Fig 2: ICA69 contributes to ferroptosis of LPS-induced RAW264.7 and H9C2 cells.A, B Alterations of COX2 and GPX4 levels in RAW264.7 cells after LPS (1 μg/mL for 0 h, 1 h, 3 h, 6 h, 12 h, 24 h) treatment (n = 3). C, D Alterations of COX2 and GPX4 levels in H9C2 myofibroblasts after LPS (1 μg/mL for 0 h, 1 h, 3 h, 6 h, 12 h, 24 h) treatment. Levels of COX2 and GPX4 intensity were normalized to the corresponding GAPDH level, and the changes are shown in the histograms (n = 3). **P < 0.05 vs. 0 h group. E Cell viability was detected by an CCK8 assay in the indicated groups (n = 3). *P < 0.001 vs. Control group, #P < 0.05 vs. LPS group. F MDA levels in RAW264.7 cells stimulated by LPS (1 μg/mL) for 12 h after ICA69 siRNA transfection (n = 3). G The level of SOD in the indicated group RAW264.7 cells (n = 3). H, I The protein levels of COX2 and GPX4 in RAW264.7 cells transfected with ICA69 siRNA and then stimulated by LPS (1 μg/mL) for 12 h. Levels of COX2 and GPX4 intensity were normalized to the corresponding GAPDH level, and the changes are shown in the histograms (n = 3). J, K The mRNA levels of PTGS2 and GPX4 in RAW264.7 cells transfected with ICA69 siRNA and then stimulated by LPS (1 μg/mL) for 12 h (n = 3). The data are expressed as the mean ± standard error of the mean. *P < 0.05 vs. Vehicle + LPS group, **P < 0.05 vs. ICA69 siRNA + LPS group, ns, no significance. L JC-1 staining for mitochondrial membrane potential and cellular morphology under a phase-contrast microscope. (scale bar = 10 μm). M Intracellular ROS level was detected by flow cytometry.

Fig 3: ICA69 deficiency improved the survival and cardiac function of LPS-treated mice and suppressed cardiac inflammation.A Survival curves of ICA69-deficient (KO) and wild-type(WT) mice after LPS challenge. Male ICA69 KO and WT mice at 8 weeks of age were stimulated with LPS or PBS, respectively. The mice were observed for their health status a few times per day for 1 week. Data were analyzed by Kaplan–Meier log-rank test. n = 10 per group. *P < 0.05 vs. Sham + WT group, #P < 0.05 vs. LPS + WT group. B–D The mRNA levels of TNF-α, IL-1β, and IL-6 in myocardial tissues of each group (n = 8/6/6). E, F Effect of ICA69 deficiency on LPS-induced LDH release in cardiac tissue and serum (n = 6/5). G, H Effect of ICA69 deficiency on LPS-induced CK-MB release in cardiac tissue and serum (n = 7/5). I–L Effect of ICA69 deficiency on left ventricle ejection fraction, left ventricle fractional shortening, and heart rate of each group of each group after Sham or LPS injection (n = 7). The data are expressed as the mean ± standard error of the mean. *P < 0.05 vs. LPS group, **P < 0.05 vs. ICA69-KO + LPS group. M Representative images of the morphological analysis and inflammatory cells infiltration as reflected by the H&E staining, and immunohistochemistry staining for CD68 protein. The arrow marks inflammatory cells (n = 3, 10 + fields per heart).

Fig 4: ICA69 affected the expression level of STING.A Representative immunoblots and densitometric analysis of total STING in cardiac tissue of ICA69-deficient (KO) and wild-type (WT) mice after LPS challenge. Levels of total STING intensity were normalized to the corresponding TUBULIN level, and the changes are shown in the histograms (n = 6). B, C The mRNA levels of STING and IFN-β in indicated groups that were stimulated with LPS or PBS for 12 h (n = 6). The data are expressed as the mean ± standard error of the mean.*P < 0.05 vs. LPS group, **P < 0.05 vs. ICA69-KO + LPS group, ns no significance. D The effect of LPS for the alterations of ICA69 and STING interaction in cardiac tissues. Representative western blot bands of ICA69 and STING in cardiac tissue by co-IP (n = 3 biologically independent experiments). E Representative images of immunofluorescence of ICA69 and STING in RAW264.7 cells stimulated by LPS (1 μg/mL) for 12 h. (scale bar = 50 μm).

Fig 5: Increased ICA69 levels were positively correlated with the severity of sepsis.A Correlations of ICA69 expression with Acute Physiologic and Chronic Health Evaluation II scores in the septic patients (r = 0.6682, P < 0.001). Data were analyzed by Spearman correlation test. B–D The mRNA expression of ICA1, STING, and GPX4 were measured in PBMCs of 52 septic patients and 60 healthy controls by qRT-PCR. E–G ICA69, STING, and GPX4 levels were measured in PBMCs of septic patients and healthy controls by western blot. Relative expression was normalized to GAPDH levels. Corresponding histograms of the changes are shown (n = 4). The data are expressed as the mean ± standard error of the mean. **P < 0.05 vs. corresponding healthy controls, ns no significance.