Kat2a-Mediated Histone Lactylation Promotes Ferroptosis and Inflammation in Sepsis-Associated Lung Injury

Kat2a-Mediated Histone Lactylation Promotes Ferroptosis and Inflammation in Sepsis-Associated Lung Injury | 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 Kat2a-Mediated Histone Lactylation Promotes Ferroptosis and Inflammation in Sepsis-Associated Lung Injury Changhan Ouyang, Jie Cheng, Mingqun Chen, Zeye Lin, Guangyao Zhu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8069085/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 The mortality rate of sepsis-associated acute lung injury (ALI) remains high, with its core mechanisms closely linked to macrophage-mediated inflammation and ferroptosis. Lipopolysaccharide (LPS), a key factor in Gram-negative bacterial infections, can activate epigenetic modifications and induce ferroptosis. However, the role of Kat2a-mediated histone lactylation (H3K18la) in ALI and its interaction with ferroptosis remain unclear. This study aims to investigate the pathogenic mechanism of the Kat2a-H3K18la axis in ALI through its regulation of the cholesterol metabolism enzyme CH25H and the ferroptosis pathway, as well as to evaluate targeted intervention strategies. Our study revealed LPS significantly upregulated Kat2a expression and H3K18la modification levels in lung tissues and macrophages. Inhibition of the Kat2a-H3K18la axis by MB-3 alleviated pulmonary hemorrhage, edema, fibrosis, and inflammatory infiltration while reducing NLRP3 inflammasome activation and M1 macrophage polarization. Kat2a-H3K18la epigenetically regulated ferroptosis bidirectionally, promoting transferrin receptor (Tfrc) transcription while suppressing protective genes Slc7a11/GPX4. Finally, we revealed that the exogenous lactate specifically reversed MB-3 inhibitory effect on ferroptosis, uncovering a novel "lactate metabolism-H3K18la modification-ferroptosis" pathway. In summary, our research reveals a new function for the Kat2a-ferroptosis axis in acute lung injury, revealing important information for possible therapeutic approaches. Biological sciences/Molecular biology/Epigenetics Biological sciences/Molecular biology/Post-translational modifications Biological sciences/Cell biology Epigenetic modification Histone lactylation Kat2a Acute lung injury Ferroptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Sepsis is a life-threatening illness characterized by organ failure caused by an inappropriate host response to infection. It affects millions of individuals globally each year. Its consistently high death rate places a significant strain on society and families. Acute lung injury (ALI) is one of the most common and severe consequences of sepsis, affecting up to 50% of patients. Numerous studies show that Gram-negative bacterial infections are the leading cause of acute lung injury (ALI), and that lipopolysaccharide (LPS), a critical component of Gram-negative bacteria's outer membrane, plays an important role in causing inflammation and lung damage [1-4] . Macrophages, the most prevalent immune cell in the lungs, are important for sepsis-induced acute lung damage [5–6] . Due to the absence of targeted treatment approaches, the clinical management of ALI is currently fraught with difficulties [7] . Thus, it is essential to further clarify the pathophysiology of ALI. Ferroptosis is a recently discovered controlled cell death mechanism that relies on iron and lipid peroxides, as opposed to apoptosis, autophagy, and other kinds of cell death [8–10] . Recent research indicates that ferroptosis may be caused by increased lipid ROS and inhibition of glutathione peroxidase 4 (GPX4) [11] . Numerous illnesses, such as cancer, Alzheimer's, and intracerebral hemorrhage, have been linked to it [12] . Recent research has demonstrated the critical role ferroptosis plays in the development of sepsis-induced lung injury [13] . Our comprehension of the intricate regulatory pathways of ferroptosis in ALI is still lacking, despite earlier studies showing that LPS causes ferroptosis in alveolar epithelial cells and aggravates sepsis-related lung injury [14] . Thus, the goal of this study is to get a better understanding of the role of ferroptosis in ALI and the regulatory mechanisms that underpin it. Epigenetic alterations, such as histone modifications, DNA methylation, and chromatin remodeling, affect gene expression without modifying DNA sequences and are important processes for macrophage pro-inflammatory polarization [15] . For instance, LPS activates TLR4 signaling, leading to kinase-mediated recruitment of histone methyltransferases that deposit activating H3K4me3 marks at pro-inflammatory gene promoters, enhancing chromatin accessibility and transcription factor binding to amplify inflammatory responses [16] . The newly discovered histone (and non-histone) lactylation modification is a rapid and dynamic epigenetic mark whose dysregulation is linked to transcriptional dysfunction and disease, making it a potential diagnostic marker or therapeutic target [17] . However, the specific host factors mediating lactylation-dependent inflammatory regulation in macrophages and their role in ferroptosis remain unclear. Therefore, this study seeks to elucidate the function of lactylation modification in ALI through its regulation of ferroptosis and its underlying mechanisms. Lysine acetyltransferase 2A (Kat2a, alternatively referred to as GCN5) was first identified as a key component of the histone acetyltransferase (HAT) family in yeast and is highly conserved among mammals, especially in humans and mice. Emerging evidence suggests that aberrant gene expression driven by the synergistic regulation of transcription and epigenetic factors may play a pivotal role in lung injury [18] . However, the role of Kat2a in ALI remains largely unexplored. Here, we demonstrate that treatment with the selective Kat2a inhibitor MB-3 significantly ameliorates inflammatory damage in ALI models. According to our findings, Kat2a represents a unique therapeutic target in the metabolic and epigenetic reprogramming of macrophages, providing a method of modulating inflammatory responses both at the start and as ALI progresses. 2. Materials and Methods 2.1 Reagents and Antibodies We ordered MB-3 (M2449), lactate (LA, 71718), lipopolysaccharide (LPS, L2880), the lactate dehydrogenase inhibitor dichloroacetate (DCA, 347795), and dimethyl sulfoxide (DMSO) from Sigma-Aldrich (USA). DMEM high-glucose medium, fetal bovine serum (FBS), and penicillin/streptomycin solution (100×) were sourced from Gibco (USA). Cyclodextrin and the CCK-8 assay kit were purchased from Meilun Biotechnology (Dalian, China). The bicinchoninic acid (BCA) protein quantification kit was supplied by KeriBio (Wuhan, China). Antibodies against Kat2a (#3305) and α-Tubulin (#3873) were procured from Cell Signaling Technology (CST, USA). Antibodies targeting Slc7a11 (A2413), Tfrc (A5865), CH25H (A24299), IL-1β (A16288), and NLRP3 (A5652) were obtained from ABclonal Biotechnology (Wuhan, China). Antibodies for GPX4 (bs-3884R), Caspase-1 (bs-0169R), and GAPDH (bs-10900R) were sourced from Bioss Antibodies (Beijing, China). The H3K18la (PTM-1406RM) antibody and Pan-Kla (PTM-1401RM) was provided by PTM BIO, and the β-actin (ABL1011) antibody was obtained from Abbkine. 2.2 Sepsis-Induced Acute Lung Injury (ALI) Animal Model Male C57BL/6 adult mice (8–10 weeks old, weighing 20–22 g) kept in specified pathogen-free (SPF) conditions were acquired for this investigation from the Hubei Provincial Laboratory Animal Research Center. These mice were housed in an SPF environment with a 12-hour light/dark cycle, temperature, and humidity control. Every experimental procedure was carried out in accordance with the Laboratory Animal Management Regulations of Hubei Province. There were six mice in each of the four groups that the animals were randomly assigned to. Among them, 12 C57BL/6 mice with endotoxic shock models were first pretreated by either MB-3 (10mg/kg body weight, intraperitoneal administration) or DMSO, and then received an intraperitoneal injection of LPS (10 mg/kg body weight). Eight hours after injection, serum samples were taken for ELISA analysis. Hematoxylin and eosin (H&E) staining and immunohistochemical testing were performed on lung tissues that had been paraformaldehyde-fixed [19] . Furthermore, fresh tissue samples that weren't utilized for fixation were kept in liquid nitrogen for later molecular biology tests like ELISA and Western blot. 2.3 Cell Culture and Stimulation Procell Life Science & Technology Co., Ltd. (Wuhan, China) supplied the mouse monocyte-macrophage cell line Raw264.7. These cells were kept in complete DMEM medium (pH 7.2–7.4) supplemented with 1% penicillin–streptomycin and 10% fetal bovine serum. They were then raised in a humidified incubator with 5% CO₂ at 37°C. After reaching 60–70% confluence, Raw264.7 cells were cultivated in serum-free media containing LPS for 24 hours prior to additional analysis. To stimulate macrophages, LPS was given at a concentration of 1 μg/mL for the specified durations, unless otherwise specified [20] . 2.4 Western blotting With the use of Beyotime's RIPA lysis buffer, which contains phosphatase and protease inhibitors, total protein was extracted from lung tissues and cells. Protein quantities were measured using a BCA assay kit. In order to heat denaturize the protein samples, they were then incubated for 10 minutes at 98°C in a metal bath. SDS-PAGE electrophoresis was then used to separate the proteins after they had been moved to PVDF membranes (Merck Millipore, Billerica, MA, USA). Following a two-hour blocking period at room temperature using 5% skim milk, primary and secondary antibodies conjugated with horseradish peroxidase were applied to the membranes. Lastly, enhanced chemiluminescence reagents (MeilunBio) were used to visualize the blots. 2.5 ROS measurement Create a stock solution with a concentration of 10 mM, DCFH-DA (Beyotime, S0033S) was dissolved in DMSO. The stock solution was then further diluted using serum-free medium (at a 1:1000 dilution) to a working concentration of 10 μM. Raw 264.7 cells were seeded into six-well plates, and the designated chemicals were applied to the cells once they adhered. The cells were then exposed to the DCFH-DA working solution for 30 minutes at 37°C. Lastly, fluorescence microscopy was used to detect the intracellular ROS levels. 2.6 Measurement of lactate levels Serum samples were obtained from the orbital venous plexus of septic mice, subjected to centrifugation at 3000 rpm for 15 minutes, and then analyzed for lactate levels via ELISA following the manufacturer's instructions [21] . 2.7 Cell viability assay Cell viability was assessed using the CCK-8 test as directed by the manufacturer. 2.8 H&E staining and MASSON staining After being treated with a 4% paraformaldehyde solution, lung tissues were embedded in paraffin. Lung tissue was processed in serial sections and then stained with Masson's trichrome and hematoxylin-eosin (H&E). The left lungs, which had been fixed in 4% paraformaldehyde, were specifically paraffin-embedded, divided into sections that were 5 μm thick, baked for 60 minutes at 60°C, and then dewaxed with fresh xylene. These lung tissue sections were then processed for pathological staining with H&E and Masson's reagents. 2.9 Immunohistochemical staining Consistent with previous descriptions, 5 μm-thick lung sections were dewaxed with xylene and then processed through graded ethanol solutions (100%, 90%, 70%) followed by water. After dewaxing, the sections underwent antigen retrieval by microwave heating in citrate buffer for 30 minutes, were cooled to room temperature, and rinsed with PBS for 2 minutes. Inhibiting endogenous peroxidase activity was accomplished with 10% goat serum and 3% H2O2. After that, the sections were treated with Kat2a antibody (diluted 1:500 in PBS) for a whole night at 4°C. They were then incubated with anti-rabbit reagent for an hour at 37°C. After three PBS washes and five minutes of DAB preparation, the slices were inspected under a microscope. 2.10 TUNEL staining As previously described, 5μm-thick lung sections were processed with proteinase K and incubated at ambient temperature for 25 minutes. Permeabilization buffer was then added to fully cover the tissue sections, after which Tdt and dUTP reagents (mixed at a 1:9 ratio) were added and incubated at 37°C for 2 hours. Following the addition of 3% hydrogen peroxide solution and 25 minutes of incubation in the dark, converter-POD was applied and incubated at 37°C for 30 minutes, with subsequent development using DAB solution. After that, a microscope was used to study and take pictures of the sections. 2.11 Lung injury score The extent of lung damage, which included thickening of the alveolar septum, inflammation, bleeding, and swelling, was evaluated and graded in a blinded way. There were four ratings for alveolar septal thickening, bleeding, and edema: none (score = 0), mild (score = 1), moderate (score = 2), severe (score = 3), and extremely severe (score = 4). By calculating the total number of inflammatory cells per 100 x 100 field, inflammation was measured. 2.12 RNA-seq The samples were treated using the TRIzol Reagent to extract total RNA. The KC-Digital Stranded mRNA Library Prep Kit for Illumina (cat. no. DR08502, Servicebio, China) was used to construct strand-specific RNA sequencing libraries from 2 μg of total RNA, following the manufacturer's instructions [22] . This kit eliminates duplication bias from PCR and sequencing by applying unique molecular identifiers (UMIs) with eight random bases to pre-amplified cDNA molecules. Prior to final sequencing, library products in the 200-500 bp range were enriched and quantified using the PE150 mode on the DNBSEQ-T7 platform (MGI Tech, China). Servicebio (Wuhan, China) carried out library preparation and sequencing. 2.13 Determination of lipid peroxidation Raw264.7 cells were seeded in 6-well plates. The following day the medium was changed, and medications were added for a 24 hours treatment. The medium was thrown away after three PBS washes. Following that, 10 μM of C11-BODIPY581/591 (Beyotime, S0043S) was added, and the mixture was incubated for 30 minutes in the presence of light. The cells were subjected to three PBS washes before being viewed, photographed, and recorded using a fluorescence microscope. 2.14 Statistical analysis GraphPad Prism 9.5 was used to perform statistical analyses. All numerical data is presented as mean ± SD. To analyze differences between numerous groups, a one-way ANOVA with the Bonferroni post-hoc test was employed, while the Student's t-test was utilized for two groups. Each experiment had biological replicates, and a P-value of less than 0.05 was considered statistically significant. To ensure the repeatability of the findings, each experiment was performed at least three times. 3. Results 3.1 Kat2a and Histone Lactylation Levels are Increased in Sepsis-Associated Lung Injury Our work was particularly concerned with determining the functional involvement of Kat2a and H3K18la in ALI and the underlying regulatory mechanisms. Initial examination of Kat2a expression levels in mouse lungs revealed a significant increase in Kat2a protein levels in lung tissues by Western blot analysis, Similar results were confirmed at the histopathological level, with immunohistochemical staining demonstrating increased Kat2a-positive cells in LPS-treated animals compared to controls (Figures 1A and B). To study the impact of lactylation modification in acute lung damage, we used a Western blot test to determine H3K18la expression levels. The study confirmed a trend similar to the global lactylation level — Lactylation levels were higher in the model group compared to the blank group. Meanwhile, immunofluorescence staining showed a significant increase in both global lactylation and H3K18la levels(Figures 1C-E). We found that in the single-cell sequencing of LPS-induced lung injury, numerous cells were expressed. Among them, Raw264.7 cells showed the highest expression, The results in Figure 1F show that the gene expression of Kat2a protein in Raw264.7 cells increases with the elevation of LPS concentration, so we used Raw264.7 cells for subsequent cell experiments(Figure 1G). At the cellular level, elevated Kat2a and H3K18la protein expression was identified in LPS-treated Raw264.7 murine monocyte-macrophage cells compared to controls (Figure 1H). These findings collectively demonstrate that Kat2a and H3K18la expression is elevated in ALI, suggesting that Kat2a and H3K18la may serve as a crucial molecular regulator during ALI pathogenesis. 3.2 Kat2a Inhibition Ameliorates Sepsis-Associated Acute Lung Injury We reduced Kat2a levels in mice by intraperitoneal injection of MB-3 and its control solvent DMSO. Two hours later, we established a sepsis model by intraperitoneal injection of LPS(Figure 2B). We found that LPS treatment induced weight loss, lethargy, and growth retardation in mice, while MB-3 administration prevented these physiological alterations [23] . MB-3 pretreatment significantly attenuated LPS-induced ALI in comparison to the LPS group, as seen by lower tissue damage, interstitial edema, pulmonary hemorrhage, and thinner alveolar walls. H&E staining revealed severe structural disruption in ALI mouse lungs at 6 hours post-LPS stimulation, characterized by alveolar collapse, architectural disorganization with fused alveoli, thickened septa, extensive inflammatory cell infiltration, and increased hemorrhagic foci (indicated by arrows). MB-3 pretreatment markedly alleviated these pathological changes. Masson's trichrome staining demonstrated aggravated pulmonary fibrosis in LPS-treated mice, which was significantly ameliorated by MB-3 (Figure 2A). The increased lung wet/dry weight ratio was reversed by MB-3 treatment (Figure 2C). Given that uncontrolled inflammation represents a key pathological mechanism of ALI, blinded inflammatory scoring showed MB-3 substantially reduced pulmonary inflammation scores (Figure 2D), indicating its protective effect against LPS-induced lung injury. The results of CD68 immunohistochemistry showed that the LPS group had a considerably greater M1 macrophage marker than the blank group. Pretreatment with MB-3, however, reduced the positive expression of CD68(Figure 2E). Immunofluorescence staining confirmed that MB-3 significantly suppressed LPS-induced ROS overproduction (Figure 2F). 3.3 Kat2a Inhibition controls lipopolysaccharide (LPS)-induced systemic inflammation in vivo We investigated LPS-induced global gene expression profiles in Raw264.7 using RNA-seq. To study the direct involvement of Kat2a in the macrophage-mediated inflammatory response, seven murine macrophages were treated with either DMSO or MB-3. The TNF-α signaling pathway, ferroptosis, and rheumatoid arthritis were found to have upregulated genes, according to the KEGG pathway (Figure 3A). To study the possible mechanisms behind the therapeutic benefits of Kat2a on LPS-induced pulmonary inflammatory damage, we assessed the influence of MB-3 on inflammation and immunological function in mice models. Administration of the selective Kat2a inhibitor MB-3 to LPS-challenged mice revealed through Western blot analysis that pulmonary inflammatory factors (Caspase-1, IL-1β, NLRP3) were significantly elevated in the LPS group compared to controls, while MB-3 treatment markedly suppressed their expression(Figures 3B-E). Similarly, in vitro experiments confirmed that MB-3 substantially prevented LPS-induced macrophage inflammatory responses by reducing protein levels of NLRP3, IL-1β, and Caspase-1 (Figures 3F-I). Additionally, MB-3 decreased the protein expression of M1 macrophage marker INOS, demonstrating anti-inflammatory effects(Figures 3J and K). These findings indicate that the Kat2a inhibitor MB-3 significantly alleviates pathological damage in sepsis-associated acute lung injury, suggesting Kat2a as a critical pathogenic factor in this condition. To examine the effect of Kat2a on the viability of LPS-stimulated inflammatory macrophages, we modulated Kat2a levels in Raw264.7 cells using MB-3 and further assessed its interference with LPS-induced cell viability. MB-3 (50 μM) significantly inhibited the LPS-induced increase in RAW264.7 cell viability(Figures 3L). Cholesterol-25-hydroxylase(CH25H), the biosynthetic enzyme for 25-hydroxycholesterol (25-HC), exhibits its highest expression in the lungs, though its role in pulmonary biology remains unclear [24] . Lipopolysaccharide (LPS) induces dramatic increases in both 25-HC (>80-fold) and its synthase CH25H (>100-fold) in macrophages [25] . To investigate MB-3's effect on ferroptosis in ALI, we evaluated its therapeutic potential in LPS-induced ALI using a murine model. CH25H protein expression was significantly upregulated in lung tissues following LPS administration, while MB-3 pretreatment markedly suppressed this increase. Consistent results were observed in vitro(Figures 3M and N). 3.4 Kat2a Inhibition Modulates Ferroptosis via targeting Tfrc and Slc7a11 To investigate Kat2a’s direct role in macrophage-mediated ferroptosis, we used RNA-seq to analyze LPS-induced global gene expression profiles in DMSO or MB-3 treated Raw264.7 murine macrophages. The Kat2a inhibitor MB-3 substantially reduced several ferroptosis-promoting genes (Hmox1, Tfrc, Txnrd1, and Fth1) while increasing expression of ferroptosis-inhibitory genes (Slc7a11, GPX4, PPard, and Gch1) (Figures 4A and B). We evaluated MB-3's therapeutic effect on LPS-induced ALI by examining expression levels of established ferroptosis markers GPX4 and Slc7a11 in lung tissue and macrophages. LPS administration decreased GPX4 and Slc7a11 protein expression while MB-3 pretreatment significantly restored their expression in lung tissues (Figures 4C-E). The Tfrc gene was elevated in the LPS group of macrophages, and its expression was downregulated following MB-3 pretreatment, according to Western blot data. The LPS group showed downregulation of the ferroptosis-inhibitory genes Slc7a11 and GPX4, whereas MB-3 boosted their expression(Figures 4F-I). As a regulated form of necrosis, ferroptosis inhibition may reduce cell death [26-27] . TUNEL staining demonstrated decreased apoptotic cell numbers following MB-3 treatment. LPS-challenged lung tissues showed significantly increased apoptotic cells, whereas MB-3 administration reduced cell apoptosis, suggesting that MB-3 alleviates LPS-induced lung injury potentially by suppressing ferroptosis, thereby mitigating pulmonary damage and fibrosis in ALI mice(Figure S1A). In the meantime, we discovered that MB-3 significantly reduced the rise in lipid peroxidation brought on by LPS (Figure 4J). These findings indicate that Kat2a promotes ferroptosis induction upon LPS stimulation, which appears essential for initiating ferroptosis in vitro. 3.5 Histone Lactylation Modulates Ferroptosis and inflammation To investigate the effect of lactylation modification on ferroptosis gene expression, we manipulated the histone lactylation level in Raw264.7 cells by treating them with exogenous lactic acid or the lactate dehydrogenase inhibitor DCA. Stimulation of Raw264.7 cells with lipopolysaccharide (LPS) led to an increase in H3K18la level. After adding exogenous lactic acid, we found that the protein expression level of histone H3K18la increased in a lactic acid concentration-dependent manner(Figure 5A). Additionally, the addition of exogenous lactic acid resulted in elevated intracellular lactic acid level and histone H3K18la level(Figure 5B). Meanwhile, DCA significantly inhibited intracellular lactic acid and H3K18la levels in Raw264.7(Figure 5C). Furthermore, we analyzed the effect of H3K18la on ferroptosis genes in macrophages in vitro. First, we treated macrophages with lactic acid at different concentration gradients and found that the ferroptosis marker genes (Tfrc, Slc7a11) showed a concentration-dependent response, while GPX4 exhibited little change(Figures 5D and E). Subsequently, in in vitro experiments, on the basis of LPS stimulation, we added exogenous lactic acid and observed that lactic acid increased the protein expression of Tfrc but decreased the protein expression of Slc7a11 and GPX4. This indicates that lactic acid can promote ferroptosis to a certain extent(Figures 5F-I). Therefore, we further added the lactic acid inhibitor DCA, and contrary to the previous findings, DCA significantly inhibited ferroptosis(Figures 5J-L). Building on prior findings demonstrating LPS-induced CH25H upregulation. Studies have shown that LPS elevates CH25H expression, leading to increased 25-HC production, which subsequently depletes intracellular glutathione (GSH) [28] . This GSH reduction compromises cellular antioxidant capacity, exacerbating ferroptosis and ultimately triggering ALI. Notably, the MB-3 counteracts this process by downregulating CH25H expression via Kat2a inhibition, thereby restoring GSH levels. This regulatory effect suppresses ferroptosis, as evidenced by upregulated ferroptosis markers GPX4 and Slc7a11, ultimately mitigating alveolar epithelial cell damage and inflammatory responses to ameliorate sepsis-associated ALI. To clarify the regulatory effect of histone lactylation modification on CH25H, we employed exogenous lactate and the lactate dehydrogenase inhibitor dichloroacetate (DCA) in LPS-induced cellular models. Exogenous lactate treatment significantly increased CH25H protein expression (p<0.01), whereas DCA treatment produced the opposite effect (p<0.05) (Figures S2). Coupled with previous evidence that lactate promotes ferroptosis, these findings suggest MB-3 may inhibit KAT2A-mediated histone lactylation to downregulate CH25H expression, thereby attenuating CH25H-driven ferroptosis and achieving therapeutic protection against lung injury. The data above show that lactylation modification plays an essential role in controlling the ferroptosis process. 3.6 Kat2a elevates Histone Lactylation Promotes Ferroptosis and Inflammation Ferroptosis is a key factor in acute lung damage. Previous research has demonstrated that the Kat2a inhibitor MB-3 can suppress the expression of ferroptosis genes in acute lung damage, whereas lactylation modification can enhance ferroptosis. Therefore, we can conclude that both can regulate the ferroptosis pathway, but the regulatory relationship between Kat2a and H3K18la requires further investigation. Our previous differential gene expression analysis of macrophage RNA-seq data revealed significant alterations in iron-related genes following MB-3 pretreatment prior to LPS stimulation, Through volcano plots and heatmaps, we found that MB-3 can regulate lactic acid concentration through glycolysis-related enzymes. Among them, the expression of hexokinase 2 (HK2) was significant, and compared with the LPS group, MB-3 could reduce its expression(Figures 6A and B). Meanwhile, the same trend was confirmed by Western blot(Figure 6C). According to clinical data, septic patients who have high serum lactate levels have a worse prognosis and more organ damage. ELISA results demonstrated significantly higher serum lactate levels in ALI mice and macrophages than in controls, while MB-3 treatment reduced lactate concentrations, indicating that MB-3 regulates histone H3K18la expression by suppressing lactate production (Figures 6D and E). We also looked at the protein expression of histone H3K18la in lung tissues and macrophages, and found a substantial rise in H3K18la levels in the LPS-induced ALI model group compared to the controls. MB-3 administration markedly reduced histone H3K18la levels in both ALI lung tissues and macrophages(Figures 6F and G). The results of immunofluorescence staining were consistent with the above(Figure 6H). Through Western blot and lipid peroxide fluorescence staining, we found that on the basis of LPS-induced ferroptosis in Raw264.7 cells, the addition of exogenous lactic acid could reverse the ferroptosis-inhibiting effect of MB-3(Figure 6I). The same effect was also observed in immunofluorescence staining for lipid peroxides(Figure 6J). Therefore, we conclude that the Kat2a inhibitor MB-3 reduces lactic acid concentration by inhibiting the enzymatic activity of HK2, leading to decreased protein expression of histone H3K18la and thereby suppressing ferroptosis. However, this effect can be reversed by the addition of exogenous lactic acid. Discussion Sepsis-associated acute lung injury (ALI), a potentially fatal clinical disease, is defined by breakdown of the alveolar-capillary barrier, neutrophil infiltration, and a cytokine storm that eventually leads to progressive respiratory failure. This study reveals that lysine acetyltransferase Kat2a plays a central role in septic ALI by mediating histone H3K18 lactylation (H3K18la) to regulate the ferroptosis pathway. Experimental results demonstrate that LPS stimulation significantly upregulates Kat2a expression in lung tissues and macrophages, accompanied by elevated H3K18la modification and increased serum lactate levels. This lactylation modification serves as a critical bridge connecting metabolic dysregulation with epigenetic control, directly participating in gene transcriptional regulation by modulating chromatin accessibility [29-30] . Notably, the selective inhibitor MB-3 effectively reduces H3K18la levels by inhibiting Kat2a enzymatic activity, thereby ameliorating pulmonary pathological damage, inflammation, and fibrosis, offering a novel epigenetic perspective for targeted ALI therapy [31] . Ferroptosis, an iron-dependent programmed cell death caused by lipid peroxide buildup, is critical in ALI development [32-33] . Mechanistic studies demonstrate that the Kat2a-H3K18la axis drives lung injury through dual regulation of ferroptosis key molecules. RNA-seq and protein validation confirm that Kat2a promotes transcriptional activation of the pro-ferroptosis gene Tfrc while suppressing protective genes Slc7a11 and GPX4 via H3K18la modification. Furthermore, exogenous lactate specifically upregulates Tfrc and downregulates GPX4/Slc7a11, whereas the lactate dehydrogenase inhibitor DCA completely reverses this effect, establishing for the first time a "lactate metabolism-histone lactylation-ferroptosis" cascade regulatory axis(Figure 7A). A groundbreaking finding of this study is the identification of cholesterol-25-hydroxylase (CH25H) as a central player in the Kat2a-H3K18la regulatory network. CH25H, highly expressed in lung tissue, generates 25-hydroxycholesterol (25-HC), which exacerbates oxidative damage by depleting glutathione (GSH) [34] . LPS stimulation induces over 100-fold upregulation of CH25H, while MB-3 significantly suppresses its expression in a lactate-dependent manner—exogenous lactate increases whereas DCA decreases CH25H protein levels [35-36] . This not only positions CH25H as a key node linking cholesterol metabolism to ferroptosis but also exemplifies direct epigenetic regulation of metabolic enzyme expression, providing experimental evidence for the "metabolism-epigenetics-cell death" interplay in ALI. Therapeutically, MB-3 exhibits multi-dimensional protective effects. Beyond inhibiting ferroptosis, it significantly reduces pulmonary ROS accumulation and TUNEL-positive cells, suppresses LPS-induced macrophage hyperactivation, and inhibits NLRP3/caspase-1/IL-1β inflammasome activation and M1 macrophage polarization. This multi-targeting advantage surpasses conventional anti-inflammatory strategies, offering new therapeutic avenues for septic ALI. Importantly, this study pioneers the incorporation of CH25H into the Kat2a-H3K18la network, revealing a novel mechanism whereby epigenetic modifications mediate ferroptosis through metabolic enzymes and providing a theoretical framework for metabolism-epigenetics crosstalk [37-39] . Despite these advances, limitations remain: The impact of MB-3 on non-histone protein lactylation has not yet been evaluated. Future studies should employ lactylome profiling to systematically analyze MB-3's effects on lactylation patterns in macrophages and lung tissues, and focus on verifying the research on the Kat2a-H3K18la axis regarding CH25H's regulation of the ferroptosis pathway to fully elucidate the molecular mechanisms of MB-3-mediated lactylation regulation. In conclusion, Kat2a orchestrates septic ALI progression by dynamically regulating ferroptosis through histone lactylation. Targeting this axis not only advances our understanding of ALI pathogenesis but also lays an experimental foundation for developing precision therapeutic strategies. Declarations Acknowledgement This work was strongly supported by the National Natural Science Foundation of China Project (Grant Number: 82073852) and Innovation Team Project of Hubei University of Science and Technology (Grant Number:2022T01). Author Contributions JC and CHOY have given substantial contributions to the conception and design of the manuscript. JC, MQC , ZYL, WJZ and XLD to acquisition, analysis, and interpretation of the data. All authors have participated in drafting the manuscript. JC and CHOY revised it critically. All authors read and approved the final version of the manuscript. Funding This work was strongly supported by the National Natural Science Foundation of China Project (Grant Number: 82073852) and Innovation Team Project of Hubei University of Science and Technology (Grant Number:2022T01). Ethics approval and consent to participate Not applicable. Consent for publication All authors agree to publish this article. Competing interests The authors have declared that no competing interest exists. Ethical approval All animal experiments were approved by the Hubei Provincial Laboratory Animal Research Center. Data availability All data generated or analyzed during this study are included in this published article and its supplementary information files. References Hsieh YH, Deng JS, Pan HP, Liao JC, Huang SS, Huang GJ. Sclareol ameliorate lipopolysaccharide-induced acute lung injury through inhibition of MAPK and induction of HO-1 signaling. Int Immunopharmacol . 2017; 44 :16–25. Zhou Y, Liu T, Duan JX, Li P, Sun GY, Liu YP, et al. Soluble epoxide hydrolase inhibitor attenuates lipopolysaccharide-induced acute lung injury and improves survival in mice. Shock . 2017; 47 (5):638–45. Yang JX, Li M, Chen XO, Lian QQ, Wang Q, Gao F, et al. 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Nrf2 attenuates ferroptosis-mediated IIR-ALI by modulating TERT and Slc7a11. Cell Death Dis. 2021; 12 (11): 1027. Liu, Y, Wei, Z, Ma, X, et al. 25-Hydroxycholesterol activates the expression of cholesterol 25-hydroxylase in an LXR-dependent mechanism. J LIPID RES. 2018; 59 (3): 439-451. Romero, J, Toral-Rios, D, Yu, J, et al. 25-hydroxycholesterol promotes brain cytokine production and leukocyte infiltration in a mouse model of lipopolysaccharide-induced neuroinflammation. J Neuroinflammation. 2024; 21 (1): 251. Diczfalusy, U, Olofsson, KE, Carlsson, AM, et al. Marked upregulation of cholesterol 25-hydroxylase expression by lipopolysaccharide. J LIPID RES. 2009; 50 (11): 2258-64. Xiao, J, Wang, S, Chen, L, et al. 25-Hydroxycholesterol regulates lysosome AMP kinase activation and metabolic reprogramming to educate immunosuppressive macrophages. IMMUNITY. 2024; 57 (5): 1087-1104.e7. Li, Y, Xiao, G, Fu, X, et al. CH25H/25-HC promotes pulmonary fibrosis via AMPK/STAT6 pathway-dependent M2 macrophage polarization in COPD. Immunobiology. 2025; 230 Immunobiology. Canfrán-Duque, A, Rotllan, N, Zhang, X, et al. Macrophage-Derived 25-Hydroxycholesterol Promotes Vascular Inflammation, Atherogenesis, and Lesion Remodeling. CIRCULATION . 2023; 147 CIRCULATION. Additional Declarations There is no conflict of interest Supplementary Files WBOriginal.docx Original material FigureS1.png Fig. S1 Kat2a Inhibition Modulates Ferroptosis. S1A TUNEL staining in lung tissues (N = 6 mice/group). Scale bar = 50 μm. FigureS2.png Fig. S2 Histone lactylation modification promotes ferroptosis and inflammation. S1A-B The expression of CH25H protein varies with the concentration of LA(N=3). B The protein expression of CH25H after LA pre-administration (N = 3 independent experiments). C-E The protein expression of CH25H after DCA pre-administration (N = 3 independent experiments). Significance tested using One-way ANOVA. Statistical significance is shown as *p < 0.05, **p < 0.01, ***p < 0.001. 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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Scale bar = 20 μm. \u003cstrong\u003eB \u003c/strong\u003eKat2a protein expression in lung tissues (N = 3 independent experiments).\u003cstrong\u003eC\u003c/strong\u003e The Pan Kla immunoblots of lung tissues (N = 3 independent experiments). \u003cstrong\u003eD\u003c/strong\u003e H3K18la expression in lung tissues of control and ALI mice was investigated by IF staining (N = 6 mice/group). Scale bar = 20 μm. \u003cstrong\u003eE\u003c/strong\u003e H3K18la protein expression in lung tissues (N = 3 independent experiments). \u003cstrong\u003eF\u003c/strong\u003e The expression of Kat2a protein varies with the concentration of LPS(N=3). \u003cstrong\u003eG\u003c/strong\u003e Single-cell sequencing of acute lung injury. \u003cstrong\u003eH\u003c/strong\u003eKat2a and H3K18la protein expression in Raw264.7 (N = 3 independent experiments). Significance tested using One-way ANOVA. Statistical significance is shown as *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8069085/v1/4dc87c22bcb2ab254b1f178c.png"},{"id":97442451,"identity":"dd3ccdde-5b39-48f7-88e2-adf049088867","added_by":"auto","created_at":"2025-12-04 12:09:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":27876040,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKat2a Inhibition significantly alleviates ALI in vivo.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Representative images of H\u0026amp;E and Masson staining of lung tissue (N = 6 mice/group). Scale bar = 50 μm. \u003cstrong\u003eB \u003c/strong\u003eThe experimental design of drug treatment for ALI model mice. \u003cstrong\u003eC\u003c/strong\u003e Lung wet-to-dry weight ratio and was determined in all groups (n = 6). \u003cstrong\u003eD\u003c/strong\u003e Semiquantitative histological scores of lung injury in groups described in panel (n = 6). \u003cstrong\u003eE\u003c/strong\u003e Representative images of CD68 staining of lung tissue (N = 6 mice/group). Scale bar = 20 μm. \u003cstrong\u003eF\u003c/strong\u003e The ROS staining in Raw264.7 (N = 3 independent experiments). Scale bar = 20 μm .Significance tested using One-way ANOVA. Statistical significance is shown as *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8069085/v1/14e3c2d7b69b51914ce2761e.png"},{"id":97442386,"identity":"1047db83-fba8-43ce-a456-b09112077adb","added_by":"auto","created_at":"2025-12-04 12:09:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4271356,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe inflammatory mechanism by which Kat2a inhibitors regulate systemic responses.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e KEGG pathway analysis in Raw264.7 pretreated with MB-3 (50 μM) or DMSO followed by stimulation with LPS (100 ng/mL) for 4 h. \u003cstrong\u003eB-E \u003c/strong\u003eNLRP3,Caspase-1 and IL-1βprotein expression in lung tissues (N = 3 independent experiments).\u003cstrong\u003eF-I\u003c/strong\u003e NLRP3,Caspase-1 and IL-1βprotein expression in Raw264.7 (N = 3 independent experiments).\u003cstrong\u003eJ-K\u003c/strong\u003e INOS protein expression in Raw264.7 (N = 3 independent experiments). \u003cstrong\u003eL\u003c/strong\u003eCell viability of Raw264.7. \u003cstrong\u003eM-N\u003c/strong\u003e CH25H protein expression in lung tissues and Raw264.7. (N = 3 independent experiments). Significance tested using One-way ANOVA. Statistical significance is shown as *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8069085/v1/dc1dfdc2e065098c72664961.png"},{"id":97442375,"identity":"6beee3a0-6758-43d6-9093-40de9183cb3d","added_by":"auto","created_at":"2025-12-04 12:09:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":7714941,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKat2a Inhibition Modulates Ferroptosis.\u003c/strong\u003e \u003cstrong\u003eA-B\u003c/strong\u003e Scatter plots and Heatmap of gene expression in Raw264.7 pretreated with MB-3 (50 μM) or DMSO followed by stimulation with LPS (100 ng/mL) for 4 h. Red: upregulated genes. Blue: downregulated genes. \u003cstrong\u003eC-E \u003c/strong\u003eSlc7a11 and GPX4 protein expression in lung tissues (N = 3 independent experiments). \u003cstrong\u003eF-I\u003c/strong\u003e Tfrc, Slc7a11 and GPX4 protein expression in Raw264.7 (N = 3 independent experiments). \u003cstrong\u003eJ \u003c/strong\u003eLipid peroxidation staining of Raw264.7 cells(N = 3 independent experiments). Scale bar = 20 μm. Significance tested using One-way ANOVA. Statistical significance is shown as *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8069085/v1/2dd49cda64a9810ff7712c5a.png"},{"id":97442371,"identity":"e23b560b-ff5b-425b-8fda-3852ced44ab3","added_by":"auto","created_at":"2025-12-04 12:09:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3256983,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistone lactylation modification promotes ferroptosis and inflammation.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e The expression of H3K18la protein varies with the concentration of LA(N=3). \u003cstrong\u003eB \u003c/strong\u003eThe protein expression of H3K18la after LA pre-administration (N = 3 independent experiments). \u003cstrong\u003eC\u003c/strong\u003e The protein expression of H3K18la after DCA pre-administration (N = 3 independent experiments). \u003cstrong\u003eD-E \u003c/strong\u003eThe expression of Tfrc, Slc7a11 and GPX4 protein varies with the concentration of LA(N=3). \u003cstrong\u003eF-I\u003c/strong\u003e The protein expression of Tfrc, Slc7a11 and GPX4 after LA pre-administration (N = 3 independent experiments). \u003cstrong\u003eJ-L\u003c/strong\u003e The protein expression of Tfrc, Slc7a11 and GPX4 after DCA pre-administration (N = 3 independent experiments). Significance tested using One-way ANOVA. Statistical significance is shown as *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8069085/v1/9275e46ad046b2349cea425e.png"},{"id":97442448,"identity":"a1591eec-54ea-490f-b451-3188c80c481e","added_by":"auto","created_at":"2025-12-04 12:09:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":23586015,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKat2a elevates Histone Lactylation Promotes Ferroptosis and Inflammation.\u003c/strong\u003e \u003cstrong\u003eA-B\u003c/strong\u003e Scatter plots and Heatmap of gene expression in Raw264.7 pretreated with MB-3 (50 μM) or DMSO followed by stimulation with LPS (100 ng/mL) for 4 h. Red: upregulated genes. Blue: downregulated genes. \u003cstrong\u003eC \u003c/strong\u003eHK2 protein expression in lung tissues (N = 3 independent experiments). \u003cstrong\u003eD\u003c/strong\u003e The lactic acid concentration in lung tissue (N=4). \u003cstrong\u003eE\u003c/strong\u003e The lactic acid concentration in Raw264.7 (N=4). \u003cstrong\u003eF-G\u003c/strong\u003e H3K18la protein expression in lung tissues and Raw264.7 pretreated with the indicated amounts of MB-3 followed by stimulation with LPS. (N = 3 independent experiments). \u003cstrong\u003eH \u003c/strong\u003eImmunofluorescence staining for H3K18la in lung tissues (N = 6 mice/group). Scale bar = 20 μm.\u003cstrong\u003e I\u003c/strong\u003e Tfrc, Slc7a11 and GPX4 protein expression in Raw264.7 pretreated with the indicated amounts of MB-3 and LA followed by stimulation with LPS. (N = 3 independent experiments).\u003cstrong\u003e J\u003c/strong\u003e Lipid peroxidation staining of Raw264.7 cells (N = 3 independent experiments). Scale bar = 20 μm. Significance tested using One-way ANOVA. Statistical significance is shown as *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8069085/v1/d1452b60c902c03d73462552.png"},{"id":97667612,"identity":"408d6df8-67c4-4c9c-a4b2-02878de54c63","added_by":"auto","created_at":"2025-12-08 09:23:55","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2744097,"visible":true,"origin":"","legend":"\u003cp\u003eThis protocol describes that the Kat2a-H3K18la axis alleviates LPS-induced acute lung injury by inhibiting ferroptosis and inflammation.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8069085/v1/41c1251e40008a253c0e583d.png"},{"id":98628223,"identity":"a8bf27e1-12d1-4152-85d2-95b413d0461d","added_by":"auto","created_at":"2025-12-19 17:11:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":73542393,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8069085/v1/3e414f88-5c3b-4a82-a13a-a15b371ef698.pdf"},{"id":97442452,"identity":"e470bca6-cb72-42c5-9160-95ba756e3949","added_by":"auto","created_at":"2025-12-04 12:09:08","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15703132,"visible":true,"origin":"","legend":"Original material","description":"","filename":"WBOriginal.docx","url":"https://assets-eu.researchsquare.com/files/rs-8069085/v1/967ab0299d603370d4bac02a.docx"},{"id":97442443,"identity":"30cabdc9-0585-47e6-83d7-6abed5fce3ad","added_by":"auto","created_at":"2025-12-04 12:09:07","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12988704,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S1 Kat2a Inhibition Modulates Ferroptosis. S1A \u003c/strong\u003eTUNEL staining in lung tissues (N = 6 mice/group). Scale bar = 50 μm.\u003c/p\u003e","description":"","filename":"FigureS1.png","url":"https://assets-eu.researchsquare.com/files/rs-8069085/v1/1076d209cd2f72c65511e76a.png"},{"id":97442370,"identity":"e9cb819c-de6e-4625-985b-c81153367ffd","added_by":"auto","created_at":"2025-12-04 12:09:05","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1017217,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S2 Histone lactylation modification promotes ferroptosis and inflammation. S1A-B \u003c/strong\u003eThe expression of CH25H protein varies with the concentration of LA(N=3). \u003cstrong\u003eB \u003c/strong\u003eThe protein expression of CH25H after LA pre-administration (N = 3 independent experiments). \u003cstrong\u003eC-E\u003c/strong\u003e The protein expression of CH25H after DCA pre-administration (N = 3 independent experiments). Significance tested using One-way ANOVA. Statistical significance is shown as *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"FigureS2.png","url":"https://assets-eu.researchsquare.com/files/rs-8069085/v1/073fb6b8aa441ce7b60f8fab.png"}],"financialInterests":"There is no conflict of interest","formattedTitle":"Kat2a-Mediated Histone Lactylation Promotes Ferroptosis and Inflammation in Sepsis-Associated Lung Injury","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSepsis is a life-threatening illness characterized by organ failure caused by an inappropriate host response to infection. It affects millions of individuals globally each year. Its consistently high death rate places a significant strain on society and families. Acute lung injury (ALI) is one of the most common and severe consequences of sepsis, affecting up to 50% of patients. Numerous studies show that Gram-negative bacterial infections are the leading cause of acute lung injury (ALI), and that lipopolysaccharide (LPS), a critical component of Gram-negative bacteria\u0026apos;s outer membrane, plays an important role in causing inflammation and lung damage \u003csup\u003e[1-4]\u003c/sup\u003e. Macrophages, the most prevalent immune cell in the lungs, are important for sepsis-induced acute lung damage\u003csup\u003e[5\u0026ndash;6]\u003c/sup\u003e. Due to the absence of targeted treatment approaches, the clinical management of ALI is currently fraught with difficulties \u003csup\u003e[7]\u003c/sup\u003e. Thus, it is essential to further clarify the pathophysiology of ALI.\u003c/p\u003e\n\u003cp\u003eFerroptosis is a recently discovered controlled cell death mechanism that relies on iron and lipid peroxides, as opposed to apoptosis, autophagy, and other kinds of cell death\u003csup\u003e[8\u0026ndash;10]\u003c/sup\u003e. Recent research indicates that ferroptosis may be caused by increased lipid ROS and inhibition of glutathione peroxidase 4 (GPX4) \u003csup\u003e[11]\u003c/sup\u003e. Numerous illnesses, such as cancer, Alzheimer\u0026apos;s, and intracerebral hemorrhage, have been linked to it \u003csup\u003e[12]\u003c/sup\u003e. Recent research has demonstrated the critical role ferroptosis plays in the development of sepsis-induced lung injury\u003csup\u003e[13]\u003c/sup\u003e. Our comprehension of the intricate regulatory pathways of ferroptosis in ALI is still lacking, despite earlier studies showing that LPS causes ferroptosis in alveolar epithelial cells and aggravates sepsis-related lung injury \u003csup\u003e[14]\u003c/sup\u003e. Thus, the goal of this study is to get a better understanding of the role of ferroptosis in ALI and the regulatory mechanisms that underpin it.\u003c/p\u003e\n\u003cp\u003eEpigenetic alterations, such as histone modifications, DNA methylation, and chromatin remodeling, affect gene expression without modifying DNA sequences and are important processes for macrophage pro-inflammatory polarization\u003csup\u003e[15]\u003c/sup\u003e. For instance, LPS activates TLR4 signaling, leading to kinase-mediated recruitment of histone methyltransferases that deposit activating H3K4me3 marks at pro-inflammatory gene promoters, enhancing chromatin accessibility and transcription factor binding to amplify inflammatory responses\u003csup\u003e[16]\u003c/sup\u003e. The newly discovered histone (and non-histone) lactylation modification is a rapid and dynamic epigenetic mark whose dysregulation is linked to transcriptional dysfunction and disease, making it a potential diagnostic marker or therapeutic target\u003csup\u003e[17]\u003c/sup\u003e. However, the specific host factors mediating lactylation-dependent inflammatory regulation in macrophages and their role in ferroptosis remain unclear. Therefore, this study seeks to elucidate the function of lactylation modification in ALI through its regulation of ferroptosis and its underlying mechanisms.\u003c/p\u003e\n\u003cp\u003eLysine acetyltransferase 2A (Kat2a, alternatively referred to as GCN5) was first identified as a key component of the histone acetyltransferase (HAT) family in yeast and is highly conserved among mammals, especially in humans and mice. Emerging evidence suggests that aberrant gene expression driven by the synergistic regulation of transcription and epigenetic factors may play a pivotal role in lung injury\u003csup\u003e[18]\u003c/sup\u003e. However, the role of Kat2a in ALI remains largely unexplored. Here, we demonstrate that treatment with the selective Kat2a inhibitor MB-3 significantly ameliorates inflammatory damage in ALI models. According to our findings, Kat2a represents a unique therapeutic target in the metabolic and epigenetic reprogramming of macrophages, providing a method of modulating inflammatory responses both at the start and as ALI progresses.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Reagents and Antibodies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe ordered MB-3 (M2449), lactate (LA, 71718), lipopolysaccharide (LPS, L2880), the lactate dehydrogenase inhibitor dichloroacetate (DCA, 347795), and dimethyl sulfoxide (DMSO) from Sigma-Aldrich (USA). DMEM high-glucose medium, fetal bovine serum (FBS), and penicillin/streptomycin solution (100\u0026times;) were sourced from Gibco (USA). Cyclodextrin and the CCK-8 assay kit were purchased from Meilun Biotechnology (Dalian, China). The bicinchoninic acid (BCA) protein quantification kit was supplied by KeriBio (Wuhan, China). Antibodies against Kat2a (#3305) and \u0026alpha;-Tubulin (#3873) were procured from Cell Signaling Technology (CST, USA). Antibodies targeting Slc7a11 (A2413), Tfrc (A5865), CH25H (A24299), IL-1\u0026beta; (A16288), and NLRP3 (A5652) were obtained from ABclonal Biotechnology (Wuhan, China). Antibodies for GPX4 (bs-3884R), Caspase-1 (bs-0169R), and GAPDH (bs-10900R) were sourced from Bioss Antibodies (Beijing, China). The H3K18la (PTM-1406RM) antibody and Pan-Kla (PTM-1401RM) was provided by PTM BIO, and the \u0026beta;-actin (ABL1011) antibody was obtained from Abbkine.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Sepsis-Induced Acute Lung Injury (ALI) Animal Model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMale C57BL/6 adult mice (8\u0026ndash;10 weeks old, weighing 20\u0026ndash;22 g) kept in specified pathogen-free (SPF) conditions were acquired for this investigation from the Hubei Provincial Laboratory Animal Research Center. These mice were housed in an SPF environment with a 12-hour light/dark cycle, temperature, and humidity control. Every experimental procedure was carried out in accordance with the Laboratory Animal Management Regulations of Hubei Province. There were six mice in each of the four groups that the animals were randomly assigned to. Among them, 12 C57BL/6 mice with endotoxic shock models were first pretreated by either MB-3 (10mg/kg body weight, intraperitoneal administration) or DMSO, and then received an intraperitoneal injection of LPS (10 mg/kg body weight). Eight hours after injection, serum samples were taken for ELISA analysis. Hematoxylin and eosin (H\u0026amp;E) staining and immunohistochemical testing were performed on lung tissues that had been paraformaldehyde-fixed\u003csup\u003e[19]\u003c/sup\u003e. Furthermore, fresh tissue samples that weren\u0026apos;t utilized for fixation were kept in liquid nitrogen for later molecular biology tests like ELISA and Western blot.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Cell Culture and Stimulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProcell Life Science \u0026amp; Technology Co., Ltd. (Wuhan, China) supplied the mouse monocyte-macrophage cell line Raw264.7. These cells were kept in complete DMEM medium (pH 7.2\u0026ndash;7.4) supplemented with 1% penicillin\u0026ndash;streptomycin and 10% fetal bovine serum. They were then raised in a humidified incubator with 5% CO₂ at 37\u0026deg;C. After reaching 60\u0026ndash;70% confluence, Raw264.7 cells were cultivated in serum-free media containing LPS for 24 hours prior to additional analysis. To stimulate macrophages, LPS was given at a concentration of 1 \u0026mu;g/mL for the specified durations, unless otherwise specified \u003csup\u003e[20]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Western blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWith the use of Beyotime\u0026apos;s RIPA lysis buffer, which contains phosphatase and protease inhibitors, total protein was extracted from lung tissues and cells. Protein quantities were measured using a BCA assay kit. In order to heat denaturize the protein samples, they were then incubated for 10 minutes at 98\u0026deg;C in a metal bath. SDS-PAGE electrophoresis was then used to separate the proteins after they had been moved to PVDF membranes (Merck Millipore, Billerica, MA, USA). Following a two-hour blocking period at room temperature using 5% skim milk, primary and secondary antibodies conjugated with horseradish peroxidase were applied to the membranes. Lastly, enhanced chemiluminescence reagents (MeilunBio) were used to visualize the blots.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 ROS measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCreate a stock solution with a concentration of 10 mM, DCFH-DA (Beyotime, S0033S) was dissolved in DMSO. The stock solution was then further diluted using serum-free medium (at a 1:1000 dilution) to a working concentration of 10 \u0026mu;M. Raw 264.7 cells were seeded into six-well plates, and the designated chemicals were applied to the cells once they adhered. The cells were then exposed to the DCFH-DA working solution for 30 minutes at 37\u0026deg;C. Lastly, fluorescence microscopy was used to detect the intracellular ROS levels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Measurement of lactate levels\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum samples were obtained from the orbital venous plexus of septic mice, subjected to centrifugation at 3000 rpm for 15 minutes, and then analyzed for lactate levels via ELISA following the manufacturer\u0026apos;s instructions\u003csup\u003e[21]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Cell viability assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell viability was assessed using the CCK-8 test as directed by the manufacturer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8 H\u0026amp;E staining and MASSON staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter being treated with a 4% paraformaldehyde solution, lung tissues were embedded in paraffin. Lung tissue was processed in serial sections and then stained with Masson\u0026apos;s trichrome and hematoxylin-eosin (H\u0026amp;E). The left lungs, which had been fixed in 4% paraformaldehyde, were specifically paraffin-embedded, divided into sections that were 5\u0026nbsp;\u0026mu;m thick, baked for 60 minutes at 60\u0026deg;C, and then dewaxed with fresh xylene. These lung tissue sections were then processed for pathological staining with H\u0026amp;E and Masson\u0026apos;s reagents.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9 Immunohistochemical staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsistent with previous descriptions, 5\u0026nbsp;\u0026mu;m-thick lung sections were dewaxed with xylene and then processed through graded ethanol solutions (100%, 90%, 70%) followed by water. After dewaxing, the sections underwent antigen retrieval by microwave heating in citrate buffer for 30 minutes, were cooled to room temperature, and rinsed with PBS for 2 minutes. Inhibiting endogenous peroxidase activity was accomplished with 10% goat serum and 3% H2O2. After that, the sections were treated with Kat2a antibody (diluted 1:500 in PBS) for a whole night at 4\u0026deg;C. They were then incubated with anti-rabbit reagent for an hour at 37\u0026deg;C. After three PBS washes and five minutes of DAB preparation, the slices were inspected under a microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10 TUNEL staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs previously described, 5\u0026mu;m-thick lung sections were processed with proteinase K and incubated at ambient temperature for 25 minutes. Permeabilization buffer was then added to fully cover the tissue sections, after which Tdt and dUTP reagents (mixed at a 1:9 ratio) were added and incubated at 37\u0026deg;C for 2 hours. Following the addition of 3% hydrogen peroxide solution and 25 minutes of incubation in the dark, converter-POD was applied and incubated at 37\u0026deg;C for 30 minutes, with subsequent development using DAB solution. After that, a microscope was used to study and take pictures of the sections.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.11 Lung injury score\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe extent of lung damage, which included thickening of the alveolar septum, inflammation, bleeding, and swelling, was evaluated and graded in a blinded way. There were four ratings for alveolar septal thickening, bleeding, and edema: none (score = 0), mild (score = 1), moderate (score = 2), severe (score = 3), and extremely severe (score = 4). By calculating the total number of inflammatory cells per 100 x 100 field, inflammation was measured.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.12 RNA-seq\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe samples were treated using the TRIzol Reagent to extract total RNA. The KC-Digital Stranded mRNA Library Prep Kit for Illumina (cat. no. DR08502, Servicebio, China) was used to construct strand-specific RNA sequencing libraries from 2 \u0026mu;g of total RNA, following the manufacturer\u0026apos;s instructions \u003csup\u003e[22]\u003c/sup\u003e. This kit eliminates duplication bias from PCR and sequencing by applying unique molecular identifiers (UMIs) with eight random bases to pre-amplified cDNA molecules. Prior to final sequencing, library products in the 200-500 bp range were enriched and quantified using the PE150 mode on the DNBSEQ-T7 platform (MGI Tech, China). Servicebio (Wuhan, China) carried out library preparation and sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.13 Determination of lipid peroxidation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw264.7 cells were seeded in 6-well plates. The following day the medium was changed, and medications were added for a 24 hours treatment. The medium was thrown away after three PBS washes. Following that, 10 \u0026mu;M of C11-BODIPY581/591 (Beyotime, S0043S) was added, and the mixture was incubated for 30 minutes in the presence of light. The cells were subjected to three PBS washes before being viewed, photographed, and recorded using a fluorescence microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.14 Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGraphPad Prism 9.5 was used to perform statistical analyses. All numerical data is presented as mean \u0026plusmn; SD. To analyze differences between numerous groups, a one-way ANOVA with the Bonferroni post-hoc test was employed, while the Student\u0026apos;s t-test was utilized for two groups. Each experiment had biological replicates, and a P-value of less than 0.05 was considered statistically significant. To ensure the repeatability of the findings, each experiment was performed at least three times.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Kat2a and Histone Lactylation Levels are Increased in Sepsis-Associated Lung Injury\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur work was particularly concerned with determining the functional involvement of Kat2a and H3K18la in ALI and the underlying regulatory mechanisms. Initial examination of Kat2a expression levels in mouse lungs revealed a significant increase in Kat2a protein levels in lung tissues by Western blot analysis, Similar results were confirmed at the histopathological level, with immunohistochemical staining demonstrating increased Kat2a-positive cells in LPS-treated animals compared to controls (Figures 1A and B). To study the impact of lactylation modification in acute lung damage, we used a Western blot test to determine H3K18la expression levels. The study confirmed a trend similar to the global lactylation level \u0026mdash; Lactylation levels were higher in the model group compared to the blank group. Meanwhile, immunofluorescence staining showed a significant increase in both global lactylation and H3K18la levels(Figures 1C-E).\u003c/p\u003e\n\u003cp\u003eWe found that in the single-cell sequencing of LPS-induced lung injury, numerous cells were expressed. Among them, Raw264.7 cells showed the highest expression, The results in Figure 1F show that the gene expression of Kat2a protein in Raw264.7 cells increases with the elevation of LPS concentration, so we used Raw264.7 cells for subsequent cell experiments(Figure 1G). At the cellular level, elevated Kat2a and H3K18la protein expression was identified in LPS-treated Raw264.7 murine monocyte-macrophage cells compared to controls (Figure 1H).\u003c/p\u003e\n\u003cp\u003eThese findings collectively demonstrate that Kat2a and H3K18la expression is elevated in ALI, suggesting that Kat2a and H3K18la may serve as a crucial molecular regulator during ALI pathogenesis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Kat2a Inhibition Ameliorates Sepsis-Associated Acute Lung Injury\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe reduced Kat2a levels in mice by intraperitoneal injection of MB-3 and its control solvent DMSO. Two hours later, we established a sepsis model by intraperitoneal injection of LPS(Figure 2B). We found that LPS treatment induced weight loss, lethargy, and growth retardation in mice, while MB-3 administration prevented these physiological alterations\u003csup\u003e[23]\u003c/sup\u003e. MB-3 pretreatment significantly attenuated LPS-induced ALI in comparison to the LPS group, as seen by lower tissue damage, interstitial edema, pulmonary hemorrhage, and thinner alveolar walls. H\u0026amp;E staining revealed severe structural disruption in ALI mouse lungs at 6 hours post-LPS stimulation, characterized by alveolar collapse, architectural disorganization with fused alveoli, thickened septa, extensive inflammatory cell infiltration, and increased hemorrhagic foci (indicated by arrows). MB-3 pretreatment markedly alleviated these pathological changes. Masson\u0026apos;s trichrome staining demonstrated aggravated pulmonary fibrosis in LPS-treated mice, which was significantly ameliorated by MB-3 (Figure 2A). The increased lung wet/dry weight ratio was reversed by MB-3 treatment (Figure 2C). Given that uncontrolled inflammation represents a key pathological mechanism of ALI, blinded inflammatory scoring showed MB-3 substantially reduced pulmonary inflammation scores\u0026nbsp;(Figure 2D), indicating its protective effect against LPS-induced lung injury. The results of CD68 immunohistochemistry showed that the LPS group had a considerably greater M1 macrophage marker than the blank group. Pretreatment with MB-3, however, reduced the positive expression of CD68(Figure 2E). Immunofluorescence staining confirmed that MB-3 significantly suppressed LPS-induced ROS overproduction (Figure 2F).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Kat2a Inhibition controls lipopolysaccharide (LPS)-induced systemic inflammation in vivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe investigated LPS-induced global gene expression profiles in Raw264.7 using RNA-seq. To study the direct involvement of Kat2a in the macrophage-mediated inflammatory response, seven murine macrophages were treated with either DMSO or MB-3. The TNF-\u0026alpha;\u0026nbsp;signaling pathway, ferroptosis, and rheumatoid arthritis were found to have upregulated genes, according to the KEGG pathway (Figure 3A). To study the possible mechanisms behind the therapeutic benefits of Kat2a on LPS-induced pulmonary inflammatory damage, we assessed the influence of MB-3 on inflammation and immunological function in mice models. Administration of the selective Kat2a inhibitor MB-3 to LPS-challenged mice revealed through Western blot analysis that pulmonary inflammatory factors (Caspase-1, IL-1\u0026beta;, NLRP3) were significantly elevated in the LPS group compared to controls, while MB-3 treatment markedly suppressed their expression(Figures 3B-E). Similarly, in vitro experiments confirmed that MB-3 substantially prevented LPS-induced macrophage inflammatory responses by reducing protein levels of NLRP3, IL-1\u0026beta;, and Caspase-1 (Figures 3F-I). Additionally, MB-3 decreased the protein expression of M1 macrophage marker INOS, demonstrating anti-inflammatory effects(Figures 3J and K). These findings indicate that the Kat2a inhibitor MB-3 significantly alleviates pathological damage in sepsis-associated acute lung injury, suggesting Kat2a as a critical pathogenic factor in this condition. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo examine the effect of Kat2a on the viability of LPS-stimulated inflammatory macrophages, we modulated Kat2a levels in Raw264.7 cells using MB-3 and further assessed its interference with LPS-induced cell viability. MB-3 (50 \u0026mu;M) significantly inhibited the LPS-induced increase in RAW264.7 cell viability(Figures 3L).\u003c/p\u003e\n\u003cp\u003eCholesterol-25-hydroxylase(CH25H), the biosynthetic enzyme for 25-hydroxycholesterol (25-HC), exhibits its highest expression in the lungs, though its role in pulmonary biology remains unclear \u003csup\u003e[24]\u003c/sup\u003e. Lipopolysaccharide (LPS) induces dramatic increases in both 25-HC (\u0026gt;80-fold) and its synthase CH25H (\u0026gt;100-fold) in macrophages \u003csup\u003e[25]\u003c/sup\u003e. To investigate MB-3\u0026apos;s effect on ferroptosis in ALI, we evaluated its therapeutic potential in LPS-induced ALI using a murine model. CH25H protein expression was significantly upregulated in lung tissues following LPS administration, while MB-3 pretreatment markedly suppressed this increase. Consistent results were observed in vitro(Figures 3M and N).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Kat2a Inhibition Modulates Ferroptosis via targeting Tfrc and Slc7a11\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate Kat2a\u0026rsquo;s direct role in macrophage-mediated ferroptosis, we used RNA-seq to analyze LPS-induced global gene expression profiles in DMSO or MB-3 treated Raw264.7 murine macrophages. The Kat2a inhibitor MB-3 substantially reduced several ferroptosis-promoting genes (Hmox1, Tfrc, Txnrd1, and Fth1) while increasing expression of ferroptosis-inhibitory genes (Slc7a11, GPX4, PPard, and Gch1) (Figures 4A and B). We evaluated MB-3\u0026apos;s therapeutic effect on LPS-induced ALI by examining expression levels of established ferroptosis markers GPX4 and Slc7a11 in lung tissue and macrophages. LPS administration decreased GPX4 and Slc7a11 protein expression while MB-3 pretreatment significantly restored their expression in lung tissues (Figures 4C-E). The Tfrc gene was elevated in the LPS group of macrophages, and its expression was downregulated following MB-3 pretreatment, according to Western blot data. The LPS group showed downregulation of the ferroptosis-inhibitory genes Slc7a11 and GPX4, whereas MB-3 boosted their expression(Figures 4F-I).\u003c/p\u003e\n\u003cp\u003eAs a regulated form of necrosis, ferroptosis inhibition may reduce cell death\u003csup\u003e[26-27]\u003c/sup\u003e. TUNEL staining demonstrated decreased apoptotic cell numbers following MB-3 treatment. LPS-challenged lung tissues showed significantly increased apoptotic cells, whereas MB-3 administration reduced cell apoptosis, suggesting that MB-3 alleviates LPS-induced lung injury potentially by suppressing ferroptosis, thereby mitigating pulmonary damage and fibrosis in ALI mice(Figure S1A). In the meantime, we discovered that MB-3 significantly reduced the rise in lipid peroxidation brought on by LPS (Figure 4J).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese findings indicate that Kat2a promotes ferroptosis induction upon LPS stimulation, which appears essential for initiating ferroptosis in vitro.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Histone Lactylation Modulates Ferroptosis and inflammation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the effect of lactylation modification on ferroptosis gene expression, we manipulated the histone lactylation level in Raw264.7 cells by treating them with exogenous lactic acid or the lactate dehydrogenase inhibitor DCA. Stimulation of Raw264.7 cells with lipopolysaccharide (LPS) led to an increase in H3K18la level. After adding exogenous lactic acid, we found that the protein expression level of histone H3K18la increased in a lactic acid concentration-dependent manner(Figure 5A). Additionally, the addition of exogenous lactic acid resulted in elevated intracellular lactic acid level and histone H3K18la level(Figure 5B). Meanwhile, DCA significantly inhibited intracellular lactic acid and H3K18la levels in Raw264.7(Figure 5C).\u003c/p\u003e\n\u003cp\u003eFurthermore, we analyzed the effect of H3K18la on ferroptosis genes in macrophages in vitro. First, we treated macrophages with lactic acid at different concentration gradients and found that the ferroptosis marker genes (Tfrc, Slc7a11) showed a concentration-dependent response, while GPX4 exhibited little change(Figures 5D and E). Subsequently, in in vitro experiments, on the basis of LPS stimulation, we added exogenous lactic acid and observed that lactic acid increased the protein expression of Tfrc but decreased the protein expression of Slc7a11 and GPX4. This indicates that lactic acid can promote ferroptosis to a certain extent(Figures 5F-I). Therefore, we further added the lactic acid inhibitor DCA, and contrary to the previous findings, DCA significantly inhibited ferroptosis(Figures 5J-L).\u003c/p\u003e\n\u003cp\u003eBuilding on prior findings demonstrating LPS-induced CH25H upregulation. Studies have shown that LPS elevates CH25H expression, leading to increased 25-HC production, which subsequently depletes intracellular glutathione (GSH)\u003csup\u003e[28]\u003c/sup\u003e. This GSH reduction compromises cellular antioxidant capacity, exacerbating ferroptosis and ultimately triggering ALI. Notably, the MB-3 counteracts this process by downregulating CH25H expression via Kat2a inhibition, thereby restoring GSH levels. This regulatory effect suppresses ferroptosis, as evidenced by upregulated ferroptosis markers GPX4 and Slc7a11, ultimately mitigating alveolar epithelial cell damage and inflammatory responses to ameliorate sepsis-associated ALI.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo clarify the regulatory effect of histone lactylation modification on CH25H, we employed exogenous lactate and the lactate dehydrogenase inhibitor dichloroacetate (DCA) in LPS-induced cellular models. Exogenous lactate treatment significantly increased CH25H protein expression (p\u0026lt;0.01), whereas DCA treatment produced the opposite effect (p\u0026lt;0.05) (Figures S2). Coupled with previous evidence that lactate promotes ferroptosis, these findings suggest MB-3 may inhibit KAT2A-mediated histone lactylation to downregulate CH25H expression, thereby attenuating CH25H-driven ferroptosis and achieving therapeutic protection against lung injury.\u003c/p\u003e\n\u003cp\u003eThe data above show that lactylation modification plays an essential role in controlling the ferroptosis process.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003e3.6 Kat2a elevates Histone Lactylation Promotes Ferroptosis and Inflammation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFerroptosis is a key factor in acute lung damage. Previous research has demonstrated that the Kat2a inhibitor MB-3 can suppress the expression of ferroptosis genes in acute lung damage, whereas lactylation modification can enhance ferroptosis. Therefore, we can conclude that both can regulate the ferroptosis pathway, but the regulatory relationship between Kat2a and H3K18la requires further investigation. Our previous differential gene expression analysis of macrophage RNA-seq data revealed significant alterations in iron-related genes following MB-3 pretreatment prior to LPS stimulation, Through volcano plots and heatmaps, we found that MB-3 can regulate lactic acid concentration through glycolysis-related enzymes. Among them, the expression of hexokinase 2 (HK2) was significant, and compared with the LPS group, MB-3 could reduce its expression(Figures 6A and B). Meanwhile, the same trend was confirmed by Western blot(Figure 6C). According to clinical data, septic patients who have high serum lactate levels have a worse prognosis and more organ damage. ELISA results demonstrated significantly higher serum lactate levels in ALI mice and macrophages \u0026nbsp;than in controls, while MB-3 treatment reduced lactate concentrations, indicating that MB-3 regulates histone H3K18la expression by suppressing lactate production (Figures 6D and E).\u003c/p\u003e\n\u003cp\u003eWe also looked at the protein expression of histone H3K18la in lung tissues and macrophages, and found a substantial rise in H3K18la levels in the LPS-induced ALI model group compared to the controls. MB-3 administration markedly reduced histone H3K18la levels in both ALI lung tissues and macrophages(Figures 6F and G). The results of immunofluorescence staining were consistent with the above(Figure 6H).\u003c/p\u003e\n\u003cp\u003eThrough Western blot and lipid peroxide fluorescence staining, we found that on the basis of LPS-induced ferroptosis in Raw264.7 cells, the addition of exogenous lactic acid could reverse the ferroptosis-inhibiting effect of MB-3(Figure 6I). The same effect was also observed in immunofluorescence staining for lipid peroxides(Figure 6J).\u003c/p\u003e\n\u003cp\u003eTherefore, we conclude that the Kat2a inhibitor MB-3 reduces lactic acid concentration by inhibiting the enzymatic activity of HK2, leading to decreased protein expression of histone H3K18la and thereby suppressing ferroptosis. However, this effect can be reversed by the addition of exogenous lactic acid.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSepsis-associated acute lung injury (ALI), a potentially fatal clinical disease, is defined by breakdown of the alveolar-capillary barrier, neutrophil infiltration, and a cytokine storm that eventually leads to progressive respiratory failure. This study reveals that lysine acetyltransferase Kat2a plays a central role in septic ALI by mediating histone H3K18 lactylation (H3K18la) to regulate the ferroptosis pathway. Experimental results demonstrate that LPS stimulation significantly upregulates Kat2a expression in lung tissues and macrophages, accompanied by elevated H3K18la modification and increased serum lactate levels. This lactylation modification serves as a critical bridge connecting metabolic dysregulation with epigenetic control, directly participating in gene transcriptional regulation by modulating chromatin accessibility\u003csup\u003e[29-30]\u003c/sup\u003e. Notably, the selective inhibitor MB-3 effectively reduces H3K18la levels by inhibiting Kat2a enzymatic activity, thereby ameliorating pulmonary pathological damage, inflammation, and fibrosis, offering a novel epigenetic perspective for targeted ALI therapy\u003csup\u003e[31]\u003c/sup\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFerroptosis, an iron-dependent programmed cell death caused by lipid peroxide buildup, is critical in ALI development\u003csup\u003e[32-33]\u003c/sup\u003e. Mechanistic studies demonstrate that the Kat2a-H3K18la axis drives lung injury through dual regulation of ferroptosis key molecules. RNA-seq and protein validation confirm that Kat2a promotes transcriptional activation of the pro-ferroptosis gene Tfrc while suppressing protective genes Slc7a11 and GPX4 via H3K18la modification. Furthermore, exogenous lactate specifically upregulates Tfrc and downregulates GPX4/Slc7a11, whereas the lactate dehydrogenase inhibitor DCA completely reverses this effect, establishing for the first time a \u0026quot;lactate metabolism-histone lactylation-ferroptosis\u0026quot; cascade regulatory axis(Figure 7A). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA groundbreaking finding of this study is the identification of cholesterol-25-hydroxylase (CH25H) as a central player in the Kat2a-H3K18la regulatory network. CH25H, highly expressed in lung tissue, generates 25-hydroxycholesterol (25-HC), which exacerbates oxidative damage by depleting glutathione (GSH)\u003csup\u003e[34]\u003c/sup\u003e. LPS stimulation induces over 100-fold upregulation of CH25H, while MB-3 significantly suppresses its expression in a lactate-dependent manner\u0026mdash;exogenous lactate increases whereas DCA decreases CH25H protein levels\u003csup\u003e[35-36]\u003c/sup\u003e. This not only positions CH25H as a key node linking cholesterol metabolism to ferroptosis but also exemplifies direct epigenetic regulation of metabolic enzyme expression, providing experimental evidence for the \u0026quot;metabolism-epigenetics-cell death\u0026quot; interplay in ALI. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTherapeutically, MB-3 exhibits multi-dimensional protective effects. Beyond inhibiting ferroptosis, it significantly reduces pulmonary ROS accumulation \u0026nbsp;and TUNEL-positive cells, suppresses LPS-induced macrophage hyperactivation, and inhibits NLRP3/caspase-1/IL-1\u0026beta; inflammasome activation and M1 macrophage polarization. This multi-targeting advantage surpasses conventional anti-inflammatory strategies, offering new therapeutic avenues for septic ALI. Importantly, this study pioneers the incorporation of CH25H into the Kat2a-H3K18la network, revealing a novel mechanism whereby epigenetic modifications mediate ferroptosis through metabolic enzymes and providing a theoretical framework for metabolism-epigenetics crosstalk\u003csup\u003e[37-39]\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDespite these advances, limitations remain: The impact of MB-3 on non-histone protein lactylation has not yet been evaluated. Future studies should employ lactylome profiling to systematically analyze MB-3\u0026apos;s effects on lactylation patterns in macrophages and lung tissues, and focus on verifying the research on the Kat2a-H3K18la axis regarding CH25H\u0026apos;s regulation of the ferroptosis pathway to fully elucidate the molecular mechanisms of MB-3-mediated lactylation regulation. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn conclusion, Kat2a orchestrates septic ALI progression by dynamically regulating ferroptosis through histone lactylation. Targeting this axis not only advances our understanding of ALI pathogenesis but also lays an experimental foundation for developing precision therapeutic strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was strongly supported by the National Natural Science Foundation of China Project (Grant Number: 82073852) and Innovation Team Project of Hubei University of Science and Technology (Grant Number:2022T01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJC and CHOY have given substantial contributions to the conception and design of the manuscript. JC, MQC , ZYL, WJZ and XLD to acquisition, analysis, and interpretation of the data. All authors have participated in drafting the manuscript. JC and CHOY revised it critically. All authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was strongly supported by the National Natural Science Foundation of China Project (Grant Number: 82073852) and Innovation Team Project of Hubei University of Science and Technology (Grant Number:2022T01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors agree to publish this article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared that no competing interest exists.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Hubei Provincial Laboratory Animal Research Center.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col class=\"decimal_type\"\u003e\n \u003cli\u003eHsieh YH, Deng JS, Pan HP, Liao JC, Huang SS, Huang GJ. 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Macrophage-Derived 25-Hydroxycholesterol Promotes Vascular Inflammation, Atherogenesis, and Lesion Remodeling. \u003cem\u003eCIRCULATION\u003c/em\u003e. 2023; \u003cstrong\u003e147\u003c/strong\u003e CIRCULATION.\u0026nbsp;\u003c/li\u003e\n\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":"Epigenetic modification, Histone lactylation, Kat2a, Acute lung injury, Ferroptosis","lastPublishedDoi":"10.21203/rs.3.rs-8069085/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8069085/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The mortality rate of sepsis-associated acute lung injury (ALI) remains high, with its core mechanisms closely linked to macrophage-mediated inflammation and ferroptosis. Lipopolysaccharide (LPS), a key factor in Gram-negative bacterial infections, can activate epigenetic modifications and induce ferroptosis. However, the role of Kat2a-mediated histone lactylation (H3K18la) in ALI and its interaction with ferroptosis remain unclear. This study aims to investigate the pathogenic mechanism of the Kat2a-H3K18la axis in ALI through its regulation of the cholesterol metabolism enzyme CH25H and the ferroptosis pathway, as well as to evaluate targeted intervention strategies. Our study revealed LPS significantly upregulated Kat2a expression and H3K18la modification levels in lung tissues and macrophages. Inhibition of the Kat2a-H3K18la axis by MB-3 alleviated pulmonary hemorrhage, edema, fibrosis, and inflammatory infiltration while reducing NLRP3 inflammasome activation and M1 macrophage polarization. Kat2a-H3K18la epigenetically regulated ferroptosis bidirectionally, promoting transferrin receptor (Tfrc) transcription while suppressing protective genes Slc7a11/GPX4. Finally, we revealed that the exogenous lactate specifically reversed MB-3 inhibitory effect on ferroptosis, uncovering a novel \"lactate metabolism-H3K18la modification-ferroptosis\" pathway. In summary, our research reveals a new function for the Kat2a-ferroptosis axis in acute lung injury, revealing important information for possible therapeutic approaches.","manuscriptTitle":"Kat2a-Mediated Histone Lactylation Promotes Ferroptosis and Inflammation in Sepsis-Associated Lung Injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-04 12:08:41","doi":"10.21203/rs.3.rs-8069085/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":"0a8b6fe8-4528-4fda-be2f-190bca68c611","owner":[],"postedDate":"December 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":58937955,"name":"Biological sciences/Molecular biology/Epigenetics"},{"id":58937956,"name":"Biological sciences/Molecular biology/Post-translational modifications"},{"id":58937957,"name":"Biological sciences/Cell biology"}],"tags":[],"updatedAt":"2025-12-19T10:41:34+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-04 12:08:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8069085","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8069085","identity":"rs-8069085","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}