Up-regulated FOXO3/IL-10 Axis Inhibits Mitochondria-Associated Ferroptosis in Sepsis-Induced Diaphragm Dysfunction

Up-regulated FOXO3/IL-10 Axis Inhibits Mitochondria-Associated Ferroptosis in Sepsis-Induced Diaphragm Dysfunction | 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 Research Article Up-regulated FOXO3/IL-10 Axis Inhibits Mitochondria-Associated Ferroptosis in Sepsis-Induced Diaphragm Dysfunction Hua Liu, Dongdong Chai, Xiang Lyu, Bin Zhao, Nan Zhi, Yaqiong Yang, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4539738/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 Sepsis can lead to diaphragm dysfunction and atrophy known as sepsis-induced diaphragm dysfunction (SIDD), a major cause of mortality in the ICU. Our present study aimed to investigate whether ferroptosis is implicated in the pathogenesis of SIDD and the underlying molecular mechanism. The results demonstrated that in both in vivo and in vitro septic models, indicators such as the oxygen consumption rate (OCR), extracellular acidification rate (ECAR), reactive oxygen species (ROS), and complex I-V levels, alongside Transmission Electron Microscope (TEM) imaging, revealed mitochondria-associated changes. These alterations were mitigated by the ferroptosis inhibitor Ferrostatin (Fer-1), confirming that ferroptosis—a mitochondria-linked form of programmed cell death, plays a crucial role in SIDD. Through RNA sequencing (RNA-seq), transposase-accessible chromatin sequencing (ATAC-seq), and Dual-Luciferase Reporter Assay, we found that the FOXO3/IL-10 axis was suppressed in septic mice yet can be reactivated through administration of Fer-1. Furthermore, overexpression of FOXO3 shielded the diaphragm against sepsis-induced ferroptosis by boosting IL-10 production and enhancing the expression of Nrf2-mediated antioxidative genes such as GPX4. This reduced lipid peroxidation and concurrently ameliorated mitochondrial damage. Therefore, activating FOXO3 or administering IL-10 could offer a promising approach for treating SIDD. Sepsis-induced diaphragm dysfunction Ferroptosis Oxidative stress Mitochondrial dysfunction FOXO3/IL-10 axis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Sepsis is a critical condition triggered by the body's excessive immune response to infection, leading to widespread organ damage and potentially causing long-term disabilities for survivors [ 1 , 2 ]. Sepsis can cause diaphragm weakness and atrophy defined as sepsis-induced diaphragm dysfunction (SIDD), leading to respiratory failure in patients requiring mechanical ventilation. This condition significantly extends the duration of mechanical ventilation and escalates mortality rates in intensive care units (ICU) [ 3 ] , [ 4 – 6 ]. Previous studies have unveiled that the molecular underpinnings of SIDD span a range of processes [ 4 , 7 – 9 ], including inflammation [ 10 – 12 ], oxidative stress [ 13 , 14 ], metabolic imbalances [ 15 ], mitochondrial impairment [ 5 , 16 – 19 ], and disruptions in protein synthesis and degradation [ 20 ]. Although treatments targeting antioxidants and mitochondrial function have been identified as promising strategies for mitigating SIDD [ 5 , 8 , 21 , 22 ], these studies mainly focused on animal experiments, lacking specific therapeutic targets and facing challenges in clinical translation. Therefore, our current focus is on filling this research gap by identifying potential therapeutic targets with clinical applicability. In recent years, the study of ferroptosis, a regulated form of cell death, has gained considerable attention due to its involvement in a range of conditions including cancer [ 23 ], ischemia/reperfusion injury [ 24 ], neurodegenerative diseases [ 25 ], and acute lung injury [ 26 ]. Emerging evidence suggests ferroptosis also plays a role in sepsis. For instance, it has been shown that YAP1 can mitigate sepsis-induced acute lung injury by inhibiting ferritinophagy, a process linked to ferroptosis [ 27 ]. Further research indicates that Irisin's ability to suppress ferroptosis can protect against sepsis-induced brain dysfunction by activating the Nrf2/GPX4 signaling pathway [ 28 ] and preventing sepsis-induced acute kidney injury through the SIRT1/Nrf2 pathway [ 29 ]. Despite these insights, the connection between ferroptosis and sepsis-induced diaphragm dysfunction (SIDD) remains an area ripe for more detailed exploration. Studies have identified the malonylation of VDAC2 as a contributor to sepsis-induced myocardial dysfunction, implicating mitochondrial-related ferroptosis in this process [ 30 ], Our research found a surge in plasma Fe 2+ concentrations and the elevation of markers indicative of muscle cell damage alongside the decrease of antioxidative enzymes and GSH in patients with sepsis, suggesting ferroptosis may be involved in SIDD. By exploring the role of ferroptosis in modulating inflammation, oxidative stress, and mitochondrial imbalance in SIDD, potential therapeutic targets could be uncovered. Therefore, further research is essential to deepen our understanding of ferroptosis's impact on SIDD and its viability as a therapeutic target. We conducted a comprehensive analysis, assessing various parameters such as ferroptosis related protein expression levels, markers of inflammation, oxidative stress, and mitochondrial health, to uncover the potential molecular dynamics at play. We first identified that ferroptosis is the main death style in SIDD. To further elucidate the target molecular, we performed RNA-seq, ATAC-seq, along with protein-protein interaction (PPI) analysis. Our findings revealed that IL-10 is a critical molecular and FOXO3 is its regulatory transcription factors in the diaphragm of septic mice treated with Fer-1. So, we specifically honed in on the role of the FOXO3/IL-10/ferroptosis axis in SIDD, aiming to understand how enhancing the expression of FOXO3 and IL-10 influences diaphragm dysfunction in sepsis. 2. Results 2.1 Total Fe and Fe2+, indicators of muscle injury and oxidative stress increased while GSH, CAT, and SOD decreased in the serum of patients with sepsis To evaluate the pathological changes among patients with sepsis compared to normal individuals, the levels of various markers were measured among both groups. ELISA detection showed increased levels of CK-MB, Myo, and AST in serum of patients with sepsis compared to normal individuals (Fig. 1 A). Through detecting biochemical indexes, we found that patients with sepsis had lower GSH, CAT, and SOD levels in their serum compared to normal individuals (Fig. 1 B). Inversely, compared to healthy individuals, increased concentrations of LDH and MDA were measured in the serum samples of patients with sepsis (Fig. 1 B). Moreover, the ELISA assay indicated that the levels of total Fe and Fe 2+ were significantly increased while no significant change in Fe 3+ was observed, implying disturbance in iron metabolism in patients with sepsis (Fig. 1 C). 2.2 Ferroptosis is a main death type in LPS-induced C2C12 muscle cell To explore whether ferroptosis occurs in C2C12 muscle cells in response to sepsis, these cells were subjected to single or combined treatment of LPS with different cell death inhibitors. The CCK8 assay indicated that, compared to the control untreated cells, cell viability was markedly decreased by single treatment with LPS (p < 0.001, Fig. 2 A). In addition, the combination of LPS treatment with Fer-1, Z-VAD-FMK, Boc-D-FMK, necrostatin-1, or N-acetyl cysteine partially reversed the effect of LPS group (p < 0.001, Fig. 2 A). Interestingly, the combined treatment with LPS and Fer-1 showed the most pronounced reversal of cell viability (p < 0.001, Fig. 2 A), indicating that Fer-1 may effectively counteract LPS-induced ferroptosis. Moreover, immunofluorescence analysis indicated that the treatment with LPS alone resulted in a significant decrease in GPX4 expression (p < 0.001, Fig. 2 B) and a notable increase in ACSL4 expression (p < 0.001, Fig. 2 C). Decreased GPX4 expression and elevated ACSL4 levels induced by LPS-induced were reverted by the ferroptosis inhibitors (Figs. 2 B and 2 C). Fer-1 treatment showed the most significant partial reversal of protein expression of GPX4 and ACSL4 (p < 0.001, Figs. 2 B and 2 C). These findings suggest that Fer-1 has the potential to rescue the detrimental effects of LPS-induced cell death by restoring GPX4 expression and reducing ACSL4 levels, indicating that ferroptosis is a main death type in LPS-induced C2C12 muscle cell. 2.3 Mitochondria-associated ferroptosis is involved in LPS-induced C2C12 muscle cell We found that cell viability by CCK8 assay was significantly decreased in the LPS and Erastin groups compared to the control group, while treatment with Fer-1 partially rescued the effect of LPS and Erastin (Fig. 3 A). Moreover, decreased levels of GSH, and increased levels of LDH and MDA were recorded in the LPS and Erastin groups compared to the control group; however, combined treatment with Fer-1 counteracted LPS- and Erastin- inhibition of cell viability (Fig. 3 B). In addition, increased total Fe and Fe 2+ contents in the LPS and Erastin groups were recorded compared to the control group, while no remarkable difference in groups was recorded for Fe 3+ (Fig. 3 C). Moreover, Fer-1 inhibited the effect of LPS and Erastin on the content of Fe and Fe 2+ (Fig. 3 C). In addition, compared to the control group, OCR assay indicated a significant decrease in OCR levels in the LPS and Erastin groups, while Fer-1 partially mitigated OCR levels in LPS + Fer-1 group (Fig. 3 D). Furthermore, the detection of ECAR levels indicated an increase in ECAR levels in LPS and Erastin groups (Fig. 3 E). Interestingly, compared to the LPS and Erastin groups, Fer-1 treatment displayed a partial reversal in ECAR levels, which suggested a potential protective effect against LPS-induced glycolytic alterations (Fig. 3 E). The levels of mitochondrial complexes I-V were measured to detect the percentage of cell metabolic activity (Fig. 3 F). The treatment with LPS or Erastin was accompanied by a significant decrease in the activity of mitochondrial complexes I-V relatively to the control group (Fig. 3 F). In the LPS + Fer-1 and Erastin + Fer-1 groups, the treatment of Fer-1 partially reversed the activity of mitochondrial complexes I-V compared to the LPS and Erastin groups (Fig. 3 F). C11-BODIPY staining was used to evaluate the level of lipid peroxidation in C2C12 muscle cell (Fig. 3 G). A significant increase in C11-BODIPY fluorescence intensity was observed in the LPS and Erastin groups comparatively to the control group, but this trend was counteracted by Fer-1 in the LPS + Fer-1 and Erastin + Fer-1 groups (Fig. 3 G). Immunofluorescence analysis revealed decreased protein content of GPX4 in the LPS and Erastin groups compared to the control group (Fig. 3 H). Combined treatment with Fer-1 counteracted the effect of LPS and Erastin on the expression of GPX4 (Fig. 3 H). In addition, the protein content of ACSL4 was significantly increased in the LPS and Erastin groups while this effect was counteracted by Fer-1 treatment (Fig. 3 I). Moreover, the fluorescence probe FerroOrange staining showed increased levels of intracellular free Fe 2+ in the LPS and Erastin groups compared to the control group, but Fer-1 reversed these trends (Fig. 3 J). TEM was used to study the ultrastructural changes in C2C12 muscle cell across different treatment groups. LPS and Erastin groups showed significant changes compared to the control group. For example, Mitochondrial swelling, mitochondrial skeleton damage and mitochondrial respiratory chain function impaired; Mitochondrial cristae degeneration and morphological changes were observed. Mitochondrial morphological changes can lead to functional abnormalitie. The LPS + Fer-1 and Erastin + Fer-1 groups showed improvements compared to their respective groups. Fer-1 action partly restored LPS-Erastin-induced changes in ultrastructural features and alleviated the effects of LPS and Erastin on sarcomeres, mitochondria, and the cytoplasm (Fig. 3 K). Flow cytometry analysis indicated a remarkable increase in ROS levels in the LPS and Erastin groups compared to the control group; treatment with Fer-1 reversed this effect in the LPS + Fer-1 and Erastin + Fer-1 groups (Fig. 3 L). Furthermore, CFDA-SE labeling was performed to analyze cell viability in the different treatment groups (Fig. 3 M). Compared to the control group, a significant decrease in cell viability in the LPS group was observed (Fig. 3 M). However, combined treatment with Fer-1 in the LPS + Fer-1 and Erastin + Fer-1 groups led to a marked improvement in cell viability compared to the LPS group (Fig. 3 M). We also analyzed the gene expression levels of NRF2, GPX4, ACSL4, IL-10, and FOXO3 after exposing the cells to various treatments (Fig. 3 N and Figure S5A) . The LPS and Erastin groups showed significant differences in gene expression compared to the control group, with NRF2 and GPX4, IL-10, and FOXO3 being upregulated while ACSL4 was downregulated (Fig. 3 N and Figure S5A) . In contrast, combined treatment with Fer-1 partly restored gene expression in C2C12 cells, leading to decreased expression of ACSL4 and upregulation of NRF2 and GPX4, IL-10 and FOXO3 in both the LPS + Fer-1 and Erastin + Fer-1 groups (Fig. 3 N Figure S5A ). Moreover, in LPS and Erastin groups, western blotting revealed the upregulation of ACSL4 and the downregulation of GPX4 and NRF2, IL-10 and FOXO3; these expression trends were partially reversed by combined treatment with Fer-1 (Fig. 3 O and Figure S5B ). 2.4 Ferroptosis and Mitochondria Impairment play a vital role in in the diaphragm of septic mice To further explore the mechanism underlying ferroptosis in SIDD, we developed a mouse model of CLP (Fig. 4 A), and performed a series of experiments. As shown in Fig. 4 B, the diaphragm muscle contraction tension was measured in different groups and the results showed that the tension was significantly decreased in CLP group whereas Fer-1 treatment markedly alleviated this effect (Fig. 4 B). Assays were performed to detect the levels of Myo and CK-MB, as well as biochemical markers MDA and GSH in the muscle (Fig. 4 C). The levels of Myo, CK-MB, and MDA was significantly increased whereas GSH was markedly decreased in the CLP group (Fig. 4 C). Interestingly, Fer-1 treatment counteracted the effect of CLP (Fig. 4 C). In addition, we measured the levels of MDA, SOD and GSH in the serum of mice and found that MDA was significantly increased whereas GSH and SOD were markedly decreased in the CLP group, which was reversed by Fer-1 ( Figure S1 A ). Moreover, ELISA assay results revealed that CLP significantly increased the levels of Total Fe and Fe 2+ in the muscle (Fig. 4 D) and serum ( Figure S1 B ) of mice, and these effects were inhibited by Fer-1 treatment. Next, oxygen consumption rate (OCR) was measured (Fig. 4 E) and the results showed that CLP markedly decreased OCR levels, while Fer-1 treatment counteracted this effect (Fig. 4 E). The measurement of ECAR also indicated CLP markedly decreased ECAR level, but this effect was counteracted by Fer-1 treatment (Fig. 4 F). HE staining of diaphragm muscle tissue indicated that, compared to the sham and Control groups, the CLP group exhibited abnormal histopathological changes, including disruption of muscle fibers, the presence of infiltrating inflammatory cells, necrotic areas, and interstitial edema in the diaphragm muscle tissue (Fig. 4 G). In the CLP + Fer-1 group, diaphragm muscle tissue showed improvements in histopathological features compared to the CLP group (Fig. 4 G). These improvements included reduced inflammatory cell infiltration, less disruption of muscle fibers, decreased interstitial edema, and smaller necrotic areas (Fig. 4 H). TEM examination was used to examine the ultrastructural changes in diaphragm muscle tissue (Fig. 4 H). Compared to the Control and Sham groups, the CLP group exhibited significant ultrastructural alterations in diaphragm muscle tissue, including disorganized myofibrils, disrupted Z-lines, swollen mitochondria with loss of cristae, and increased cytoplasmic vacuolization (Fig. 4 H). In contrast, in the CLP + Fer-1 group, diaphragm muscle tissue showed improvements including better organization of myofibrils, clearer Z-lines, reduced mitochondrial swelling, and decreased cytoplasmic vacuolization in ultrastructural features compared to the CLP group (Fig. 4 H). Real-time PCR analysis was performed to measure changes in gene expression levels of GPX4, ACSL4 and NRF2 in each group (Fig. 4 I). The results showed that GPX4, and NRF2 were downregulated by CLP but Fer-1 treatment reversely upregulated the expression of these genes (Fig. 4 I). Inverse trends were observed for ACSL4 mRNA expression (Fig. 4 I). Western blot analysis was used to measure changes in the protein content of GPX4, ACSL4, NRF2 and P-NRF2 in each group (Fig. 4 J). The results showed that GPX4 and p-NRF2 were dramatically downregulated in CLP while ACSL4 was upregulated comparatively to the sham and control groups, but Fer-1 treatment reversed the expression tendency of these markers (Fig. 4 J). Furthermore, the results of the immunofluorescence assay indicated that CLP significantly decreased the expression of GPX4 ( Figure S1 C ) and α-SMA ( Figure S1 D ) while increased expression of ACSL4 was observed ( Figure S1 E ); these results were reversed by the treatment with Fer-1. Overall, the study suggests that Fer-1 treatment may protect SIDD from restraining metabolic changes and mitochondrial impairment, hinting that ferroptosis and mitochondria impairment play a vital role in in the diaphragm of septic mice. 2.5 FOXO3/IL-10 axis was upregulated in the diaphragm of septic mice treated with Fer-1 To further elucidated the molecular mechanism, we performed RNA-seq analysis using diaphragm tissues from Sham, CLP, and CLP + Fer-1 mice. The heatmap (Fig. 5 A) shows the differentially expressed genes among the three groups. Figure 5 B illustrates the number of common differentially expressed genes across Sham vs. CLP, Sham vs. Fer-1, and CLP vs. Fer-1 groups. A total of 355 common genes were identified. The functional enrichment analysis of differentially expressed genes is shown in Fig. 5 C which demonstrates the Gene Ontology categories that were significantly enriched among the Sham vs. CLP and CLP vs. Fer-1 comparisons. We found that the biological processes of “steroid metabolic process”, “fatty acid metabolic process”, “small molecule catabolic process”, “wound healing”, and “organic acid biosynthetic process” were the most enriched terms in both the Sham vs. CLP and CLP vs. Fer-1 comparisons (Fig. 5 C). Furthermore, KEGG pathway enrichment analysis of differentially expressed genes among the two comparisons was performed, and the results are depicted in Fig. 5 D. We found that the “complement and coagulation cascade”, “retinol metabolism”, and “steroid hormone biosynthesis” were the pathways significantly affected by both CLP and Fer-1 treatments (Fig. 5 D). These results suggest that Fer-1 treatment prevents the differential expression of several genes that were altered in response to CLP. To identify the genes that were responsive to both CLP and Fer-1, we performed clustering analysis to identify gene expression profiles. Figure 6 A displays profiles ranked by the significance of the number of genes assigned compared to expected, while Fig. 6 B presents profiles ranked by the number of genes assigned. Notably, profiles 1 and 5, containing genes affected by CLP that can be restored by Fer-1, were identified (Figs. 6 A and 6 B). The gene expression changes were illustrated in Fig. 6 C, while the PPI network of the Fer-1-target genes in CLP was showcased in Fig. 6 D. The PPI network indicated strong interactions among the Fer-1-target genes in CLP (Fig. 6 D). Lastly, we identified the hub Fer-1-target genes in CLP using MCODE (Fig. 6 E). The results indicated that 29 genes could be considered as hub genes, among which figured IL10 and other immune- and inflammation-related genes (Fig. 6 E). To understand how epigenetic regulation plays a role in ferroptosis in sepsis, we performed ATAC-seq analysis using diaphragm muscle tissue collected from Sham, CLP, and CLP + Fer-1 mice. The results showed that each group had different gene segments, including promoter, 5'UTR, 3'UTR, and other gene content (Fig. 7 A). In each category, we noticed variations in the way genes were distributed across different chromatin states (Fig. 7 B). In addition, we conducted GO (Fig. 7 C) and KEGG pathway analyses (Fig. 7 D). The results showed that “actin filament organization”, “actin filament organization”, “muscle system process”, “regulation of actin filament-based process”, and “regulation of vasculature development” were the biological processes mostly affected by CLP (Fig. 7 C). Moreover, the results indicated that the “negative regulation of phosphorylation”, “negative regulation of protein phosphorylation”, “response to LPS”, “response to molecule of bacterial origin”, and “regulation of actin filament-based process” were the biological processes affected by Fer-1 treatment of CLP mice (Fig. 7 C). The KEGG pathways mostly affected by CLP were “cAMP signaling pathway”, “cGMP-PKG signaling pathway”, “purine metabolism”, “lipid and atherosclerosis”, “regulation of actin cytoskeleton”, and “TNF signaling pathway”, while those affected by Fer-1 treatment of CLP mice were “herpes simplex virus 1 infection”, “Rap 1 signaling pathway”, “TNF signaling pathway”, and “toxoplasmosis”. The integrated analysis of mRNA-seq and ATAC-seq data revealed significant changes in gene expression and chromatin accessibility in response to CLP and Fer-1 treatment (Fig. 8 A). The quadrant plot showed differential gene expression in the four groups: control, CLP model, and CLP + Fer-1 (Fig. 8 A). The Venn diagrams of highly and lowly expressed genes in the first (Fig. 8 B) and fourth (Fig. 8 C) quadrants, respectively, were analyzed for GO and KEGG pathway analysis. The PPI network of transcription factors identified by ATAC-seq and Fer-1-target genes in CLP was established (Fig. 8 D), and hub Fer-1-target genes in CLP (Fig. 8 E) were identified, indicating the strong interactions among Fer-1-target proteins in CLP in the central role of IL10 in this process. Finally, IL10 and FOXO3 were selected for dual luciferase assays, which validated the interaction among between both proteins (Figs. 8 F and 8 G). 2.6 Up regulated FOXO3/IL-10 axis prevents diaphragm from sepsis-induced ferroptosis by activating Nrf2/GPX4 signaling Immunofluorescence analysis indicated significantly downregulated expression of GPX4 expression in the CLP group, which was notably reversed by combined treatment with mIL-10 or Fer-1 ( Figure S2A ). On the contrary, 4HNE expression was upregulated in the CLP group compared to the Sham group; however, combined treatment with mIL-10 or Fer-1 led to a decrease in 4HNE expression compared to the CLP group ( Figure S2B ). Additionally, there was an upregulation of ACSL4 expression in the CLP group compared to the Sham group. However, treatment with mIL-10 or Fer-1 resulted in a decreased expression of ACSL4 in the CLP + mIL-10 and CLP + Fer-1 groups ( Figure S2C ). Through TEM analysis of muscle tissue, it was observed that the CLP group displayed notable morphological changes in their mitochondria compared to the Sham group. The changes observed in the CLP group were indicative of cellular stress and damage, including disrupted cristae, irregular shape, and a swollen appearance ( Figure S2D ). However, treatment with either mlL-10 or Fer-1 had a restorative effect on the mitochondria in the CLP group ( Figure S2D ). Indeed, following treatment, the mitochondria showed improved structure and integrity. with the disrupted cristae appearing more organized and defined. The irregular shape and swelling of the mitochondria were also reduced, indicating a mitigation of cellular stress and a partial restoration of normal mitochondrial morphology ( Figure S2D ). Furthermore, qRT-PCR of ACSL4, GPX4, NRF2, IL-10, and FOXO3 in diaphragm tissue from the different groups was detected ( Figure S2E ). In the CLP group, GPX4, NRF2, IL-10, and FOXO3 expression were decreased while ACSL4 was upregulated. Indeed, CLP mice treated with Fer-1 or mIL-10 showed marked reversal of these changes with upregulation of GPX4, NRF2, IL-10, and FOXO3 and downregulation of ACSL4 expression ( Figure S2E ). Furthermore, the western blot analysis showed that in the CLP group, GPX4, pNRF2, IL-10, and FOXO3 decreased, but these effects were reversed by treatment with mIL-10 or Fer-1. Contrary results were recorded for ACSL4, while no significant difference was recorded for NRF2 among groups ( Figure S2F ). To investigate the effect of FOXO3/IL10 axis in SIDD, different experiments were performed in vivo and in vitro (Fig. 9 and Fig. 10 ). Analysis of immunofluorescence in CLP or LPS groups versus controls showed a decrease in GPX4 MFI. Comparatively, the overexpression of FOXO3 and combination of mIL-10 led to an increase in the MFI of GPX4 when contrasted with the CLP or LPS groups and their respective negative counterparts (Fig. 9 A and Fig. 10 A). The MFI of 4HNE protein was elevated in the CLP or LPS groups compared to the control groups, according to immunofluorescence analysis shown in Fig. 9 B and Fig. 10 B. The MFI of 4HNE in the CLP or LPS groups were higher than those in the FOXO3 overexpression group. The combination of mIL-10 and FOXO3 promoted their effect, manifested in decreased 4HNE MFI. As revealed by the immunofluorescence analysis, ACSL4 MFI was greater in the CLP and LPS groups than in their corresponding control groups. FOXO3 overexpression caused a decrease in ACSL4 MFI compared to CLP or LPS groups and their respective controls (OE-NC), as shown in Figs. 9 C and 10 C. mIL-10 incorporation in the therapy resulted in reduced ACSL4 MFI due to increased FOXO3 overexpression. Mitochondrial alterations were observed via TEM in both animal and in vitro models following ferroptosis. The experimental conditions brought to light structural modifications and possible mitochondrial damage (Fig. 9 D and 10 F). We performed C11-Bodipy assay to assess variations of C2C12 muscle cells in different experimental groups. In the LPS group C11-Bodipy fluorescence increased significantly, suggesting lipid peroxidation and oxidative stress. Nevertherless, lipid peroxidation was significantly decreased in the LPS + OE-FOXO3 group. Furthermore, when we co-administrated LPS + Fer-1, Fer-1 clearly attenuated the LPS-induced lipid peroxidation, which had no significant difference in the LPS + OE-FOXO3 group. Moreover, OE-IL-10 + OE-FOXO3 combined overexpression produced an additive effect on reducing lipid peroxidation (Fig. 10 D). The FerroOrange staining was performed to assess the Fe 2+ alterations in C2C12 muscle cells in different experimental group. The LPS group displayed a significant increase in FerroOrange fluorescence, suggesting significantly higher levels of Fe 2+ . Indeed, the fluorescence intensity was kept high in the LPS + OE-NC group, suggesting that overexpression of the negative control gene had no effect on the LPS-induced changes. However, we observed a decreased FerroOrange fluorescence in the LPS + OE-FOXO3 group, which suggested a decrease in Fe 2+ . In similar manner, the administration of Fer-1 reversed LPS-induced increase of Fe 2+ in the LPS + Fer-1 group. Interestingly, in the LPS + OE-IL-10 + OE-FOXO3 group, the simultaneous overexpression of IL-10 and FOXO3 resulted in an additive effect on the reduction of Fe 2+ (Fig. 10 E). ROS was conducted in C2C12 muscle cells within various experimental groups (Fig. 10 G). We observed a significant increase in ROS in the LPS group, showing oxidative stress. The LPS + OE-NC group showed a very high ROS level which demonstrated that transfection of the negative control gene didn’t affect the ROS level induced by LPS. However, the LPS + OE-FOXO3 group exhibited a marked reduction in ROS levels. In addition, Fer-1 treatment significantly suppressed the ROS level in the LPS + Fer-1 group, revealing the antioxidative effect of Fer-1 against LPS-induced oxidative stress. Additionally, in the LPS + OE-IL-10 + OE-FOXO3 group, ROS level was even decreased furtherly, indicating that the co-overexpression of IL-10 and FOXO3 produced an additive effect on reducing oxidative stress (Fig. 10 G). Furthermore, the CFDA-SE staining was done to observe the viability of C2C12 muscle cells in different experimental groups (Fig. 10 H). In the LPS group, a significant reduction of CFDA-SE fluorescence was observed compared to the control group, indicative of reduced cell survival. However, the CFDA-SE fluorescence intensity was significantly increased in the LPS + OE-FOXO3 group, meaning an obvious improvement of cell viability. Likewise, in the LPS + Fer-1 group, the fluorescence intensity was remarkably increased, suggesting that Fer-1 treatment could efficiently bring cells back to viability. In particular, there was an enhancement of CFDA-SE fluorescence in the LPS + OE-IL-10 + OE-FOXO3 group, which indicated that there was a synergistic effect with the simultaneous overexpression of FOXO3 and IL-10 (Fig. 10 H). RT-PCR and western blotting were performed to evaluate mRNA expression levels (Fig. 9 E and Fig. 10 I) and protein levels (Fig. 9 F and Fig. 10 J) in each group. The CLP and LPS groups showed increased ACSL4 mRNA and protein expression compared to the control groups, while the overexpression of FOXO3 resulted in decreased ACSL4 mRNA and protein expression relative to both the CLP or LPS groups and their corresponding controls. Furthermore, the combination with mIL-10 led to decreased ACSL4 mRNA and protein expression. The expression level of GPX4, NRF2/P-NRF2, IL-10, and FOXO3 genes and proteins showed opposite trends of ACSL4 (Fig. 9 E and Fig. 10 I) and (Fig. 9 F and Fig. 10 J). Providing a comprehensive overview of the protein expression changes in vivo and in vitro models, these findings illustrated possible effects of FOXO3 overexpression and mIL-10 treatment on cellular processes related to oxidative stress, inflammation and survival. To scrutinize the function of IL-10 and FOXO3 in SIDD, C2C12 muscle cells were transfected with IL-10 or FOXO3 vectors. Western blot analysis demonstrated the overexpression of IL-10 ( Figure S3A ) and FOXO3 ( Figure S3B ), indicating that the transfection of IL-10 and FOXO3 expression vectors were successfully performed. Using immunofluorescence assay, we found remarkably reduced GPX4 expression in the LPS treatment group compared to the control ( Figure S4A ). In addition, treatment with Fer-1 or IL-10 overexpression reversed the LPS-mediated decrease in GPX4 gene expression ( Figure S4A ). Immunofluorescence staining for 4HNE revealed that the increase in 4HNE expression in the LPS group was abolished by either Fer-1 treatment or overexpression of IL-10 ( Figure S4B ). The mRNA expression of ACSL4 was upregulated in the LPS group compared with the control group on immunofluorescence staining. In contrast, overexpression of IL-10 or Fer-1 led to downregulation of ACSL4 expression in the LPS + OE-IL-10 and LPS + Fer-1 groups vs. the LPS group ( Figure S4C ). As revealed by the C11-BODIPY assay, lipids were more peroxidated in the LPS group. However, administration of Fer-1 or overexpression of IL-10 mitigated the effect of LPS on lipid peroxidation ( Figure S4D ). Additionally, the Ferrorange assay demonstrated increased iron accumulation in the LPS group compared to the control group. Notably, overexpression of IL-10 or treatment with Fer-1 led to a decrease in iron accumulation in the LPS + OE-IL-10 and LPS + Fer-1 groups, respectively, compared to the LPS group. The NC group exhibited similar levels of iron accumulation as the control group ( Figure S4E ). TEM analysis showed that LPS caused mitochondria morphology changes, which were reversed by either IL-10 overexpression or Fer-1 treatment ( Figure S4F ). Flow cytometry analysis indicated that ROS levels significantly increased in the LPS group compared to Control. However, overexpression of IL-10 in LPS + OE-IL-10 or treatment with Fer-1 in LPS + Fer-1 significantly decreased ROS levels compared to the LPS group ( Figure S4G ). These findings indicated that both IL-10 overexpression and Fer-1 treatment effectively reduced ROS levels in LPS-treated C2C12 muscle cells. Moreover, CFDA-SE staining showed that LPS reduced cell viability. However, IL-10 overexpression or Fer-1 treatment reversed the effect of LPS on cell viability ( Figure S4H ). These findings suggested that IL-10 overexpression and Fer-1 treatment can promote cell viability in LPS-induced C2C12 muscle cells. Moreover, qRT-PCR of GPX4, NRF2, IL-10, and FOXO3 indicated the downregulation of these genes in the LPS group compared to the control group while ACSL4 mRNA level showed opposite trends. However, treatment with Fer-1 or overexpressing IL-10 reversed the effect of LPS on the expression levels of these genes ( Figure S4I ). Furthermore, western blot analysis showed similar trends in protein expression ( Figure S4J ). These findings suggested that the up-regulated FOXO3/IL-10 axis prevents the diaphragm from sepsis-induced ferroptosis by activating Nrf2/GPX4 signaling. 3. Discussion Our study's findings revealed elevated markers of muscle cell injury across in vitro and in vivo sepsis models as well as in clinical samples, alongside diminished diaphragm muscle contraction tension in septic mice, confirming the presence of sepsis-induced diaphragm dysfunction (SIDD). These observations align with prior research indicating compromised diaphragm function under various conditions. Specifically, it has been reported that 40–50% of critically ill patients experience diaphragm atrophy, with about 50–60% of these patients suffering from diaphragm dysfunction [ 31 , 32 ]. Additionally, septic patients display significant diaphragm muscle degradation, leading to more pronounced atrophy and dysfunction compared to non-septic individuals [ 33 ]. Given that SIDD can precipitate respiratory failure and potentially fatal outcomes in sepsis patients, it is critical to unravel the molecular underpinnings and develop corresponding therapeutic strategies. However, the precise mechanisms driving SIDD remain to be fully elucidated. Numerous studies have established a connection between ferroptosis and sepsis, suggesting ferroptosis as a critical pathological form of cell death in sepsis. For example, ferritinophagy-mediated ferroptosis has been implicated in sepsis-induced cardiac damage [ 34 ]. Recent findings indicate that Fer-1 and Irisin can counteract neuronal ferroptosis through the Nrf2/HO-1, Nrf2/GPX4, and glutamate excitotoxicity signaling pathways, offering potential therapeutic avenues for sepsis-induced encephalopathy [ 28 , 35 , 36 ]. These insights suggest that ferroptosis might represent a novel pathophysiological mechanism in sepsis, contributing to the damage of various organs during the condition. However, the specific involvement of ferroptosis in the development of sep-sis-induced diaphragm dysfunction (SIDD) has been less clear. Our research fills this gap by elucidating the role of ferroptosis in the pathogenesis of SIDD, marking the first demonstration that inhibiting ferroptosis can shield against SIDD. This protection is achieved by diminishing oxidative stress, ameliorating mitochondrial dysfunction, and correcting metabolic disturbances associated with ferroptosis. Sepsis is characterized by systemic disturbances that lead to organ dysfunction [ 37 ], with the diaphragm being notably affected. It undergoes morphological alterations and suffers mitochondrial damage, resulting in diminished function [ 16 ]. The accumulation of Fe 2+ within mitochondria can escalate the production of reactive oxygen species (ROS), promoting lipid peroxidation and cell death [ 38 ]. Concurrently, the excess ROS can lead to the depolarization of mitochondrial membrane potential and the opening of permeability transition pores, further compromising mitochondrial structure and function. This sequence of events highlights a significant link between mitochondrial health and ferroptosis, underscoring the importance of our findings in the context of SIDD and sepsis treatment strategies [ 39 ]. Although it's well-documented that diaphragm damage during sepsis is closely linked to mitochondrial dysfunction, the specific interplay between mitochondrial dysfunction and ferroptosis in the context of SIDD remained unclear until now. Our study unveiled those measurements such as oxygen consumption rate (OCR), extracellular acidification rate (ECAR), mitochondrial complex activity, and ultrastructural integrity exhibited comparable alterations in C2C12 muscle cells subjected to either LPS induction or treatment with the ferroptosis inducer Erastin. These findings suggest that mitochondria-related ferroptosis plays a role in the development of SIDD. In the setting of sepsis, an infectious challenge triggers the upregulation of nuclear receptor coactivator 4 (NCOA4), which specifically targets ferritin for autophagic degradation. This process releases a substantial amount of Fe 3+ into the cytoplasm, increasing the free iron concentration and facilitating iron-induced cell death[ 34 , 40 ]. Characteristics of this cell death include cellular membrane rupture, mitochondrial volume reduction, increased density of the mitochondrial double membrane, and diminished or absent mitochondrial cristae—observations that align with our experimental results. Throughout this process [ 41 ], expression of critical proteins such as transferrin receptor (TFR), GPX4, ACSL4, and Ferritin are altered, while the Nrf2 signaling pathway emerges as a regulatory mechanism controlling the initiation and progression of iron-induced cell death [ 38 ]. The results of our research demonstrate a notable reduction in GPX4 and Nrf2 expression levels, alongside an increase in Fe2+, ROS, and ACSL4 expression within the diaphragm of septic mice. These observations underline the significance of iron-induced cell death in the pathogenesis of SIDD. Additionally, the observed reduction in mitochondrial cristae, alongside diminished oxygen consumption rate (OCR), heightened extracellular acidification rate (ECAR), and increased ROS production, further indicate mitochondrial dysfunction as a pivotal factor in promoting iron-induced cell death. This study confirms the occurrence of mitochondria-associated ferroptosis in the diaphragm during sepsis, suggesting that targeting the balance of cellular homeostasis could be crucial for addressing both the pathological and physiological impacts of sepsis. These in-sights pave the way for new therapeutic strategies in sepsis management and open novel pathways for drug development. To elucidate how iron-induced cell death contributes to SIDD, we employed RNA sequencing (RNA-seq) and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), complemented by protein-protein interaction (PPI) analysis. Our investigation uncovered that the FOXO3/IL-10 regulatory axis is suppressed in septic mice, yet can be reactivated through the administration of Fer-1. Further analysis identified FOXO3 as a key transcription factor for IL-10, significantly boosting IL-10 expression both in vitro and in vivo. FOXO3 is recognized for its crucial role in regulation of cell metabolism managing dysfunction [ 42 ] and stress response [ 43 ]. Our findings indicated that levels of both FOXO3 and IL-10 were diminished in C2C12 cells subjected to LPS/Erastin; however, Fer-1 treatment successfully reinstated the FOXO3/IL-10 axis. To explore the interaction between FOXO3 and mitochondria-associated ferroptosis in SIDD more deeply, our results revealed that FOXO3 overexpression markedly reduced lipid peroxidation in the diaphragm of mice and in LPS-stimulated C2C12 cells, achieved through cell transfection techniques. Consistent with our observations, recent research has demonstrated that depletion of FoxO3a results in heightened membrane potential, increased oxygen consumption, and accumulation of lipid peroxides. On the contrary, activation of the FoxO3a/HIF1α pathway correlates with diminished levels of 4-HNE staining and MDA in brain tissues [ 44 ]. Furthermore, our studies reveal that overexpression of FOXO3 effectively counteracts the surge of ROS in C2C12 cells triggered by LPS exposure. Notably, FOXO3 overexpression not only reinstates the diminished expression of key genes such as Nrf2 and GPX4 but also reduces the elevated expression of ACSL4. It concurrently enhances the ultrastructural integrity of mitochondria in both mouse diaphragms and LPS-challenged C2C12 cells, mirroring the effects seen with Fer-1 treatment. These insights underscore the pivotal role of FOXO3 in safeguarding against ferroptosis in SIDD through its regulatory impact on mitochondrial function and oxidative stress. This study marks the first to demonstrate that FOXO3 activation can thwart mitochondria-associated ferroptosis in SIDD, heralding new therapeutic avenues for addressing this condition. Our study uniquely demonstrates that FOXO3 mitigates ferroptosis in SIDD by binding to the promoters of IL-10, thereby enhancing its expression. This mechanism underscores the therapeutic potential of IL-10 in combatting ferroptosis, as evidenced by our findings that administering mice with mIL-10 or overexpressing IL-10 in LPS-stimulated C2C12 cells significantly curbs ferroptosis. This aligns with previous research suggesting ferroptosis's involvement in infectious diseases, notably affecting host immune responses and the inflammatory cascade [ 45 ]. Administration of mIL-10 to mice was observed to significantly enhance diaphragmatic force production [ 46 ], highlighting the beneficial impact of IL-10 on diaphragm function. Despite these advancements, the precise dynamics of diaphragm muscle response to IL-10 remain to be fully elucidated. Additionally, our data indicates that the reduction of IL-10 in SIDD can be partially reversed by Fer-1, suggesting a complex interplay between IL-10 and ferroptosis. This raises intriguing questions about the potential of targeting IL-10 in ferroptosis-based therapeutic strategies. However, it remains uncertain whether a portion of IL-10 is secreted by immune cells migrating from the blood. Further research is essential to shed light on this aspect in the future. 4. Material and methods 4.1 Patients We recruited a cohort of 60 participants, comprising 20 individuals without any reported medical conditions and 40 patients diagnosed with sepsis, who ranged in age from 30 to 95. The recruitment and sample collection process were conducted at Xinhua 96 Hospital from November 2021 to September 2022 and was carried out following the Ethics Committee approval number XHEC-SHDC-2020-049. 4.2 Animals and experimental design The Animal Care and Use Committee of Xinhua Hospital, School of Medicine, Shang hai Jiao Tong University, Shanghai granted approval for our study (Grant number SYXK2018-0038), which utilized C57BL/6 mice weighing 20 ± 2 g and aged 8 weeks. These mice were procured from Charles River Laboratories Animal Co., Ltd. (Beijing, China) and were provided with food and water ad libitum in a controlled environment for a week, with a 12 h light-dark cycle at 25 ± 1°C and 50 ± 10% relative humidity. In order to induce sepsis, cecal ligation and puncture (CLP) surgery was performed on the mice while under anesthesia (sevoflurane). The midline of the abdomen was cut, and the caecum was ligated to a length of approximately 0.5 cm and punctured using a 23-gauge needle. The abdomen was then sutured, and subcutaneous injection of 1 ml of normal saline was administered [ 47 ]. Control mice were subjected to sham surgery, involving only incisions to the abdomen without ligation or puncture of the caecum. All mice were sacrificed 72 hours after CLP. 4.3 Animal grouping and treatments Following the surgery, we divided the animals into five groups: Control (no surgery), Sham (surgery without ligation and puncture), Sham + Fer-1 (surgery without ligation and puncture but with Ferrostatin 1 (Fer-1) treatment), CLP (surgery with ligation and puncture), and CLP + Fer-1 (surgery with ligation and puncture plus Fer-1 treatment). We administered Fer-1 (Selleckchem, cat. No. S7243) at 10 mg/kg through injection after the surgery repeating every 12 hours throughout the experiment. During administration, the Sham and Control groups received an amount of vehicle solution (0.9% saline). We observed the animals closely for any discomfort for 72 hours before performing euthanasia using carbon dioxide asphyxiation. We collected blood samples to analyze the levels of cytokines, with all chemicals used being of quality and obtained from Sigma Aldrich unless otherwise stated. 4.4 Muscle tension test The detection method employed by Coombes et al. [ 48 ] involved vertical suspensions of diaphragm strips in a McBurney bath filled with Krebs solution kept at a temperature of 26°C and continuously supplied with a mixture of 95% O2 and 5% CO2. The rib end of the strip was fixed to the bottom of the bathtub, while the central tendon end was connected to a tension transducer, with platinum wire electrodes placed on either side of the muscle strip near the rib end. The diaphragm strip was adjusted to its optimal initial length (optimal fiber length, Lo) using small voltage stimulation and left to stabilize for 20 minutes. Subsequently, the electronic stimulator stimulated the diaphragm strips at a supramaximal voltage of approximately 20V, while the output signal of the tension sensor was recorded and analyzed using a biological signal acquisition and processing system. This methodology was adopted to enable accurate analysis and measurement of the resultant output signal. 4.5 ATAC-seq ATAC-seq was conducted to identify genes and transcription factors involved in regulating enzymes related to glucose metabolism and indicators of ferroptosis, such as NCOA4 or FTMT. In this process, we inserted sequencing adapters into chromatin regions accessible using a transposase. The resulting library was then. Sequenced using Illumina HiSeq. To analyze the data obtained from the ATAC seq experiment, we employed bioinformatics methods, which included peak calling with MACS2 annotating peaks using Homer software and performing gene ontology analysis using Metascape. We employed DESeq2 to identify genes and transcription factors that showed expression. The results were effectively visualized using ggplot2. 4.6 RNA-Seq RNA was extracted from samples using TRIzol, and quality was assessed with NanoDrop. TruSeq Stranded mRNA Library Prep Kit was used to generate RNA-seq libraries sequenced on Illumina HiSeq2500. Raw sequencing reads were processed with Trimmomatic and aligned to the human genome using HISAT2. DESeq2 was used for differential gene expression analysis. Gene ontology analysis was performed with DAVID. TFs regulating metabolic enzymes or ferroptosis indicators were identified using the Enrichr database. Gene expression levels were compared between experimental groups and validated with qRT-PCR. 4.7 C2C12 culture and treatment The C2C12 cell lines were procured from the American Type Culture Collection (ATCC) and cultivated in Dulbecco's modified Eagle's medium (DMEM; 11995065, Gibco, Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) from Biological Industries, Beit Haemek, Israel. The cells were then incubated in a humidified chamber at 37°C with 5% CO2. Prior to their utilization, the C2C12 cells underwent routine screening for mycoplasma using the Mycoplasma Detection Kit-QuickTest (Biotool, Houston, TX, USA). Following this, the cells were differentiated into muscle cells using DMEM enriched with 2% horse serum from Biological Industries for a period of five days. 4.8 Plasmid construction The present study involved the construction of plasmids for the expression of full-length open reading frames of interleukin-10 (IL-10) and forkhead box O3 (FOXO3). The IL-10 wild-type 3′-untranslated region (UTR) was cloned into the pGL3-basic vector from Promega (Madison, WI), while site-directed mutagenesis using the QuikChange™ kit from Stratagene (La Jolla, CA) was performed to generate the mutated IL-10 3′-UTR (Mut) after performing FoxO3 seed sequence analysis. 4.9 Transient transfection C2C12 cells (5 × 104 cells/cm2) underwent transient transfection with 5ug of the pcDNA3.1-IL-10 and/or negative controls (Thermo Fisher Scientific, Waltham, MA) or plasmids via the Dharmafect transfection reagent (Dharmacon, Lafayette, CO), following the manufacturer's recommendations. The medium was refreshed every 3 days, and the cells were subsequently harvested for analysis. 4.10 Dual-Luciferase Reporter Assay In order to validate the in-silico prediction results, the dual-luciferase reporter assay was conducted. The promoter regions of IL-10, including both the wild-type (WT) and mutant (MUT) forms, were amplified and cloned into the pGL3-basic luciferase vector, resulting in the construction of the pGL3-IL-10-promoter plasmid. As a control, the pGL3-basic luciferase vector without the IL-10 promoter was utilized. Additionally, a specific effector plasmid of FOXO3 (pCDH-FOXO3) was developed. The transfection process was performed in 293T cell lines by using Lipofectamine 3000 (Invitrogen, USA). After a 24-hour incubation period, the luciferase activity was evaluated using the Dual-Luciferase R Reporter 1000 Assay system (Promega, Madison, WI, USA) to detect the promoter activities. The ratio of Firefly to Renilla luciferase activity was utilized to express the luciferase activity. 4.11 Cell viability assay The CCK-8 (Dojindo) method was utilized as a reference to assess cell viability. Specifically, C2C12 cells were seeded into a 96-well plate at a concentration of 5 ×104 cells/well and cultured for 24 hours. Afterward, the cells were exposed to varying concentrations of LPS (10 µg/mL, Sigma), Fer-1 (10 µM, MCE), Erastin (5 µM, Cayman), Z-VAD-FMK (40 µM, Selleck), Boc-D(OMe)-FMK (50 µM, Enzo), Necrostatin-1 (10 µM, Enzo), and N-Acetyl-L-cysteine (1mM, Sigma) for 12 hours. Subsequently, 20 µl of CCK-8 solution were directly added to the medium (200 µl per well), and the cells were incubated at 37°C for an additional 4 hours. The absorbances (Abs) of different groups were then measured at 450 nm (n = 3). 4.12 Western blotting We used RIPA buffer (Thermo Fisher, #89901) supplemented with protease and phosphatase inhibitors to prepare protein extracts from the samples. The protein concentration was determined using a BCA protein assay kit (Thermo Fisher, #23225) following the instructions provided by the manufacturer. Afterward, we proceeded with a technique called sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) using 10% resolving gels (Bio-Rad, #4561034). Then we transferred the proteins onto nitrocellulose membranes (Bio-Rad, #1620115). Next, we blocked the membranes by using a solution consisting of 5% milk, in Tris-buffered saline, containing 0.1% Tween 20 (TBST). This blocking process lasted for an hour at room temperature. After that, we incubated the membranes overnight at a temperature of 4°C with antibodies. These antibodies targeted GPX4 (Abcam, #ab125066), ACSL4 (Abcam, #ab155282), p-NRF2(Santa Cruz Biotechnology, #sc-293123), NRF2 (Abcam, #ab62352), FOXO3 (Thermo Fisher Scientific, # PA5-27243), IL-10 (R&D Systems, #MAB417), and Recombinant mouse IL-10 (Cat: 417-ML-025, R&D Systems, Minneapolis, MN, USA) was reconstituted in sterile endotoxin-free PBS at 100 ug/mL. Following a wash, with TBST the membranes were incubated at room temperature for an hour with antibodies that were linked to peroxidase. To visualize the protein bands, a chemiluminescence (ECL) kit (Thermo Fisher Scientific #32106) was used, along with an imaging system from Bio-Rad. The expression levels of the proteins were quantified using ImageJ software (NIH). Normalized to β-actin as a reference, for loading control. This entire process was carried out in triplicate. 4.13 RT-PCR The cells were treated with TRIzol Life Technologies) to extract RNA according to the manufacturer's instructions. Next, the RNA was reverse transcribed using the QuantiTect Reverse Transcription kit (Qiagen) following the provided protocol. The cDNA was amplified using gene primers and the SYBR Green PCR Master Mix (Applied Biosystems). The reaction mixture included 10 µL of SYBR Green PCR Master Mix, 1 µL of cDNA template, 0.5 µL of forward and reverse primers (10 µM), and 8 µL of nuclease water. The thermal cycling conditions were set at 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. To assess the expression levels of the target genes accurately, they were normalized against GAPDH using the 2 − ΔΔCt method. The RT-PCR primers were bought from Sigma Aldrich; their sequences can be found in Table 1 . We optimized the primer concentrations to 10 µM. We conducted all experiments three times. Table 1 Primers used in this study Primer name Primer sequense M-GPX4-F 3’-CCTCCCCAGTACTGCAACAG-5’ M-GPX4-R 3’-GTGACGATGCACACGAAACC-5’ M-ACSL4-F 3’-GAAAGGCTATGACGCCCCTC-5’ M-ACSL4-R 3’-ATCATGCGGACATTCCCTCC-5’ M-NRF2-F 3’-GTTGCCCACTTGGTGGATTG-5’ M-NRF2-R 3’-TTCTGCGTGCTCAGAAACCT-5’ M-IL-10-F 3’-CAGTACAGCCGGGAAGACAA-5’ M-IL-10-R 3’-AGGCTTGGCAACCCAAGTAA-5’ M-FOXO3-F 3’-ACCTACGCATCCAGTGTGAG-5’ M- FOXO3-R 3’-GCAAAGAAAAGGAGGGGGTC-5’ M-ACTIN-F 3’-ACCCTAAGGCCAACCGTGAAA-5’ M-ACTIN-R 3’-ATGGCGTGAGGGAGAGCATA-5’ 4.14 Immunofluorescence assay To investigate the expression levels of GPX4, ACSL4, and 4HNE, we prepared the cells and tissues by fixing them with 4% paraformaldehyde for 15 minutes at room temperature. Next, we made the cells permeable using 0.1% Triton X 100. Blocked them with a solution of 1% BSA, in PBS. Subsequently, we applied the antibodies. Allowed them to incubate overnight. Following this we rinsed the cells with PBS. Treated them with Alexa Fluor 488 and anti-Fluor 594 antibodies, which were diluted at a ratio of 1:500. We further stained the cells with DAPI. Mounted them using Thermo Fisher Scientifics mounting medium. Finally, we analyzed the cells using ImageJ software. 4.15 Detection of muscle cell function indicators AST, LDH, CK, and Myo We utilized kits (AST/LDH/CK/Myo) to assess the status of muscle cell function. The instructions provided by the manufacturers were followed diligently including the use of recommended concentrations. The AST kit was obtained from Abcam (ab102523), the LDH kit from Cayman Chemical (601170), the CK kit from BioVision (K758 100), and the Myo kit from Roche (05067990190). To carry out the assays, we added the reagents to our samples, allowed them to incubate for the specified duration, and then employed a microplate reader to measure absorbance. By comparing the absorbance values of our samples against curves generated with the provided standards we could ascertain each indicator's concentrations. 4.16 ROS detection To detect reactive oxygen species (ROS) in our samples we utilized a fluorescent probe called DCFH-DA. We added 10 µM of DCFH-DA (Cayman Chemical, 10004331) to the samples and allowed them to incubate at 37°C, for 30 minutes. Subsequently, we rinsed the samples with phosphate-buffered saline (PBS) to eliminate any remaining DCFH-DA and extracted the resulting product, known as dichlorofluorescein (DCF) using a lysis buffer. Finally, we determined the brightness of the samples using a microplate reader. To quantify the concentration of ROS, in each sample we compared the brightness to a curve generated using hydrogen peroxide (H2O2). 4.17 Iron Assay To assess the iron levels in C2C12 cells, a FerroOrange assay was performed. The cells were exposed to FerroOrange (1 µMol/L, Dojindo, Japan) and incubated at 37°C with 5% CO2 for 30 minutes. A BioTek Cytation 5 fluorescence microscope (BioTek, USA) was then utilized to visualize the cells and quantify the amount of iron present. The FerroOrange assay functions by binding iron ions specifically, producing a fluorescent signal that can be detected and calculated. 4.18 Measurement of the oxygen consumption rate (OCR) The extracellular flux analyzer, specifically the seahorse Bioscience X96 model from Agilent Technologies, was utilized to quantify the rate of dissolved O2 in the immediate vicinity of adherent cells that had been cultured in the XF96 V3 cell culture microplate developed by Seahorse Bioscience. The C2C12 cells, which had been cultured in DMEM supplemented with 0.5% FBS, were seeded into the XF96 V3 cell culture microplate at a density of 0.8 × 104 cells per well. Following this, the cells were washed and left to incubate in the base medium created by Agilent Technologies at 37°C for an hour. The mitochondria stress test kit was employed to measure the oxygen consumption rates (OCR, pmol.min-1) in real-time, in accordance with the manufacturer's guidelines. To assess the glycolytic activity, sequential compound injections, including oligomycin A (1 µM), FCCP (1 µM), and Rotenone/antimycin A (0.5 µM), were administered onto the microplate. 4.19 Transmission electron microscopy The cells and tissues were stimulated and then fixed in electron microscope fixative at 4 ℃ for a duration of 15 minutes. Next, the cells were carefully scraped off using a cell scraper and transferred to 1.5 ml centrifuge tubes. The fixative was replaced with fresh fixative and the samples were left in it for 4 hours at 4 ℃. The cells were then dehydrated using ethanol through gradient elution for 15 minutes each time. Afterward, the cells were permeabilized at 37 ℃ for 8–12 hours and embedded at 60 ℃ for 48 hours. Finally, the samples were sectioned at 80–100 nm using an ultramicrotome and stained. TEM (FEI Tecnai G20 TWIN, USA) was used to photograph all samples. 4.20 C11-BODIPY assay To detect lipid peroxidation, an in-situ lipid peroxidation sensor C11-BODIPY (581/591) from Thermo Fisher Scientific was utilized. The cells were pre-incubated in a fresh culture medium containing 5 µM of the probe at 37°C for 30 minutes. Afterward, they were washed twice with PBS and once with culture medium. The cells were further incubated with fresh medium for another 30 minutes at 37°C, and their observation was conducted via flow cytometry and confocal microscope. 4.21 MitoTox Complete OXPHOS Activity Assay To assess the impact of each treatment on the electron transport chain complexes (I, II, III, IV and V), the MitoTox Complete OXPHOS Activity Assay Panel (Abcam, Cambridge, MA, USA) was used according to the manufacturer's instructions. The assay measured the direct effect of ketamine on each of the complexes, which were obtained from isolated mitochondria that were in their functionally active state. Highly specific monoclonal antibodies were attached to 96-well microplates for each complex. To determine the activity of each complex, the decrease in absorbance in milli-optical density per min was measured at room temperature or 37°C, as outlined by the manufacturer. Using a FLUOstar OPTIMA-6 (BMG Labtech, Durham, NC, USA) microplate reader, absorbance was measured every minute for two hours in kinetic mode. 4.22 Extracellular acidification rate (ECAR) analysis The Seahorse Extracellular Flux (XF-96) analyzer from Seahorse Bioscience in Chicopee, MA was used to determine the glycolysis and glycolytic capacity of cells. Cells were seeded for 2 hours in a medium devoid of glucose. The analyzer provided extracellular acidification (ECAR) associated with glycolysis, maximum glycolytic capacity, and non-glycolytic ECAR after three sequential injections of D-glucose (2 g/L), oligomycin (1 µM), and 2-Deoxyglucose (100 mM). The ECAR after the addition of D-glucose was used to define glycolysis, while the ECAR after the addition of oligomycin was used to define maximum glycolytic capacity. Non-glycolytic activity was associated with the ECAR after treatment with 2-Deoxyglucose. 4.23 CFDA-SE labeling A solution of 5mM CFDA-SE stock (2µL) was mixed with 1mL of PBS and then added to cells (4.5×10 6 ) that were washed thrice with PBS. The cells were incubated for 5 minutes at room temperature at a concentration of 10 µM. The labeled cells were washed using 10 volumes of 20°C PBS containing 5% heat-inactivated FBS and centrifuged at 280×g for 5 minutes at 20°C. After the supernatant was discarded, the cells were washed twice and then seeded at a density of 2×10 5 cells/mL in RPMI containing 10% FBS. Finally, the cells were treated with S-N-, S-N+, S + N-, S + N+, Pre, and Post and incubated for 40 hours. 4.24 Statistical analysis The experimental data underwent a one-way analysis of variance (ANOVA), two-way ANOVA and the Tukey HSD test using GraphPad Prism version 9 (GraphPad Software Inc., San Diego, CA, USA). The data was checked for significant differences, with a statistical significance level of 0.05. The data were presented as mean ± standard error of the mean (SEM). 5. Conclusions In conclusion, our research offers groundbreaking insights into the role of mitochondria-associated ferroptosis in driving cell death and facilitating the development of sepsis-induced diaphragm dysfunction (SIDD), a novel discovery. Furthermore, we highlight the protective functions of the FOXO3/IL-10 axis against SIDD, attributed to its ability to counteract ferroptosis. Based on these findings, we suggest potential therapeutic avenues, including the use of ferroptosis inhibitors, agents targeting mitochondrial oxidative stress, and activators of the FOXO3/IL-10 signaling pathway, as promising strategies to combat SIDD. Declarations Conflicts of Interest: The authors declare that there are no conflict of interests, we do not have any possible conflicts of interest. Funding: This work was supported by grants from the National Natural Science Foundation of China (grant no. 82102237; no.82172159; no.82201320) and the Interdisciplinary Projects of Shanghai Jiao Tong University (grant no. YG2021QN56) and the project of Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine (grant no. JYZZ131). Author Contributions : Conceptualization, Xiaojian Weng and Hong Jiang; methodology, Weiwen Zhang; software, Yi Jin; validation, Yudi Liao, Hui Dong. and Xuhui Zhou; formal analysis, Yaqiong Yang.; investigation, Nan Zhi; resources, Xiaojian Weng; data curation, Bin Zhao; writing—original draft preparation, Hua Liu; writing—review and editing, Dongdong Chai.; visualization, Xiang Lyu; supervision, Xiang Lyu.; project administration, Hong Jiang; funding acquisition, Xiaojian Weng, Hua Liu. All authors have read and agreed to the published version of the manuscript. Data Availability Statement: The data underlying this article are available in the article and in its online supplementary material. Ethical Approval: The Animal Care and Use Committee of Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai approved our study (Grant number SYXK2018-0038) Consent for publication: Not applicable Acknowledgements: Not applicable References Purcarea A, Sovaila S (2020) Sepsis, a 2020 review for the internist. Rom J Intern Med 58:129–137 Cecconi M, Evans L, Levy M, Rhodes A (2018) Sepsis and septic shock. Lancet 392:75–82 Fleischmann C, Scherag A, Adhikari NKJ, Hartog CS, Tsaganos T, Schlattmann P, Angus DC, Reinhart K (2016) Int Forum Acute Care, Assessment of Global Incidence and Mortality of Hospital-treated Sepsis. Am J Respir Crit Care Med 193:259–272 Supinski GS, Schroder EA, Wang L, Morris AJ, Callahan LAP (2021) Mitoquinone mesylate (MitoQ) prevents sepsis-induced diaphragm dysfunction. J Appl Physiol 131:778–787 Supinski GS, Wang L, Schroder EA, Callahan LAP (2020) MitoTEMPOL, a mitochondrial targeted antioxidant, prevents sepsis-induced diaphragm dysfunction. Am J Physiology-Lung Cell Mol Physiol 319:228–238 Saccheri C, Morawiec E, Delemazure J, Mayaux J, Dube B-P, Similowski T, Demoule A, Dres M (2020) ICU-acquired weakness, diaphragm dysfunction and long-term outcomes of critically ill patients. Ann Intensiv Care 10:1–9 Moreno-Ruperez A, Priego T, Gonzalez-Nicolas Ma, Lopez-Calderon A, Lazaro A, Martin AI (2022) Role of Glucocorticoid Signaling and HDAC4 Activation in Diaphragm and Gastrocnemius Proteolytic Activity in Septic Rats. Int J Mol Sci 23:3641 Supinski GS, Wang L, Schroder EA, Callahan LAP (2020) SS31, a mitochondrially targeted antioxidant, prevents sepsis-induced reductions in diaphragm strength and endurance. J Appl Physiol 128:463–472 Wu J, Liu H, Chu T, Jiang P, Li S-t (2019) Neuregulin-1β attenuates sepsis-induced diaphragm atrophy by activating the PI3K/Akt signaling pathway. J Muscle Res Cell Motil 40:43–51 Demoule A, Divangahi M, Yahiaoui L, Danialou G, Gvozdic D, Labbe K, Bao W, Petrof BJ (2006) Endotoxin triggers nuclear factor-κB-dependent up-regulation of multiple proinflammatory genes in the diaphragm. Am J Respir Crit Care Med 174:646–653 Okazaki T, Liang F, Li T, Lemaire C, Danialou G, Shoelson SE, Petrof BJ (2014) Muscle-Specific Inhibition of the Classical Nuclear Factor-κB Pathway Is Protective Against Diaphragmatic Weakness in Murine Endotoxemia. Crit Care Med 42:501–509 Labbe K, Danialou G, Gvozdic D, Demoule A, Divangahi M, Boyd JH, Petrof BJ (2010) Inhibition of monocyte chemoattractant protein-1 prevents diaphragmatic inflammation and maintains contractile function during endotoxemia. Crit Care 14:187 Javesghani D, Magder SA, Barreiro E, Quinn MT, Hussain SNA (2002) Molecular characterization of a superoxide-generating NAD(P)H oxidase in the ventilatory muscles. Am J Respir Crit Care Med 165:412–418 Callahan LA, Nethery D, Stofan D, DiMarco A, Supinski G (2001) Free radical-induced contractile protein dysfunction in endotoxin-induced sepsis. Am J Respir Cell Mol Biol 24:210–217 Supinski GS, Alimov AP, Wang L, Song X-H, Callahan LA (2016) Calcium-dependent phospholipase A2 modulates infection-induced diaphragm dysfunction. Am J Physiology-Lung Cell Mol Physiol 310:975–984 Oliveira TS, Santos AT, Vieira Andrade CB, Silva JD, Blanco N, Roche NdN, Woyames J, Silva PL, Macedo PR, Rocco WS, da-Silva TM, Ortiga-Carvalho FF (2021) Bloise, Sepsis Disrupts Mitochondrial Function and Diaphragm Morphology. Front Physiol 12:704044 Supinski GS, Callahan LA (2006) Hemin prevents cardiac and diaphragm mitochondrial dysfunction in sepsis. Free Radic Biol Med 40:127–137 Bloise FF, van der Spek AH, Surovtseva OV, Ortiga-Carvalho TM, Fliers E, Boelen A (2016) Differential Effects of Sepsis and Chronic Inflammation on Diaphragm Muscle Fiber Type, Thyroid Hormone Metabolism, and Mitochondrial Function, Thyroid, 26 600–609 Bloise FF, Santos AT, de Brito J, Vieira de Andrade CB, Oliveira TS, Pereira de Souza AF, Fontes KN, Silva JD, Blanco N, Silva PL, Macedo Rocco PR, Fliers E, Boelen A, da-Silva WS (2020) T.M. Ortiga-Carvalho, Sepsis Impairs Thyroid Hormone Signaling and Mitochondrial Function in the Mouse Diaphragm, Thyroid, 30 1079–1090 Jude B, Tissier F, Dubourg A, Droguet M, Castel T, Leon K, Giroux-Metges M-A, Pennec J-P (2020) TGF-β Pathway Inhibition Protects the Diaphragm From Sepsis-Induced Wasting and Weakness in Rat, Shock, 53 772–778 Fujimura N, Sumita S, Aimono M, Masuda Y, Shichinohe Y, Narimatsu E, Namiki A (2000) Effect of free radical scavengers on diaphragmatic contractility in septic peritonitis. Am J Respir Crit Care Med 162:2159–2165 Powers SK, Hudson MB, Nelson WB, Talbert EE, Min K, Szeto HH, Kavazis AN, Smuder AJ (2011) Mitochondria-targeted antioxidants protect against mechanical ventilation-induced diaphragm weakness. Crit Care Med 39:1749–1759 Kim R, Hashimoto A, Markosyan N, Tyurin VA, Tyurina YY, Kar G, Fu S, Sehgal M, Garcia-Gerique L, Kossenkov A, Gebregziabher BA, Tobias JW, Hicks K, Halpin RA, Cvetesic N, Deng H, Donthireddy L, Greenberg A, Nam B, Vonderheide RH, Nefedova Y, Kagan VE, Gabrilovich DI (2022) Ferroptosis of tumour neutrophils causes immune suppression in cancer. Nature 612:338–346 Tuo Q-z, Liu Y, Xiang Z, Yan H-F, Zou T, Shu Y, Ding X-l, Zou J-j, Xu S, Tang F, Gong Y-q, Li X-l, Yang Z-z, Li W-j, Zhang S-t, Ayton S, Bush AI, Xu H, Dai L, Dong B, Lei P (2022) Thrombin induces ACSL4-dependent ferroptosis during cerebral ischemia/reperfusion, Signal Transduction and Targeted Therapy, 7 59 Ryan SK, Zelic M, Han Y, Teeple E, Chen L, Sadeghi M, Shankara S, Guo L, Li C, Pontarelli F, Jensen EH, Comer AL, Kumar D, Zhang M, Gans J, Zhang B, Proto JD, Saleh J, Dodge JC, Savova V, Rajpal D, Ofengeim D, Hammond TR (2023) Microglia ferroptosis is regulated by SEC24B and contributes to neurodegeneration. Nat Neurosci 26:12–26 Zhang H, Liu J, Zhou Y, Qu M, Wang Y, Guo K, Shen R, Sun Z, Cata JP, Yang S, Chen W, Miao C (2022) Neutrophil extracellular traps mediate m6A modification and regulates sepsis-associated acute lung injury by activating ferroptosis in alveolar epithelial cells. Int J Biol Sci 18:3337–3357 Zhang J, Zheng Y, Wang Y, Wang J, Sang A, Song X, Li X (2022) YAP1 alleviates sepsis-induced acute lung injury via inhibiting ferritinophagy-mediated ferroptosis. Front Immunol 13:884362 Wang J, Zhu Q, Wang Y, Peng J, Shao L, Li X (2022) Irisin protects against sepsis-associated encephalopathy by suppressing ferroptosis via activation of the Nrf2/GPX4 signal axis. Free Radic Biol Med 187:171–184 Qiongyue Z, Xin Y, Meng P, Sulin M, Yanlin W, Xinyi L, Xuemin S (2022) Post-treatment With Irisin Attenuates Acute Kidney Injury in Sepsis Mice Through Anti-Ferroptosis via the SIRT1/Nrf2 Pathway. Front Pharmacol 13:857067 She H, Tan L, Du Y, Zhou Y, Guo N, Zhang J, Du Y, Wang Y, Wu Z, Ma C, Li Q, Mao Q, Hu Y, Liu L, Li T (2023) VDAC2 malonylation participates in sepsis-induced myocardial dysfunction via mitochondrial-related ferroptosis. Int J Biol Sci 19:3143–3158 Le Neindre A, Wormser J, Luperto M, Bruel C, Misset B, Bouhemad B, Philippart F (2023) Diaphragm function in patients with sepsis and septic shock: A longitudinal ultrasound study. Australian Crit Care 36:239–246 Demoule A, Molinari N, Jung B, Prodanovic H, Chanques G, Matecki S, Mayaux J, Similowski T, Jaber S (2016) Patterns of diaphragm function in critically ill patients receiving prolonged mechanical ventilation: a prospective longitudinal study. Ann Intensiv Care 6:75 Lecronier M, Jung B, Molinari N, Pinot J, Similowski T, Jaber S, Demoule A, Dres M (2022) Severe but reversible impaired diaphragm function in septic mechanically ventilated patients. Ann Intensiv Care 12:34 Li N, Wang W, Zhou H, Wu Q, Duan M, Liu C, Wu H, Deng W, Shen D, Tang Q (2020) Ferritinophagy-mediated ferroptosis is involved in sepsis-induced cardiac injury. Free Radic Biol Med 160:303–318 Wang J, Yang S, Jing G, Wang Q, Zeng C, Song X, Li X (2023) Inhibition of ferroptosis protects sepsis-associated encephalopathy. Cytokine 161:156078 Xie Z, Xu M, Xie J, Liu T, Xu X, Gao W, Li Z, Bai X, Liu X (2022) Inhibition of Ferroptosis Attenuates Glutamate Excitotoxicity and Nuclear Autophagy in a CLP Septic Mouse Model, Shock, 57 694–702 Esper AM, Martin GS (2009) Extending international sepsis epidemiology: the impact of organ dysfunction. Crit Care 13:120 Fang X, Wang H, Han D, Xie E, Yang X, Wei J, Gu S, Gao F, Zhu N, Yin X, Cheng Q, Zhang P, Dai W, Chen J, Yang F, Yang H-T, Linkermann A, Gu W, Min J, Wang F (2019) Ferroptosis as a target for protection against cardiomyopathy. Proc Natl Acad Sci USA 116:2672–2680 Chan S, Lian Q, Chen M-P, Jiang D, Ho JTK, Cheung Y-F, Chan GC-F (2018) Deferiprone inhibits iron overload-induced tissue factor bearing endothelial microparticle generation by inhibition oxidative stress induced mitochondrial injury, and apoptosis. Toxicol Appl Pharmcol 338:148–158 Liu Q, Wu J, Zhang X, Wu X, Zhao Y, Ren J (2021) Iron homeostasis and disorders revisited in the sepsis. Free Radic Biol Med 165:1–13 Park E, Chung SW (2019) ROS-mediated autophagy increases intracellular iron levels and ferroptosis by ferritin and transferrin receptor regulation. Cell Death Dis 10:822 Manley S, Ni H-M, Williams JA, Kong B, DiTacchio L, Guo G, Ding W-X (2014) Farnesoid X receptor regulates forkhead Box O3a activation in ethanol-induced autophagy and hepatotoxicity. Redox Biol 2:991–1002 Bernardo VS, Torres FF, Da Silva DGH (2023) FoxO3 and oxidative stress: a multifaceted role in cellular adaptation. J Mol Medicine-Jmm 101:83–99 Zhong S, Chen W, Wang B, Gao C, Liu X, Song Y, Qi H, Liu H, Wu T, Wang R, Chen B (2023) Energy stress modulation of AMPK/FoxO3 signaling inhibits mitochondria-associated ferroptosis. Redox Biol 63:102760 Bagayoko S, Meunier E (2022) Emerging roles of ferroptosis in infectious diseases. FEBS J 289:7869–7890 Divangahi M, Demoule A, Danialou G, Yahiaoui L, Bao W, Xing Z, Petrof BJ (2007) Impact of IL-10 on diaphragmatic cytokine expression and contractility during Pseudomonas Infection. Am J Respir Cell Mol Biol 36:504–512 Rittirsch D, Huber-Lang MS, Flierl MA, Ward PA (2009) Immunodesign of experimental sepsis by cecal ligation and puncture. Nat Protoc 4:31–36 Coombes SA, Cauraugh JH, Janelle CM (2006) Emotion and movement: Activation of defensive circuitry alters the magnitude of a sustained muscle contraction. Neurosci Lett 396:192–196 Supplementary Files SupplementalMaterial.pdf 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. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4539738","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":314223443,"identity":"162450a4-61cd-41c0-826a-b2fb76eab53a","order_by":0,"name":"Hua Liu","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine Affiliated Ninth People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hua","middleName":"","lastName":"Liu","suffix":""},{"id":314223444,"identity":"05347b9e-88a4-40b9-bd72-d44440a63058","order_by":1,"name":"Dongdong Chai","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine Affiliated Ninth People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Dongdong","middleName":"","lastName":"Chai","suffix":""},{"id":314223445,"identity":"ad5e34bf-1ae8-40c6-b64f-8a14fd035050","order_by":2,"name":"Xiang Lyu","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine Affiliated Ninth People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Lyu","suffix":""},{"id":314223446,"identity":"66f47fb4-f0ef-4297-80fc-7c0409138e34","order_by":3,"name":"Bin Zhao","email":"","orcid":"","institution":"Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine: Shanghai Jiaotong University School of Medicine Xinhua Hospital","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Zhao","suffix":""},{"id":314223447,"identity":"e9d68ba4-7d65-4d3f-93f0-9aa5c8f192ea","order_by":4,"name":"Nan Zhi","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Nan","middleName":"","lastName":"Zhi","suffix":""},{"id":314223448,"identity":"109e4f71-3d25-4b2e-87b9-b8080c044410","order_by":5,"name":"Yaqiong Yang","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine Affiliated Ninth People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yaqiong","middleName":"","lastName":"Yang","suffix":""},{"id":314223449,"identity":"d03377b9-f16d-4e14-a795-7898fd5f13cb","order_by":6,"name":"Xuhui Zhou","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine Affiliated Ninth People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xuhui","middleName":"","lastName":"Zhou","suffix":""},{"id":314223450,"identity":"f5f6a1e2-8642-4727-bcf4-a6c726a30163","order_by":7,"name":"Hui 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Hospital","correspondingAuthor":false,"prefix":"","firstName":"Weiwen","middleName":"","lastName":"Zhang","suffix":""},{"id":314223453,"identity":"1912671a-7f51-4068-a2a1-0abd51d125f9","order_by":10,"name":"Yi Jin","email":"","orcid":"","institution":"Second Affiliated Hospital of Naval Medical Universty","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Jin","suffix":""},{"id":314223454,"identity":"c0cd8391-727a-497a-95cd-08bb5f3e285c","order_by":11,"name":"Hong Jiang","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine Affiliated Ninth People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Jiang","suffix":""},{"id":314223455,"identity":"1fc36484-7749-4f9d-9023-81ec8218610d","order_by":12,"name":"Xiaojian Weng","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-5629-4894","institution":"Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine: Shanghai Jiaotong University School of Medicine Xinhua Hospital","correspondingAuthor":true,"prefix":"","firstName":"Xiaojian","middleName":"","lastName":"Weng","suffix":""}],"badges":[],"createdAt":"2024-06-06 10:47:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4539738/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4539738/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60184654,"identity":"b5c0e6f6-6b6c-4e97-973a-ce729ec1e2b2","added_by":"auto","created_at":"2024-07-12 18:38:05","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":899721,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of muscle injury markers, anti-oxidative enzyme activity, MDA, GSH content and iron concentration in clinical samples.\u003c/strong\u003e (A) ELISA detection of the Myo and CK-MB and biochemical detection of AST in serum samples from patients with and without sepsis. (B) Biochemical detection of MDA, GSH, CAT, SOD, and LDH in the serum samples from patients with and without CLP. (C) ELISA detection of total Fe, Fe\u003csup\u003e2+\u003c/sup\u003e, and Fe\u003csup\u003e3+\u003c/sup\u003e in the serum samples from patients with and without sepsis. ***p\u0026lt;0.001 compared to healthy individuals.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4539738/v1/9dd6387f3c9616c4852e5a48.jpg"},{"id":60184653,"identity":"d9add0fa-e194-4ab0-b16d-014fb56b4e11","added_by":"auto","created_at":"2024-07-12 18:38:05","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4588246,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell viability, immunofluorescence staining of GPX4 and ACSL4 expression in LPS-induced C2C12 muscle cell treated with different cell death inhibitors. \u003c/strong\u003e(A) Cell viability, as measured by the CCK 8 assay. (B) Immunofluorescence analysis of GPX4 expression in different treatment groups. (C) Immunofluorescence staining of ACSL4 expression in different treatment groups. ***p \u0026lt; 0.001, and ****p\u0026lt;0.0001 among the compared groups.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4539738/v1/e84b62cdf2a6b929360cf69f.jpg"},{"id":60186421,"identity":"ad5110f8-a72d-4a1b-b779-39eacca2be56","added_by":"auto","created_at":"2024-07-12 18:54:05","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":18529931,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of mitochondria-associated ferroptosis on LPS-induced C2C12 muscle cells.\u003c/strong\u003e (A) CCK8 assay for detection of the viability of C2C12 cells after treatment with different drugs. (B) Biochemical analysis of LDH, MDA, and GSH levels in the cell supernatant. (C) ELISA assay was used to determine the levels of Total Fe, Fe\u003csup\u003e2+\u003c/sup\u003e, and Fe\u003csup\u003e3+\u003c/sup\u003e. (D) Detection of OCR. (E) Detection of ECAR. (F) Detection of the activity of the mitochondrial respiratory chain complexes. (G) C11-BODIPY assay detection of lipid peroxidation. (H) Immunofluorescence analysis was conducted to determine the protein content of GPX4. (I) Immunofluorescence analysis was conducted to determine the protein content of ACSL4. (J) Fluorescence probe FerroOrange was used for immunofluorescence confocal microscopy to measure the levels of intracellular free Fe\u003csup\u003e2+\u003c/sup\u003e. (K) Transmission electron microscopy was used to observe mitochondrial ultrastructure. (L) Flow cytometry analysis was performed to measure the levels of ROS. (M) CFDA-SE staining for detection of cell viability. (N) Real-time PCR was performed to measure the changes in gene expression levels of GPX4, ACSL4, NRF2. (O) Western blot analysis was conducted to measure the protein content changes of GPX4, ACSL4, NRF2. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, and ****p\u0026lt;0.0001 among the compared groups.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4539738/v1/3e30e406f12f9ef33ee8fb27.jpg"},{"id":60184655,"identity":"4ee6a971-c96f-4dfd-b03b-4a7f57d30098","added_by":"auto","created_at":"2024-07-12 18:38:05","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":9345971,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRole of mitochondria-associated ferroptosis in the diaphragm of septic mice\u003c/strong\u003e. (A) Schematic diagram of experiments after Mouse CLP model establishment. (B) Measurement of diaphragm muscle contraction tension. (C) Serum ELISA assay to detect levels of Myo and CK-MB, as well as biochemical markers MDA and GSH. (D) ELISA assay to determine the levels of Total Fe, Fe\u003csup\u003e2+\u003c/sup\u003e, and Fe\u003csup\u003e3+\u003c/sup\u003e in the diaphragm. (E) Measurement of OCR in the diaphragm. (F) Measurement of ECAR in the diaphragm. (G) Histopathological examination of diaphragm muscle tissue. (H) Transmission electron microscopy observation of subcellular mitochondrial changes in diaphragm muscle tissue. (I) Real-time PCR analysis to measure changes in gene expression levels of GPX4, ACSL4 and NRF2. (J) Western blot analysis to measure changes in protein content of GPX4, ACSL4 and NRF2/P-NRF2. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, and ****p\u0026lt;0.0001 among the compared groups.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4539738/v1/f9fc5b821bc3d5fafe668cd8.jpg"},{"id":60186122,"identity":"43beb43e-5cb6-44b3-9fb5-cd3f49d3465c","added_by":"auto","created_at":"2024-07-12 18:46:05","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2492909,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRNA-seq and differential expression of genes in diaphragm tissue from Sham, CLP and CLP+Fer-1 groups. \u003c/strong\u003e(A) Heatmap showing differentially expressed genes in diaphragm tissue Sham, CLP and CLP+Fer-1 mice. (B) Ven diagram showing the number of common differentially expressed genes (355 common genes) identified in Sham vs. CLP, Sham vs. Fer-1, and CLP vs. Fer-1 comparisons. (C) Gene Ontology functional enrichment analysis of differentially expressed genes among the Sham vs. CLP and CLP vs. Fer-1 comparisons. (D) KEGG pathway enrichment analysis of differentially expressed genes among the Sham vs. CLP and CLP vs. Fer-1 comparisons.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4539738/v1/1f06187ed13b6869cfa0aed1.jpg"},{"id":60184661,"identity":"3fa4a009-b1a0-444b-b2af-cbe9fed9fce2","added_by":"auto","created_at":"2024-07-12 18:38:05","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4177340,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of Fer-1-target genes in CLP\u003c/strong\u003e(A) Profiles ordered based on the p-value significance of number of genes assigned versus expected. (B) Profiles ordered based on the number of genes assigned. Profiles 1 and 5 were found to be significant profiles containing genes altered by CLP and that could be restored by Fer-1. (C) Expression change. (D) Protein-protein interaction (PPI) network of Fer-1-target genes in CLP. (E) Identification of hub Fer-1-target genes in CLP.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4539738/v1/30dff7a641164cc7c43fc02a.jpg"},{"id":60184663,"identity":"2157a43b-6b73-4299-bfb8-5fcc6fef815f","added_by":"auto","created_at":"2024-07-12 18:38:05","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3346432,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eATAC-seq analysis of diaphragm muscle tissue collected from Sham, CLP model, CLP+Fer-1 groups.\u003c/strong\u003e (A) Different gene segments in each group, including promoter, 5'UTR, 3'UTR, and other gene content. (B) Distribution of genes in different chromatin states in each group. (C) Gene Ontology (GO) analysis. (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4539738/v1/11dbc8b860ac41eba3eb91d3.jpg"},{"id":60186121,"identity":"66c13b97-4f6c-4c5f-ba94-cbf8f4e9746a","added_by":"auto","created_at":"2024-07-12 18:46:05","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":5317560,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntegrated analysis of mRNA-seq and ATAC-seq data. \u003c/strong\u003e(A) Quadrant plot showing the combined analysis of differentially expressed genes using mRNA-seq and ATAC-seq. (B) Venn diagram of highly expressed genes in the first quadrant, with corresponding Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. (C) Venn diagram of lowly expressed genes in the fourth quadrant, with corresponding GO and KEGG pathway analysis. (D) Protein-protein interaction (PPI) network of transcription factors identified by ATAC-seq and Fer-1-target genes in CLP. (E) Identification of hub Fer-1-target genes in CLP. (F-G) Validation results using dual luciferase assays. **p \u0026lt; 0.01 compared to NC.\u003c/p\u003e","description":"","filename":"Fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4539738/v1/12cd65e0f125447307533617.jpg"},{"id":60184658,"identity":"9c32a0f6-67aa-4152-ac24-baf24268450c","added_by":"auto","created_at":"2024-07-12 18:38:05","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":14447786,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effects of FOXO3 overexpression with or without mIL-10 treatment on ferroptosis and related downstream signaling protein expression in the diaphragm of septic mice.\u003c/strong\u003e (A) Immunofluorescence detection of GPX4 protein levels. (B) Immunofluorescence detection of 4HNE protein levels. (C) Immunofluorescence detection of ACSL4 protein levels. (D) Transmission electron microscopy observation of mitochondrial changes. (E) Real-time PCR detection of changes in GPX4, ACSL4, NRF2, IL-10, and FOXO3 gene expression levels. (F) Western blot detection of changes in GPX4, ACSL4, NRF2/P-NRF2, IL-10, and FOXO3 protein levels. OE-FOXO3 = FOXO3 overexpression,\u003cstrong\u003e \u003c/strong\u003eOE-NC= negative control for overexpression. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, and ****p\u0026lt;0.0001 among the compared groups.\u003c/p\u003e","description":"","filename":"Fig9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4539738/v1/916921b002d31a6bcb51ee8d.jpg"},{"id":60184664,"identity":"b09dcbf7-86fa-49b1-b53e-9dab5552c919","added_by":"auto","created_at":"2024-07-12 18:38:05","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":23641252,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effects of FOXO3 overexpression with or without IL-10 overexpression treatment on ferroptosis and related downstream signaling protein expression in LPS-induced C2C12 muscle cell.\u003c/strong\u003e (A) Immunofluorescence detection of GPX4 protein levels. (B) Immunofluorescence detection of 4HNE protein levels. (C) Immunofluorescence detection of ACSL4 protein levels. (D) C11-BODIPY assay detection of lipid peroxidation. (E) FerroOrange staining for detection of Fe\u003csup\u003e2+\u003c/sup\u003e. (F) Transmission electron microscopy observation of mitochondrial changes.\u0026nbsp; (G) ROS detection by flow cytometry. (H) CFDA-SE staining for detection of cell viability. (I) Real-time PCR detection of changes in GPX4, ACSL4, NRF2, IL-10, and FOXO3 gene expression levels in each group. (J) Western blot detection of changes in GPX4, ACSL4, NRF2/P-NRF2, IL-10, and FOXO3 protein levels in each group. OE-FOXO3 = FOXO3 overexpression, OE-NC= negative control for overexpression. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, and ****p\u0026lt;0.0001 among the compared groups.\u003c/p\u003e","description":"","filename":"Fig10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4539738/v1/288fb40c2424cf5072b798da.jpg"},{"id":60350909,"identity":"709e9db2-5450-48f4-baa7-3c402e1cfc30","added_by":"auto","created_at":"2024-07-15 22:26:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":88026590,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4539738/v1/95e416eb-d408-4f76-9459-71a2a3755203.pdf"},{"id":60184656,"identity":"2d5c3c22-ee8c-47a9-81ac-75d64b197f4d","added_by":"auto","created_at":"2024-07-12 18:38:05","extension":"pdf","order_by":20,"title":"","display":"","copyAsset":false,"role":"supplement","size":779120,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4539738/v1/26883f250b8da20d41827f06.pdf"}],"financialInterests":"","formattedTitle":"Up-regulated FOXO3/IL-10 Axis Inhibits Mitochondria-Associated Ferroptosis in Sepsis-Induced Diaphragm Dysfunction","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSepsis is a critical condition triggered by the body's excessive immune response to infection, leading to widespread organ damage and potentially causing long-term disabilities for survivors [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Sepsis can cause diaphragm weakness and atrophy defined as sepsis-induced diaphragm dysfunction (SIDD), leading to respiratory failure in patients requiring mechanical ventilation. This condition significantly extends the duration of mechanical ventilation and escalates mortality rates in intensive care units (ICU) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003csup\u003e,\u003c/sup\u003e [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePrevious studies have unveiled that the molecular underpinnings of SIDD span a range of processes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], including inflammation [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], oxidative stress [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], metabolic imbalances [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], mitochondrial impairment [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and disruptions in protein synthesis and degradation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Although treatments targeting antioxidants and mitochondrial function have been identified as promising strategies for mitigating SIDD [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], these studies mainly focused on animal experiments, lacking specific therapeutic targets and facing challenges in clinical translation. Therefore, our current focus is on filling this research gap by identifying potential therapeutic targets with clinical applicability.\u003c/p\u003e \u003cp\u003eIn recent years, the study of ferroptosis, a regulated form of cell death, has gained considerable attention due to its involvement in a range of conditions including cancer [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], ischemia/reperfusion injury [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], neurodegenerative diseases [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and acute lung injury [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Emerging evidence suggests ferroptosis also plays a role in sepsis. For instance, it has been shown that YAP1 can mitigate sepsis-induced acute lung injury by inhibiting ferritinophagy, a process linked to ferroptosis [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Further research indicates that Irisin's ability to suppress ferroptosis can protect against sepsis-induced brain dysfunction by activating the Nrf2/GPX4 signaling pathway [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and preventing sepsis-induced acute kidney injury through the SIRT1/Nrf2 pathway [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Despite these insights, the connection between ferroptosis and sepsis-induced diaphragm dysfunction (SIDD) remains an area ripe for more detailed exploration.\u003c/p\u003e \u003cp\u003eStudies have identified the malonylation of VDAC2 as a contributor to sepsis-induced myocardial dysfunction, implicating mitochondrial-related ferroptosis in this process [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], Our research found a surge in plasma Fe\u003csup\u003e2+\u003c/sup\u003e concentrations and the elevation of markers indicative of muscle cell damage alongside the decrease of antioxidative enzymes and GSH in patients with sepsis, suggesting ferroptosis may be involved in SIDD. By exploring the role of ferroptosis in modulating inflammation, oxidative stress, and mitochondrial imbalance in SIDD, potential therapeutic targets could be uncovered. Therefore, further research is essential to deepen our understanding of ferroptosis's impact on SIDD and its viability as a therapeutic target.\u003c/p\u003e \u003cp\u003eWe conducted a comprehensive analysis, assessing various parameters such as ferroptosis related protein expression levels, markers of inflammation, oxidative stress, and mitochondrial health, to uncover the potential molecular dynamics at play. We first identified that ferroptosis is the main death style in SIDD. To further elucidate the target molecular, we performed RNA-seq, ATAC-seq, along with protein-protein interaction (PPI) analysis. Our findings revealed that IL-10 is a critical molecular and FOXO3 is its regulatory transcription factors in the diaphragm of septic mice treated with Fer-1. So, we specifically honed in on the role of the FOXO3/IL-10/ferroptosis axis in SIDD, aiming to understand how enhancing the expression of FOXO3 and IL-10 influences diaphragm dysfunction in sepsis.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cp\u003e \u003cb\u003e2.1 Total Fe and Fe2+, indicators of muscle injury and oxidative stress increased while GSH, CAT, and SOD decreased in the serum of patients with sepsis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the pathological changes among patients with sepsis compared to normal individuals, the levels of various markers were measured among both groups. ELISA detection showed increased levels of CK-MB, Myo, and AST in serum of patients with sepsis compared to normal individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Through detecting biochemical indexes, we found that patients with sepsis had lower GSH, CAT, and SOD levels in their serum compared to normal individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Inversely, compared to healthy individuals, increased concentrations of LDH and MDA were measured in the serum samples of patients with sepsis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Moreover, the ELISA assay indicated that the levels of total Fe and Fe\u003csup\u003e2+\u003c/sup\u003e were significantly increased while no significant change in Fe\u003csup\u003e3+\u003c/sup\u003e was observed, implying disturbance in iron metabolism in patients with sepsis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Ferroptosis is a main death type in LPS-induced C2C12 muscle cell\u003c/h2\u003e \u003cp\u003eTo explore whether ferroptosis occurs in C2C12 muscle cells in response to sepsis, these cells were subjected to single or combined treatment of LPS with different cell death inhibitors. The CCK8 assay indicated that, compared to the control untreated cells, cell viability was markedly decreased by single treatment with LPS (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In addition, the combination of LPS treatment with Fer-1, Z-VAD-FMK, Boc-D-FMK, necrostatin-1, or N-acetyl cysteine partially reversed the effect of LPS group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Interestingly, the combined treatment with LPS and Fer-1 showed the most pronounced reversal of cell viability (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), indicating that Fer-1 may effectively counteract LPS-induced ferroptosis. Moreover, immunofluorescence analysis indicated that the treatment with LPS alone resulted in a significant decrease in GPX4 expression (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and a notable increase in ACSL4 expression (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Decreased GPX4 expression and elevated ACSL4 levels induced by LPS-induced were reverted by the ferroptosis inhibitors (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Fer-1 treatment showed the most significant partial reversal of protein expression of GPX4 and ACSL4 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). These findings suggest that Fer-1 has the potential to rescue the detrimental effects of LPS-induced cell death by restoring GPX4 expression and reducing ACSL4 levels, indicating that ferroptosis is a main death type in LPS-induced C2C12 muscle cell.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Mitochondria-associated ferroptosis is involved in LPS-induced C2C12 muscle cell\u003c/h2\u003e \u003cp\u003eWe found that cell viability by CCK8 assay was significantly decreased in the LPS and Erastin groups compared to the control group, while treatment with Fer-1 partially rescued the effect of LPS and Erastin (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Moreover, decreased levels of GSH, and increased levels of LDH and MDA were recorded in the LPS and Erastin groups compared to the control group; however, combined treatment with Fer-1 counteracted LPS- and Erastin- inhibition of cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In addition, increased total Fe and Fe\u003csup\u003e2+\u003c/sup\u003e contents in the LPS and Erastin groups were recorded compared to the control group, while no remarkable difference in groups was recorded for Fe\u003csup\u003e3+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Moreover, Fer-1 inhibited the effect of LPS and Erastin on the content of Fe and Fe\u003csup\u003e2+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). In addition, compared to the control group, OCR assay indicated a significant decrease in OCR levels in the LPS and Erastin groups, while Fer-1 partially mitigated OCR levels in LPS\u0026thinsp;+\u0026thinsp;Fer-1 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Furthermore, the detection of ECAR levels indicated an increase in ECAR levels in LPS and Erastin groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Interestingly, compared to the LPS and Erastin groups, Fer-1 treatment displayed a partial reversal in ECAR levels, which suggested a potential protective effect against LPS-induced glycolytic alterations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). The levels of mitochondrial complexes I-V were measured to detect the percentage of cell metabolic activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). The treatment with LPS or Erastin was accompanied by a significant decrease in the activity of mitochondrial complexes I-V relatively to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). In the LPS\u0026thinsp;+\u0026thinsp;Fer-1 and Erastin\u0026thinsp;+\u0026thinsp;Fer-1 groups, the treatment of Fer-1 partially reversed the activity of mitochondrial complexes I-V compared to the LPS and Erastin groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). C11-BODIPY staining was used to evaluate the level of lipid peroxidation in C2C12 muscle cell (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). A significant increase in C11-BODIPY fluorescence intensity was observed in the LPS and Erastin groups comparatively to the control group, but this trend was counteracted by Fer-1 in the LPS\u0026thinsp;+\u0026thinsp;Fer-1 and Erastin\u0026thinsp;+\u0026thinsp;Fer-1 groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Immunofluorescence analysis revealed decreased protein content of GPX4 in the LPS and Erastin groups compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Combined treatment with Fer-1 counteracted the effect of LPS and Erastin on the expression of GPX4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). In addition, the protein content of ACSL4 was significantly increased in the LPS and Erastin groups while this effect was counteracted by Fer-1 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). Moreover, the fluorescence probe FerroOrange staining showed increased levels of intracellular free Fe\u003csup\u003e2+\u003c/sup\u003e in the LPS and Erastin groups compared to the control group, but Fer-1 reversed these trends (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ). TEM was used to study the ultrastructural changes in C2C12 muscle cell across different treatment groups. LPS and Erastin groups showed significant changes compared to the control group. For example, Mitochondrial swelling, mitochondrial skeleton damage and mitochondrial respiratory chain function impaired; Mitochondrial cristae degeneration and morphological changes were observed. Mitochondrial morphological changes can lead to functional abnormalitie. The LPS\u0026thinsp;+\u0026thinsp;Fer-1 and Erastin\u0026thinsp;+\u0026thinsp;Fer-1 groups showed improvements compared to their respective groups. Fer-1 action partly restored LPS-Erastin-induced changes in ultrastructural features and alleviated the effects of LPS and Erastin on sarcomeres, mitochondria, and the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK). Flow cytometry analysis indicated a remarkable increase in ROS levels in the LPS and Erastin groups compared to the control group; treatment with Fer-1 reversed this effect in the LPS\u0026thinsp;+\u0026thinsp;Fer-1 and Erastin\u0026thinsp;+\u0026thinsp;Fer-1 groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL). Furthermore, CFDA-SE labeling was performed to analyze cell viability in the different treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM). Compared to the control group, a significant decrease in cell viability in the LPS group was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM). However, combined treatment with Fer-1 in the LPS\u0026thinsp;+\u0026thinsp;Fer-1 and Erastin\u0026thinsp;+\u0026thinsp;Fer-1 groups led to a marked improvement in cell viability compared to the LPS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM). We also analyzed the gene expression levels of NRF2, GPX4, ACSL4, IL-10, and FOXO3 after exposing the cells to various treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eN \u003cb\u003eand Figure S5A)\u003c/b\u003e. The LPS and Erastin groups showed significant differences in gene expression compared to the control group, with NRF2 and GPX4, IL-10, and FOXO3 being upregulated while ACSL4 was downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eN \u003cb\u003eand Figure S5A)\u003c/b\u003e. In contrast, combined treatment with Fer-1 partly restored gene expression in C2C12 cells, leading to decreased expression of ACSL4 and upregulation of NRF2 and GPX4, IL-10 and FOXO3 in both the LPS\u0026thinsp;+\u0026thinsp;Fer-1 and Erastin\u0026thinsp;+\u0026thinsp;Fer-1 groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eN \u003cb\u003eFigure S5A\u003c/b\u003e). Moreover, in LPS and Erastin groups, western blotting revealed the upregulation of ACSL4 and the downregulation of GPX4 and NRF2, IL-10 and FOXO3; these expression trends were partially reversed by combined treatment with Fer-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eO \u003cb\u003eand Figure S5B\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Ferroptosis and Mitochondria Impairment play a vital role in in the diaphragm of septic mice\u003c/h2\u003e \u003cp\u003eTo further explore the mechanism underlying ferroptosis in SIDD, we developed a mouse model of CLP (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), and performed a series of experiments. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, the diaphragm muscle contraction tension was measured in different groups and the results showed that the tension was significantly decreased in CLP group whereas Fer-1 treatment markedly alleviated this effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Assays were performed to detect the levels of Myo and CK-MB, as well as biochemical markers MDA and GSH in the muscle (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The levels of Myo, CK-MB, and MDA was significantly increased whereas GSH was markedly decreased in the CLP group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Interestingly, Fer-1 treatment counteracted the effect of CLP (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In addition, we measured the levels of MDA, SOD and GSH in the serum of mice and found that MDA was significantly increased whereas GSH and SOD were markedly decreased in the CLP group, which was reversed by Fer-1 (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA\u003c/b\u003e). Moreover, ELISA assay results revealed that CLP significantly increased the levels of Total Fe and Fe\u003csup\u003e2+\u003c/sup\u003e in the muscle (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) and serum (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB\u003c/b\u003e) of mice, and these effects were inhibited by Fer-1 treatment. Next, oxygen consumption rate (OCR) was measured (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eE) and the results showed that CLP markedly decreased OCR levels, while Fer-1 treatment counteracted this effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). The measurement of ECAR also indicated CLP markedly decreased ECAR level, but this effect was counteracted by Fer-1 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). HE staining of diaphragm muscle tissue indicated that, compared to the sham and Control groups, the CLP group exhibited abnormal histopathological changes, including disruption of muscle fibers, the presence of infiltrating inflammatory cells, necrotic areas, and interstitial edema in the diaphragm muscle tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). In the CLP\u0026thinsp;+\u0026thinsp;Fer-1 group, diaphragm muscle tissue showed improvements in histopathological features compared to the CLP group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). These improvements included reduced inflammatory cell infiltration, less disruption of muscle fibers, decreased interstitial edema, and smaller necrotic areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). TEM examination was used to examine the ultrastructural changes in diaphragm muscle tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Compared to the Control and Sham groups, the CLP group exhibited significant ultrastructural alterations in diaphragm muscle tissue, including disorganized myofibrils, disrupted Z-lines, swollen mitochondria with loss of cristae, and increased cytoplasmic vacuolization (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). In contrast, in the CLP\u0026thinsp;+\u0026thinsp;Fer-1 group, diaphragm muscle tissue showed improvements including better organization of myofibrils, clearer Z-lines, reduced mitochondrial swelling, and decreased cytoplasmic vacuolization in ultrastructural features compared to the CLP group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Real-time PCR analysis was performed to measure changes in gene expression levels of GPX4, ACSL4 and NRF2 in each group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). The results showed that GPX4, and NRF2 were downregulated by CLP but Fer-1 treatment reversely upregulated the expression of these genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). Inverse trends were observed for ACSL4 mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). Western blot analysis was used to measure changes in the protein content of GPX4, ACSL4, NRF2 and P-NRF2 in each group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). The results showed that GPX4 and p-NRF2 were dramatically downregulated in CLP while ACSL4 was upregulated comparatively to the sham and control groups, but Fer-1 treatment reversed the expression tendency of these markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). Furthermore, the results of the immunofluorescence assay indicated that CLP significantly decreased the expression of GPX4 (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC\u003c/b\u003e) and α-SMA (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD\u003c/b\u003e) while increased expression of ACSL4 was observed (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE\u003c/b\u003e); these results were reversed by the treatment with Fer-1. Overall, the study suggests that Fer-1 treatment may protect SIDD from restraining metabolic changes and mitochondrial impairment, hinting that ferroptosis and mitochondria impairment play a vital role in in the diaphragm of septic mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.5 FOXO3/IL-10 axis was upregulated in the diaphragm of septic mice treated with Fer-1\u003c/h2\u003e \u003cp\u003eTo further elucidated the molecular mechanism, we performed RNA-seq analysis using diaphragm tissues from Sham, CLP, and CLP\u0026thinsp;+\u0026thinsp;Fer-1 mice. The heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) shows the differentially expressed genes among the three groups. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eB illustrates the number of common differentially expressed genes across Sham vs. CLP, Sham vs. Fer-1, and CLP vs. Fer-1 groups. A total of 355 common genes were identified. The functional enrichment analysis of differentially expressed genes is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eC which demonstrates the Gene Ontology categories that were significantly enriched among the Sham vs. CLP and CLP vs. Fer-1 comparisons. We found that the biological processes of \u0026ldquo;steroid metabolic process\u0026rdquo;, \u0026ldquo;fatty acid metabolic process\u0026rdquo;, \u0026ldquo;small molecule catabolic process\u0026rdquo;, \u0026ldquo;wound healing\u0026rdquo;, and \u0026ldquo;organic acid biosynthetic process\u0026rdquo; were the most enriched terms in both the Sham vs. CLP and CLP vs. Fer-1 comparisons (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Furthermore, KEGG pathway enrichment analysis of differentially expressed genes among the two comparisons was performed, and the results are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eD. We found that the \u0026ldquo;complement and coagulation cascade\u0026rdquo;, \u0026ldquo;retinol metabolism\u0026rdquo;, and \u0026ldquo;steroid hormone biosynthesis\u0026rdquo; were the pathways significantly affected by both CLP and Fer-1 treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). These results suggest that Fer-1 treatment prevents the differential expression of several genes that were altered in response to CLP. To identify the genes that were responsive to both CLP and Fer-1, we performed clustering analysis to identify gene expression profiles. Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eA displays profiles ranked by the significance of the number of genes assigned compared to expected, while Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eB presents profiles ranked by the number of genes assigned. Notably, profiles 1 and 5, containing genes affected by CLP that can be restored by Fer-1, were identified (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). The gene expression changes were illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, while the PPI network of the Fer-1-target genes in CLP was showcased in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eD. The PPI network indicated strong interactions among the Fer-1-target genes in CLP (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Lastly, we identified the hub Fer-1-target genes in CLP using MCODE (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). The results indicated that 29 genes could be considered as hub genes, among which figured IL10 and other immune- and inflammation-related genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo understand how epigenetic regulation plays a role in ferroptosis in sepsis, we performed ATAC-seq analysis using diaphragm muscle tissue collected from Sham, CLP, and CLP\u0026thinsp;+\u0026thinsp;Fer-1 mice. The results showed that each group had different gene segments, including promoter, 5'UTR, 3'UTR, and other gene content (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). In each category, we noticed variations in the way genes were distributed across different chromatin states (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). In addition, we conducted GO (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eC) and KEGG pathway analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). The results showed that \u0026ldquo;actin filament organization\u0026rdquo;, \u0026ldquo;actin filament organization\u0026rdquo;, \u0026ldquo;muscle system process\u0026rdquo;, \u0026ldquo;regulation of actin filament-based process\u0026rdquo;, and \u0026ldquo;regulation of vasculature development\u0026rdquo; were the biological processes mostly affected by CLP (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Moreover, the results indicated that the \u0026ldquo;negative regulation of phosphorylation\u0026rdquo;, \u0026ldquo;negative regulation of protein phosphorylation\u0026rdquo;, \u0026ldquo;response to LPS\u0026rdquo;, \u0026ldquo;response to molecule of bacterial origin\u0026rdquo;, and \u0026ldquo;regulation of actin filament-based process\u0026rdquo; were the biological processes affected by Fer-1 treatment of CLP mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). The KEGG pathways mostly affected by CLP were \u0026ldquo;cAMP signaling pathway\u0026rdquo;, \u0026ldquo;cGMP-PKG signaling pathway\u0026rdquo;, \u0026ldquo;purine metabolism\u0026rdquo;, \u0026ldquo;lipid and atherosclerosis\u0026rdquo;, \u0026ldquo;regulation of actin cytoskeleton\u0026rdquo;, and \u0026ldquo;TNF signaling pathway\u0026rdquo;, while those affected by Fer-1 treatment of CLP mice were \u0026ldquo;herpes simplex virus 1 infection\u0026rdquo;, \u0026ldquo;Rap 1 signaling pathway\u0026rdquo;, \u0026ldquo;TNF signaling pathway\u0026rdquo;, and \u0026ldquo;toxoplasmosis\u0026rdquo;.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe integrated analysis of mRNA-seq and ATAC-seq data revealed significant changes in gene expression and chromatin accessibility in response to CLP and Fer-1 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). The quadrant plot showed differential gene expression in the four groups: control, CLP model, and CLP\u0026thinsp;+\u0026thinsp;Fer-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). The Venn diagrams of highly and lowly expressed genes in the first (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003eB) and fourth (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003eC) quadrants, respectively, were analyzed for GO and KEGG pathway analysis. The PPI network of transcription factors identified by ATAC-seq and Fer-1-target genes in CLP was established (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003eD), and hub Fer-1-target genes in CLP (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003eE) were identified, indicating the strong interactions among Fer-1-target proteins in CLP in the central role of IL10 in this process. Finally, IL10 and FOXO3 were selected for dual luciferase assays, which validated the interaction among between both proteins (Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003eF and \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Up regulated FOXO3/IL-10 axis prevents diaphragm from sepsis-induced ferroptosis by activating Nrf2/GPX4 signaling\u003c/h2\u003e \u003cp\u003eImmunofluorescence analysis indicated significantly downregulated expression of GPX4 expression in the CLP group, which was notably reversed by combined treatment with mIL-10 or Fer-1 (\u003cb\u003eFigure S2A\u003c/b\u003e). On the contrary, 4HNE expression was upregulated in the CLP group compared to the Sham group; however, combined treatment with mIL-10 or Fer-1 led to a decrease in 4HNE expression compared to the CLP group (\u003cb\u003eFigure S2B\u003c/b\u003e). Additionally, there was an upregulation of ACSL4 expression in the CLP group compared to the Sham group. However, treatment with mIL-10 or Fer-1 resulted in a decreased expression of ACSL4 in the CLP\u0026thinsp;+\u0026thinsp;mIL-10 and CLP\u0026thinsp;+\u0026thinsp;Fer-1 groups (\u003cb\u003eFigure S2C\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThrough TEM analysis of muscle tissue, it was observed that the CLP group displayed notable morphological changes in their mitochondria compared to the Sham group. The changes observed in the CLP group were indicative of cellular stress and damage, including disrupted cristae, irregular shape, and a swollen appearance (\u003cb\u003eFigure S2D\u003c/b\u003e). However, treatment with either mlL-10 or Fer-1 had a restorative effect on the mitochondria in the CLP group (\u003cb\u003eFigure S2D\u003c/b\u003e). Indeed, following treatment, the mitochondria showed improved structure and integrity. with the disrupted cristae appearing more organized and defined. The irregular shape and swelling of the mitochondria were also reduced, indicating a mitigation of cellular stress and a partial restoration of normal mitochondrial morphology (\u003cb\u003eFigure S2D\u003c/b\u003e). Furthermore, qRT-PCR of ACSL4, GPX4, NRF2, IL-10, and FOXO3 in diaphragm tissue from the different groups was detected (\u003cb\u003eFigure S2E\u003c/b\u003e). In the CLP group, GPX4, NRF2, IL-10, and FOXO3 expression were decreased while ACSL4 was upregulated. Indeed, CLP mice treated with Fer-1 or mIL-10 showed marked reversal of these changes with upregulation of GPX4, NRF2, IL-10, and FOXO3 and downregulation of ACSL4 expression (\u003cb\u003eFigure S2E\u003c/b\u003e). Furthermore, the western blot analysis showed that in the CLP group, GPX4, pNRF2, IL-10, and FOXO3 decreased, but these effects were reversed by treatment with mIL-10 or Fer-1. Contrary results were recorded for ACSL4, while no significant difference was recorded for NRF2 among groups (\u003cb\u003eFigure S2F\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eTo investigate the effect of FOXO3/IL10 axis in SIDD, different experiments were performed \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e9\u003c/span\u003e \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e10\u003c/span\u003e). Analysis of immunofluorescence in CLP or LPS groups versus controls showed a decrease in GPX4 MFI. Comparatively, the overexpression of FOXO3 and combination of mIL-10 led to an increase in the MFI of GPX4 when contrasted with the CLP or LPS groups and their respective negative counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e9\u003c/span\u003eA \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e10\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe MFI of 4HNE protein was elevated in the CLP or LPS groups compared to the control groups, according to immunofluorescence analysis shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e9\u003c/span\u003eB \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e10\u003c/span\u003eB. The MFI of 4HNE in the CLP or LPS groups were higher than those in the FOXO3 overexpression group. The combination of mIL-10 and FOXO3 promoted their effect, manifested in decreased 4HNE MFI. As revealed by the immunofluorescence analysis, ACSL4 MFI was greater in the CLP and LPS groups than in their corresponding control groups. FOXO3 overexpression caused a decrease in ACSL4 MFI compared to CLP or LPS groups and their respective controls (OE-NC), as shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e9\u003c/span\u003eC and \u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e10\u003c/span\u003eC. mIL-10 incorporation in the therapy resulted in reduced ACSL4 MFI due to increased FOXO3 overexpression. Mitochondrial alterations were observed via TEM in both animal and in vitro models following ferroptosis. The experimental conditions brought to light structural modifications and possible mitochondrial damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e9\u003c/span\u003eD and \u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e10\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eWe performed C11-Bodipy assay to assess variations of C2C12 muscle cells in different experimental groups. In the LPS group C11-Bodipy fluorescence increased significantly, suggesting lipid peroxidation and oxidative stress. Nevertherless, lipid peroxidation was significantly decreased in the LPS\u0026thinsp;+\u0026thinsp;OE-FOXO3 group. Furthermore, when we co-administrated LPS\u0026thinsp;+\u0026thinsp;Fer-1, Fer-1 clearly attenuated the LPS-induced lipid peroxidation, which had no significant difference in the LPS\u0026thinsp;+\u0026thinsp;OE-FOXO3 group. Moreover, OE-IL-10\u0026thinsp;+\u0026thinsp;OE-FOXO3 combined overexpression produced an additive effect on reducing lipid peroxidation (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e10\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eThe FerroOrange staining was performed to assess the Fe\u003csup\u003e2+\u003c/sup\u003e alterations in C2C12 muscle cells in different experimental group. The LPS group displayed a significant increase in FerroOrange fluorescence, suggesting significantly higher levels of Fe\u003csup\u003e2+\u003c/sup\u003e. Indeed, the fluorescence intensity was kept high in the LPS\u0026thinsp;+\u0026thinsp;OE-NC group, suggesting that overexpression of the negative control gene had no effect on the LPS-induced changes. However, we observed a decreased FerroOrange fluorescence in the LPS\u0026thinsp;+\u0026thinsp;OE-FOXO3 group, which suggested a decrease in Fe\u003csup\u003e2+\u003c/sup\u003e. In similar manner, the administration of Fer-1 reversed LPS-induced increase of Fe\u003csup\u003e2+\u003c/sup\u003e in the LPS\u0026thinsp;+\u0026thinsp;Fer-1 group. Interestingly, in the LPS\u0026thinsp;+\u0026thinsp;OE-IL-10\u0026thinsp;+\u0026thinsp;OE-FOXO3 group, the simultaneous overexpression of IL-10 and FOXO3 resulted in an additive effect on the reduction of Fe\u003csup\u003e2+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e10\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eROS was conducted in C2C12 muscle cells within various experimental groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e10\u003c/span\u003eG). We observed a significant increase in ROS in the LPS group, showing oxidative stress. The LPS\u0026thinsp;+\u0026thinsp;OE-NC group showed a very high ROS level which demonstrated that transfection of the negative control gene didn\u0026rsquo;t affect the ROS level induced by LPS. However, the LPS\u0026thinsp;+\u0026thinsp;OE-FOXO3 group exhibited a marked reduction in ROS levels. In addition, Fer-1 treatment significantly suppressed the ROS level in the LPS\u0026thinsp;+\u0026thinsp;Fer-1 group, revealing the antioxidative effect of Fer-1 against LPS-induced oxidative stress. Additionally, in the LPS\u0026thinsp;+\u0026thinsp;OE-IL-10\u0026thinsp;+\u0026thinsp;OE-FOXO3 group, ROS level was even decreased furtherly, indicating that the co-overexpression of IL-10 and FOXO3 produced an additive effect on reducing oxidative stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e10\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eFurthermore, the CFDA-SE staining was done to observe the viability of C2C12 muscle cells in different experimental groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e10\u003c/span\u003eH). In the LPS group, a significant reduction of CFDA-SE fluorescence was observed compared to the control group, indicative of reduced cell survival. However, the CFDA-SE fluorescence intensity was significantly increased in the LPS\u0026thinsp;+\u0026thinsp;OE-FOXO3 group, meaning an obvious improvement of cell viability. Likewise, in the LPS\u0026thinsp;+\u0026thinsp;Fer-1 group, the fluorescence intensity was remarkably increased, suggesting that Fer-1 treatment could efficiently bring cells back to viability. In particular, there was an enhancement of CFDA-SE fluorescence in the LPS\u0026thinsp;+\u0026thinsp;OE-IL-10\u0026thinsp;+\u0026thinsp;OE-FOXO3 group, which indicated that there was a synergistic effect with the simultaneous overexpression of FOXO3 and IL-10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e10\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003eRT-PCR and western blotting were performed to evaluate mRNA expression levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e9\u003c/span\u003eE \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e10\u003c/span\u003eI) and protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e9\u003c/span\u003eF \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e10\u003c/span\u003eJ) in each group. The CLP and LPS groups showed increased ACSL4 mRNA and protein expression compared to the control groups, while the overexpression of FOXO3 resulted in decreased ACSL4 mRNA and protein expression relative to both the CLP or LPS groups and their corresponding controls. Furthermore, the combination with mIL-10 led to decreased ACSL4 mRNA and protein expression. The expression level of GPX4, NRF2/P-NRF2, IL-10, and FOXO3 genes and proteins showed opposite trends of ACSL4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e9\u003c/span\u003eE \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e10\u003c/span\u003eI) and (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e9\u003c/span\u003eF \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e10\u003c/span\u003eJ).\u003c/p\u003e \u003cp\u003eProviding a comprehensive overview of the protein expression changes \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e models, these findings illustrated possible effects of FOXO3 overexpression and mIL-10 treatment on cellular processes related to oxidative stress, inflammation and survival. To scrutinize the function of IL-10 and FOXO3 in SIDD, C2C12 muscle cells were transfected with IL-10 or FOXO3 vectors. Western blot analysis demonstrated the overexpression of IL-10 (\u003cb\u003eFigure S3A\u003c/b\u003e) and FOXO3 (\u003cb\u003eFigure S3B\u003c/b\u003e), indicating that the transfection of IL-10 and FOXO3 expression vectors were successfully performed. Using immunofluorescence assay, we found remarkably reduced GPX4 expression in the LPS treatment group compared to the control (\u003cb\u003eFigure S4A\u003c/b\u003e). In addition, treatment with Fer-1 or IL-10 overexpression reversed the LPS-mediated decrease in GPX4 gene expression (\u003cb\u003eFigure S4A\u003c/b\u003e). Immunofluorescence staining for 4HNE revealed that the increase in 4HNE expression in the LPS group was abolished by either Fer-1 treatment or overexpression of IL-10 (\u003cb\u003eFigure S4B\u003c/b\u003e). The mRNA expression of ACSL4 was upregulated in the LPS group compared with the control group on immunofluorescence staining. In contrast, overexpression of IL-10 or Fer-1 led to downregulation of ACSL4 expression in the LPS\u0026thinsp;+\u0026thinsp;OE-IL-10 and LPS\u0026thinsp;+\u0026thinsp;Fer-1 groups vs. the LPS group (\u003cb\u003eFigure S4C\u003c/b\u003e). As revealed by the C11-BODIPY assay, lipids were more peroxidated in the LPS group. However, administration of Fer-1 or overexpression of IL-10 mitigated the effect of LPS on lipid peroxidation (\u003cb\u003eFigure S4D\u003c/b\u003e). Additionally, the Ferrorange assay demonstrated increased iron accumulation in the LPS group compared to the control group. Notably, overexpression of IL-10 or treatment with Fer-1 led to a decrease in iron accumulation in the LPS\u0026thinsp;+\u0026thinsp;OE-IL-10 and LPS\u0026thinsp;+\u0026thinsp;Fer-1 groups, respectively, compared to the LPS group. The NC group exhibited similar levels of iron accumulation as the control group (\u003cb\u003eFigure S4E\u003c/b\u003e). TEM analysis showed that LPS caused mitochondria morphology changes, which were reversed by either IL-10 overexpression or Fer-1 treatment (\u003cb\u003eFigure S4F\u003c/b\u003e). Flow cytometry analysis indicated that ROS levels significantly increased in the LPS group compared to Control. However, overexpression of IL-10 in LPS\u0026thinsp;+\u0026thinsp;OE-IL-10 or treatment with Fer-1 in LPS\u0026thinsp;+\u0026thinsp;Fer-1 significantly decreased ROS levels compared to the LPS group (\u003cb\u003eFigure S4G\u003c/b\u003e). These findings indicated that both IL-10 overexpression and Fer-1 treatment effectively reduced ROS levels in LPS-treated C2C12 muscle cells. Moreover, CFDA-SE staining showed that LPS reduced cell viability. However, IL-10 overexpression or Fer-1 treatment reversed the effect of LPS on cell viability (\u003cb\u003eFigure S4H\u003c/b\u003e). These findings suggested that IL-10 overexpression and Fer-1 treatment can promote cell viability in LPS-induced C2C12 muscle cells. Moreover, qRT-PCR of GPX4, NRF2, IL-10, and FOXO3 indicated the downregulation of these genes in the LPS group compared to the control group while ACSL4 mRNA level showed opposite trends. However, treatment with Fer-1 or overexpressing IL-10 reversed the effect of LPS on the expression levels of these genes (\u003cb\u003eFigure S4I\u003c/b\u003e). Furthermore, western blot analysis showed similar trends in protein expression (\u003cb\u003eFigure S4J\u003c/b\u003e). These findings suggested that the up-regulated FOXO3/IL-10 axis prevents the diaphragm from sepsis-induced ferroptosis by activating Nrf2/GPX4 signaling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eOur study's findings revealed elevated markers of muscle cell injury across in vitro and in vivo sepsis models as well as in clinical samples, alongside diminished diaphragm muscle contraction tension in septic mice, confirming the presence of sepsis-induced diaphragm dysfunction (SIDD). These observations align with prior research indicating compromised diaphragm function under various conditions. Specifically, it has been reported that 40\u0026ndash;50% of critically ill patients experience diaphragm atrophy, with about 50\u0026ndash;60% of these patients suffering from diaphragm dysfunction [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Additionally, septic patients display significant diaphragm muscle degradation, leading to more pronounced atrophy and dysfunction compared to non-septic individuals [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Given that SIDD can precipitate respiratory failure and potentially fatal outcomes in sepsis patients, it is critical to unravel the molecular underpinnings and develop corresponding therapeutic strategies. However, the precise mechanisms driving SIDD remain to be fully elucidated.\u003c/p\u003e \u003cp\u003eNumerous studies have established a connection between ferroptosis and sepsis, suggesting ferroptosis as a critical pathological form of cell death in sepsis. For example, ferritinophagy-mediated ferroptosis has been implicated in sepsis-induced cardiac damage [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Recent findings indicate that Fer-1 and Irisin can counteract neuronal ferroptosis through the Nrf2/HO-1, Nrf2/GPX4, and glutamate excitotoxicity signaling pathways, offering potential therapeutic avenues for sepsis-induced encephalopathy [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. These insights suggest that ferroptosis might represent a novel pathophysiological mechanism in sepsis, contributing to the damage of various organs during the condition. However, the specific involvement of ferroptosis in the development of sep-sis-induced diaphragm dysfunction (SIDD) has been less clear. Our research fills this gap by elucidating the role of ferroptosis in the pathogenesis of SIDD, marking the first demonstration that inhibiting ferroptosis can shield against SIDD. This protection is achieved by diminishing oxidative stress, ameliorating mitochondrial dysfunction, and correcting metabolic disturbances associated with ferroptosis. Sepsis is characterized by systemic disturbances that lead to organ dysfunction [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], with the diaphragm being notably affected. It undergoes morphological alterations and suffers mitochondrial damage, resulting in diminished function [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The accumulation of Fe\u003csup\u003e2+\u003c/sup\u003e within mitochondria can escalate the production of reactive oxygen species (ROS), promoting lipid peroxidation and cell death [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Concurrently, the excess ROS can lead to the depolarization of mitochondrial membrane potential and the opening of permeability transition pores, further compromising mitochondrial structure and function. This sequence of events highlights a significant link between mitochondrial health and ferroptosis, underscoring the importance of our findings in the context of SIDD and sepsis treatment strategies [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough it's well-documented that diaphragm damage during sepsis is closely linked to mitochondrial dysfunction, the specific interplay between mitochondrial dysfunction and ferroptosis in the context of SIDD remained unclear until now. Our study unveiled those measurements such as oxygen consumption rate (OCR), extracellular acidification rate (ECAR), mitochondrial complex activity, and ultrastructural integrity exhibited comparable alterations in C2C12 muscle cells subjected to either LPS induction or treatment with the ferroptosis inducer Erastin. These findings suggest that mitochondria-related ferroptosis plays a role in the development of SIDD. In the setting of sepsis, an infectious challenge triggers the upregulation of nuclear receptor coactivator 4 (NCOA4), which specifically targets ferritin for autophagic degradation. This process releases a substantial amount of Fe\u003csup\u003e3+\u003c/sup\u003e into the cytoplasm, increasing the free iron concentration and facilitating iron-induced cell death[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Characteristics of this cell death include cellular membrane rupture, mitochondrial volume reduction, increased density of the mitochondrial double membrane, and diminished or absent mitochondrial cristae\u0026mdash;observations that align with our experimental results. Throughout this process [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], expression of critical proteins such as transferrin receptor (TFR), GPX4, ACSL4, and Ferritin are altered, while the Nrf2 signaling pathway emerges as a regulatory mechanism controlling the initiation and progression of iron-induced cell death [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe results of our research demonstrate a notable reduction in GPX4 and Nrf2 expression levels, alongside an increase in Fe2+, ROS, and ACSL4 expression within the diaphragm of septic mice. These observations underline the significance of iron-induced cell death in the pathogenesis of SIDD. Additionally, the observed reduction in mitochondrial cristae, alongside diminished oxygen consumption rate (OCR), heightened extracellular acidification rate (ECAR), and increased ROS production, further indicate mitochondrial dysfunction as a pivotal factor in promoting iron-induced cell death. This study confirms the occurrence of mitochondria-associated ferroptosis in the diaphragm during sepsis, suggesting that targeting the balance of cellular homeostasis could be crucial for addressing both the pathological and physiological impacts of sepsis. These in-sights pave the way for new therapeutic strategies in sepsis management and open novel pathways for drug development.\u003c/p\u003e \u003cp\u003eTo elucidate how iron-induced cell death contributes to SIDD, we employed RNA sequencing (RNA-seq) and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), complemented by protein-protein interaction (PPI) analysis. Our investigation uncovered that the FOXO3/IL-10 regulatory axis is suppressed in septic mice, yet can be reactivated through the administration of Fer-1. Further analysis identified FOXO3 as a key transcription factor for IL-10, significantly boosting IL-10 expression both in vitro and in vivo. FOXO3 is recognized for its crucial role in regulation of cell metabolism managing dysfunction [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] and stress response [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Our findings indicated that levels of both FOXO3 and IL-10 were diminished in C2C12 cells subjected to LPS/Erastin; however, Fer-1 treatment successfully reinstated the FOXO3/IL-10 axis. To explore the interaction between FOXO3 and mitochondria-associated ferroptosis in SIDD more deeply, our results revealed that FOXO3 overexpression markedly reduced lipid peroxidation in the diaphragm of mice and in LPS-stimulated C2C12 cells, achieved through cell transfection techniques.\u003c/p\u003e \u003cp\u003eConsistent with our observations, recent research has demonstrated that depletion of FoxO3a results in heightened membrane potential, increased oxygen consumption, and accumulation of lipid peroxides. On the contrary, activation of the FoxO3a/HIF1α pathway correlates with diminished levels of 4-HNE staining and MDA in brain tissues [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Furthermore, our studies reveal that overexpression of FOXO3 effectively counteracts the surge of ROS in C2C12 cells triggered by LPS exposure. Notably, FOXO3 overexpression not only reinstates the diminished expression of key genes such as Nrf2 and GPX4 but also reduces the elevated expression of ACSL4. It concurrently enhances the ultrastructural integrity of mitochondria in both mouse diaphragms and LPS-challenged C2C12 cells, mirroring the effects seen with Fer-1 treatment. These insights underscore the pivotal role of FOXO3 in safeguarding against ferroptosis in SIDD through its regulatory impact on mitochondrial function and oxidative stress. This study marks the first to demonstrate that FOXO3 activation can thwart mitochondria-associated ferroptosis in SIDD, heralding new therapeutic avenues for addressing this condition.\u003c/p\u003e \u003cp\u003eOur study uniquely demonstrates that FOXO3 mitigates ferroptosis in SIDD by binding to the promoters of IL-10, thereby enhancing its expression. This mechanism underscores the therapeutic potential of IL-10 in combatting ferroptosis, as evidenced by our findings that administering mice with mIL-10 or overexpressing IL-10 in LPS-stimulated C2C12 cells significantly curbs ferroptosis. This aligns with previous research suggesting ferroptosis's involvement in infectious diseases, notably affecting host immune responses and the inflammatory cascade [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Administration of mIL-10 to mice was observed to significantly enhance diaphragmatic force production [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], highlighting the beneficial impact of IL-10 on diaphragm function. Despite these advancements, the precise dynamics of diaphragm muscle response to IL-10 remain to be fully elucidated. Additionally, our data indicates that the reduction of IL-10 in SIDD can be partially reversed by Fer-1, suggesting a complex interplay between IL-10 and ferroptosis. This raises intriguing questions about the potential of targeting IL-10 in ferroptosis-based therapeutic strategies. However, it remains uncertain whether a portion of IL-10 is secreted by immune cells migrating from the blood. Further research is essential to shed light on this aspect in the future.\u003c/p\u003e"},{"header":"4. Material and methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Patients\u003c/h2\u003e \u003cp\u003eWe recruited a cohort of 60 participants, comprising 20 individuals without any reported medical conditions and 40 patients diagnosed with sepsis, who ranged in age from 30 to 95. The recruitment and sample collection process were conducted at Xinhua 96 Hospital from November 2021 to September 2022 and was carried out following the Ethics Committee approval number XHEC-SHDC-2020-049.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Animals and experimental design\u003c/h2\u003e \u003cp\u003eThe Animal Care and Use Committee of Xinhua Hospital, School of Medicine, Shang hai Jiao Tong University, Shanghai granted approval for our study (Grant number SYXK2018-0038), which utilized C57BL/6 mice weighing 20\u0026thinsp;\u0026plusmn;\u0026thinsp;2 g and aged 8 weeks. These mice were procured from Charles River Laboratories Animal Co., Ltd. (Beijing, China) and were provided with food and water ad libitum in a controlled environment for a week, with a 12 h light-dark cycle at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C and 50\u0026thinsp;\u0026plusmn;\u0026thinsp;10% relative humidity. In order to induce sepsis, cecal ligation and puncture (CLP) surgery was performed on the mice while under anesthesia (sevoflurane). The midline of the abdomen was cut, and the caecum was ligated to a length of approximately 0.5 cm and punctured using a 23-gauge needle. The abdomen was then sutured, and subcutaneous injection of 1 ml of normal saline was administered [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Control mice were subjected to sham surgery, involving only incisions to the abdomen without ligation or puncture of the caecum. All mice were sacrificed 72 hours after CLP.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Animal grouping and treatments\u003c/h2\u003e \u003cp\u003eFollowing the surgery, we divided the animals into five groups: Control (no surgery), Sham (surgery without ligation and puncture), Sham\u0026thinsp;+\u0026thinsp;Fer-1 (surgery without ligation and puncture but with Ferrostatin 1 (Fer-1) treatment), CLP (surgery with ligation and puncture), and CLP\u0026thinsp;+\u0026thinsp;Fer-1 (surgery with ligation and puncture plus Fer-1 treatment). We administered Fer-1 (Selleckchem, cat. No. S7243) at 10 mg/kg through injection after the surgery repeating every 12 hours throughout the experiment. During administration, the Sham and Control groups received an amount of vehicle solution (0.9% saline). We observed the animals closely for any discomfort for 72 hours before performing euthanasia using carbon dioxide asphyxiation. We collected blood samples to analyze the levels of cytokines, with all chemicals used being of quality and obtained from Sigma Aldrich unless otherwise stated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Muscle tension test\u003c/h2\u003e \u003cp\u003eThe detection method employed by Coombes et al. [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] involved vertical suspensions of diaphragm strips in a McBurney bath filled with Krebs solution kept at a temperature of 26\u0026deg;C and continuously supplied with a mixture of 95% O2 and 5% CO2. The rib end of the strip was fixed to the bottom of the bathtub, while the central tendon end was connected to a tension transducer, with platinum wire electrodes placed on either side of the muscle strip near the rib end. The diaphragm strip was adjusted to its optimal initial length (optimal fiber length, Lo) using small voltage stimulation and left to stabilize for 20 minutes. Subsequently, the electronic stimulator stimulated the diaphragm strips at a supramaximal voltage of approximately 20V, while the output signal of the tension sensor was recorded and analyzed using a biological signal acquisition and processing system. This methodology was adopted to enable accurate analysis and measurement of the resultant output signal.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.5 ATAC-seq\u003c/h2\u003e \u003cp\u003eATAC-seq was conducted to identify genes and transcription factors involved in regulating enzymes related to glucose metabolism and indicators of ferroptosis, such as NCOA4 or FTMT. In this process, we inserted sequencing adapters into chromatin regions accessible using a transposase. The resulting library was then. Sequenced using Illumina HiSeq.\u0026nbsp;To analyze the data obtained from the ATAC seq experiment, we employed bioinformatics methods, which included peak calling with MACS2 annotating peaks using Homer software and performing gene ontology analysis using Metascape. We employed DESeq2 to identify genes and transcription factors that showed expression. The results were effectively visualized using ggplot2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.6 RNA-Seq\u003c/h2\u003e \u003cp\u003eRNA was extracted from samples using TRIzol, and quality was assessed with NanoDrop. TruSeq Stranded mRNA Library Prep Kit was used to generate RNA-seq libraries sequenced on Illumina HiSeq2500. Raw sequencing reads were processed with Trimmomatic and aligned to the human genome using HISAT2. DESeq2 was used for differential gene expression analysis. Gene ontology analysis was performed with DAVID. TFs regulating metabolic enzymes or ferroptosis indicators were identified using the Enrichr database. Gene expression levels were compared between experimental groups and validated with qRT-PCR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.7 C2C12 culture and treatment\u003c/h2\u003e \u003cp\u003eThe C2C12 cell lines were procured from the American Type Culture Collection (ATCC) and cultivated in Dulbecco's modified Eagle's medium (DMEM; 11995065, Gibco, Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) from Biological Industries, Beit Haemek, Israel. The cells were then incubated in a humidified chamber at 37\u0026deg;C with 5% CO2. Prior to their utilization, the C2C12 cells underwent routine screening for mycoplasma using the Mycoplasma Detection Kit-QuickTest (Biotool, Houston, TX, USA). Following this, the cells were differentiated into muscle cells using DMEM enriched with 2% horse serum from Biological Industries for a period of five days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.8 Plasmid construction\u003c/h2\u003e \u003cp\u003eThe present study involved the construction of plasmids for the expression of full-length open reading frames of interleukin-10 (IL-10) and forkhead box O3 (FOXO3). The IL-10 wild-type 3\u0026prime;-untranslated region (UTR) was cloned into the pGL3-basic vector from Promega (Madison, WI), while site-directed mutagenesis using the QuikChange\u0026trade; kit from Stratagene (La Jolla, CA) was performed to generate the mutated IL-10 3\u0026prime;-UTR (Mut) after performing FoxO3 seed sequence analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.9 Transient transfection\u003c/h2\u003e \u003cp\u003eC2C12 cells (5 \u0026times; 104 cells/cm2) underwent transient transfection with 5ug of the pcDNA3.1-IL-10 and/or negative controls (Thermo Fisher Scientific, Waltham, MA) or plasmids via the Dharmafect transfection reagent (Dharmacon, Lafayette, CO), following the manufacturer's recommendations. The medium was refreshed every 3 days, and the cells were subsequently harvested for analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.10 Dual-Luciferase Reporter Assay\u003c/h2\u003e \u003cp\u003eIn order to validate the \u003cem\u003ein-silico\u003c/em\u003e prediction results, the dual-luciferase reporter assay was conducted. The promoter regions of IL-10, including both the wild-type (WT) and mutant (MUT) forms, were amplified and cloned into the pGL3-basic luciferase vector, resulting in the construction of the pGL3-IL-10-promoter plasmid. As a control, the pGL3-basic luciferase vector without the IL-10 promoter was utilized. Additionally, a specific effector plasmid of FOXO3 (pCDH-FOXO3) was developed. The transfection process was performed in 293T cell lines by using Lipofectamine 3000 (Invitrogen, USA). After a 24-hour incubation period, the luciferase activity was evaluated using the Dual-Luciferase R Reporter 1000 Assay system (Promega, Madison, WI, USA) to detect the promoter activities. The ratio of Firefly to Renilla luciferase activity was utilized to express the luciferase activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.11 Cell viability assay\u003c/h2\u003e \u003cp\u003eThe CCK-8 (Dojindo) method was utilized as a reference to assess cell viability. Specifically, C2C12 cells were seeded into a 96-well plate at a concentration of 5 \u0026times;104 cells/well and cultured for 24 hours. Afterward, the cells were exposed to varying concentrations of LPS (10 \u0026micro;g/mL, Sigma), Fer-1 (10 \u0026micro;M, MCE), Erastin (5 \u0026micro;M, Cayman), Z-VAD-FMK (40 \u0026micro;M, Selleck), Boc-D(OMe)-FMK (50 \u0026micro;M, Enzo), Necrostatin-1 (10 \u0026micro;M, Enzo), and N-Acetyl-L-cysteine (1mM, Sigma) for 12 hours. Subsequently, 20 \u0026micro;l of CCK-8 solution were directly added to the medium (200 \u0026micro;l per well), and the cells were incubated at 37\u0026deg;C for an additional 4 hours. The absorbances (Abs) of different groups were then measured at 450 nm (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e4.12 Western blotting\u003c/h2\u003e \u003cp\u003eWe used RIPA buffer (Thermo Fisher, #89901) supplemented with protease and phosphatase inhibitors to prepare protein extracts from the samples. The protein concentration was determined using a BCA protein assay kit (Thermo Fisher, #23225) following the instructions provided by the manufacturer. Afterward, we proceeded with a technique called sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) using 10% resolving gels (Bio-Rad, #4561034). Then we transferred the proteins onto nitrocellulose membranes (Bio-Rad, #1620115). Next, we blocked the membranes by using a solution consisting of 5% milk, in Tris-buffered saline, containing 0.1% Tween 20 (TBST). This blocking process lasted for an hour at room temperature. After that, we incubated the membranes overnight at a temperature of 4\u0026deg;C with antibodies. These antibodies targeted GPX4 (Abcam, #ab125066), ACSL4 (Abcam, #ab155282), p-NRF2(Santa Cruz Biotechnology, #sc-293123), NRF2 (Abcam, #ab62352), FOXO3 (Thermo Fisher Scientific, # PA5-27243), IL-10 (R\u0026amp;D Systems, #MAB417), and Recombinant mouse IL-10 (Cat: 417-ML-025, R\u0026amp;D Systems, Minneapolis, MN, USA) was reconstituted in sterile endotoxin-free PBS at 100 ug/mL. Following a wash, with TBST the membranes were incubated at room temperature for an hour with antibodies that were linked to peroxidase. To visualize the protein bands, a chemiluminescence (ECL) kit (Thermo Fisher Scientific #32106) was used, along with an imaging system from Bio-Rad. The expression levels of the proteins were quantified using ImageJ software (NIH). Normalized to β-actin as a reference, for loading control. This entire process was carried out in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e4.13 RT-PCR\u003c/h2\u003e \u003cp\u003eThe cells were treated with TRIzol Life Technologies) to extract RNA according to the manufacturer's instructions. Next, the RNA was reverse transcribed using the QuantiTect Reverse Transcription kit (Qiagen) following the provided protocol. The cDNA was amplified using gene primers and the SYBR Green PCR Master Mix (Applied Biosystems). The reaction mixture included 10 \u0026micro;L of SYBR Green PCR Master Mix, 1 \u0026micro;L of cDNA template, 0.5 \u0026micro;L of forward and reverse primers (10 \u0026micro;M), and 8 \u0026micro;L of nuclease water. The thermal cycling conditions were set at 95\u0026deg;C for 10 minutes followed by 40 cycles of 95\u0026deg;C for 15 seconds and 60\u0026deg;C for 60 seconds. To assess the expression levels of the target genes accurately, they were normalized against GAPDH using the 2\u0026thinsp;\u0026minus;\u0026thinsp;ΔΔCt method. The RT-PCR primers were bought from Sigma Aldrich; their sequences can be found in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. We optimized the primer concentrations to 10 \u0026micro;M. We conducted all experiments three times.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimers used in this study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimer name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimer sequense\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM-GPX4-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u0026rsquo;-CCTCCCCAGTACTGCAACAG-5\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM-GPX4-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u0026rsquo;-GTGACGATGCACACGAAACC-5\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM-ACSL4-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u0026rsquo;-GAAAGGCTATGACGCCCCTC-5\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM-ACSL4-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u0026rsquo;-ATCATGCGGACATTCCCTCC-5\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM-NRF2-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u0026rsquo;-GTTGCCCACTTGGTGGATTG-5\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM-NRF2-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u0026rsquo;-TTCTGCGTGCTCAGAAACCT-5\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM-IL-10-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u0026rsquo;-CAGTACAGCCGGGAAGACAA-5\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM-IL-10-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u0026rsquo;-AGGCTTGGCAACCCAAGTAA-5\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM-FOXO3-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u0026rsquo;-ACCTACGCATCCAGTGTGAG-5\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM- FOXO3-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u0026rsquo;-GCAAAGAAAAGGAGGGGGTC-5\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM-ACTIN-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u0026rsquo;-ACCCTAAGGCCAACCGTGAAA-5\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM-ACTIN-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u0026rsquo;-ATGGCGTGAGGGAGAGCATA-5\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e4.14 Immunofluorescence assay\u003c/h2\u003e \u003cp\u003eTo investigate the expression levels of GPX4, ACSL4, and 4HNE, we prepared the cells and tissues by fixing them with 4% paraformaldehyde for 15 minutes at room temperature. Next, we made the cells permeable using 0.1% Triton X 100. Blocked them with a solution of 1% BSA, in PBS. Subsequently, we applied the antibodies. Allowed them to incubate overnight. Following this we rinsed the cells with PBS. Treated them with Alexa Fluor 488 and anti-Fluor 594 antibodies, which were diluted at a ratio of 1:500. We further stained the cells with DAPI. Mounted them using Thermo Fisher Scientifics mounting medium. Finally, we analyzed the cells using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e4.15 Detection of muscle cell function indicators AST, LDH, CK, and Myo\u003c/h2\u003e \u003cp\u003eWe utilized kits (AST/LDH/CK/Myo) to assess the status of muscle cell function. The instructions provided by the manufacturers were followed diligently including the use of recommended concentrations. The AST kit was obtained from Abcam (ab102523), the LDH kit from Cayman Chemical (601170), the CK kit from BioVision (K758 100), and the Myo kit from Roche (05067990190). To carry out the assays, we added the reagents to our samples, allowed them to incubate for the specified duration, and then employed a microplate reader to measure absorbance. By comparing the absorbance values of our samples against curves generated with the provided standards we could ascertain each indicator's concentrations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e4.16 ROS detection\u003c/h2\u003e \u003cp\u003eTo detect reactive oxygen species (ROS) in our samples we utilized a fluorescent probe called DCFH-DA. We added 10 \u0026micro;M of DCFH-DA (Cayman Chemical, 10004331) to the samples and allowed them to incubate at 37\u0026deg;C, for 30 minutes. Subsequently, we rinsed the samples with phosphate-buffered saline (PBS) to eliminate any remaining DCFH-DA and extracted the resulting product, known as dichlorofluorescein (DCF) using a lysis buffer. Finally, we determined the brightness of the samples using a microplate reader. To quantify the concentration of ROS, in each sample we compared the brightness to a curve generated using hydrogen peroxide (H2O2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e4.17 Iron Assay\u003c/h2\u003e \u003cp\u003eTo assess the iron levels in C2C12 cells, a FerroOrange assay was performed. The cells were exposed to FerroOrange (1 \u0026micro;Mol/L, Dojindo, Japan) and incubated at 37\u0026deg;C with 5% CO2 for 30 minutes. A BioTek Cytation 5 fluorescence microscope (BioTek, USA) was then utilized to visualize the cells and quantify the amount of iron present. The FerroOrange assay functions by binding iron ions specifically, producing a fluorescent signal that can be detected and calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e4.18 Measurement of the oxygen consumption rate (OCR)\u003c/h2\u003e \u003cp\u003eThe extracellular flux analyzer, specifically the seahorse Bioscience X96 model from Agilent Technologies, was utilized to quantify the rate of dissolved O2 in the immediate vicinity of adherent cells that had been cultured in the XF96 V3 cell culture microplate developed by Seahorse Bioscience. The C2C12 cells, which had been cultured in DMEM supplemented with 0.5% FBS, were seeded into the XF96 V3 cell culture microplate at a density of 0.8 \u0026times; 104 cells per well. Following this, the cells were washed and left to incubate in the base medium created by Agilent Technologies at 37\u0026deg;C for an hour. The mitochondria stress test kit was employed to measure the oxygen consumption rates (OCR, pmol.min-1) in real-time, in accordance with the manufacturer's guidelines. To assess the glycolytic activity, sequential compound injections, including oligomycin A (1 \u0026micro;M), FCCP (1 \u0026micro;M), and Rotenone/antimycin A (0.5 \u0026micro;M), were administered onto the microplate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e4.19 Transmission electron microscopy\u003c/h2\u003e \u003cp\u003eThe cells and tissues were stimulated and then fixed in electron microscope fixative at 4 ℃ for a duration of 15 minutes. Next, the cells were carefully scraped off using a cell scraper and transferred to 1.5 ml centrifuge tubes. The fixative was replaced with fresh fixative and the samples were left in it for 4 hours at 4 ℃. The cells were then dehydrated using ethanol through gradient elution for 15 minutes each time. Afterward, the cells were permeabilized at 37 ℃ for 8\u0026ndash;12 hours and embedded at 60 ℃ for 48 hours. Finally, the samples were sectioned at 80\u0026ndash;100 nm using an ultramicrotome and stained. TEM (FEI Tecnai G20 TWIN, USA) was used to photograph all samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e4.20 C11-BODIPY assay\u003c/h2\u003e \u003cp\u003eTo detect lipid peroxidation, an in-situ lipid peroxidation sensor C11-BODIPY (581/591) from Thermo Fisher Scientific was utilized. The cells were pre-incubated in a fresh culture medium containing 5 \u0026micro;M of the probe at 37\u0026deg;C for 30 minutes. Afterward, they were washed twice with PBS and once with culture medium. The cells were further incubated with fresh medium for another 30 minutes at 37\u0026deg;C, and their observation was conducted via flow cytometry and confocal microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e4.21 MitoTox Complete OXPHOS Activity Assay\u003c/h2\u003e \u003cp\u003eTo assess the impact of each treatment on the electron transport chain complexes (I, II, III, IV and V), the MitoTox Complete OXPHOS Activity Assay Panel (Abcam, Cambridge, MA, USA) was used according to the manufacturer's instructions. The assay measured the direct effect of ketamine on each of the complexes, which were obtained from isolated mitochondria that were in their functionally active state. Highly specific monoclonal antibodies were attached to 96-well microplates for each complex. To determine the activity of each complex, the decrease in absorbance in milli-optical density per min was measured at room temperature or 37\u0026deg;C, as outlined by the manufacturer. Using a FLUOstar OPTIMA-6 (BMG Labtech, Durham, NC, USA) microplate reader, absorbance was measured every minute for two hours in kinetic mode.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e4.22 Extracellular acidification rate (ECAR) analysis\u003c/h2\u003e \u003cp\u003eThe Seahorse Extracellular Flux (XF-96) analyzer from Seahorse Bioscience in Chicopee, MA was used to determine the glycolysis and glycolytic capacity of cells. Cells were seeded for 2 hours in a medium devoid of glucose. The analyzer provided extracellular acidification (ECAR) associated with glycolysis, maximum glycolytic capacity, and non-glycolytic ECAR after three sequential injections of D-glucose (2 g/L), oligomycin (1 \u0026micro;M), and 2-Deoxyglucose (100 mM). The ECAR after the addition of D-glucose was used to define glycolysis, while the ECAR after the addition of oligomycin was used to define maximum glycolytic capacity. Non-glycolytic activity was associated with the ECAR after treatment with 2-Deoxyglucose.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003e4.23 CFDA-SE labeling\u003c/h2\u003e \u003cp\u003eA solution of 5mM CFDA-SE stock (2\u0026micro;L) was mixed with 1mL of PBS and then added to cells (4.5\u0026times;10\u003csup\u003e6\u003c/sup\u003e) that were washed thrice with PBS. The cells were incubated for 5 minutes at room temperature at a concentration of 10 \u0026micro;M. The labeled cells were washed using 10 volumes of 20\u0026deg;C PBS containing 5% heat-inactivated FBS and centrifuged at 280\u0026times;g for 5 minutes at 20\u0026deg;C. After the supernatant was discarded, the cells were washed twice and then seeded at a density of 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/mL in RPMI containing 10% FBS. Finally, the cells were treated with S-N-, S-N+, S\u0026thinsp;+\u0026thinsp;N-, S\u0026thinsp;+\u0026thinsp;N+, Pre, and Post and incubated for 40 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec33\" class=\"Section2\"\u003e \u003ch2\u003e4.24 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe experimental data underwent a one-way analysis of variance (ANOVA), two-way ANOVA and the Tukey HSD test using GraphPad Prism version 9 (GraphPad Software Inc., San Diego, CA, USA). The data was checked for significant differences, with a statistical significance level of 0.05. The data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM).\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn conclusion, our research offers groundbreaking insights into the role of mitochondria-associated ferroptosis in driving cell death and facilitating the development of sepsis-induced diaphragm dysfunction (SIDD), a novel discovery. Furthermore, we highlight the protective functions of the FOXO3/IL-10 axis against SIDD, attributed to its ability to counteract ferroptosis. Based on these findings, we suggest potential therapeutic avenues, including the use of ferroptosis inhibitors, agents targeting mitochondrial oxidative stress, and activators of the FOXO3/IL-10 signaling pathway, as promising strategies to combat SIDD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare that there are no conflict of interests, we do not have any possible conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported by grants from the National Natural Science Foundation of China (grant no. 82102237; no.82172159; no.82201320) and the Interdisciplinary Projects of Shanghai Jiao Tong University (grant no. YG2021QN56) and the project of Shanghai Ninth People\u0026rsquo;s Hospital, Shanghai Jiao Tong University School of Medicine (grant no. JYZZ131).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e: Conceptualization, Xiaojian Weng and Hong Jiang; methodology, Weiwen Zhang; software, Yi Jin; validation, Yudi Liao, Hui Dong. and Xuhui Zhou; formal analysis, Yaqiong Yang.; investigation, Nan Zhi; resources, Xiaojian Weng; data curation, Bin Zhao; writing\u0026mdash;original draft preparation, Hua Liu; writing\u0026mdash;review and editing, Dongdong Chai.; visualization, Xiang Lyu; supervision, Xiang Lyu.; project administration, Hong Jiang; funding acquisition, Xiaojian Weng, Hua Liu. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e The data underlying this article are available in the article and in its online supplementary material.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval:\u0026nbsp;\u003c/strong\u003eThe Animal Care and Use Committee of Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai approved our study (Grant number SYXK2018-0038)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e Not applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePurcarea A, Sovaila S (2020) Sepsis, a 2020 review for the internist. Rom J Intern Med 58:129\u0026ndash;137\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCecconi M, Evans L, Levy M, Rhodes A (2018) Sepsis and septic shock. Lancet 392:75\u0026ndash;82\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFleischmann C, Scherag A, Adhikari NKJ, Hartog CS, Tsaganos T, Schlattmann P, Angus DC, Reinhart K (2016) Int Forum Acute Care, Assessment of Global Incidence and Mortality of Hospital-treated Sepsis. Am J Respir Crit Care Med 193:259\u0026ndash;272\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSupinski GS, Schroder EA, Wang L, Morris AJ, Callahan LAP (2021) Mitoquinone mesylate (MitoQ) prevents sepsis-induced diaphragm dysfunction. J Appl Physiol 131:778\u0026ndash;787\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSupinski GS, Wang L, Schroder EA, Callahan LAP (2020) MitoTEMPOL, a mitochondrial targeted antioxidant, prevents sepsis-induced diaphragm dysfunction. Am J Physiology-Lung Cell Mol Physiol 319:228\u0026ndash;238\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaccheri C, Morawiec E, Delemazure J, Mayaux J, Dube B-P, Similowski T, Demoule A, Dres M (2020) ICU-acquired weakness, diaphragm dysfunction and long-term outcomes of critically ill patients. Ann Intensiv Care 10:1\u0026ndash;9\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoreno-Ruperez A, Priego T, Gonzalez-Nicolas Ma, Lopez-Calderon A, Lazaro A, Martin AI (2022) Role of Glucocorticoid Signaling and HDAC4 Activation in Diaphragm and Gastrocnemius Proteolytic Activity in Septic Rats. Int J Mol Sci 23:3641\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSupinski GS, Wang L, Schroder EA, Callahan LAP (2020) SS31, a mitochondrially targeted antioxidant, prevents sepsis-induced reductions in diaphragm strength and endurance. J Appl Physiol 128:463\u0026ndash;472\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu J, Liu H, Chu T, Jiang P, Li S-t (2019) Neuregulin-1β attenuates sepsis-induced diaphragm atrophy by activating the PI3K/Akt signaling pathway. J Muscle Res Cell Motil 40:43\u0026ndash;51\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDemoule A, Divangahi M, Yahiaoui L, Danialou G, Gvozdic D, Labbe K, Bao W, Petrof BJ (2006) Endotoxin triggers nuclear factor-κB-dependent up-regulation of multiple proinflammatory genes in the diaphragm. Am J Respir Crit Care Med 174:646\u0026ndash;653\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOkazaki T, Liang F, Li T, Lemaire C, Danialou G, Shoelson SE, Petrof BJ (2014) Muscle-Specific Inhibition of the Classical Nuclear Factor-κB Pathway Is Protective Against Diaphragmatic Weakness in Murine Endotoxemia. Crit Care Med 42:501\u0026ndash;509\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLabbe K, Danialou G, Gvozdic D, Demoule A, Divangahi M, Boyd JH, Petrof BJ (2010) Inhibition of monocyte chemoattractant protein-1 prevents diaphragmatic inflammation and maintains contractile function during endotoxemia. Crit Care 14:187\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJavesghani D, Magder SA, Barreiro E, Quinn MT, Hussain SNA (2002) Molecular characterization of a superoxide-generating NAD(P)H oxidase in the ventilatory muscles. Am J Respir Crit Care Med 165:412\u0026ndash;418\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCallahan LA, Nethery D, Stofan D, DiMarco A, Supinski G (2001) Free radical-induced contractile protein dysfunction in endotoxin-induced sepsis. Am J Respir Cell Mol Biol 24:210\u0026ndash;217\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSupinski GS, Alimov AP, Wang L, Song X-H, Callahan LA (2016) Calcium-dependent phospholipase A2 modulates infection-induced diaphragm dysfunction. Am J Physiology-Lung Cell Mol Physiol 310:975\u0026ndash;984\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOliveira TS, Santos AT, Vieira Andrade CB, Silva JD, Blanco N, Roche NdN, Woyames J, Silva PL, Macedo PR, Rocco WS, da-Silva TM, Ortiga-Carvalho FF (2021) Bloise, Sepsis Disrupts Mitochondrial Function and Diaphragm Morphology. Front Physiol 12:704044\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSupinski GS, Callahan LA (2006) Hemin prevents cardiac and diaphragm mitochondrial dysfunction in sepsis. Free Radic Biol Med 40:127\u0026ndash;137\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBloise FF, van der Spek AH, Surovtseva OV, Ortiga-Carvalho TM, Fliers E, Boelen A (2016) Differential Effects of Sepsis and Chronic Inflammation on Diaphragm Muscle Fiber Type, Thyroid Hormone Metabolism, and Mitochondrial Function, Thyroid, 26 600\u0026ndash;609\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBloise FF, Santos AT, de Brito J, Vieira de Andrade CB, Oliveira TS, Pereira de Souza AF, Fontes KN, Silva JD, Blanco N, Silva PL, Macedo Rocco PR, Fliers E, Boelen A, da-Silva WS (2020) T.M. Ortiga-Carvalho, Sepsis Impairs Thyroid Hormone Signaling and Mitochondrial Function in the Mouse Diaphragm, Thyroid, 30 1079\u0026ndash;1090\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJude B, Tissier F, Dubourg A, Droguet M, Castel T, Leon K, Giroux-Metges M-A, Pennec J-P (2020) TGF-β Pathway Inhibition Protects the Diaphragm From Sepsis-Induced Wasting and Weakness in Rat, Shock, 53 772\u0026ndash;778\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFujimura N, Sumita S, Aimono M, Masuda Y, Shichinohe Y, Narimatsu E, Namiki A (2000) Effect of free radical scavengers on diaphragmatic contractility in septic peritonitis. Am J Respir Crit Care Med 162:2159\u0026ndash;2165\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePowers SK, Hudson MB, Nelson WB, Talbert EE, Min K, Szeto HH, Kavazis AN, Smuder AJ (2011) Mitochondria-targeted antioxidants protect against mechanical ventilation-induced diaphragm weakness. Crit Care Med 39:1749\u0026ndash;1759\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim R, Hashimoto A, Markosyan N, Tyurin VA, Tyurina YY, Kar G, Fu S, Sehgal M, Garcia-Gerique L, Kossenkov A, Gebregziabher BA, Tobias JW, Hicks K, Halpin RA, Cvetesic N, Deng H, Donthireddy L, Greenberg A, Nam B, Vonderheide RH, Nefedova Y, Kagan VE, Gabrilovich DI (2022) Ferroptosis of tumour neutrophils causes immune suppression in cancer. Nature 612:338\u0026ndash;346\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTuo Q-z, Liu Y, Xiang Z, Yan H-F, Zou T, Shu Y, Ding X-l, Zou J-j, Xu S, Tang F, Gong Y-q, Li X-l, Yang Z-z, Li W-j, Zhang S-t, Ayton S, Bush AI, Xu H, Dai L, Dong B, Lei P (2022) Thrombin induces ACSL4-dependent ferroptosis during cerebral ischemia/reperfusion, Signal Transduction and Targeted Therapy, 7 59\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRyan SK, Zelic M, Han Y, Teeple E, Chen L, Sadeghi M, Shankara S, Guo L, Li C, Pontarelli F, Jensen EH, Comer AL, Kumar D, Zhang M, Gans J, Zhang B, Proto JD, Saleh J, Dodge JC, Savova V, Rajpal D, Ofengeim D, Hammond TR (2023) Microglia ferroptosis is regulated by SEC24B and contributes to neurodegeneration. Nat Neurosci 26:12\u0026ndash;26\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang H, Liu J, Zhou Y, Qu M, Wang Y, Guo K, Shen R, Sun Z, Cata JP, Yang S, Chen W, Miao C (2022) Neutrophil extracellular traps mediate m6A modification and regulates sepsis-associated acute lung injury by activating ferroptosis in alveolar epithelial cells. Int J Biol Sci 18:3337\u0026ndash;3357\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang J, Zheng Y, Wang Y, Wang J, Sang A, Song X, Li X (2022) YAP1 alleviates sepsis-induced acute lung injury via inhibiting ferritinophagy-mediated ferroptosis. Front Immunol 13:884362\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J, Zhu Q, Wang Y, Peng J, Shao L, Li X (2022) Irisin protects against sepsis-associated encephalopathy by suppressing ferroptosis via activation of the Nrf2/GPX4 signal axis. Free Radic Biol Med 187:171\u0026ndash;184\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQiongyue Z, Xin Y, Meng P, Sulin M, Yanlin W, Xinyi L, Xuemin S (2022) Post-treatment With Irisin Attenuates Acute Kidney Injury in Sepsis Mice Through Anti-Ferroptosis via the SIRT1/Nrf2 Pathway. Front Pharmacol 13:857067\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShe H, Tan L, Du Y, Zhou Y, Guo N, Zhang J, Du Y, Wang Y, Wu Z, Ma C, Li Q, Mao Q, Hu Y, Liu L, Li T (2023) VDAC2 malonylation participates in sepsis-induced myocardial dysfunction via mitochondrial-related ferroptosis. Int J Biol Sci 19:3143\u0026ndash;3158\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLe Neindre A, Wormser J, Luperto M, Bruel C, Misset B, Bouhemad B, Philippart F (2023) Diaphragm function in patients with sepsis and septic shock: A longitudinal ultrasound study. Australian Crit Care 36:239\u0026ndash;246\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDemoule A, Molinari N, Jung B, Prodanovic H, Chanques G, Matecki S, Mayaux J, Similowski T, Jaber S (2016) Patterns of diaphragm function in critically ill patients receiving prolonged mechanical ventilation: a prospective longitudinal study. Ann Intensiv Care 6:75\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLecronier M, Jung B, Molinari N, Pinot J, Similowski T, Jaber S, Demoule A, Dres M (2022) Severe but reversible impaired diaphragm function in septic mechanically ventilated patients. Ann Intensiv Care 12:34\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi N, Wang W, Zhou H, Wu Q, Duan M, Liu C, Wu H, Deng W, Shen D, Tang Q (2020) Ferritinophagy-mediated ferroptosis is involved in sepsis-induced cardiac injury. Free Radic Biol Med 160:303\u0026ndash;318\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J, Yang S, Jing G, Wang Q, Zeng C, Song X, Li X (2023) Inhibition of ferroptosis protects sepsis-associated encephalopathy. Cytokine 161:156078\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie Z, Xu M, Xie J, Liu T, Xu X, Gao W, Li Z, Bai X, Liu X (2022) Inhibition of Ferroptosis Attenuates Glutamate Excitotoxicity and Nuclear Autophagy in a CLP Septic Mouse Model, Shock, 57 694\u0026ndash;702\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEsper AM, Martin GS (2009) Extending international sepsis epidemiology: the impact of organ dysfunction. Crit Care 13:120\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFang X, Wang H, Han D, Xie E, Yang X, Wei J, Gu S, Gao F, Zhu N, Yin X, Cheng Q, Zhang P, Dai W, Chen J, Yang F, Yang H-T, Linkermann A, Gu W, Min J, Wang F (2019) Ferroptosis as a target for protection against cardiomyopathy. Proc Natl Acad Sci USA 116:2672\u0026ndash;2680\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChan S, Lian Q, Chen M-P, Jiang D, Ho JTK, Cheung Y-F, Chan GC-F (2018) Deferiprone inhibits iron overload-induced tissue factor bearing endothelial microparticle generation by inhibition oxidative stress induced mitochondrial injury, and apoptosis. Toxicol Appl Pharmcol 338:148\u0026ndash;158\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Q, Wu J, Zhang X, Wu X, Zhao Y, Ren J (2021) Iron homeostasis and disorders revisited in the sepsis. Free Radic Biol Med 165:1\u0026ndash;13\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark E, Chung SW (2019) ROS-mediated autophagy increases intracellular iron levels and ferroptosis by ferritin and transferrin receptor regulation. Cell Death Dis 10:822\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eManley S, Ni H-M, Williams JA, Kong B, DiTacchio L, Guo G, Ding W-X (2014) Farnesoid X receptor regulates forkhead Box O3a activation in ethanol-induced autophagy and hepatotoxicity. Redox Biol 2:991\u0026ndash;1002\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBernardo VS, Torres FF, Da Silva DGH (2023) FoxO3 and oxidative stress: a multifaceted role in cellular adaptation. J Mol Medicine-Jmm 101:83\u0026ndash;99\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhong S, Chen W, Wang B, Gao C, Liu X, Song Y, Qi H, Liu H, Wu T, Wang R, Chen B (2023) Energy stress modulation of AMPK/FoxO3 signaling inhibits mitochondria-associated ferroptosis. Redox Biol 63:102760\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBagayoko S, Meunier E (2022) Emerging roles of ferroptosis in infectious diseases. FEBS J 289:7869\u0026ndash;7890\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDivangahi M, Demoule A, Danialou G, Yahiaoui L, Bao W, Xing Z, Petrof BJ (2007) Impact of IL-10 on diaphragmatic cytokine expression and contractility during Pseudomonas Infection. Am J Respir Cell Mol Biol 36:504\u0026ndash;512\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRittirsch D, Huber-Lang MS, Flierl MA, Ward PA (2009) Immunodesign of experimental sepsis by cecal ligation and puncture. Nat Protoc 4:31\u0026ndash;36\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoombes SA, Cauraugh JH, Janelle CM (2006) Emotion and movement: Activation of defensive circuitry alters the magnitude of a sustained muscle contraction. Neurosci Lett 396:192\u0026ndash;196\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":"Sepsis-induced diaphragm dysfunction, Ferroptosis, Oxidative stress, Mitochondrial dysfunction, FOXO3/IL-10 axis","lastPublishedDoi":"10.21203/rs.3.rs-4539738/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4539738/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSepsis can lead to diaphragm dysfunction and atrophy known as sepsis-induced diaphragm dysfunction (SIDD), a major cause of mortality in the ICU. Our present study aimed to investigate whether ferroptosis is implicated in the pathogenesis of SIDD and the underlying molecular mechanism. The results demonstrated that in both in vivo and in vitro septic models, indicators such as the oxygen consumption rate (OCR), extracellular acidification rate (ECAR), reactive oxygen species (ROS), and complex I-V levels, alongside Transmission Electron Microscope (TEM) imaging, revealed mitochondria-associated changes. These alterations were mitigated by the ferroptosis inhibitor Ferrostatin (Fer-1), confirming that ferroptosis\u0026mdash;a mitochondria-linked form of programmed cell death, plays a crucial role in SIDD. Through RNA sequencing (RNA-seq), transposase-accessible chromatin sequencing (ATAC-seq), and Dual-Luciferase Reporter Assay, we found that the FOXO3/IL-10 axis was suppressed in septic mice yet can be reactivated through administration of Fer-1. Furthermore, overexpression of FOXO3 shielded the diaphragm against sepsis-induced ferroptosis by boosting IL-10 production and enhancing the expression of Nrf2-mediated antioxidative genes such as GPX4. This reduced lipid peroxidation and concurrently ameliorated mitochondrial damage. Therefore, activating FOXO3 or administering IL-10 could offer a promising approach for treating SIDD.\u003c/p\u003e","manuscriptTitle":"Up-regulated FOXO3/IL-10 Axis Inhibits Mitochondria-Associated Ferroptosis in Sepsis-Induced Diaphragm Dysfunction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-12 18:38:00","doi":"10.21203/rs.3.rs-4539738/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":"dea75083-ce4b-4662-a7a3-2efc53809cba","owner":[],"postedDate":"July 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-15T22:17:28+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-12 18:38:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4539738","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4539738","identity":"rs-4539738","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}