeting the sites of inflammation. Furthermore, FGF21, which was initially identified and cloned in 2000, performs various biological roles, including promoting tissue repair and regulating metabolism (147). Research conducted by Gao et al (148) suggested that the administration of exogenous FGF21 leads to a reduction in lung inflammation and apoptosis by influencing the TLR4/MyD88/NF-κB signaling pathway, thereby mitigating SALI. As a result, the relevance of FGF21 in severe conditions such as ALI and ARDS has gained interest (149).