Liver plays a key role in sepsis, a systemic inflammatory response syndrome caused by infection. Ferroptosis is involved in sepsis-induced liver injury. We aimed to assess the changes in ferroptosis in cecal ligation and puncture (CLP)-induced septic mice, and determine the role of lipocalin-2 (LCN2) in liver ferroptosis. CLP was used to induce sepsis in mice. The morphological changes in liver tissues and mitochondrial structure were observed using hematoxylin and eosin staining and transmission electron microscopy. The levels of serum alanine transaminase, aspartate aminotransferase, superoxide dismutase, and malondialdehyde were detected using the corresponding kits. The changes of reactive oxygen species level in liver tissues were detected using dihydroethidium as a fluorescence probe. LCN2, cysteine-glutamate reverse transport system, and dihydroorotate dehydrogenase protein levels in the liver were detected by western blotting. The ferroptosis inhibitor ferrostatin-1 (Fer-1), iron chelator dexrazoxane (DXZ), iron-dextran, and LCN2 knockdown studies were performed to determine role of ferroptosis and LCN2 in liver injury during sepsis. Ferroptosis levels increased in the liver tissues of CLP-induced septic mice. Both Fer-1 and DXZ suppressed ferroptosis and attenuated liver injury following sepsis challenge, whereas iron-dextran increased ferroptosis and liver injury in mice with sepsis. LCN2 knockdown suppressed ferroptosis and reduced oxidative stress in the liver. Ferroptosis inhibition attenuates septic liver injury. LCN2 knockdown alleviates sepsis-induced liver injury by inhibiting ferroptosis and reducing oxidative stress.

To investigate the role of knockdown of lipocalin-2 (LCN2) on DHODH mediated mitochondrial ferroptosis in sepsis-induced myocardial injury (SIMI) in mouse and cardiomyocyte injury model, and investigate the likely mechanism of STAT3. In this study, we established an sepsis induced cardiomyopathy (SIMI) model using cecal ligation and puncture (CLP) in mice. Mouse cardiac samples were used to identify the association between the serum lipocalin 2 (LCN2) level and spesis progression. Chemical inhibition of ferroptosis were conducted to illustrate the effect of ferroptosis upon SIMI.Then, mouse models with knockdown of LCN2 were used to ascertain the role of LCN2 upon ferroptosis and spesis. To interaction mechanism between LCN2 and DHODH was investigated. we overexpressed DHODH in mouse HL-1 cardiomyocytes using lentivirus, and established a lipopolysaccharide (LPS)-induced cardiomyocyte injury model. The results showed that in septic mice, the increase of LCN2 was correlated positively with myocardial severity, glutathione peroxidase 4 (GPX4) and dihydroorotate dehydrogenase (DHODH) mediated ferroptosis both occurred in SIMI. Inhibiting ferroptosis reduced LCN2 expression, thereby alleviated SIMI. Molecular docking and immunoprecipitation revealed strong binding between LCN2 and DHODH. Knockdown of LCN2 inhibited DHODH mediated ferroptosis, decreased phosphorylated STAT3 (p-STAT3) protein expression level, improved mitochondrial function, and mitigated SIMI. Meanwhile, exogenous recombinant LCN2 protein induced mouse cardiomyocyte ferroptosis. Overexpression of DHODH protected against LPS-induced cardiomyocyte injury. Inhibition of DHODH abolished knockdown-LCN2 induced decrease of p-STAT3 protein expression. However, after inhibition of mitochondrial GPX4 expression by Fin56, p-STAT3 protein was not changed. In summary, our study confirmed knockdown of LCN2 inhibited DHODH mediated ferroptosis in SIMI, inhibiting DHODH attenuated the protection of knockdown-LCN2 through inhibiting STAT3 phosphorylation. It provides the new insight for preventing sepsis-induced myocardial injury.

This study aimed to investigate the regulatory effect of lipocalin-2 (LCN2) knockdown on DHODH-mediated mitochondrial ferroptosis in sepsis-induced myocardial injury (SIMI) and clarify the underlying STAT3-related molecular mechanism in a cecal ligation and puncture (CLP) mouse model and lipopolysaccharide (LPS)-stimulated HL-1 cardiomyocyte injury model. We established a classic cecal ligation and puncture (CLP)-induced SIMI mouse model. Pharmacological suppression of ferroptosis was performed to confirm the pathogenic role of ferroptosis in SIMI progression. Subsequently, LCN2-knockdown mice were utilized to explore the biological function of LCN2 in modulating myocardial ferroptosis and septic cardiac injury, and the direct protein interaction between LCN2 and DHODH was verified via molecular docking and co-immunoprecipitation assays. In vitro, we constructed stable DHODH-overexpressing HL-1 cardiomyocytes via lentiviral transfection and established a lipopolysaccharide (LPS)-induced cardiomyocyte injury model. The results showed that LCN2 expression was markedly upregulated in SIMI. Both GPX4- and DHODH-dependent mitochondrial ferroptosis were significantly activated during SIMI. Lentivirus-mediated DHODH overexpression exerted prominent protective effects against LPS-induced cardiomyocyte injury. Importantly, pharmacological blockade of DHODH by Brequinar reversed the reduction of p-STAT3 induced by LCN2 silencing. However, Fin56-mediated specific inhibition of mitochondrial GPX4 exhibited no significant effect on STAT3 phosphorylation level, revealing distinct regulatory mechanisms for these two pathways. In summary, LCN2 knockdown inhibits DHODH-mediated mitochondrial ferroptosis to alleviate SIMI. Pharmacological inhibition of DHODH abrogates the cardioprotective effect of LCN2 knockdown by restoring STAT3 phosphorylation. These findings provide novel insights into the prevention and treatment of sepsis-induced myocardial injury.