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. 1A). 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. 1B). Inversely, compared to healthy individuals, increased concentrations of LDH and MDA were measured in the serum samples of patients with sepsis (Fig. 1B). Moreover, the ELISA assay indicated that the levels of total Fe and Fe2+ were significantly increased while no significant change in Fe3+ was observed, implying disturbance in iron metabolism in patients with sepsis (Fig. 1C).
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. 2A). 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. 2A). Interestingly, the combined treatment with LPS and Fer-1 showed the most pronounced reversal of cell viability (p < 0.001, Fig. 2A), 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. 2B) and a notable increase in ACSL4 expression (p < 0.001, Fig. 2C). Decreased GPX4 expression and elevated ACSL4 levels induced by LPS-induced were reverted by the ferroptosis inhibitors (Figs. 2B and 2C). Fer-1 treatment showed the most significant partial reversal of protein expression of GPX4 and ACSL4 (p < 0.001, Figs. 2B and 2C). 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. 3A). 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. 3B). In addition, increased total Fe and Fe2+ contents in the LPS and Erastin groups were recorded compared to the control group, while no remarkable difference in groups was recorded for Fe3+ (Fig. 3C). Moreover, Fer-1 inhibited the effect of LPS and Erastin on the content of Fe and Fe2+ (Fig. 3C). 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. 3D). Furthermore, the detection of ECAR levels indicated an increase in ECAR levels in LPS and Erastin groups (Fig. 3E). 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. 3E). The levels of mitochondrial complexes I-V were measured to detect the percentage of cell metabolic activity (Fig. 3F). 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. 3F). 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. 3F). C11-BODIPY staining was used to evaluate the level of lipid peroxidation in C2C12 muscle cell (Fig. 3G). 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. 3G). Immunofluorescence analysis revealed decreased protein content of GPX4 in the LPS and Erastin groups compared to the control group (Fig. 3H). Combined treatment with Fer-1 counteracted the effect of LPS and Erastin on the expression of GPX4 (Fig. 3H). 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. 3I). Moreover, the fluorescence probe FerroOrange staining showed increased levels of intracellular free Fe2+ in the LPS and Erastin groups compared to the control group, but Fer-1 reversed these trends (Fig. 3J). 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. 3K). 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. 3L). Furthermore, CFDA-SE labeling was performed to analyze cell viability in the different treatment groups (Fig. 3M). Compared to the control group, a significant decrease in cell viability in the LPS group was observed (Fig. 3M). 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. 3M). We also analyzed the gene expression levels of NRF2, GPX4, ACSL4, IL-10, and FOXO3 after exposing the cells to various treatments (Fig. 3N 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. 3N 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. 3N 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. 3O 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. 4A), and performed a series of experiments. As shown in Fig. 4B, 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. 4B). Assays were performed to detect the levels of Myo and CK-MB, as well as biochemical markers MDA and GSH in the muscle (Fig. 4C). The levels of Myo, CK-MB, and MDA was significantly increased whereas GSH was markedly decreased in the CLP group (Fig. 4C). Interestingly, Fer-1 treatment counteracted the effect of CLP (Fig. 4C). 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 S1A). Moreover, ELISA assay results revealed that CLP significantly increased the levels of Total Fe and Fe2+ in the muscle (Fig. 4D) and serum (Figure S1B) of mice, and these effects were inhibited by Fer-1 treatment. Next, oxygen consumption rate (OCR) was measured (Fig. 4E) and the results showed that CLP markedly decreased OCR levels, while Fer-1 treatment counteracted this effect (Fig. 4E). The measurement of ECAR also indicated CLP markedly decreased ECAR level, but this effect was counteracted by Fer-1 treatment (Fig. 4F). 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. 4G). In the CLP + Fer-1 group, diaphragm muscle tissue showed improvements in histopathological features compared to the CLP group (Fig. 4G). These improvements included reduced inflammatory cell infiltration, less disruption of muscle fibers, decreased interstitial edema, and smaller necrotic areas (Fig. 4H). TEM examination was used to examine the ultrastructural changes in diaphragm muscle tissue (Fig. 4H). 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. 4H). 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. 4H). Real-time PCR analysis was performed to measure changes in gene expression levels of GPX4, ACSL4 and NRF2 in each group (Fig. 4I). The results showed that GPX4, and NRF2 were downregulated by CLP but Fer-1 treatment reversely upregulated the expression of these genes (Fig. 4I). Inverse trends were observed for ACSL4 mRNA expression (Fig. 4I). Western blot analysis was used to measure changes in the protein content of GPX4, ACSL4, NRF2 and P-NRF2 in each group (Fig. 4J). 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. 4J). Furthermore, the results of the immunofluorescence assay indicated that CLP significantly decreased the expression of GPX4 (Figure S1C) and α-SMA (Figure S1D) while increased expression of ACSL4 was observed (Figure S1E); 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. 5A) shows the differentially expressed genes among the three groups. Figure 5B 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. 5C 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. 5C). Furthermore, KEGG pathway enrichment analysis of differentially expressed genes among the two comparisons was performed, and the results are depicted in Fig. 5D. 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. 5D). 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 6A displays profiles ranked by the significance of the number of genes assigned compared to expected, while Fig. 6B 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. 6A and 6B). The gene expression changes were illustrated in Fig. 6C, while the PPI network of the Fer-1-target genes in CLP was showcased in Fig. 6D. The PPI network indicated strong interactions among the Fer-1-target genes in CLP (Fig. 6D). Lastly, we identified the hub Fer-1-target genes in CLP using MCODE (Fig. 6E). The results indicated that 29 genes could be considered as hub genes, among which figured IL10 and other immune- and inflammation-related genes (Fig. 6E).
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. 7A). In each category, we noticed variations in the way genes were distributed across different chromatin states (Fig. 7B). In addition, we conducted GO (Fig. 7C) and KEGG pathway analyses (Fig. 7D). 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. 7C). 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. 7C). 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. 8A). The quadrant plot showed differential gene expression in the four groups: control, CLP model, and CLP + Fer-1 (Fig. 8A). The Venn diagrams of highly and lowly expressed genes in the first (Fig. 8B) and fourth (Fig. 8C) 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. 8D), and hub Fer-1-target genes in CLP (Fig. 8E) 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. 8F and 8G).
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. 9A and Fig. 10A).
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. 9B and Fig. 10B. 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. 9C and 10C. 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. 9D and 10F).
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. 10D).
The FerroOrange staining was performed to assess the Fe2+ alterations in C2C12 muscle cells in different experimental group. The LPS group displayed a significant increase in FerroOrange fluorescence, suggesting significantly higher levels of Fe2+. 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 Fe2+. In similar manner, the administration of Fer-1 reversed LPS-induced increase of Fe2+ 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 Fe2+ (Fig. 10E).
ROS was conducted in C2C12 muscle cells within various experimental groups (Fig. 10G). 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. 10G).
Furthermore, the CFDA-SE staining was done to observe the viability of C2C12 muscle cells in different experimental groups (Fig. 10H). 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. 10H).
RT-PCR and western blotting were performed to evaluate mRNA expression levels (Fig. 9E and Fig. 10I) and protein levels (Fig. 9F and Fig. 10J) 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. 9E and Fig. 10I) and (Fig. 9F and Fig. 10J).
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.