BAM15 treatment mitigates lipopolysaccharide-induced acute lung injury by preserving mitochondrial dynamic equilibrium and modulating inflammatory responses via the cGAS–STING signalling pathway

BAM15 treatment mitigates lipopolysaccharide-induced acute lung injury by preserving mitochondrial dynamic equilibrium and modulating inflammatory responses via the cGAS–STING signalling pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article BAM15 treatment mitigates lipopolysaccharide-induced acute lung injury by preserving mitochondrial dynamic equilibrium and modulating inflammatory responses via the cGAS–STING signalling pathway Chao Cao, Xiaoli Wang, Rui Tian, Ranran Li, Ruoming Tan, Tingting Pan, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7622689/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Acute lung injury (ALI) is a life-threatening condition that is often induced by endotoxins and leads to severe inflammation and mitochondrial dysfunction. In this study, we investigated the protective effects of BAM15 {C 16 H 10 F 2 N 6 O; also known as N5,N6-bis(2-fluorophenyl)-[ 1 , 2 , 5 ]oxadiazolo[3,4-b]pyrazine-5,6-diamine}, a mitochondrial uncoupler, in lipopolysaccharide (LPS)-induced ALI, focussing on its role in the regulation of mitochondrial dynamics and immune responses. Using a mouse model of LPS-induced ALI, we assessed mitochondrial dynamics by imaging and biochemical assays, and analysed cyclic GMP-AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway activation using western blotting and immunofluorescence. We found that BAM15 treatment resulted in a significant reduction in mortality and attenuated lung inflammation, oxidative stress and autophagy. Moreover, BAM15 contributes to the maintenance of mitochondrial dynamics by promoting mitochondrial fusion and inhibiting fission whilst downregulating cGAS–STING activation, thereby rectifying delayed neutrophil apoptosis and attenuating inflammation. BAM15 protects against LPS-induced ALI by maintaining mitochondrial homeostasis, reducing the generation of mitochondrial reactive oxygen species, and limiting excessive autophagy. It also reduces mitochondrial DNA release and prevents excessive cGAS–STING activation. On the basis of these findings, we propose that BAM15 could represent a promising therapeutic candidate for the treatment of LPS-induced ALI. Biological sciences/Biochemistry/Immunochemistry Biological sciences/Immunology/Immune cell death BAM15 acute lung injury mitochondrial dynamics neutrophil apoptosis cGAS-STING pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Acute lung injury (ALI) and its more severe form, acute respiratory failure (ARDS), are debilitating respiratory conditions, characterised by widespread lung inflammation, increased alveolar and capillary permeability, and severe hypoxaemia that are not attributable to cardiogenic pulmonary oedema [ 1 , 2 ]. These adverse conditions can arise from diverse direct and indirect insults to the lungs, including trauma, sepsis, pneumonia, and the inhalation of toxic substances [ 3 ]. However, despite significant advances in our understanding of the pathophysiology of ALI, it poses significant clinical challenges, owing to its high incidence and associated mortality [ 4 ]. The prevalence of ARDS in intensive care units is estimated to be approximately 10%, with a mortality rate as high as 46% [ 5 , 6 ]. These alarming findings highlight the urgent need for effective therapeutic strategies that can be applied to reduce the burden of ALI on public health systems. Mitochondria, as cellular powerhouses, play pivotal roles in maintaining energy metabolism, redox balance, and regulating cell death pathways [ 7 ]. Mitochondrial dysfunction has recently emerged as a key factor in the pathogenesis of infectious diseases, and a growing body of evidence supports the role of mitochondria in lung epithelial and immune cells, which are particularly susceptible to oxidative stress and damage, notable hallmarks of ALI [ 8 – 10 ]. Disruption of mitochondrial dynamics, specifically the equilibrium between mitochondrial fusion and fission, has been identified as a contributory factor in disease progression, with excessive mitochondrial fission resulting in mitochondrial fragmentation, an elevated production of reactive oxygen species (ROS), and the release of mitochondrial DNA (mtDNA) [ 11 , 12 ]. Clinical studies have shown that mtDNA levels are associated with the severity and prognosis of ARDS [ 13 ], and it has been established that a disruption of mitochondrial function can promote the activation of inflammatory pathways, including the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signalling pathway, which is associated with innate immune responses and exacerbates lung inflammation and injury by influencing neutrophil function [ 14 ]. Subsequently, this cascade activates inflammatory responses via interferon regulatory factor 3 (IRF3) and nuclear factor κB (NF-κB) signalling, which are central to the innate immune response and the pathogenesis of numerous inflammatory diseases[ 15 , 16 ]. Nevertheless, few studies have sought to assess the therapeutic strategies targeting mitochondrial dysfunction and the cGAS-STING pathway, which potentially represent promising avenues for new interventions. BAM15 has recently attracted considerable interest in different disease models, particularly those involving mitochondrial dysfunction [ 17 – 20 ]. BAM15 is a selective mitochondrial uncoupler with distinctive chemical characteristics facilitating efficient mitochondrial membrane penetration. By uncoupling oxidative phosphorylation without causing significant mitochondrial depolarisation or cytotoxic effects, BAM15 can specifically modulate mitochondrial respiration and enhance metabolic flexibility, whilst maintaining membrane integrity. Mitochondrial uncouplers disrupt the proton gradient across the mitochondrial membrane, thereby reducing the membrane potential and ROS production without compromising ATP synthesis. This distinctive mechanism enables BAM15 to alleviate oxidative stress and prevent mitochondrial injury, thus establishing it as a promising candidate for treating conditions in which mitochondrial-associated dysfunction is a pivotal factor [ 21 , 22 ]. Moreover, recent findings have indicated that mitochondrial uncoupling may contribute to the regulation of mitochondrial dynamics, promote mitochondrial fusion, and prevent excessive fission, all of which are key factors for the maintenance of mitochondrial homeostasis [ 23 , 24 ]. Thus, by restoring the equilibrium between mitochondrial fusion and fission, BAM15 may contribute to the preservation of lung cell function and a reduction in the inflammatory responses associated with ALI. In this study, we sought to examine the effects of BAM15 on LPS-induced ALI, with a particular emphasis on its capacity to maintain mitochondrial dynamic equilibrium and modulate neutrophil apoptosis. By elucidating these fundamental mechanisms, we provide new insights into potential therapeutic strategies for reducing ALI-induced morbidity and mortality in clinical settings. Results BAM15 treatment reduces mortality and attenuates lung injury in mice with LPS-induced ALI We established that BAM15 had no discernible effects on liver, kidney, or cardiac function, with enzyme and biomarker levels remaining within normal ranges (Fig. S1 ). These findings accordingly confirmed that BAM15 is well tolerated at the selected dose and had no significant adverse impacts on the overall physiological health of mice. BAM15 treatment was found to have significant protective effects in a murine model of LPS-induced ALI. Initially, we found that BAM15 treatment contributed to a marked improvement in survival, as illustrated in Fig. 1 A, with mice in the LPS + BAM15 group being characterised by a significantly higher survival than those in the LPS + Vehicle group. Histopathological analysis, based on haematoxylin and eosin staining, revealed severe lung damage among the LPS + Vehicle group mice, including evidence of alveolar destruction, oedema, and tissue congestion. However, BAM15 treatment significantly attenuated these pathological changes, resulting in a more preserved alveolar structure and a reduced number of inflammatory cells (Fig. 1 B). In addition, compared with the LPS + Vehicle group, we also detected significantly lower lung W/D ratios and protein contents (which quantify damage based on specific pathological criteria) in the LPS + BAM15 mice, thereby further confirming the protective effects of BAM15 on lung tissues (Fig. 1 C). As shown in Fig. 1 D, BAM15 was also found to enhance respiratory function, as evidenced by an increase in arterial oxygen partial pressure (PaO₂) in LPS + BAM15 mice compared with those in the LPS + Vehicle group, thereby indicating better oxygenation and reduced respiratory distress. To assess the metabolic disturbances and tissue damage in ALI, we determined the levels of lactate and lactate dehydrogenase, the former of which were significantly lower in LPS + BAM15 mice compared with those in the LPS + Vehicle group, indicating improved metabolic function and enhanced tissue oxygenation. Similarly, a significant reduction in lactate dehydrogenase levels was observed in the BAM15-treated mice, indicating a notable improvement in tissue integrity. Moreover, serum TAS levels, an indicator of the protection of lung tissues from oxidative damage via the scavenging of ROS, were reduced in the LPS + Vehicle mice compared with those in the sham-operated group. In addition, the levels of serum TOS, a sensitive indicator of oxidative stress, and the TOS/TAS ratio, which represents the redox balance between antioxidant and oxidative states (i.e., OSI), were elevated in LPS + Vehicle mice. BAM15 was found to inhibit the elevation of TOS and OSI levels, whilst augmenting the release of TAS (Fig. 1 E). From the perspective of consistency with clinical treatment protocols, we conducted further investigations on the administration of BAM15 at 6 h after disease onset, the findings of which indicated that the administration of BAM15 is an effective treatment for lung injury and improves survival outcomes, even after disease onset (Fig. S2 ). Collectively, our findings indicate that BAM15 treatment can contribute to a significant mitigation of lung injury, promotes oxygenation, and alleviates oxidative damage, thereby providing evidence for its potential application as a therapeutic intervention for LPS-induced ALI. BAM15 inhibits delayed neutrophil apoptosis, thereby suppressing inflammatory infiltration and attenuating lung injury Infections induce immune system activation, including the recruitment of a range of immune-related cell types, among which neutrophils play pivotal roles as an initial line of defence. We have previously demonstrated a significant delay in neutrophil apoptosis during lung injury, which as a consequence was significantly exacerbated [ 25 ]. Flow cytometric analysis of BALF revealed that BAM15 treatment resulted in moderate reductions in neutrophil infiltration and the proportion of neutrophils in the BALF. However, there were no significant changes in the number of macrophages or monocytes (Fig. 2 A, B). The results also revealed that BAM15 can significantly alleviate the delay in neutrophil apoptosis when used for lung injury treatment (Fig. 2 C, D). This suggests that BAM15's anti-inflammatory effects primarily involve maintaining neutrophil viability during the acute phase, rather than inducing apoptosis. Furthermore, immunofluorescence analysis revealed a reduction in the number of neutrophils within the lung tissues following BAM15 treatment, thereby indicating a notable reduction in tissue infiltration (Fig. 2 E), and that BAM15 may alleviate tissue injury by regulating neutrophil apoptosis. BAM15 maintains tight junction integrity in lung tissue by mitigating mitochondrial damage and reducing mtDNA release in LPS-challenged mice Acute lung injury is characterised by an excessive production of ROS, leading to increases in oxidative stress, which in turn causes mtDNA damage and abnormalities in mitochondrial ultrastructure. As shown in Fig. 3 A, compared with mice in the LPS + Vehicle group, those treated with BAM15 were characterised by a significant reduction in mitochondrial oxidative stress, thereby highlighting the protective role of this uncoupler in preserving mitochondrial function. Electron microscopy revealed extensive mitochondrial damage and endoplasmic reticulum expansion in the lung tissues of LPS + Vehicle group mice, whereas the morphology of mitochondria in the LPS + BAM15 mice remained relatively well intact (Fig. 3 B). Further analysis indicated that exposure to LPS resulted in a marked increase in mtDNA content over 24 h, which was significantly attenuated by BAM15 treatment (Fig. 3 C), whereas immunofluorescence staining revealed a significant enhancement of the structural integrity of tight junctions in BAM15-treated mice, as indicated by an increase in occludin expression (Fig. 3 D). Additionally, we established that BAM15 inhibits excessive autophagy in lung tissue cells, thereby preventing the overactivation of autophagic processes in response to lung injury, and western blot analysis revealed substantial changes in autophagy-related proteins, with a significant upregulation of pro-apoptotic markers, such as p62, PINK1, and PCG-1α, being detected in the lung injury group, whereas there was a corresponding reduction in the expression of LC3B-II (Fig. 3 E). Notably, these expression profiles were reversed in the LPS + BAM15 mice, suggesting that the protective effects BAM15 are mediated via an inhibition of autophagy and subsequent reduction in mtDNA release, thereby mitigating mitochondrial oxidative stress. BAM15 modulates mitochondrial homeostasis via the cGAS/STING signalling pathway The release of mtDNA into the cytoplasm following mitochondrial damage is detected by cGAS, which activates the downstream cGAS-STING pathway [ 13 ]. In the present study, we demonstrated that BAM15 regulates mitochondrial autophagy by modulating this pathway. As illustrated in Fig. 4 A, compared with mice in the LPS + Vehicle group, key proteins in this pathway, namely, cGAS, TBK1, and STING, were significantly downregulated in the BAM15-treated mice during the lung injury phase. Furthermore, we observed an elevation in the expression of the regulatory proteins USP18 and RNF5, indicating that the effects of BAM15 are mediated via a modulation of these upstream regulators. As illustrated in Fig. 4 B, compared with the LPS + Vehicle group mice, qPCR analysis revealed a significant reduction in cGAS and STING expression among those in the LPS + BAM15 group. Immunohistochemical staining revealed a significant increase in cGAS and STING expression during lung injury, which was markedly reduced in response to BAM15 treatment (Fig. 4 C). Moreover, the administration of BAM15 resulted in a notable reduction in the levels of pro-inflammatory cytokines, including IFN-β, TNF-α, and IL-6, which is indicative of its anti-inflammatory properties (Fig. 4 D). In conclusion, these findings indicate that BAM15 alleviates mitochondrial oxidative stress and reduces inflammation by inhibiting the cGAS-STING signalling pathway. BAM15 protects against mitochondrial damage and maintains intercellular junction stability via the STING pathway in ALI Building on previous studies, we further examined the influence of BAM15-regulated autophagy on lung injury using a STING −/− mouse model. Notably, in the absence of STING, we observed an abrogation of the protective effects of BAM15. As illustrated in Fig. 5 A, compared with mice in the wild-type (WT) group, the BAM15-treated STING −/− mice were characterised by significantly lower rates of survival. Moreover, we failed to detect any improvement in lung injury, as assessed by haematoxylin and eosin staining, in the STING −/− group following BAM15 treatment (Fig. 5 B). Similarly, there were no significant improvements in lung injury scores, lung wet-to-dry ratios, or oxygen partial pressures in the BAM15-treated STING −/− mice compared with those in the WT group (Fig. 5 C). Consistently, immunofluorescence analysis revealed that in the absence of STING, BAM15 failed to restore lung tissue integrity (Fig. 5 D), whereas electron microscopy revealed no notable mitochondrial protection in BAM15-treated STING −/− mice (Fig. 5 E), and relative to that in the WT mice, mitochondrial stress remained elevated (Fig. 5 F). These findings thus highlight the pivotal role play by the STING pathway in mediating the protective effects of BAM15 against lung injury and mitochondrial damage. BAM15 regulates autophagy and modulates inflammation via the cGAS-STING pathway, thereby reducing mtDNA release and cGAS-STING activation Further investigations were conducted to determine the mechanisms underlying the observed effects of BAM15. Following LPS stimulation and BAM15 intervention, STING −/− mice were found to be characterised by elevated levels of autophagy and lysosomal activity compared with those in the WT group, indicating that a deficiency of STING hampers the ability of BAM15 to fully regulate autophagy and lysosomal dynamics (Fig. 6 A). Additionally, our observation that mtDNA levels in STING −/− mice remained elevated and higher than those in the WT group tends to indicate that STING is essential for BAM15-mediated mtDNA clearance, given that its absence result in a reduction in the efficiency of mtDNA reduction (Fig. 6 B). Furthermore, immunohistochemical analysis (Fig. 6 C) revealed that compared with normal mice, cGAS levels were higher in those with STING-knockout following BAM15 treatment. However, the levels of cGAMP, a secondary messenger produced in response to cGAS activation, remained relatively stable, which would tend to indicate that despite the reduction in cGAS levels, the deletion of STING disrupted a feedback loop required for significant changes in cGAMP production. Compared with WT mice, in STING −/− model mice, we detected a reduction in the capacity of BAM15 to regulate the delay in neutrophil apoptosis, as indicated by a significant increase in the proportion of neutrophils in these latter mice (Fig. 6 D). Moreover, the overall rate of neutrophil apoptosis was observed to be markedly elevated, thereby providing evidence that the STING pathway plays a pivotal role in mediating the effects of BAM15 with respect to neutrophil survival (Fig. 6 E). These findings would thus appear to indicate that an absence of STING may compromise the efficacy of BAM15 in preventing the delay in neutrophil apoptosis, and also inhibits the enhancement of neutrophil infiltration in lung tissue (Fig. 6 F), thereby highlighting the key role of the STING signalling pathway in this process. With respect to inflammatory markers, we found that compared with the BAM15-treated WT mice, the levels of IFN-β, IL-6, and IL-1β remained elevated in the STING −/− mice treated with BAM15 (Fig. 6 G), thus indicating that a deficiency of STING diminished the efficacy of BAM15 in controlling inflammation, leading to a persistent expression of inflammatory cytokines. Collectively, these findings highlight the essential role played by the STING pathway in promoting the BAM15-mediated autophagy and the regulation of inflammatory responses. Discussion Our findings in this study, in which we investigated the effects of BAM15, a mitochondrial uncoupler, on mitochondrial function and the cGAS-STING signalling pathway, provide substantial evidence to indicate that by preserving mitochondrial dynamics and suppressing inflammatory pathways, BAM15 can confer significant protection against LPS-induced ALI. Specifically, BAM15 was found to mitigate key pathological features of ALI, including apoptosis, inflammation, oxidative stress, and autophagy, whilst restoring mitochondrial homeostasis (Fig. 7 ). These findings accordingly highlight the potential applicability of BAM15 as a promising therapeutic agent for the treatment of ALI/ARDS, thereby addressing the pressing need for targeted interventions that go beyond conventional supportive therapies. Compared with other uncouplers, such as carbonyl cyanide- p -trifluoromethoxyphenylhydrazone and 2,4-dinitrophenol, which induce mitochondrial depolarisation and are frequently associated with cytotoxic effects, BAM15 has lower cytotoxicity and fewer off-target effects on plasma membrane depolarisation [ 26 ]. These distinctive characteristics highlight the potential therapeutic applicability of BAM15 as a safer mitochondrial uncoupler. Although the therapeutic potential of mitochondrial uncouplers has previously been investigated with respect to the treatment of cardiovascular diseases [ 17 ], diabetes [ 18 ], non-alcoholic steatohepatitis [ 19 ], and sepsis [ 20 ], to the best of our knowledge, there have been no studies that have assessed the efficacy of these uncouplers in the treatment of ALI or ARDS. A significant finding in this study was that BAM15 can effectively contribute to maintenance of the equilibrium of mitochondrial dynamics in ALI, particularly the balance between mitochondrial fusion and fission, which are of paramount importance in maintaining mitochondrial integrity and function [ 27 ]. BAM15 promotes fusion, whilst inhibiting excessive fission, thereby preventing mitochondrial fragmentation, suppressing ROS production, and reducing mtDNA release, all of which can contribute to an alleviation of cellular damage and inflammation [ 21 , 28 , 29 ]. By maintaining this balance, BAM15 mitigates the cellular damage commonly associated with mitochondrial dysfunction and excessive ROS production in ALI. Autophagy is an adaptive and protective cellular process that mitigates cellular damage and inhibits apoptosis during sepsis [ 30 ], and during the early stages of sepsis, autophagy contributes to protecting the host from MODS by preserving immune cell viability, balancing pro- and anti-inflammatory cytokine production, and facilitating clearance of microbial infections [ 31 ]. In the present study, we detected a reduction in the levels of autophagic markers in the lung tissues of mice treated with BAM15, thus substantiating its protective function in lung epithelial cells and overall lung performance during ALI. Furthermore, we established that BAM15 plays a pivotal role in reversing the delay in neutrophil apoptosis. In this regard, although neutrophils play key roles in the initial defence against pathogens, their prolonged activation and delayed apoptosis can result in significant tissue damage and exacerbate lung injury [ 32 , 33 ]. To prevent excessive tissue damage, neutrophils normally undergo apoptosis following the resolution phase. However, in ALI, multiple factors, including inflammatory cytokines and hypoxia, can cause a delay in the apoptotic process, resulting in the sustained survival of neutrophils and an increased risk of tissue damage. This prolonged survival of neutrophils can intensify the inflammatory environment, leading to further damage to the lung tissues and a prolongation of recovery. Delayed neutrophil apoptosis regulation may accordingly represent an effective means of restoring homeostasis and attenuating the release of inflammatory infiltrates, whilst also reducing tissue damage associated with excessive inflammation. Consequently, the neutrophil apoptotic pathway could serve as a useful target for alleviating inflammatory responses in ALI. The cGAS-STING pathway plays a pivotal role in the innate immune response against diverse pathogens, including viruses, bacteria, and parasites [ 34 , 35 ]. However, the function of the STING pathway is not merely restricted to antimicrobial defence. In response to cellular stress, mtDNA is released into the cytoplasm, wherein it activates the cGAS-STING pathway, thereby initiating an inflammatory response [ 36 , 37 ]. Furthermore, abnormal activation of the cGAS-STING pathway has been identified as a key factor contributing to lung injury and as an inflammatory mediator [ 38 ]. A deficiency in either cGAS or STING has been demonstrated to alleviate silica-induced lung inflammation [ 39 ], and in the present study, we found that BAM15 efficiently suppresses mtDNA release and mitigates cellular autophagy and mitochondrial damage via the cGAS-STING pathway, thereby exerting a protective effect against ALI. Consequently, exploiting the ability of BAM15 to modulate mitochondrial dynamics, reduce ROS production, inhibit mtDNA release, and downregulate the cGAS-STING pathway may represent a novel therapeutic approach that could complement existing treatments or serve as a stand-alone therapy, particularly in patients unresponsive to current interventions. However, despite these encouraging findings, further investigations are required to address the limitations of this study. Notably, although our murine model provides valuable insights into the protective effects of BAM15, its applicability to human ALI and ARDS remains uncertain. The complexity of human ALI, which frequently involves comorbidities and triggers other than LPS exposure, necessitates further studies in clinical settings or more complex animal models to fully assess the translational potential of BAM15. Although the regulation of mitochondrial dynamics and the cGAS-STING pathway by BAM15 is evident, the precise molecular mechanisms underlying these effects remain unclear. Future research should accordingly focus on identifying the upstream regulators and downstream effectors involved in the BAM15-mediated mitochondrial protection and resolution of inflammation. Collectively, our findings in this study provide convincing evidence that BAM15 can aid in protecting against LPS-induced ALI by preserving the mitochondrial dynamic balance, reducing delayed neutrophil apoptosis, attenuating oxidative stress and autophagy, and inhibiting the cGAS-STING signalling pathway. These findings identify BAM15 as a promising therapeutic candidate for the treatment of induced ALI and address the significant shortcomings of current therapies by targeting both mitochondrial dysfunction and excessive inflammation. Further studies are, nevertheless, required to elucidate the molecular mechanisms underlying the protective effects of BAM15 and to evaluate its safety and efficacy in clinical settings. Materials and methods Animal model Male C57BL/6 and STING –/– mice, aged 8 to 10 weeks and weighing 20 to 25 g. C57BL/6 mice were obtained from Charles River (Beijing, China), and STING –/– mice were obtained from the Model Animal Research Center of Nanjing University (Nanjing, China). All mice were of a C57BL/6 genetic background. The genotyping of the knockout mice was conducted through polymerase chain reaction (PCR) analysis of DNA extracted from tail biopsy samples. The animals were randomly assigned to experimental groups. No animals were excluded from the study on the grounds of illness, and the animal experiments were conducted in a blinded manner. The mice were housed in a controlled environment maintained at a temperature and humidity of 24–26℃ and 55%–60%, respectively, under a 12 h light:12 h dark cycle, with free access to food and water. ALI model Lipopolysaccharide (LPS) (LPS, L2630, E. coli 0111: B4; Sigma Aldrich, MA, USA) in 0.9% saline was administered via intratracheal injection with a single dose of 10 mg/kg LPS to generate a model of ALI. This relatively high-dose LPS model has been established to induce a robust inflammatory response and severe pulmonary injury, closely mirroring the pathological features of human acute lung injury [ 40 , 41 ]. The BAM15-treated groups were administered BAM15 intraperitoneally at the time of the LPS challenge. Based on the findings of previous studies [ 21 , 26 ], mice were intraperitoneally administered a 5 mg/kg dose of BAM15 to assess potential drug toxicity. The control and BAM15-only groups received equivalent volumes of vehicle, comprising 40 mL/kg 3% dimethyl sulphoxide (DMSO) diluted with 0.9% saline. The C57BL/6 mice were randomly assigned to four groups. The experimental groups were as follows: Sham + Vehicle, Sham + BAM15, LPS + Vehicle, and LPS + BAM15. Following a 24 h period of LPS challenge, the mice were sacrificed and blood or bronchoalveolar lavage (BAL) samples were collected. The organs were either frozen immediately or fixed in formalin for subsequent analysis. The frozen organs were stored at − 80°C. To ensure the reliability of the study findings, the investigators were blinded to the group allocation for analysis. Survival analysis The survival of the mice was evaluated at 24 h intervals following exposure, and percentage survival was determined at 7 d after LPS administration. At the end of the observation period, surviving mice were euthanised via CO₂ asphyxiation. Histological and immunohistochemical analyses Mice were euthanised via CO 2 asphyxiation. Lung tissues were harvested, fixed in 10% neutral buffered formalin, embedded in paraffin and sectioned at 5 µm thickness. Haematoxylin and eosin staining was performed to assess the degree of lung injury, with light microscopy performed to obtain non-overlapping fields of view (20 µm) for each animal, including alveolar thickening, inflammatory cell infiltration, and oedema. Wet/dry ratio determination Freshly obtained right lungs were weighed and then heated in a 68°C oven for 72 h. The weight of the lung tissues was recorded until no further change was observed. The wet/dry ratio was calculated to quantify the severity of pulmonary oedema. Analysis of arterial blood gas The animals were anaesthetised at a timepoint of 24 h. The carotid artery of each animal was cannulated, and arterial blood was collected into a syringe. Blood gases (PaO₂ and PaCO₂), lactate, and lactate dehydrogenase were measured using a blood gas analyzer (GemPremier 3000; Beckman, USA). The remaining blood was stored for further biochemical analysis. Subsequently, the lungs were meticulously harvested, taking care to ensure that both right and left lungs were collected. The left lung tissue was rapidly frozen in liquid nitrogen and stored at − 80°C for subsequent analysis. The right lung tissue was divided into two portions and separately placed into glutaraldehyde and buffered 10% formalin solutions for histological evaluation. Measurement of oxidative stress The oxidative stress index (OSI) was calculated as the ratio of total oxidant status (TOS) to total antioxidant status (TAS). The levels of TOS and TAS in serum were determined using a commercial kit (ENZ-51011; Sigma-Aldrich, Germany) with measurements performed using a microplate spectrophotometer (NanoDrop 8000; Thermo Fisher, CA, USA) according to the manufacturer’s instructions. For TOS, the assay was calibrated using hydrogen peroxide (H 2 O 2 ) with the values obtained being expressed as µmol H 2 O 2 equivalent/L, whereas for TAS, the assay was calibrated with Trolox, and the values are expressed in terms of µmol Trolox equivalent/L. ROS levels were measured in lung homogenates using a commercially available ROS detection kit (S0035M; Beyotime, China). Flow cytometry analysis Phosphate-buffered saline (1 mL) was injected into the trachea, and having rinsed three times, sputum was collected. Cells were obtained by centrifugation at 500 × g at 4°C and collected into a centrifuge tube. Cells (5 × 10 6 per tube) were processed for flow cytometry. The cells were incubated in an Fc block (1:200: 553142; BD Biosciences, Sparks, MD) for 10 min at 4°C. Surface staining was performed in the dark for 15 min. The surface markers used for myeloid cells were as follows: BV605 rat anti-mouse CD45 (1:200: clone 30-F11, 561865; BD Biosciences), APC rat anti-mouse Ly6G (1:1000: clone 1A8, 560599; BD Biosciences), BUV737 rat anti-mouse Ly6C (1:1000; clone HK1.4. rMAb, 755201; BD Biosciences), Alexa Fluo® 568 anti-mouse CD11c (1:200: clone N418, 745852; BD Biosciences), and Alexa Fluor® 488 anti-mouse/human CD14 (1:200: clone M5E2, 555397; BD Biosciences). Apoptotic cells were detected using 7-AAD (1:200, 559925; BD Biosciences) and FITC Annexin V (1:200, 556420; BD Biosciences). Cells were analysed using a BD FACSCanto II cytometer (BD Life Sciences, San Jose, CA). To distinguish positive from negative staining cell populations, we used a fluorescence minus one (FMO) control for all gating analyses. Correction matrices were calculated and applied using FlowJo software (Tree Star, Ashland, OR). Enzyme-linked immunosorbent assay (ELISA) ELISAs were employed to determine the concentrations of cytokines [IFN-β (PI568, Beyotime, China), TNF-α (PT512, Beyotime, China), IL-1β (P5898, Beyotime, China), and IL-6 (PI326, Beyotime, China)] in plasma. The assays were performed according to the instructions provided by the manufacturer. Mitochondrial DNA content Mitochondrial DNA (mtDNA) copy number was assessed based on the ratio of mtDNA to nuclear-encoded β-actin using real-time PCR, as described by Amaral et al. [ 42 ]. DNA was extracted using a DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer’s instructions. TEM analyses Small pieces of lung tissue were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (PH 7.4). After subsequent rinsing in 0.1 M phosphate buffer, the samples were post-fixed in 1% osmium tetroxide for 2 h, dehydrated in a graded series of ethanol, embedded in acetone, and sectioned at approximately 70 nm. The preparations were viewed and image obtained using an electron microscope (Verios XHR SEM, Thermo Fisher, USA), with at least three randomly selected fields per sample being evaluated by a blinded pathologist. Immunofluorescence (IF) and immunohistochemistry (IHC) Lung tissues were fixed in 4% paraformaldehyde solution for 48 h, paraffin-embedded, and sectioned at a thickness of 5 µm. For IF microscopy, sections were deparaffinised in xylene and rehydrated using graded ethanol series, and antigen was extracted using PH 9.0 EDTA. For IF microscopy. Sections were rinsed three times (each for 5 min) with TBS, blocked with 10% donkey serum for 30 min at 37°C, and then incubated overnight at 4°C with 488-conjugated occludin recombinant antibody (1:200: CL488-80545; Proteintech, Santa Cruz, CA, USA) or anti-SQSTM1/p62 antibody (1:200: ab109012; Abcam, Cambridge, UK), anti-LC3 antibody (1:100: CL647-14600; Proteintech, Santa Cruz, CA, USA) or anti-LAMP2 (1:200: L0668; Sigma-Aldrich, Germany). After washing, the sections were incubated with diluted fluorescent-labelled secondary antibody for 30 min at room temperature and counterstained with DAPI (28718-90-3; Sigma-Aldrich, Germany) in the dark for 10 min to show the nuclei. Images were obtained using an Olympus U-25ND25 fluorescence microscope (Olympus, Tokyo, Japan). For IHC, sample sections were blocked with 5% BSA for 30 min, and then incubated with primary antibody overnight at 4°C, with secondary antibody being added after three consecutive washes with PBS. The following antibodies were used: anti-cGAS (1:200: 29958-1-AP; Proteintech, Santa Cruz, CA, USA), anti-STING (1:1000: KHC0060; Proteintech, Santa Cruz, CA, USA) and anti-p-IRF3 (1:200: ab218160; Abcam, Cambridge, UK). DAB staining was performed to show positive expression and 40,6-diamidino-2-phenylindole was added for nuclear staining. Images were obtained using a DM2500 microscope (Leica, Wetzlar, Germany,). Western blotting RIPA buffer (89901, Thermo Fisher Scientific, USA), enriched with PhosSTOP and complete protease inhibitors (Merck, Beijing, China), was used for protein extraction. The protein samples were then separated using 12.5% SDS-PAGE and subsequently transferred to polyvinylidene fluoride membranes (162–0177; Bio-Rad, Shanghai, China) following the manufacturer’s guidelines [ 43 ]. Blots were probed at 4°C overnight with appropriate primary antibodies against p62 (ab56416, Abcam, Cambridge, UK), PINK1 (23274-1-AP; Santa Cruz, CA, USA), PCG-1α (ab134047; Abcam, Cambridge, UK), LC3B-Ⅱ (ab204297; Abcam, Cambridge, UK), cGAS (29958-1-AP; Proteintech, Santa Cruz, CA, USA), TBK1 (ab289973, Abcam, Cambridge, UK), STING (KHC0060; Proteintech, Santa Cruz, CA, USA), USP18 (12153-1-AP; Proteintech, Santa Cruz, CA, USA), and RNF5 (ab200389; Abcam, Cambridge, UK). Blots were incubated with the appropriate secondary antibody (1:5000: Proteintech) and developed with horseradish peroxidase substrate (WBLUF0500; Millipore, USA). To ensure equal loading, the blots were stripped with stripping buffer (100 mmol/L 2-mercaptoethanol, 2% SDS, 62.5 mmol/L Tris, pH 6.8) and re-probed with anti-GAPDH antibody. qPCR assay RNA was extracted from lung tissues using TRIzol reagent (RR047A; Takara, Japan) following the instructions outlined in our previous study [ 43 ]. Subsequently, cDNA was synthesised and real-time PCR was conducted using a SYBR Green Premix Pro Taq HS qPCR Kit (cat. no. AG11701; Accurate Biology). Target gene transcripts were amplified from mice using a SuperScript III One-Step RT-PCR Kit (12574026; Thermo Fisher Scientific, Waltham, MA, USA) in a Roche LightCycler 480 thermocycler. RT-PCR was performed using an Eppendorf Realplex 4 instrument (Eppendorf, Hamburg, Germany). The following primers were synthesised by Univ-Bio (Shanghai, China): mtDNA: forward: 5ʹ-CACCCAAGAACAGGGTTTGT-3ʹ, reverse: 5ʹ-TGGCCATGGGTATGTTGTTA-3ʹ; cGAS: forward: 5ʹ-CAGGAAGGAACCGGACAAGC-3ʹ, reverse: 5ʹ-CCGACTCCCGTTTCTGCATT-3; and STING: forward: 5ʹ-TATACCTCAGTTGGATGTTTGGC-3ʹ, reverse: 5ʹ- CTGGAGTCAAGCTCTGAAGGC-3ʹ. Relative gene expression was calculated using β-actin as an internal reference. Results are expressed as mean ± SD of six biological replicates for each sample. Statistical analysis Data are presented as mean ± SEM. Normality and log normality were assessed using the Shapiro–Wilks test. Kaplan–Meier survival curves were analysed using the log-rank test, which was equivalent to the Mantel–Haenszel test. To determine the statistical significance of differences between independent continuous variables, a 2-tailed Student’s t -test was applied when comparing two groups for which both data sets had a normal distribution. One-way analysis of variance followed by Bonferroni’s post hoc test or the Mann–Whitney U test was performed as appropriate using Graph Prism 8.0 software (GraphPad Software, La Jolla, CA). P values of less than 0.05 were considered statistically significant (* P < 0.05; ** P < 0.01; *** P < 0.001 or # P < 0.05; ## P < 0.01; ### P < 0.001). Abbreviations ALI acute lung injury ARDS acute respiratory distress syndrome ROS reactive oxygen species mtDNA mitochondrial DNA ER endoplasmic reticulum cGAS cyclic GMP-AMP synthase STING stimulator of interferon genes cDNA complementary DNA ATP adenosine triphosphate DNA deoxyribonucleic acid NADH nicotinamide adenine dinucleotide IL interleukin LPS lipopolysaccharide MODS multiple organ dysfunction syndrome TEM transmission electron microscopy BAL bronchoalveolar lavage PaO₂ arterial oxygen partial pressure PaCO 2 partial pressure of carbon dioxide Lac lactate LDH lactate dehydrogenase OSI oxidative stress index TOS total oxidant status TAS total antioxidant status. Declarations Ethics approval and consent to participate The protocols for the animal experiments were approved by the Animal Ethics Committee of Ruijin Hospital Affiliated to Shanghai Jiaotong University School of Medicine (No. 092). Consent for publication Not applicable. Availability of data and material The data that support our findings are available from the corresponding author ( [email protected] ) upon reasonable request. Competing interests The authors declare no conflicts of interest. Funding This work was supported by the National Natural Science Foundation of China (82402510, 82372203, 82241033, 81902007), the Natural Science Foundation of Shanghai Municipality (24ZR1445800), the China Postdoctoral Science Foundation (2024M762043), and the Natural Science Foundation of Shandong Province (ZR2025MS1416), and the Research Project by the Tianjin Municipal Health Commission (2023221). Authors' contributions HPQ conceived the original idea, CC and TTP wrote the manuscript draft. CC and XLW conducted a majority of the experiments and performed data analysis. RT and RMT assisted with the animal experiments. RRL interpreted the data. HPQ and TTP revised the manuscript. All the authors have confirmed the submission of this manuscript. Acknowledgements We thank Dr. Tingting Lin for technical assistance. Graphical Abstract was created in BioRender. Cao, C. (2025) https://BioRender.com/q42p392. References Matthay, M. A., Arabi, Y., Arroliga, A. C., Bernard, G., Bersten, A. D., Brochard, L. J., Calfee, C. S., Combes, A., Daniel, B. M., Ferguson, N. D., Gong, M. N., Gotts, J. E., Herridge, M. S., Laffey, J. G., Liu, K. D., Machado, F. R., Martin, T. R., McAuley, D. F., Mercat, A., Moss, M., Mularski, R. A., Pesenti, A., Qiu, H., Ramakrishnan, N., Ranieri, V. M., Riviello, E. D., Rubin, E., Slutsky, A. S., Thompson, B. T., Twagirumugabe, T., Ware, L. B. & Wick, K. D. (2024) A New Global Definition of Acute Respiratory Distress Syndrome, Am J Respir Crit Care Med. 209, 37–47. Thompson, B. T., Chambers, R. C. & Liu, K. D. (2017) Acute Respiratory Distress Syndrome, N Engl J Med. 377, 562–572. Bos, L. D. J. & Ware, L. B. (2022) Acute respiratory distress syndrome: causes, pathophysiology, and phenotypes, Lancet. 400, 1145–1156. Fan, E., Brodie, D. & Slutsky, A. S. (2018) Acute Respiratory Distress Syndrome: Advances in Diagnosis and Treatment, JAMA. 319, 698–710. Bellani, G., Laffey, J. G., Pham, T., Fan, E., Brochard, L., Esteban, A., Gattinoni, L., van Haren, F., Larsson, A., McAuley, D. F., Ranieri, M., Rubenfeld, G., Thompson, B. T., Wrigge, H., Slutsky, A. S. & Pesenti, A. (2016) Epidemiology, Patterns of Care, and Mortality for Patients With Acute Respiratory Distress Syndrome in Intensive Care Units in 50 Countries, JAMA. 315, 788–800. Slutsky, A. S. & Ranieri, V. M. (2013) Ventilator-induced lung injury, N Engl J Med. 369, 2126–2136. Vercellino, I. & Sazanov, L. A. (2022) The assembly, regulation and function of the mitochondrial respiratory chain, Nat Rev Mol Cell Biol. 23, 141–161. Larson-Casey, J. L., He, C. & Carter, A. B. (2020) Mitochondrial quality control in pulmonary fibrosis, Redox Biol. 33, 101426. Dutra Silva, J., Su, Y., Calfee, C. S., Delucchi, K. L., Weiss, D., McAuley, D. F., O'Kane, C. & Krasnodembskaya, A. D. (2021) Mesenchymal stromal cell extracellular vesicles rescue mitochondrial dysfunction and improve barrier integrity in clinically relevant models of ARDS, Eur Respir J. 58. Chung, K.-P., Cheng, C.-N., Chen, Y.-J., Hsu, C.-L., Huang, Y.-L., Hsieh, M.-S., Kuo, H.-C., Lin, Y.-T., Juan, Y.-H., Nakahira, K., Chen, Y.-F., Liu, W.-L., Ruan, S.-Y., Chien, J.-Y., Plataki, M., Cloonan, S. M., Carmeliet, P., Choi, A. M. K., Kuo, C.-H. & Yu, C.-J. (2024) Alveolar epithelial cells mitigate neutrophilic inflammation in lung injury through regulating mitochondrial fatty acid oxidation, Nat Commun. 15, 7241. Chan, D. C. (2020) Mitochondrial Dynamics and Its Involvement in Disease, Annu Rev Pathol. 15, 235–259. Chen, W., Zhao, H. & Li, Y. (2023) Mitochondrial dynamics in health and disease: mechanisms and potential targets, Signal Transduct Target Ther. 8, 333. Faust, H. E., Reilly, J. P., Anderson, B. J., Ittner, C. A. G., Forker, C. M., Zhang, P., Weaver, B. A., Holena, D. N., Lanken, P. N., Christie, J. D., Meyer, N. J., Mangalmurti, N. S. & Shashaty, M. G. S. (2020) Plasma Mitochondrial DNA Levels Are Associated With ARDS in Trauma and Sepsis Patients, Chest. 157, 67–76. Zhao, C., Liang, F., Ye, M., Wu, S., Qin, Y., Zhao, L., Zhang, L., He, J., Cen, L. & Lin, F. (2023) GSDMD promotes neutrophil extracellular traps via mtDNA-cGAS-STING pathway during lung ischemia/reperfusion, Cell Death Discov. 9, 368. Yum, S., Li, M., Fang, Y. & Chen, Z. J. (2021) TBK1 recruitment to STING activates both IRF3 and NF-κB that mediate immune defense against tumors and viral infections, Proc Natl Acad Sci U S A. 118. Li, X., Chen, X., Zheng, L., Chen, M., Zhang, Y., Zhu, R., Chen, J., Gu, J., Yin, Q., Jiang, H., Wu, X., Ji, X., Tang, X., Dong, M., Li, Q., Gao, Y. & Chen, H. (2023) Non-canonical STING-PERK pathway dependent epigenetic regulation of vascular endothelial dysfunction via integrating IRF3 and NF-κB in inflammatory response, Acta Pharm Sin B. 13, 4765–4784. Gao, J.-L., Zhao, J., Zhu, H.-B., Peng, X., Zhu, J.-X., Ma, M.-H., Fu, Y., Hu, N., Tai, Y., Xuan, X.-C. & Dong, D.-L. (2018) Characterizations of mitochondrial uncoupling induced by chemical mitochondrial uncouplers in cardiomyocytes, Free Radic Biol Med. 124, 288–298. Axelrod, C. L., King, W. T., Davuluri, G., Noland, R. C., Hall, J., Hull, M., Dantas, W. S., Zunica, E. R., Alexopoulos, S. J., Hoehn, K. L., Langohr, I., Stadler, K., Doyle, H., Schmidt, E., Nieuwoudt, S., Fitzgerald, K., Pergola, K., Fujioka, H., Mey, J. T., Fealy, C., Mulya, A., Beyl, R., Hoppel, C. L. & Kirwan, J. P. (2020) BAM15-mediated mitochondrial uncoupling protects against obesity and improves glycemic control, EMBO Mol Med. 12, e12088. Jing, W., Jiang, M., Fu, X., Yang, J., Chen, L., Leng, F., Xu, P., Huang, W., Yu, C. & Yang, Z. (2022) Self-assembly drug-albumin nanocomposites for nonalcoholic fatty liver disease treatment, Int J Biol Macromol. 214, 697–707. Dang, C. P., Issara-Amphorn, J., Charoensappakit, A., Udompornpitak, K., Bhunyakarnjanarat, T., Saisorn, W., Sae-Khow, K. & Leelahavanichkul, A. (2021) BAM15, a Mitochondrial Uncoupling Agent, Attenuates Inflammation in the LPS Injection Mouse Model: An Adjunctive Anti-Inflammation on Macrophages and Hepatocytes, J Innate Immun. 13, 359–375. Tsuji, N., Tsuji, T., Yamashita, T., Hayase, N., Hu, X., Yuen, P. S. & Star, R. A. (2023) BAM15 treats mouse sepsis and kidney injury, linking mortality, mitochondrial DNA, tubule damage, and neutrophils, J Clin Invest. 133. Dantas, W. S., Zunica, E. R. M., Heintz, E. C., Vandanmagsar, B., Floyd, Z. E., Yu, Y., Fujioka, H., Hoppel, C. L., Belmont, K. P., Axelrod, C. L. & Kirwan, J. P. (2022) Mitochondrial uncoupling attenuates sarcopenic obesity by enhancing skeletal muscle mitophagy and quality control, J Cachexia Sarcopenia Muscle. 13, 1821–1836. Hombrebueno, J. R., Cairns, L., Dutton, L. R., Lyons, T. J., Brazil, D. P., Moynagh, P., Curtis, T. M. & Xu, H. (2019) Uncoupled turnover disrupts mitochondrial quality control in diabetic retinopathy, JCI Insight. 4. Schäfer, J. A., Bozkurt, S., Michaelis, J. B., Klann, K. & Münch, C. (2022) Global mitochondrial protein import proteomics reveal distinct regulation by translation and translocation machinery, Mol Cell. 82. Li, Y., Tan, R., Li, R., Tian, R., Liu, Z., Wang, X., Chen, E., Pan, T. & Qu, H. (2023) PKM2/STAT1-mediated PD-L1 upregulation on neutrophils during sepsis promotes neutrophil organ accumulation by serving an anti-apoptotic role, J Inflamm (Lond) . 20, 16. Kenwood, B. M., Weaver, J. L., Bajwa, A., Poon, I. K., Byrne, F. L., Murrow, B. A., Calderone, J. A., Huang, L., Divakaruni, A. S., Tomsig, J. L., Okabe, K., Lo, R. H., Cameron Coleman, G., Columbus, L., Yan, Z., Saucerman, J. J., Smith, J. S., Holmes, J. W., Lynch, K. R., Ravichandran, K. S., Uchiyama, S., Santos, W. L., Rogers, G. W., Okusa, M. D., Bayliss, D. A. & Hoehn, K. L. (2014) Identification of a novel mitochondrial uncoupler that does not depolarize the plasma membrane, Mol Metab. 3, 114–123. Tyagi, T., Yarovinsky, T. O., Faustino, E. V. S. & Hwa, J. (2024) Platelet Mitochondrial Fusion and Function in Vascular Integrity, Circ Res. 134, 162–164. Taylor, A. L., Dubuisson, O., Pandey, P., Zunica, E. R. M., Vandanmagsar, B., Dantas, W. S., Johnson, A., Axelrod, C. L. & Kirwan, J. P. (2024) Restricting bioenergetic efficiency enhances longevity and mitochondrial redox capacity in Drosophila melanogaster, Aging Cell. 23, e14107. Muhammad, W., Zhu, J., Zhai, Z., Xie, J., Zhou, J., Feng, X., Feng, B., Pan, Q., Li, S., Venkatesan, R., Li, P., Cao, H. & Gao, C. (2022) ROS-responsive polymer nanoparticles with enhanced loading of dexamethasone effectively modulate the lung injury microenvironment, Acta Biomater. 148, 258–270. Ho, J., Yu, J., Wong, S. H., Zhang, L., Liu, X., Wong, W. T., Leung, C. C. H., Choi, G., Wang, M. H. T., Gin, T., Chan, M. T. V. & Wu, W. K. K. (2016) Autophagy in sepsis: Degradation into exhaustion?, Autophagy. 12, 1073–1082. Lira Chavez, F. M., Gartzke, L. P., van Beuningen, F. E., Wink, S. E., Henning, R. H., Krenning, G. & Bouma, H. R. (2023) Restoring the infected powerhouse: Mitochondrial quality control in sepsis, Redox Biol. 68, 102968. Wang, J.-F., Wang, Y.-P., Xie, J., Zhao, Z.-Z., Gupta, S., Guo, Y., Jia, S.-H., Parodo, J., Marshall, J. C. & Deng, X.-M. (2021) Upregulated PD-L1 delays human neutrophil apoptosis and promotes lung injury in an experimental mouse model of sepsis, Blood. 138, 806–810. Song, C., Li, H., Mao, Z., Peng, L., Liu, B., Lin, F., Li, Y., Dai, M., Cui, Y., Zhao, Y., Han, D., Chen, L., Huang, X. & Pan, P. (2022) Delayed neutrophil apoptosis may enhance NET formation in ARDS, Respir Res. 23, 155. Ishikawa, H. & Barber, G. N. (2008) STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling, Nature. 455, 674–678. Ergun, S. L. & Li, L. (2020) Structural Insights into STING Signaling, Trends Cell Biol. 30, 399–407. Newman, L. E., Weiser Novak, S., Rojas, G. R., Tadepalle, N., Schiavon, C. R., Grotjahn, D. A., Towers, C. G., Tremblay, M.-È., Donnelly, M. P., Ghosh, S., Medina, M., Rocha, S., Rodriguez-Enriquez, R., Chevez, J. A., Lemersal, I., Manor, U. & Shadel, G. S. (2024) Mitochondrial DNA replication stress triggers a pro-inflammatory endosomal pathway of nucleoid disposal, Nat Cell Biol. 26, 194–206. Jiménez-Loygorri, J. I., Villarejo-Zori, B., Viedma-Poyatos, Á., Zapata-Muñoz, J., Benítez-Fernández, R., Frutos-Lisón, M. D., Tomás-Barberán, F. A., Espín, J. C., Area-Gómez, E., Gomez-Duran, A. & Boya, P. (2024) Mitophagy curtails cytosolic mtDNA-dependent activation of cGAS/STING inflammation during aging, Nat Commun. 15, 830. Decout, A., Katz, J. D., Venkatraman, S. & Ablasser, A. (2021) The cGAS-STING pathway as a therapeutic target in inflammatory diseases, Nat Rev Immunol. 21, 548–569. Benmerzoug, S., Rose, S., Bounab, B., Gosset, D., Duneau, L., Chenuet, P., Mollet, L., Le Bert, M., Lambers, C., Geleff, S., Roth, M., Fauconnier, L., Sedda, D., Carvalho, C., Perche, O., Laurenceau, D., Ryffel, B., Apetoh, L., Kiziltunc, A., Uslu, H., Albez, F. S., Akgun, M., Togbe, D. & Quesniaux, V. F. J. (2018) STING-dependent sensing of self-DNA drives silica-induced lung inflammation, Nat Commun. 9, 5226. Li, N., Liu, B., Xiong, R., Li, G., Wang, B. & Geng, Q. (2023) HDAC3 deficiency protects against acute lung injury by maintaining epithelial barrier integrity through preserving mitochondrial quality control, Redox Biol. 63, 102746. Islam, M. N., Das, S. R., Emin, M. T., Wei, M., Sun, L., Westphalen, K., Rowlands, D. J., Quadri, S. K., Bhattacharya, S. & Bhattacharya, J. (2012) Mitochondrial transfer from bone-marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury, Nat Med. 18, 759–765. Amaral, A., Ramalho-Santos, J. & St John, J. C. (2007) The expression of polymerase gamma and mitochondrial transcription factor A and the regulation of mitochondrial DNA content in mature human sperm, Hum Reprod. 22, 1585–1596. Cao, C., Memete, O., Shao, Y., Zhang, L., Liu, F., Dun, Y., He, D., Zhou, J. & Shen, J. (2022) Single-Cell RNA-Sequencing Reveals Epithelial Cell Signature of Multiple Subtypes in Chemically Induced Acute Lung Injury, Int J Mol Sci. 24. Additional Declarations (Not answered) Supplementary Files SupplementalMaterial.docx Supporting information Additional supporting information may be found online in the Supporting Information section at the end of the article. Fig. S1. Assessment of the potential effects of BAM15 on the liver, kidney, cardiac, and pancreatic function in mice. Fig. S2. Delayed BAM15 treatment improves lung injury outcomes and survival in LPS-induced ALI. WBFigure3E.pdf Original WB gel in Figure 3E WBFigure4A.pdf Original WB gel in Figure 4A Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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1","display":"","copyAsset":false,"role":"figure","size":360141,"visible":true,"origin":"","legend":"\u003cp\u003eBAM15 treatment reduces mortality and attenuates lung injury in mice with lipopolysaccharide (LPS)-induced acute lung injury (ALI). (A) Kaplan–Meier survival curves for sham-operated or ALI male mice treated with vehicle (0 h) and sham-operated or LPS-exposed mice treated with BAM15 (5 mg/kg, 0 h) over 7 days. Group sizes: Sham+Vehicle/BAM15 (\u003cem\u003en\u003c/em\u003e = 6), LPS+Vehicle (\u003cem\u003en \u003c/em\u003e= 11), and LPS+BAM15 (\u003cem\u003en\u003c/em\u003e = 10). Survival rates were higher in LPS+BAM15 than in LPS+Vehicle. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, LPS+Vehicle vs. LPS+BAM15. (B) Representative images of haematoxylin and eosin-stained lung tissues from three independent experiments (Vehicle or BAM15) 24 h post-LPS challenge. Scale bar = 20 μm. (C) Lung wet/dry ratios and bronchoalveolar lavage (BAL) protein levels in sham- and LPS-treated mice with vehicle or BAM15 administration. Data are presented as mean ± SEM. (D) Arterial blood analysis, including partial pressure of oxygen (PaO\u003csub\u003e2\u003c/sub\u003e), partial pressure of carbon dioxide (PaCO\u003csub\u003e2\u003c/sub\u003e), lactate (Lac) and lactate dehydrogenase (LDH), measured 24 h after sham or LPS treatment, followed by vehicle or BAM15 (5 mg/kg) administration. (E) Serum total antioxidant status (TAS), total oxidant status (TOS), and TOS/TAS ratio (oxidative stress index, OSI) levels were examined in Sham+Vehicle/BAM15 and LPS+Vehicle/BAM15 groups after 24 h. \u003cem\u003en\u003c/em\u003e = 10, data show mean±SEM and \u003cem\u003eP\u003c/em\u003e-value was calculated following One-way ANOVA test. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, LPS+Vehicle vs. Sham+Vehicle groups; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05,\u003csup\u003e ##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, LPS+Vehicle vs. LPS+BAM15.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7622689/v1/a409dab76c2f0315473fe16d.png"},{"id":93730984,"identity":"af42bf0f-83fb-4eb9-9973-8ae9291b9969","added_by":"auto","created_at":"2025-10-17 02:22:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":393983,"visible":true,"origin":"","legend":"\u003cp\u003eBAM15 inhibits delayed neutrophil apoptosis and attenuates infiltration during lung injury. (A) Gating of neutrophils, macrophages, and monocytes in granulocytes (CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e) of BAL at 24 h for the Sham+Vehicle, Sham+BAM15, LPS+Vehicle, and LPS+BAM15 groups. (B) Percentages of immune cells in each group. Each circle represents a log-transformed average of duplicated samples. (C) The percentages of apoptotic neutrophils in each group, and (D) apoptotic neutrophils at 24 h by flow cytometry. (E) Representative co-immunostaining images showing Ly6G (red) and EpCAM (green) as markers for inflammatory infiltration. Nuclei are stained blue with DAPI. Scale bars: 100 μm. A total of three lungs were stained in each group, and six images per lung were analysed. \u003cem\u003en\u003c/em\u003e = 6, data show mean±SEM and \u003cem\u003eP\u003c/em\u003e-value was calculated following One-way ANOVA test. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05,\u003cem\u003e \u003c/em\u003e***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, LPS+Vehicle vs. Sham+Vehicle groups; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05,\u003csup\u003e ##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, LPS+Vehicle vs. LPS+BAM15.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7622689/v1/16959dccff6a7d453de0b00b.png"},{"id":93729284,"identity":"820bb779-487c-4c98-b79a-7a5519121948","added_by":"auto","created_at":"2025-10-17 02:14:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":416136,"visible":true,"origin":"","legend":"\u003cp\u003eBAM15 preserves tight junction integrity in lung tissue by mitigating mitochondrial damage and reducing mitochondrial DNA (mtDNA) release in LPS-challenged mice. (A) Mitochondrial reactive oxygen species (ROS) production was detected spectrofluorimetrically using DCFH-DA as a fluorescent dye, and quantitative analysed in Sham+Vehicle/BAM15 and LPS+Vehicle/BAM15 mice after 24 h. \u003cem\u003en\u003c/em\u003e = 10, data show mean±SEM and \u003cem\u003eP\u003c/em\u003e-value was calculated following One-way ANOVA test. ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, LPS+Vehicle vs. Sham+Vehicle groups; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, LPS+Vehicle vs. LPS+BAM15. (B) Representative electron microscopy images showing mitochondrial ultrastructure. Extensive mitochondrial damage and endoplasmic reticulum (ER) expansion are observed (original magnification, ×5000, Scale bar: 1 μm). Red arrows indicate deformed mitochondria characterised by detached small rounded mitochondria with loss of clearly defined cristae. Experiment representative of three independent experiments. (C) Time course of plasma mtDNA levels at 24 h after LPS-treated mice were administered vehicle or BAM1. LPS exposure resulted in a significant increase in mtDNA content, which was notably reduced by subsequent BAM15 treatment. Data are represented as mean ± SEM. Analysis between groups at each time point was performed with Šidák’s multiple-comparison test following mixed-effects analysis. ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, LPS+Vehicle vs. Sham+Vehicle groups; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, LPS+Vehicle vs. LPS+BAM15. (D) Effects of BAM15 on lung tissue integrity and autophagy in LPS-induced lung injury, as analysed by immunofluorescence. Representative images of occludin- and SQSTM1-stained tissues in each group at 24 h after incubation. Green, occludin; red, SQSTM1. Nuclei were stained blue with DAPI (original magnification: ×100). Data is representative of three independent experiments. (E) Western blot analysis of pro-apoptotic markers (p62, PINK1, and PCG-1α) and an autophagy-related marker (LC3B-II). Representative blot images are shown for each group. The western blot is representative of three independent experiments.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7622689/v1/2f5f44255ddcbd51cac53de8.png"},{"id":93729293,"identity":"64b07024-d6b4-457f-be07-d309865c3687","added_by":"auto","created_at":"2025-10-17 02:14:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":425041,"visible":true,"origin":"","legend":"\u003cp\u003eBAM15 modulates the cGAS-STING signalling pathway in LPS-induced lung injury. (A) Western blot analysis of cGAS-STING pathway components, showing expression of the key proteins cGAS, TBK1, and STING and the regulatory proteins USP18 and RNF5. Each panel shown is representative of three independent experiments. (B) Quantitative PCR analysis of cGAS and STING mRNA levels. (C) Representative images of immunohistochemical staining to assess the expression of cGAS, STING, and p-IRF3. Scale bar: 100 μm. LPS-induced lung injury resulted in substantial increases in the levels of cGAS and STING, which were notably reduced following BAM15 treatment (representative of three independent repeats). (D) BAM15 reduced pro-inflammatory cytokine levels in each group. The expression of pro-inflammatory cytokines IFN-β, TNF-α, and IL-6. \u003cem\u003en\u003c/em\u003e = 6 for B and D, data show mean±SEM and \u003cem\u003eP\u003c/em\u003e-value was calculated following One-way ANOVA test. ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, LPS+Vehicle vs. Sham+Vehicle; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 or \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, LPS+Vehicle vs. LPS+BAM15.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7622689/v1/b78164568144ded9e348473e.png"},{"id":93729292,"identity":"c2f57596-88ba-429a-91a9-116b15b18172","added_by":"auto","created_at":"2025-10-17 02:14:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":391238,"visible":true,"origin":"","legend":"\u003cp\u003eThe role of the STING pathway in mediating the protective effects of BAM15 against lung injury and mitochondrial damage in LPS-induced ALI. (A) Kaplan–Meier survival curves for BAM15-treated STING\u003csup\u003e-/- \u003c/sup\u003emice compared with wild-type (WT) controls over 4 days. Group sizes: WT (\u003cem\u003en\u003c/em\u003e = 8), STING\u003csup\u003e-/-\u003c/sup\u003e (\u003cem\u003en\u003c/em\u003e = 6). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. (B) Representative haematoxylin and eosin-stained lung tissue images from BAM15-treated WT and STING\u003csup\u003e-/- \u003c/sup\u003emice at 24 h post-LPS challenge (representative of three independent repeats). Scale bar = 20 μm. (C) Lung injury score, lung wet/dry ratio and oxygen partial pressures (PaO\u003csub\u003e2\u003c/sub\u003e) were measured. Data are presented as the means ± SEM and calculated by t-test,\u003cem\u003e n\u003c/em\u003e = 6, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 or **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. (D) Representative immunofluorescence images of occludin at 24 h in BAM15-treated WT and STING\u003csup\u003e-/-\u003c/sup\u003e mice after incubation. occludin is shown in green, and nuclei are stained with blue DAPI (original magnification: ×80, scale bar = 10 μm). Data is representative of three independent experiments. (E) Representative electron microscopy images showing mitochondrial damage and ER expansion. Red arrows indicate deformed mitochondria with detached small rounded structures and loss of clearly defined cristae (original magnification: ×5000; scale bar = 1 μm). Experiment representative of three independent experiments. (F) Quantitative analysis of mitochondrial stress markers. Mitochondrial stress remained significantly higher in the STING\u003csup\u003e-/-\u003c/sup\u003e group compared with the WT group following BAM15 treatment. Data are presented as mean ± SEM of each group (Sham+BAM15/WT or STING\u003csup\u003e-/-\u003c/sup\u003e, \u003cem\u003en\u003c/em\u003e = 8; LPS+BAM15/WT, \u003cem\u003en\u003c/em\u003e = 6; LPS+BAM15/STING\u003csup\u003e-/-\u003c/sup\u003e, \u003cem\u003en\u003c/em\u003e = 5). Dunn’s multiple-comparison test following the Kruskal–Wallis test. ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, Sham+BAM15 (WT/STING\u003csup\u003e-/-\u003c/sup\u003e) vs. LPS+BAM15 (WT/STING\u003csup\u003e-/-\u003c/sup\u003e) groups; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, WT (LPS+BAM15) vs. STINT\u003csup\u003e-/-\u003c/sup\u003e (LPS+BAM15).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7622689/v1/acd290ff86b136571e0aec67.png"},{"id":93732366,"identity":"ec1ec54a-61e0-467d-984f-7a39ffee8de3","added_by":"auto","created_at":"2025-10-17 02:30:30","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":451149,"visible":true,"origin":"","legend":"\u003cp\u003eBAM15 regulates autophagy and modulates inflammation via the cGAS-STING pathway, reducing mtDNA release and cGAS-STING activation. (A) Representative immunofluorescence images of LC3 and LAMP2 at 24 h in Sham or BAM15-administered LPS-treated wild-type (WT) and STING\u003csup\u003e-/-\u003c/sup\u003e mice after incubation. LC2 is shown in green, LAMP2 in red, and nuclei are stained blue using DAPI. Scale bar: left, 20 μm; right, 10 μm. (B) The levels of mtDNA in WT and STING\u003csup\u003e-/-\u003c/sup\u003e mice after Vehicle or BAM15 treatment. Dunn’s multiple-comparison test following the Kruskal–Wallis test. ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, Sham+BAM15 (WT/STING\u003csup\u003e-/-\u003c/sup\u003e) vs. LPS+BAM15 (WT/STING\u003csup\u003e-/-\u003c/sup\u003e) groups; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, WT (LPS+BAM15) vs. STINT\u003csup\u003e-/-\u003c/sup\u003e (LPS+BAM15). (C) Immunohistochemical analysis of cGAS and cGAMP levels in WT and STING\u003csup\u003e-/-\u003c/sup\u003e mice after Vehicle or BAM15 treatment. (D) Percentage of neutrophils in the WT and STING\u003csup\u003e-/-\u003c/sup\u003e groups. (E) Apoptotic neutrophils at 24 h, and percentages of apoptotic neutrophils in each group. Data are presented as the means ± SEM and calculated by t-test,\u003cem\u003e n\u003c/em\u003e = 6, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. (F) Representative coimmunostaining images showing Ly6G (red) and EpCAM (green) as markers for inflammatory infiltration in ALI. Nuclei were stained with DAPI (blue). Scale bar: left, 20 µm; right, 10 µm. A total of three lung samples per group were stained, and six images per lung were analysed. (G) Quantification of inflammatory markers (IL-1β, IL-6, and IFN-β) in WT and STING\u003csup\u003e-/-\u003c/sup\u003e mice after BAM15 intervention. Data are presented as mean ± SEM of each group (Sham+BAM15/WT or STING\u003csup\u003e-/-\u003c/sup\u003e, \u003cem\u003en\u003c/em\u003e = 8; LPS+BAM15/WT, \u003cem\u003en\u003c/em\u003e = 6; LPS+BAM15/STING\u003csup\u003e-/-\u003c/sup\u003e, \u003cem\u003en\u003c/em\u003e = 5). Dunn’s multiple-comparison test following the Kruskal–Wallis test. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, Sham+BAM15 (WT/STING\u003csup\u003e-/-\u003c/sup\u003e) vs. LPS+BAM15 (WT/STING\u003csup\u003e-/-\u003c/sup\u003e) groups; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, WT (LPS+BAM15) vs. STINT\u003csup\u003e-/-\u003c/sup\u003e (LPS+BAM15).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7622689/v1/16acd32abdc12982ee30e69c.png"},{"id":93729296,"identity":"5cd8106d-08a1-4214-a6e3-563678240c17","added_by":"auto","created_at":"2025-10-17 02:14:31","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":176940,"visible":true,"origin":"","legend":"\u003cp\u003eA graphical summary of the mechanisms underlying the effects of BAM15 on lipopolysaccharide-induced ALI. BAM15 contributes to the maintenance of mitochondrial homeostasis by reducing mitochondrial DNA (mtDNA) release, which in turn modulates the cGAS-STING pathway and regulates key mechanisms. (1) Regulation of delayed neutrophil apoptosis: By preventing excessive mtDNA release, BAM15 ensures the timely apoptosis of neutrophils, thus reducing prolonged inflammation. (2) Reduction of ROS stress inhibits excessive autophagy: BAM15 maintains mitochondrial function, thereby reducing mitochondrial ROS production, which in turn alleviates oxidative stress and thereby prevents excessive autophagy. This contributes to an alleviation of inflammatory storms, thus reducing inflammatory reactions and protecting lung tissues. Collectively, the capacity of BAM15 to regulate mitochondrial homeostasis and control the cGAS-STING pathway provides a robust protective effect against LPS-induced ALI (created using BioRender).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7622689/v1/68c3521cd8cb038d8e491391.png"},{"id":104721043,"identity":"d17fdc50-49f4-49d8-8e90-19c99c70450b","added_by":"auto","created_at":"2026-03-16 12:21:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3728896,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7622689/v1/d3a62baa-095c-420a-8c93-ffaeb5baa107.pdf"},{"id":93729280,"identity":"2a03cbc8-c59e-4f69-9241-ad83d25f6213","added_by":"auto","created_at":"2025-10-17 02:14:30","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1286863,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupporting information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdditional supporting information may be found online in the Supporting Information section at the end of the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S1. \u003c/strong\u003eAssessment of the potential effects of BAM15 on the liver, kidney, cardiac, and pancreatic function in mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S2. \u003c/strong\u003eDelayed BAM15 treatment improves lung injury outcomes and survival in LPS-induced ALI.\u003c/p\u003e","description":"","filename":"SupplementalMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7622689/v1/5d00cb0adcba5ef7cb99175d.docx"},{"id":93729287,"identity":"506c7256-a2cc-42fa-8f81-69e1126f4f87","added_by":"auto","created_at":"2025-10-17 02:14:30","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":997634,"visible":true,"origin":"","legend":"\u003cp\u003eOriginal WB gel in Figure 3E\u003c/p\u003e","description":"","filename":"WBFigure3E.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7622689/v1/fbd500b442c513f7583c0b40.pdf"},{"id":93729285,"identity":"01f4d0b0-f196-4ca8-bc87-f6bcd4f44ec8","added_by":"auto","created_at":"2025-10-17 02:14:30","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":997689,"visible":true,"origin":"","legend":"Original WB gel in Figure 4A","description":"","filename":"WBFigure4A.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7622689/v1/f7d2023a9aa39da06c7900a0.pdf"}],"financialInterests":"(Not answered)","formattedTitle":"BAM15 treatment mitigates lipopolysaccharide-induced acute lung injury by preserving mitochondrial dynamic equilibrium and modulating inflammatory responses via the cGAS–STING signalling pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAcute lung injury (ALI) and its more severe form, acute respiratory failure (ARDS), are debilitating respiratory conditions, characterised by widespread lung inflammation, increased alveolar and capillary permeability, and severe hypoxaemia that are not attributable to cardiogenic pulmonary oedema [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. These adverse conditions can arise from diverse direct and indirect insults to the lungs, including trauma, sepsis, pneumonia, and the inhalation of toxic substances [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, despite significant advances in our understanding of the pathophysiology of ALI, it poses significant clinical challenges, owing to its high incidence and associated mortality [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The prevalence of ARDS in intensive care units is estimated to be approximately 10%, with a mortality rate as high as 46% [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These alarming findings highlight the urgent need for effective therapeutic strategies that can be applied to reduce the burden of ALI on public health systems.\u003c/p\u003e\u003cp\u003eMitochondria, as cellular powerhouses, play pivotal roles in maintaining energy metabolism, redox balance, and regulating cell death pathways [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Mitochondrial dysfunction has recently emerged as a key factor in the pathogenesis of infectious diseases, and a growing body of evidence supports the role of mitochondria in lung epithelial and immune cells, which are particularly susceptible to oxidative stress and damage, notable hallmarks of ALI [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Disruption of mitochondrial dynamics, specifically the equilibrium between mitochondrial fusion and fission, has been identified as a contributory factor in disease progression, with excessive mitochondrial fission resulting in mitochondrial fragmentation, an elevated production of reactive oxygen species (ROS), and the release of mitochondrial DNA (mtDNA) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Clinical studies have shown that mtDNA levels are associated with the severity and prognosis of ARDS [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and it has been established that a disruption of mitochondrial function can promote the activation of inflammatory pathways, including the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signalling pathway, which is associated with innate immune responses and exacerbates lung inflammation and injury by influencing neutrophil function [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Subsequently, this cascade activates inflammatory responses via interferon regulatory factor 3 (IRF3) and nuclear factor κB (NF-κB) signalling, which are central to the innate immune response and the pathogenesis of numerous inflammatory diseases[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Nevertheless, few studies have sought to assess the therapeutic strategies targeting mitochondrial dysfunction and the cGAS-STING pathway, which potentially represent promising avenues for new interventions.\u003c/p\u003e\u003cp\u003eBAM15 has recently attracted considerable interest in different disease models, particularly those involving mitochondrial dysfunction [\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. BAM15 is a selective mitochondrial uncoupler with distinctive chemical characteristics facilitating efficient mitochondrial membrane penetration. By uncoupling oxidative phosphorylation without causing significant mitochondrial depolarisation or cytotoxic effects, BAM15 can specifically modulate mitochondrial respiration and enhance metabolic flexibility, whilst maintaining membrane integrity. Mitochondrial uncouplers disrupt the proton gradient across the mitochondrial membrane, thereby reducing the membrane potential and ROS production without compromising ATP synthesis. This distinctive mechanism enables BAM15 to alleviate oxidative stress and prevent mitochondrial injury, thus establishing it as a promising candidate for treating conditions in which mitochondrial-associated dysfunction is a pivotal factor [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Moreover, recent findings have indicated that mitochondrial uncoupling may contribute to the regulation of mitochondrial dynamics, promote mitochondrial fusion, and prevent excessive fission, all of which are key factors for the maintenance of mitochondrial homeostasis [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Thus, by restoring the equilibrium between mitochondrial fusion and fission, BAM15 may contribute to the preservation of lung cell function and a reduction in the inflammatory responses associated with ALI.\u003c/p\u003e\u003cp\u003eIn this study, we sought to examine the effects of BAM15 on LPS-induced ALI, with a particular emphasis on its capacity to maintain mitochondrial dynamic equilibrium and modulate neutrophil apoptosis. By elucidating these fundamental mechanisms, we provide new insights into potential therapeutic strategies for reducing ALI-induced morbidity and mortality in clinical settings.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eBAM15 treatment reduces mortality and attenuates lung injury in mice with LPS-induced ALI\u003c/h2\u003e\u003cp\u003eWe established that BAM15 had no discernible effects on liver, kidney, or cardiac function, with enzyme and biomarker levels remaining within normal ranges (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). These findings accordingly confirmed that BAM15 is well tolerated at the selected dose and had no significant adverse impacts on the overall physiological health of mice.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBAM15 treatment was found to have significant protective effects in a murine model of LPS-induced ALI. Initially, we found that BAM15 treatment contributed to a marked improvement in survival, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, with mice in the LPS\u0026thinsp;+\u0026thinsp;BAM15 group being characterised by a significantly higher survival than those in the LPS\u0026thinsp;+\u0026thinsp;Vehicle group. Histopathological analysis, based on haematoxylin and eosin staining, revealed severe lung damage among the LPS\u0026thinsp;+\u0026thinsp;Vehicle group mice, including evidence of alveolar destruction, oedema, and tissue congestion. However, BAM15 treatment significantly attenuated these pathological changes, resulting in a more preserved alveolar structure and a reduced number of inflammatory cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In addition, compared with the LPS\u0026thinsp;+\u0026thinsp;Vehicle group, we also detected significantly lower lung W/D ratios and protein contents (which quantify damage based on specific pathological criteria) in the LPS\u0026thinsp;+\u0026thinsp;BAM15 mice, thereby further confirming the protective effects of BAM15 on lung tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, BAM15 was also found to enhance respiratory function, as evidenced by an increase in arterial oxygen partial pressure (PaO₂) in LPS\u0026thinsp;+\u0026thinsp;BAM15 mice compared with those in the LPS\u0026thinsp;+\u0026thinsp;Vehicle group, thereby indicating better oxygenation and reduced respiratory distress. To assess the metabolic disturbances and tissue damage in ALI, we determined the levels of lactate and lactate dehydrogenase, the former of which were significantly lower in LPS\u0026thinsp;+\u0026thinsp;BAM15 mice compared with those in the LPS\u0026thinsp;+\u0026thinsp;Vehicle group, indicating improved metabolic function and enhanced tissue oxygenation. Similarly, a significant reduction in lactate dehydrogenase levels was observed in the BAM15-treated mice, indicating a notable improvement in tissue integrity. Moreover, serum TAS levels, an indicator of the protection of lung tissues from oxidative damage via the scavenging of ROS, were reduced in the LPS\u0026thinsp;+\u0026thinsp;Vehicle mice compared with those in the sham-operated group. In addition, the levels of serum TOS, a sensitive indicator of oxidative stress, and the TOS/TAS ratio, which represents the redox balance between antioxidant and oxidative states (i.e., OSI), were elevated in LPS\u0026thinsp;+\u0026thinsp;Vehicle mice. BAM15 was found to inhibit the elevation of TOS and OSI levels, whilst augmenting the release of TAS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). From the perspective of consistency with clinical treatment protocols, we conducted further investigations on the administration of BAM15 at 6 h after disease onset, the findings of which indicated that the administration of BAM15 is an effective treatment for lung injury and improves survival outcomes, even after disease onset (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Collectively, our findings indicate that BAM15 treatment can contribute to a significant mitigation of lung injury, promotes oxygenation, and alleviates oxidative damage, thereby providing evidence for its potential application as a therapeutic intervention for LPS-induced ALI.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eBAM15 inhibits delayed neutrophil apoptosis, thereby suppressing inflammatory infiltration and attenuating lung injury\u003c/h3\u003e\n\u003cp\u003eInfections induce immune system activation, including the recruitment of a range of immune-related cell types, among which neutrophils play pivotal roles as an initial line of defence. We have previously demonstrated a significant delay in neutrophil apoptosis during lung injury, which as a consequence was significantly exacerbated [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Flow cytometric analysis of BALF revealed that BAM15 treatment resulted in moderate reductions in neutrophil infiltration and the proportion of neutrophils in the BALF. However, there were no significant changes in the number of macrophages or monocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). The results also revealed that BAM15 can significantly alleviate the delay in neutrophil apoptosis when used for lung injury treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). This suggests that BAM15's anti-inflammatory effects primarily involve maintaining neutrophil viability during the acute phase, rather than inducing apoptosis. Furthermore, immunofluorescence analysis revealed a reduction in the number of neutrophils within the lung tissues following BAM15 treatment, thereby indicating a notable reduction in tissue infiltration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), and that BAM15 may alleviate tissue injury by regulating neutrophil apoptosis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eBAM15 maintains tight junction integrity in lung tissue by mitigating mitochondrial damage and reducing mtDNA release in LPS-challenged mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAcute lung injury is characterised by an excessive production of ROS, leading to increases in oxidative stress, which in turn causes mtDNA damage and abnormalities in mitochondrial ultrastructure. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, compared with mice in the LPS\u0026thinsp;+\u0026thinsp;Vehicle group, those treated with BAM15 were characterised by a significant reduction in mitochondrial oxidative stress, thereby highlighting the protective role of this uncoupler in preserving mitochondrial function. Electron microscopy revealed extensive mitochondrial damage and endoplasmic reticulum expansion in the lung tissues of LPS\u0026thinsp;+\u0026thinsp;Vehicle group mice, whereas the morphology of mitochondria in the LPS\u0026thinsp;+\u0026thinsp;BAM15 mice remained relatively well intact (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Further analysis indicated that exposure to LPS resulted in a marked increase in mtDNA content over 24 h, which was significantly attenuated by BAM15 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), whereas immunofluorescence staining revealed a significant enhancement of the structural integrity of tight junctions in BAM15-treated mice, as indicated by an increase in occludin expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Additionally, we established that BAM15 inhibits excessive autophagy in lung tissue cells, thereby preventing the overactivation of autophagic processes in response to lung injury, and western blot analysis revealed substantial changes in autophagy-related proteins, with a significant upregulation of pro-apoptotic markers, such as p62, PINK1, and PCG-1α, being detected in the lung injury group, whereas there was a corresponding reduction in the expression of LC3B-II (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Notably, these expression profiles were reversed in the LPS\u0026thinsp;+\u0026thinsp;BAM15 mice, suggesting that the protective effects BAM15 are mediated via an inhibition of autophagy and subsequent reduction in mtDNA release, thereby mitigating mitochondrial oxidative stress.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eBAM15 modulates mitochondrial homeostasis via the cGAS/STING signalling pathway\u003c/h3\u003e\n\u003cp\u003eThe release of mtDNA into the cytoplasm following mitochondrial damage is detected by cGAS, which activates the downstream cGAS-STING pathway [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In the present study, we demonstrated that BAM15 regulates mitochondrial autophagy by modulating this pathway. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, compared with mice in the LPS\u0026thinsp;+\u0026thinsp;Vehicle group, key proteins in this pathway, namely, cGAS, TBK1, and STING, were significantly downregulated in the BAM15-treated mice during the lung injury phase. Furthermore, we observed an elevation in the expression of the regulatory proteins USP18 and RNF5, indicating that the effects of BAM15 are mediated via a modulation of these upstream regulators. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, compared with the LPS\u0026thinsp;+\u0026thinsp;Vehicle group mice, qPCR analysis revealed a significant reduction in cGAS and STING expression among those in the LPS\u0026thinsp;+\u0026thinsp;BAM15 group. Immunohistochemical staining revealed a significant increase in cGAS and STING expression during lung injury, which was markedly reduced in response to BAM15 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Moreover, the administration of BAM15 resulted in a notable reduction in the levels of pro-inflammatory cytokines, including IFN-β, TNF-α, and IL-6, which is indicative of its anti-inflammatory properties (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). In conclusion, these findings indicate that BAM15 alleviates mitochondrial oxidative stress and reduces inflammation by inhibiting the cGAS-STING signalling pathway.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eBAM15 protects against mitochondrial damage and maintains intercellular junction stability via the STING pathway in ALI\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBuilding on previous studies, we further examined the influence of BAM15-regulated autophagy on lung injury using a STING\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mouse model. Notably, in the absence of STING, we observed an abrogation of the protective effects of BAM15. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, compared with mice in the wild-type (WT) group, the BAM15-treated STING\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were characterised by significantly lower rates of survival. Moreover, we failed to detect any improvement in lung injury, as assessed by haematoxylin and eosin staining, in the STING\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e group following BAM15 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Similarly, there were no significant improvements in lung injury scores, lung wet-to-dry ratios, or oxygen partial pressures in the BAM15-treated STING\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice compared with those in the WT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Consistently, immunofluorescence analysis revealed that in the absence of STING, BAM15 failed to restore lung tissue integrity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), whereas electron microscopy revealed no notable mitochondrial protection in BAM15-treated STING\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), and relative to that in the WT mice, mitochondrial stress remained elevated (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). These findings thus highlight the pivotal role play by the STING pathway in mediating the protective effects of BAM15 against lung injury and mitochondrial damage.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eBAM15 regulates autophagy and modulates inflammation via the cGAS-STING pathway, thereby reducing mtDNA release and cGAS-STING activation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFurther investigations were conducted to determine the mechanisms underlying the observed effects of BAM15. Following LPS stimulation and BAM15 intervention, STING\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were found to be characterised by elevated levels of autophagy and lysosomal activity compared with those in the WT group, indicating that a deficiency of STING hampers the ability of BAM15 to fully regulate autophagy and lysosomal dynamics (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Additionally, our observation that mtDNA levels in STING\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice remained elevated and higher than those in the WT group tends to indicate that STING is essential for BAM15-mediated mtDNA clearance, given that its absence result in a reduction in the efficiency of mtDNA reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Furthermore, immunohistochemical analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) revealed that compared with normal mice, cGAS levels were higher in those with STING-knockout following BAM15 treatment. However, the levels of cGAMP, a secondary messenger produced in response to cGAS activation, remained relatively stable, which would tend to indicate that despite the reduction in cGAS levels, the deletion of STING disrupted a feedback loop required for significant changes in cGAMP production.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCompared with WT mice, in STING\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e model mice, we detected a reduction in the capacity of BAM15 to regulate the delay in neutrophil apoptosis, as indicated by a significant increase in the proportion of neutrophils in these latter mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Moreover, the overall rate of neutrophil apoptosis was observed to be markedly elevated, thereby providing evidence that the STING pathway plays a pivotal role in mediating the effects of BAM15 with respect to neutrophil survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). These findings would thus appear to indicate that an absence of STING may compromise the efficacy of BAM15 in preventing the delay in neutrophil apoptosis, and also inhibits the enhancement of neutrophil infiltration in lung tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eF), thereby highlighting the key role of the STING signalling pathway in this process. With respect to inflammatory markers, we found that compared with the BAM15-treated WT mice, the levels of IFN-β, IL-6, and IL-1β remained elevated in the STING\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice treated with BAM15 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eG), thus indicating that a deficiency of STING diminished the efficacy of BAM15 in controlling inflammation, leading to a persistent expression of inflammatory cytokines. Collectively, these findings highlight the essential role played by the STING pathway in promoting the BAM15-mediated autophagy and the regulation of inflammatory responses.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur findings in this study, in which we investigated the effects of BAM15, a mitochondrial uncoupler, on mitochondrial function and the cGAS-STING signalling pathway, provide substantial evidence to indicate that by preserving mitochondrial dynamics and suppressing inflammatory pathways, BAM15 can confer significant protection against LPS-induced ALI. Specifically, BAM15 was found to mitigate key pathological features of ALI, including apoptosis, inflammation, oxidative stress, and autophagy, whilst restoring mitochondrial homeostasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These findings accordingly highlight the potential applicability of BAM15 as a promising therapeutic agent for the treatment of ALI/ARDS, thereby addressing the pressing need for targeted interventions that go beyond conventional supportive therapies.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCompared with other uncouplers, such as carbonyl cyanide-\u003cem\u003ep\u003c/em\u003e-trifluoromethoxyphenylhydrazone and 2,4-dinitrophenol, which induce mitochondrial depolarisation and are frequently associated with cytotoxic effects, BAM15 has lower cytotoxicity and fewer off-target effects on plasma membrane depolarisation [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. These distinctive characteristics highlight the potential therapeutic applicability of BAM15 as a safer mitochondrial uncoupler. Although the therapeutic potential of mitochondrial uncouplers has previously been investigated with respect to the treatment of cardiovascular diseases [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], diabetes [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], non-alcoholic steatohepatitis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and sepsis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], to the best of our knowledge, there have been no studies that have assessed the efficacy of these uncouplers in the treatment of ALI or ARDS. A significant finding in this study was that BAM15 can effectively contribute to maintenance of the equilibrium of mitochondrial dynamics in ALI, particularly the balance between mitochondrial fusion and fission, which are of paramount importance in maintaining mitochondrial integrity and function [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. BAM15 promotes fusion, whilst inhibiting excessive fission, thereby preventing mitochondrial fragmentation, suppressing ROS production, and reducing mtDNA release, all of which can contribute to an alleviation of cellular damage and inflammation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. By maintaining this balance, BAM15 mitigates the cellular damage commonly associated with mitochondrial dysfunction and excessive ROS production in ALI. Autophagy is an adaptive and protective cellular process that mitigates cellular damage and inhibits apoptosis during sepsis [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], and during the early stages of sepsis, autophagy contributes to protecting the host from MODS by preserving immune cell viability, balancing pro- and anti-inflammatory cytokine production, and facilitating clearance of microbial infections [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In the present study, we detected a reduction in the levels of autophagic markers in the lung tissues of mice treated with BAM15, thus substantiating its protective function in lung epithelial cells and overall lung performance during ALI.\u003c/p\u003e\u003cp\u003eFurthermore, we established that BAM15 plays a pivotal role in reversing the delay in neutrophil apoptosis. In this regard, although neutrophils play key roles in the initial defence against pathogens, their prolonged activation and delayed apoptosis can result in significant tissue damage and exacerbate lung injury [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. To prevent excessive tissue damage, neutrophils normally undergo apoptosis following the resolution phase. However, in ALI, multiple factors, including inflammatory cytokines and hypoxia, can cause a delay in the apoptotic process, resulting in the sustained survival of neutrophils and an increased risk of tissue damage. This prolonged survival of neutrophils can intensify the inflammatory environment, leading to further damage to the lung tissues and a prolongation of recovery. Delayed neutrophil apoptosis regulation may accordingly represent an effective means of restoring homeostasis and attenuating the release of inflammatory infiltrates, whilst also reducing tissue damage associated with excessive inflammation. Consequently, the neutrophil apoptotic pathway could serve as a useful target for alleviating inflammatory responses in ALI.\u003c/p\u003e\u003cp\u003eThe cGAS-STING pathway plays a pivotal role in the innate immune response against diverse pathogens, including viruses, bacteria, and parasites [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. However, the function of the STING pathway is not merely restricted to antimicrobial defence. In response to cellular stress, mtDNA is released into the cytoplasm, wherein it activates the cGAS-STING pathway, thereby initiating an inflammatory response [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Furthermore, abnormal activation of the cGAS-STING pathway has been identified as a key factor contributing to lung injury and as an inflammatory mediator [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. A deficiency in either cGAS or STING has been demonstrated to alleviate silica-induced lung inflammation [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], and in the present study, we found that BAM15 efficiently suppresses mtDNA release and mitigates cellular autophagy and mitochondrial damage via the cGAS-STING pathway, thereby exerting a protective effect against ALI. Consequently, exploiting the ability of BAM15 to modulate mitochondrial dynamics, reduce ROS production, inhibit mtDNA release, and downregulate the cGAS-STING pathway may represent a novel therapeutic approach that could complement existing treatments or serve as a stand-alone therapy, particularly in patients unresponsive to current interventions.\u003c/p\u003e\u003cp\u003eHowever, despite these encouraging findings, further investigations are required to address the limitations of this study. Notably, although our murine model provides valuable insights into the protective effects of BAM15, its applicability to human ALI and ARDS remains uncertain. The complexity of human ALI, which frequently involves comorbidities and triggers other than LPS exposure, necessitates further studies in clinical settings or more complex animal models to fully assess the translational potential of BAM15. Although the regulation of mitochondrial dynamics and the cGAS-STING pathway by BAM15 is evident, the precise molecular mechanisms underlying these effects remain unclear. Future research should accordingly focus on identifying the upstream regulators and downstream effectors involved in the BAM15-mediated mitochondrial protection and resolution of inflammation.\u003c/p\u003e\u003cp\u003eCollectively, our findings in this study provide convincing evidence that BAM15 can aid in protecting against LPS-induced ALI by preserving the mitochondrial dynamic balance, reducing delayed neutrophil apoptosis, attenuating oxidative stress and autophagy, and inhibiting the cGAS-STING signalling pathway. These findings identify BAM15 as a promising therapeutic candidate for the treatment of induced ALI and address the significant shortcomings of current therapies by targeting both mitochondrial dysfunction and excessive inflammation. Further studies are, nevertheless, required to elucidate the molecular mechanisms underlying the protective effects of BAM15 and to evaluate its safety and efficacy in clinical settings.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eAnimal model\u003c/h2\u003e\u003cp\u003eMale C57BL/6 and STING\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e mice, aged 8 to 10 weeks and weighing 20 to 25 g. C57BL/6 mice were obtained from Charles River (Beijing, China), and STING\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e mice were obtained from the Model Animal Research Center of Nanjing University (Nanjing, China). All mice were of a C57BL/6 genetic background. The genotyping of the knockout mice was conducted through polymerase chain reaction (PCR) analysis of DNA extracted from tail biopsy samples. The animals were randomly assigned to experimental groups. No animals were excluded from the study on the grounds of illness, and the animal experiments were conducted in a blinded manner. The mice were housed in a controlled environment maintained at a temperature and humidity of 24\u0026ndash;26℃ and 55%\u0026ndash;60%, respectively, under a 12 h light:12 h dark cycle, with free access to food and water.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eALI model\u003c/h3\u003e\n\u003cp\u003eLipopolysaccharide (LPS) (LPS, L2630, \u003cem\u003eE. coli\u003c/em\u003e 0111: B4; Sigma Aldrich, MA, USA) in 0.9% saline was administered via intratracheal injection with a single dose of 10 mg/kg LPS to generate a model of ALI. This relatively high-dose LPS model has been established to induce a robust inflammatory response and severe pulmonary injury, closely mirroring the pathological features of human acute lung injury [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The BAM15-treated groups were administered BAM15 intraperitoneally at the time of the LPS challenge. Based on the findings of previous studies [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], mice were intraperitoneally administered a 5 mg/kg dose of BAM15 to assess potential drug toxicity. The control and BAM15-only groups received equivalent volumes of vehicle, comprising 40 mL/kg 3% dimethyl sulphoxide (DMSO) diluted with 0.9% saline. The C57BL/6 mice were randomly assigned to four groups. The experimental groups were as follows: Sham\u0026thinsp;+\u0026thinsp;Vehicle, Sham\u0026thinsp;+\u0026thinsp;BAM15, LPS\u0026thinsp;+\u0026thinsp;Vehicle, and LPS\u0026thinsp;+\u0026thinsp;BAM15. Following a 24 h period of LPS challenge, the mice were sacrificed and blood or bronchoalveolar lavage (BAL) samples were collected. The organs were either frozen immediately or fixed in formalin for subsequent analysis. The frozen organs were stored at \u0026minus;\u0026thinsp;80\u0026deg;C. To ensure the reliability of the study findings, the investigators were blinded to the group allocation for analysis.\u003c/p\u003e\n\u003ch3\u003eSurvival analysis\u003c/h3\u003e\n\u003cp\u003eThe survival of the mice was evaluated at 24 h intervals following exposure, and percentage survival was determined at 7 d after LPS administration. At the end of the observation period, surviving mice were euthanised via CO₂ asphyxiation.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eHistological and immunohistochemical analyses\u003c/h2\u003e\u003cp\u003eMice were euthanised via CO\u003csub\u003e2\u003c/sub\u003e asphyxiation. Lung tissues were harvested, fixed in 10% neutral buffered formalin, embedded in paraffin and sectioned at 5 \u0026micro;m thickness. Haematoxylin and eosin staining was performed to assess the degree of lung injury, with light microscopy performed to obtain non-overlapping fields of view (20 \u0026micro;m) for each animal, including alveolar thickening, inflammatory cell infiltration, and oedema.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eWet/dry ratio determination\u003c/h2\u003e\u003cp\u003eFreshly obtained right lungs were weighed and then heated in a 68\u0026deg;C oven for 72 h. The weight of the lung tissues was recorded until no further change was observed. The wet/dry ratio was calculated to quantify the severity of pulmonary oedema.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eAnalysis of arterial blood gas\u003c/h2\u003e\u003cp\u003eThe animals were anaesthetised at a timepoint of 24 h. The carotid artery of each animal was cannulated, and arterial blood was collected into a syringe. Blood gases (PaO₂ and PaCO₂), lactate, and lactate dehydrogenase were measured using a blood gas analyzer (GemPremier 3000; Beckman, USA). The remaining blood was stored for further biochemical analysis. Subsequently, the lungs were meticulously harvested, taking care to ensure that both right and left lungs were collected. The left lung tissue was rapidly frozen in liquid nitrogen and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for subsequent analysis. The right lung tissue was divided into two portions and separately placed into glutaraldehyde and buffered 10% formalin solutions for histological evaluation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eMeasurement of oxidative stress\u003c/h2\u003e\u003cp\u003eThe oxidative stress index (OSI) was calculated as the ratio of total oxidant status (TOS) to total antioxidant status (TAS). The levels of TOS and TAS in serum were determined using a commercial kit (ENZ-51011; Sigma-Aldrich, Germany) with measurements performed using a microplate spectrophotometer (NanoDrop 8000; Thermo Fisher, CA, USA) according to the manufacturer\u0026rsquo;s instructions. For TOS, the assay was calibrated using hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) with the values obtained being expressed as \u0026micro;mol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e equivalent/L, whereas for TAS, the assay was calibrated with Trolox, and the values are expressed in terms of \u0026micro;mol Trolox equivalent/L. ROS levels were measured in lung homogenates using a commercially available ROS detection kit (S0035M; Beyotime, China).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eFlow cytometry analysis\u003c/h2\u003e\u003cp\u003ePhosphate-buffered saline (1 mL) was injected into the trachea, and having rinsed three times, sputum was collected. Cells were obtained by centrifugation at 500 \u0026times; \u003cem\u003eg\u003c/em\u003e at 4\u0026deg;C and collected into a centrifuge tube. Cells (5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e per tube) were processed for flow cytometry. The cells were incubated in an Fc block (1:200: 553142; BD Biosciences, Sparks, MD) for 10 min at 4\u0026deg;C. Surface staining was performed in the dark for 15 min. The surface markers used for myeloid cells were as follows: BV605 rat anti-mouse CD45 (1:200: clone 30-F11, 561865; BD Biosciences), APC rat anti-mouse Ly6G (1:1000: clone 1A8, 560599; BD Biosciences), BUV737 rat anti-mouse Ly6C (1:1000; clone HK1.4. rMAb, 755201; BD Biosciences), Alexa Fluo\u0026reg; 568 anti-mouse CD11c (1:200: clone N418, 745852; BD Biosciences), and Alexa Fluor\u0026reg; 488 anti-mouse/human CD14 (1:200: clone M5E2, 555397; BD Biosciences). Apoptotic cells were detected using 7-AAD (1:200, 559925; BD Biosciences) and FITC Annexin V (1:200, 556420; BD Biosciences). Cells were analysed using a BD FACSCanto II cytometer (BD Life Sciences, San Jose, CA). To distinguish positive from negative staining cell populations, we used a fluorescence minus one (FMO) control for all gating analyses. Correction matrices were calculated and applied using FlowJo software (Tree Star, Ashland, OR).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e\u003cp\u003eELISAs were employed to determine the concentrations of cytokines [IFN-β (PI568, Beyotime, China), TNF-α (PT512, Beyotime, China), IL-1β (P5898, Beyotime, China), and IL-6 (PI326, Beyotime, China)] in plasma. The assays were performed according to the instructions provided by the manufacturer.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eMitochondrial DNA content\u003c/h2\u003e\u003cp\u003eMitochondrial DNA (mtDNA) copy number was assessed based on the ratio of mtDNA to nuclear-encoded β-actin using real-time PCR, as described by Amaral et al. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. DNA was extracted using a DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eTEM analyses\u003c/h2\u003e\u003cp\u003eSmall pieces of lung tissue were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (PH 7.4). After subsequent rinsing in 0.1 M phosphate buffer, the samples were post-fixed in 1% osmium tetroxide for 2 h, dehydrated in a graded series of ethanol, embedded in acetone, and sectioned at approximately 70 nm. The preparations were viewed and image obtained using an electron microscope (Verios XHR SEM, Thermo Fisher, USA), with at least three randomly selected fields per sample being evaluated by a blinded pathologist.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence (IF) and immunohistochemistry (IHC)\u003c/h2\u003e\u003cp\u003eLung tissues were fixed in 4% paraformaldehyde solution for 48 h, paraffin-embedded, and sectioned at a thickness of 5 \u0026micro;m. For IF microscopy, sections were deparaffinised in xylene and rehydrated using graded ethanol series, and antigen was extracted using PH 9.0 EDTA. For IF microscopy. Sections were rinsed three times (each for 5 min) with TBS, blocked with 10% donkey serum for 30 min at 37\u0026deg;C, and then incubated overnight at 4\u0026deg;C with 488-conjugated occludin recombinant antibody (1:200: CL488-80545; Proteintech, Santa Cruz, CA, USA) or anti-SQSTM1/p62 antibody (1:200: ab109012; Abcam, Cambridge, UK), anti-LC3 antibody (1:100: CL647-14600; Proteintech, Santa Cruz, CA, USA) or anti-LAMP2 (1:200: L0668; Sigma-Aldrich, Germany). After washing, the sections were incubated with diluted fluorescent-labelled secondary antibody for 30 min at room temperature and counterstained with DAPI (28718-90-3; Sigma-Aldrich, Germany) in the dark for 10 min to show the nuclei. Images were obtained using an Olympus U-25ND25 fluorescence microscope (Olympus, Tokyo, Japan).\u003c/p\u003e\u003cp\u003eFor IHC, sample sections were blocked with 5% BSA for 30 min, and then incubated with primary antibody overnight at 4\u0026deg;C, with secondary antibody being added after three consecutive washes with PBS. The following antibodies were used: anti-cGAS (1:200: 29958-1-AP; Proteintech, Santa Cruz, CA, USA), anti-STING (1:1000: KHC0060; Proteintech, Santa Cruz, CA, USA) and anti-p-IRF3 (1:200: ab218160; Abcam, Cambridge, UK). DAB staining was performed to show positive expression and 40,6-diamidino-2-phenylindole was added for nuclear staining. Images were obtained using a DM2500 microscope (Leica, Wetzlar, Germany,).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eWestern blotting\u003c/h2\u003e\u003cp\u003eRIPA buffer (89901, Thermo Fisher Scientific, USA), enriched with PhosSTOP and complete protease inhibitors (Merck, Beijing, China), was used for protein extraction. The protein samples were then separated using 12.5% SDS-PAGE and subsequently transferred to polyvinylidene fluoride membranes (162\u0026ndash;0177; Bio-Rad, Shanghai, China) following the manufacturer\u0026rsquo;s guidelines [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Blots were probed at 4\u0026deg;C overnight with appropriate primary antibodies against p62 (ab56416, Abcam, Cambridge, UK), PINK1 (23274-1-AP; Santa Cruz, CA, USA), PCG-1α (ab134047; Abcam, Cambridge, UK), LC3B-Ⅱ (ab204297; Abcam, Cambridge, UK), cGAS (29958-1-AP; Proteintech, Santa Cruz, CA, USA), TBK1 (ab289973, Abcam, Cambridge, UK), STING (KHC0060; Proteintech, Santa Cruz, CA, USA), USP18 (12153-1-AP; Proteintech, Santa Cruz, CA, USA), and RNF5 (ab200389; Abcam, Cambridge, UK). Blots were incubated with the appropriate secondary antibody (1:5000: Proteintech) and developed with horseradish peroxidase substrate (WBLUF0500; Millipore, USA). To ensure equal loading, the blots were stripped with stripping buffer (100 mmol/L 2-mercaptoethanol, 2% SDS, 62.5 mmol/L Tris, pH 6.8) and re-probed with anti-GAPDH antibody.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eqPCR assay\u003c/h2\u003e\u003cp\u003eRNA was extracted from lung tissues using TRIzol reagent (RR047A; Takara, Japan) following the instructions outlined in our previous study [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Subsequently, cDNA was synthesised and real-time PCR was conducted using a SYBR Green Premix Pro Taq HS qPCR Kit (cat. no. AG11701; Accurate Biology). Target gene transcripts were amplified from mice using a SuperScript III One-Step RT-PCR Kit (12574026; Thermo Fisher Scientific, Waltham, MA, USA) in a Roche LightCycler 480 thermocycler. RT-PCR was performed using an Eppendorf Realplex 4 instrument (Eppendorf, Hamburg, Germany). The following primers were synthesised by Univ-Bio (Shanghai, China): mtDNA: forward: 5ʹ-CACCCAAGAACAGGGTTTGT-3ʹ, reverse: 5ʹ-TGGCCATGGGTATGTTGTTA-3ʹ; cGAS: forward: 5ʹ-CAGGAAGGAACCGGACAAGC-3ʹ, reverse: 5ʹ-CCGACTCCCGTTTCTGCATT-3; and STING: forward: 5ʹ-TATACCTCAGTTGGATGTTTGGC-3ʹ, reverse: 5ʹ- CTGGAGTCAAGCTCTGAAGGC-3ʹ. Relative gene expression was calculated using β-actin as an internal reference. Results are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of six biological replicates for each sample.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Normality and log normality were assessed using the Shapiro\u0026ndash;Wilks test. Kaplan\u0026ndash;Meier survival curves were analysed using the log-rank test, which was equivalent to the Mantel\u0026ndash;Haenszel test. To determine the statistical significance of differences between independent continuous variables, a 2-tailed Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was applied when comparing two groups for which both data sets had a normal distribution. One-way analysis of variance followed by Bonferroni\u0026rsquo;s post hoc test or the Mann\u0026ndash;Whitney U test was performed as appropriate using Graph Prism 8.0 software (GraphPad Software, La Jolla, CA). \u003cem\u003eP\u003c/em\u003e values of less than 0.05 were considered statistically significant (*\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 or \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; \u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eALI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eacute lung injury\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eARDS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eacute respiratory distress syndrome\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eROS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ereactive oxygen species\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003emtDNA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emitochondrial DNA\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eER\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eendoplasmic reticulum\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ecGAS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecyclic GMP-AMP synthase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSTING\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003estimulator of interferon genes\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ecDNA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecomplementary DNA\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eATP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eadenosine triphosphate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDNA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003edeoxyribonucleic acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNADH\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003enicotinamide adenine dinucleotide\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIL\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003einterleukin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLPS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003elipopolysaccharide\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMODS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emultiple organ dysfunction syndrome\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTEM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003etransmission electron microscopy\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBAL\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ebronchoalveolar lavage\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePaO₂\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003earterial oxygen partial pressure\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePaCO\u003csub\u003e2\u003c/sub\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003epartial pressure of carbon dioxide\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLac\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003elactate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLDH\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003elactate dehydrogenase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eOSI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eoxidative stress index\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTOS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003etotal oxidant status\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTAS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003etotal antioxidant status.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protocols for the animal experiments were approved by the Animal Ethics Committee of Ruijin Hospital Affiliated to Shanghai Jiaotong University School of Medicine (No. 092).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support our findings are available from the corresponding author ([email protected]) upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (82402510, 82372203, 82241033, 81902007), the Natural Science Foundation of Shanghai Municipality (24ZR1445800), the China Postdoctoral Science Foundation (2024M762043), and the Natural Science Foundation of Shandong Province (ZR2025MS1416), and the Research Project by the Tianjin Municipal Health Commission (2023221).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHPQ conceived the original idea, CC and TTP wrote the manuscript draft. CC and XLW conducted a majority of the experiments and performed data analysis. RT and RMT assisted with the animal experiments. RRL interpreted the data. HPQ and TTP revised the manuscript. All the authors have confirmed the submission of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Tingting Lin for technical assistance. Graphical Abstract was created in BioRender. Cao, C. (2025) https://BioRender.com/q42p392.\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMatthay, M. A., Arabi, Y., Arroliga, A. C., Bernard, G., Bersten, A. D., Brochard, L. J., Calfee, C. S., Combes, A., Daniel, B. M., Ferguson, N. D., Gong, M. N., Gotts, J. E., Herridge, M. S., Laffey, J. G., Liu, K. D., Machado, F. R., Martin, T. R., McAuley, D. F., Mercat, A., Moss, M., Mularski, R. A., Pesenti, A., Qiu, H., Ramakrishnan, N., Ranieri, V. M., Riviello, E. D., Rubin, E., Slutsky, A. S., Thompson, B. T., Twagirumugabe, T., Ware, L. B. \u0026amp; Wick, K. D. (2024) A New Global Definition of Acute Respiratory Distress Syndrome, \u003cem\u003eAm J Respir Crit Care Med.\u003c/em\u003e 209, 37\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eThompson, B. T., Chambers, R. C. \u0026amp; Liu, K. D. (2017) Acute Respiratory Distress Syndrome, \u003cem\u003eN Engl J Med.\u003c/em\u003e 377, 562\u0026ndash;572.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBos, L. D. J. \u0026amp; Ware, L. B. (2022) Acute respiratory distress syndrome: causes, pathophysiology, and phenotypes, \u003cem\u003eLancet.\u003c/em\u003e 400, 1145\u0026ndash;1156.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFan, E., Brodie, D. \u0026amp; Slutsky, A. S. (2018) Acute Respiratory Distress Syndrome: Advances in Diagnosis and Treatment, \u003cem\u003eJAMA.\u003c/em\u003e 319, 698\u0026ndash;710.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBellani, G., Laffey, J. G., Pham, T., Fan, E., Brochard, L., Esteban, A., Gattinoni, L., van Haren, F., Larsson, A., McAuley, D. F., Ranieri, M., Rubenfeld, G., Thompson, B. T., Wrigge, H., Slutsky, A. S. \u0026amp; Pesenti, A. (2016) Epidemiology, Patterns of Care, and Mortality for Patients With Acute Respiratory Distress Syndrome in Intensive Care Units in 50 Countries, \u003cem\u003eJAMA.\u003c/em\u003e 315, 788\u0026ndash;800.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSlutsky, A. S. \u0026amp; Ranieri, V. M. (2013) Ventilator-induced lung injury, \u003cem\u003eN Engl J Med.\u003c/em\u003e 369, 2126\u0026ndash;2136.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVercellino, I. \u0026amp; Sazanov, L. A. (2022) The assembly, regulation and function of the mitochondrial respiratory chain, \u003cem\u003eNat Rev Mol Cell Biol.\u003c/em\u003e 23, 141\u0026ndash;161.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLarson-Casey, J. L., He, C. \u0026amp; Carter, A. B. (2020) Mitochondrial quality control in pulmonary fibrosis, \u003cem\u003eRedox Biol.\u003c/em\u003e 33, 101426.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDutra Silva, J., Su, Y., Calfee, C. S., Delucchi, K. L., Weiss, D., McAuley, D. F., O'Kane, C. \u0026amp; Krasnodembskaya, A. D. (2021) Mesenchymal stromal cell extracellular vesicles rescue mitochondrial dysfunction and improve barrier integrity in clinically relevant models of ARDS, \u003cem\u003eEur Respir J.\u003c/em\u003e 58.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChung, K.-P., Cheng, C.-N., Chen, Y.-J., Hsu, C.-L., Huang, Y.-L., Hsieh, M.-S., Kuo, H.-C., Lin, Y.-T., Juan, Y.-H., Nakahira, K., Chen, Y.-F., Liu, W.-L., Ruan, S.-Y., Chien, J.-Y., Plataki, M., Cloonan, S. M., Carmeliet, P., Choi, A. M. K., Kuo, C.-H. \u0026amp; Yu, C.-J. (2024) Alveolar epithelial cells mitigate neutrophilic inflammation in lung injury through regulating mitochondrial fatty acid oxidation, \u003cem\u003eNat Commun.\u003c/em\u003e 15, 7241.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChan, D. C. (2020) Mitochondrial Dynamics and Its Involvement in Disease, \u003cem\u003eAnnu Rev Pathol.\u003c/em\u003e 15, 235\u0026ndash;259.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen, W., Zhao, H. \u0026amp; Li, Y. (2023) Mitochondrial dynamics in health and disease: mechanisms and potential targets, \u003cem\u003eSignal Transduct Target Ther.\u003c/em\u003e 8, 333.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFaust, H. E., Reilly, J. P., Anderson, B. J., Ittner, C. A. G., Forker, C. M., Zhang, P., Weaver, B. A., Holena, D. N., Lanken, P. N., Christie, J. D., Meyer, N. J., Mangalmurti, N. S. \u0026amp; Shashaty, M. G. S. (2020) Plasma Mitochondrial DNA Levels Are Associated With ARDS in Trauma and Sepsis Patients, \u003cem\u003eChest.\u003c/em\u003e 157, 67\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhao, C., Liang, F., Ye, M., Wu, S., Qin, Y., Zhao, L., Zhang, L., He, J., Cen, L. \u0026amp; Lin, F. (2023) GSDMD promotes neutrophil extracellular traps via mtDNA-cGAS-STING pathway during lung ischemia/reperfusion, \u003cem\u003eCell Death Discov.\u003c/em\u003e 9, 368.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYum, S., Li, M., Fang, Y. \u0026amp; Chen, Z. J. (2021) TBK1 recruitment to STING activates both IRF3 and NF-κB that mediate immune defense against tumors and viral infections, \u003cem\u003eProc Natl Acad Sci U S A.\u003c/em\u003e 118.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi, X., Chen, X., Zheng, L., Chen, M., Zhang, Y., Zhu, R., Chen, J., Gu, J., Yin, Q., Jiang, H., Wu, X., Ji, X., Tang, X., Dong, M., Li, Q., Gao, Y. \u0026amp; Chen, H. (2023) Non-canonical STING-PERK pathway dependent epigenetic regulation of vascular endothelial dysfunction via integrating IRF3 and NF-κB in inflammatory response, \u003cem\u003eActa Pharm Sin B.\u003c/em\u003e 13, 4765\u0026ndash;4784.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGao, J.-L., Zhao, J., Zhu, H.-B., Peng, X., Zhu, J.-X., Ma, M.-H., Fu, Y., Hu, N., Tai, Y., Xuan, X.-C. \u0026amp; Dong, D.-L. (2018) Characterizations of mitochondrial uncoupling induced by chemical mitochondrial uncouplers in cardiomyocytes, \u003cem\u003eFree Radic Biol Med.\u003c/em\u003e 124, 288\u0026ndash;298.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAxelrod, C. L., King, W. T., Davuluri, G., Noland, R. C., Hall, J., Hull, M., Dantas, W. S., Zunica, E. R., Alexopoulos, S. J., Hoehn, K. L., Langohr, I., Stadler, K., Doyle, H., Schmidt, E., Nieuwoudt, S., Fitzgerald, K., Pergola, K., Fujioka, H., Mey, J. T., Fealy, C., Mulya, A., Beyl, R., Hoppel, C. L. \u0026amp; Kirwan, J. P. (2020) BAM15-mediated mitochondrial uncoupling protects against obesity and improves glycemic control, \u003cem\u003eEMBO Mol Med.\u003c/em\u003e 12, e12088.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJing, W., Jiang, M., Fu, X., Yang, J., Chen, L., Leng, F., Xu, P., Huang, W., Yu, C. \u0026amp; Yang, Z. (2022) Self-assembly drug-albumin nanocomposites for nonalcoholic fatty liver disease treatment, \u003cem\u003eInt J Biol Macromol.\u003c/em\u003e 214, 697\u0026ndash;707.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDang, C. P., Issara-Amphorn, J., Charoensappakit, A., Udompornpitak, K., Bhunyakarnjanarat, T., Saisorn, W., Sae-Khow, K. \u0026amp; Leelahavanichkul, A. (2021) BAM15, a Mitochondrial Uncoupling Agent, Attenuates Inflammation in the LPS Injection Mouse Model: An Adjunctive Anti-Inflammation on Macrophages and Hepatocytes, \u003cem\u003eJ Innate Immun.\u003c/em\u003e 13, 359\u0026ndash;375.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTsuji, N., Tsuji, T., Yamashita, T., Hayase, N., Hu, X., Yuen, P. S. \u0026amp; Star, R. A. (2023) BAM15 treats mouse sepsis and kidney injury, linking mortality, mitochondrial DNA, tubule damage, and neutrophils, \u003cem\u003eJ Clin Invest.\u003c/em\u003e 133.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDantas, W. S., Zunica, E. R. M., Heintz, E. C., Vandanmagsar, B., Floyd, Z. E., Yu, Y., Fujioka, H., Hoppel, C. L., Belmont, K. P., Axelrod, C. L. \u0026amp; Kirwan, J. P. (2022) Mitochondrial uncoupling attenuates sarcopenic obesity by enhancing skeletal muscle mitophagy and quality control, \u003cem\u003eJ Cachexia Sarcopenia Muscle.\u003c/em\u003e 13, 1821\u0026ndash;1836.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHombrebueno, J. R., Cairns, L., Dutton, L. R., Lyons, T. J., Brazil, D. P., Moynagh, P., Curtis, T. M. \u0026amp; Xu, H. (2019) Uncoupled turnover disrupts mitochondrial quality control in diabetic retinopathy, \u003cem\u003eJCI Insight.\u003c/em\u003e 4.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSch\u0026auml;fer, J. A., Bozkurt, S., Michaelis, J. B., Klann, K. \u0026amp; M\u0026uuml;nch, C. (2022) Global mitochondrial protein import proteomics reveal distinct regulation by translation and translocation machinery, \u003cem\u003eMol Cell.\u003c/em\u003e 82.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi, Y., Tan, R., Li, R., Tian, R., Liu, Z., Wang, X., Chen, E., Pan, T. \u0026amp; Qu, H. (2023) PKM2/STAT1-mediated PD-L1 upregulation on neutrophils during sepsis promotes neutrophil organ accumulation by serving an anti-apoptotic role, \u003cem\u003eJ Inflamm (Lond)\u003c/em\u003e. 20, 16.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKenwood, B. M., Weaver, J. L., Bajwa, A., Poon, I. K., Byrne, F. L., Murrow, B. A., Calderone, J. A., Huang, L., Divakaruni, A. S., Tomsig, J. L., Okabe, K., Lo, R. H., Cameron Coleman, G., Columbus, L., Yan, Z., Saucerman, J. J., Smith, J. S., Holmes, J. W., Lynch, K. R., Ravichandran, K. S., Uchiyama, S., Santos, W. L., Rogers, G. W., Okusa, M. D., Bayliss, D. A. \u0026amp; Hoehn, K. L. (2014) Identification of a novel mitochondrial uncoupler that does not depolarize the plasma membrane, \u003cem\u003eMol Metab.\u003c/em\u003e 3, 114\u0026ndash;123.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTyagi, T., Yarovinsky, T. O., Faustino, E. V. S. \u0026amp; Hwa, J. (2024) Platelet Mitochondrial Fusion and Function in Vascular Integrity, \u003cem\u003eCirc Res.\u003c/em\u003e 134, 162\u0026ndash;164.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTaylor, A. L., Dubuisson, O., Pandey, P., Zunica, E. R. M., Vandanmagsar, B., Dantas, W. S., Johnson, A., Axelrod, C. L. \u0026amp; Kirwan, J. P. (2024) Restricting bioenergetic efficiency enhances longevity and mitochondrial redox capacity in Drosophila melanogaster, \u003cem\u003eAging Cell.\u003c/em\u003e 23, e14107.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMuhammad, W., Zhu, J., Zhai, Z., Xie, J., Zhou, J., Feng, X., Feng, B., Pan, Q., Li, S., Venkatesan, R., Li, P., Cao, H. \u0026amp; Gao, C. (2022) ROS-responsive polymer nanoparticles with enhanced loading of dexamethasone effectively modulate the lung injury microenvironment, \u003cem\u003eActa Biomater.\u003c/em\u003e 148, 258\u0026ndash;270.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHo, J., Yu, J., Wong, S. H., Zhang, L., Liu, X., Wong, W. T., Leung, C. C. H., Choi, G., Wang, M. H. T., Gin, T., Chan, M. T. V. \u0026amp; Wu, W. K. K. (2016) Autophagy in sepsis: Degradation into exhaustion?, \u003cem\u003eAutophagy.\u003c/em\u003e 12, 1073\u0026ndash;1082.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLira Chavez, F. M., Gartzke, L. P., van Beuningen, F. E., Wink, S. E., Henning, R. H., Krenning, G. \u0026amp; Bouma, H. R. (2023) Restoring the infected powerhouse: Mitochondrial quality control in sepsis, \u003cem\u003eRedox Biol.\u003c/em\u003e 68, 102968.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang, J.-F., Wang, Y.-P., Xie, J., Zhao, Z.-Z., Gupta, S., Guo, Y., Jia, S.-H., Parodo, J., Marshall, J. C. \u0026amp; Deng, X.-M. (2021) Upregulated PD-L1 delays human neutrophil apoptosis and promotes lung injury in an experimental mouse model of sepsis, \u003cem\u003eBlood.\u003c/em\u003e 138, 806\u0026ndash;810.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSong, C., Li, H., Mao, Z., Peng, L., Liu, B., Lin, F., Li, Y., Dai, M., Cui, Y., Zhao, Y., Han, D., Chen, L., Huang, X. \u0026amp; Pan, P. (2022) Delayed neutrophil apoptosis may enhance NET formation in ARDS, \u003cem\u003eRespir Res.\u003c/em\u003e 23, 155.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIshikawa, H. \u0026amp; Barber, G. N. (2008) STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling, \u003cem\u003eNature.\u003c/em\u003e 455, 674\u0026ndash;678.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eErgun, S. L. \u0026amp; Li, L. (2020) Structural Insights into STING Signaling, \u003cem\u003eTrends Cell Biol.\u003c/em\u003e 30, 399\u0026ndash;407.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNewman, L. E., Weiser Novak, S., Rojas, G. R., Tadepalle, N., Schiavon, C. R., Grotjahn, D. A., Towers, C. G., Tremblay, M.-\u0026Egrave;., Donnelly, M. P., Ghosh, S., Medina, M., Rocha, S., Rodriguez-Enriquez, R., Chevez, J. A., Lemersal, I., Manor, U. \u0026amp; Shadel, G. S. (2024) Mitochondrial DNA replication stress triggers a pro-inflammatory endosomal pathway of nucleoid disposal, \u003cem\u003eNat Cell Biol.\u003c/em\u003e 26, 194\u0026ndash;206.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJim\u0026eacute;nez-Loygorri, J. I., Villarejo-Zori, B., Viedma-Poyatos, \u0026Aacute;., Zapata-Mu\u0026ntilde;oz, J., Ben\u0026iacute;tez-Fern\u0026aacute;ndez, R., Frutos-Lis\u0026oacute;n, M. D., Tom\u0026aacute;s-Barber\u0026aacute;n, F. A., Esp\u0026iacute;n, J. C., Area-G\u0026oacute;mez, E., Gomez-Duran, A. \u0026amp; Boya, P. (2024) Mitophagy curtails cytosolic mtDNA-dependent activation of cGAS/STING inflammation during aging, \u003cem\u003eNat Commun.\u003c/em\u003e 15, 830.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDecout, A., Katz, J. D., Venkatraman, S. \u0026amp; Ablasser, A. (2021) The cGAS-STING pathway as a therapeutic target in inflammatory diseases, \u003cem\u003eNat Rev Immunol.\u003c/em\u003e 21, 548\u0026ndash;569.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBenmerzoug, S., Rose, S., Bounab, B., Gosset, D., Duneau, L., Chenuet, P., Mollet, L., Le Bert, M., Lambers, C., Geleff, S., Roth, M., Fauconnier, L., Sedda, D., Carvalho, C., Perche, O., Laurenceau, D., Ryffel, B., Apetoh, L., Kiziltunc, A., Uslu, H., Albez, F. S., Akgun, M., Togbe, D. \u0026amp; Quesniaux, V. F. J. (2018) STING-dependent sensing of self-DNA drives silica-induced lung inflammation, \u003cem\u003eNat Commun.\u003c/em\u003e 9, 5226.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi, N., Liu, B., Xiong, R., Li, G., Wang, B. \u0026amp; Geng, Q. (2023) HDAC3 deficiency protects against acute lung injury by maintaining epithelial barrier integrity through preserving mitochondrial quality control, \u003cem\u003eRedox Biol.\u003c/em\u003e 63, 102746.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIslam, M. N., Das, S. R., Emin, M. T., Wei, M., Sun, L., Westphalen, K., Rowlands, D. J., Quadri, S. K., Bhattacharya, S. \u0026amp; Bhattacharya, J. (2012) Mitochondrial transfer from bone-marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury, \u003cem\u003eNat Med.\u003c/em\u003e 18, 759\u0026ndash;765.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAmaral, A., Ramalho-Santos, J. \u0026amp; St John, J. C. (2007) The expression of polymerase gamma and mitochondrial transcription factor A and the regulation of mitochondrial DNA content in mature human sperm, \u003cem\u003eHum Reprod.\u003c/em\u003e 22, 1585\u0026ndash;1596.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCao, C., Memete, O., Shao, Y., Zhang, L., Liu, F., Dun, Y., He, D., Zhou, J. \u0026amp; Shen, J. (2022) Single-Cell RNA-Sequencing Reveals Epithelial Cell Signature of Multiple Subtypes in Chemically Induced Acute Lung Injury, \u003cem\u003eInt J Mol Sci.\u003c/em\u003e 24.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"BAM15, acute lung injury, mitochondrial dynamics, neutrophil apoptosis, cGAS-STING pathway","lastPublishedDoi":"10.21203/rs.3.rs-7622689/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7622689/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAcute lung injury (ALI) is a life-threatening condition that is often induced by endotoxins and leads to severe inflammation and mitochondrial dysfunction. In this study, we investigated the protective effects of BAM15 {C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eF\u003csub\u003e2\u003c/sub\u003eN\u003csub\u003e6\u003c/sub\u003eO; also known as N5,N6-bis(2-fluorophenyl)-[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]oxadiazolo[3,4-b]pyrazine-5,6-diamine}, a mitochondrial uncoupler, in lipopolysaccharide (LPS)-induced ALI, focussing on its role in the regulation of mitochondrial dynamics and immune responses. Using a mouse model of LPS-induced ALI, we assessed mitochondrial dynamics by imaging and biochemical assays, and analysed cyclic GMP-AMP synthase (cGAS)\u0026ndash;stimulator of interferon genes (STING) pathway activation using western blotting and immunofluorescence. We found that BAM15 treatment resulted in a significant reduction in mortality and attenuated lung inflammation, oxidative stress and autophagy. Moreover, BAM15 contributes to the maintenance of mitochondrial dynamics by promoting mitochondrial fusion and inhibiting fission whilst downregulating cGAS\u0026ndash;STING activation, thereby rectifying delayed neutrophil apoptosis and attenuating inflammation. BAM15 protects against LPS-induced ALI by maintaining mitochondrial homeostasis, reducing the generation of mitochondrial reactive oxygen species, and limiting excessive autophagy. It also reduces mitochondrial DNA release and prevents excessive cGAS\u0026ndash;STING activation. On the basis of these findings, we propose that BAM15 could represent a promising therapeutic candidate for the treatment of LPS-induced ALI.\u003c/p\u003e","manuscriptTitle":"BAM15 treatment mitigates lipopolysaccharide-induced acute lung injury by preserving mitochondrial dynamic equilibrium and modulating inflammatory responses via the cGAS–STING signalling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-17 02:14:25","doi":"10.21203/rs.3.rs-7622689/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"478e19b8-16a6-47bf-89b7-32f81596b463","owner":[],"postedDate":"October 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":55731410,"name":"Biological sciences/Biochemistry/Immunochemistry"},{"id":55731411,"name":"Biological sciences/Immunology/Immune cell death"}],"tags":[],"updatedAt":"2026-03-16T12:20:48+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-17 02:14:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7622689","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7622689","identity":"rs-7622689","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}