Heat stroke (HS) is a severe condition associated with prolonged exposure to high temperatures, leading to systemic inflammation and multi-organ damage. Disruption of the intestinal barrier plays a crucial role in HS progression by allowing endotoxins to enter circulation and trigger widespread inflammation. Lactoferrin (LF), known for its immune-modulating and barrier-protective properties, shows promise in mitigating HS-related damage. This study investigated the protective effects of LF on the intestinal barrier under HS conditions using in vitro and in vivo models. In Caco-2 cell monolayers exposed to heat stress, LF improved barrier integrity by increasing transepithelial electrical resistance (TEER) and maintaining tight junctions. In mice, LF supplementation enhanced heat tolerance, delayed HS onset, and reduced multi-organ damage, whereas LF knockout (KO) mice exhibited exacerbated damage. Further analysis revealed that heat stress induced ferroptosis by suppressing GPX4 and SLC7A11, contributing to intestinal injury. LF alleviated ferroptosis by upregulating these proteins and reducing Fe²⁺ accumulation, reactive oxygen species (ROS) generation, and lipid peroxidation. Additionally, LF inhibited heat stress-induced activation of the MAPK pathway by suppressing the phosphorylation of p38, ERK, and JNK, suggesting that MAPK signaling mediates its protective effects. These findings suggest that LF supplementation may enhance heat tolerance and prevent HS-induced intestinal barrier damage through ferroptosis inhibition and MAPK pathway regulation.

Introduction: Immunosuppression is the main cause of the high mortality rate in patients with sepsis. The decrease in the number and dysfunction of CD4⁺ T lymphocytes is crucial to the immunosuppressed state of sepsis, in turn affecting the development and prognosis of sepsis. Autophagy has been shown to play an important role in the immune imbalance exhibited during sepsis.Methods: In this study, we modulate the expression of miR-223 in CD4⁺ T lymphocytes, via the transfection of a mimic or an inhibitor of miR-223 to establish cell models of miR-223 overexpression and knockdown, respectively. Levels of autophagy were monitored using a double-labeled lentivirus (mRFP-GFP-LC3) and electron microscopy, and western blot analysis was used to estimate the levels of autophagy-related proteins and FOXO1 in the two cell models after co-treatment with lipopolysaccharide (LPS) and siRNA against FOXO1.Results: We found that when the expression of miR-223 increased, FOXO1 expression decreased and autophagy decreased; whereas, when FOXO1 expression was inhibited, autophagy decreased significantly in different cell models after LPS induction.Conclusion: Thus, this study proved that miR-223 participate in the regulation of LPS-induced autophagy via the regulation of FOXO1 expression in CD4⁺ T lymphocytes which shed a new light for the diagnosis and treatment of sepsis.

Introduction: Excessive oxidative burst and dysregulated neutrophil extracellular trap (NET) formation contribute to tissue damage in acute lung injury (ALI) and are largely driven by the combined actions of NADPH oxidase 2 (NOX2) and myeloperoxidase (MPO). While lactoferrin (LTF) is a known multifunctional immunomodulatory glycoprotein, its precise role in modulating the NOX2–MPO–NETosis axis in ALI remains undefined. Methods: We employed a time-course model of lipopolysaccharide (LPS)-induced ALI in C57BL/6N mice, combined with quantitative label-free lung proteomics and downstream bioinformatic analyses to map dynamic molecular changes. At the inflammatory peak, aerosolized bovine lactoferrin (bLF) was administered in vivo, and histological lung injury, pulmonary inflammatory cytokine levels, neutrophil infiltration, and markers related to the NOX2–MPO–NETosis axis were evaluated. Results: LPS induced typical ALI pathology that peaked between days 1 and 3 (D1–D3). Proteomic and network analyses consistently highlighted NET formation as a centrally enriched early KEGG pathway and identified LTF as a key protein–protein interaction hub closely connected to p47phox (encoded by Ncf1) and MPO. Evaluation of aerosolized bLF demonstrated significant mitigation of ALI pathology, reducing lung injury, pro-inflammatory cytokines, and neutrophil recruitment. Mechanistically, bLF suppressed NETosis by reducing p47phox and MPO expression and, crucially, diminished p47phox phosphorylation in vivo, consistent with reduced NOX2 activation. Discussion: These findings identify LTF as a critical dynamic regulator of the p47phox–MPO–NETosis axis in LPS-induced ALI. They also highlight bLF as a promising candidate for further translational evaluation and support the rationale for developing bioengineered, lactoferrin-based nanomedicines aimed at modulating innate immunity and mitigating neutrophil-driven lung injury in respiratory infectious diseases.

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