Nicotinamide mononucleotide mitigates hyperoxia-aggravated septic lung injury via the GPx4-mediated anti-ferroptosis signaling pathway in alveolar epithelial cells

Background

The molecular mechanisms and optimal treatment strategies underlying hyperoxia-aggravated septic lung injury remain elusive. We explored the effects and mechanisms of nicotinamide mononucleotide (NMN) on hyperoxia-aggravated septic lung injury.

Methods

The rat and cellular models of sepsis-induced lung injury were established and subjected to hyperoxygenation treatment, followed by treatment with NMN, ferroptosis promoter, or inhibitor separately. The extent of lung injury was assessed based on histological examination, lung histological injury scores, wet/dry weight ratio of lung tissues, oxygenation indexes, TNF-ɑ and IL-6 levels, and cell viability. Meanwhile, ferroptosis was assessed through various methods. The levels of glutathione peroxidase 4 (GPx4) and 4-hydroxynonenal (4-HNE) in lung tissues were determined by immunohistochemistry, while iron deposition was evaluated using Prussian blue staining. Fe²⁺, MDA, and GSH levels were also detected with the respective kits. The reactive oxygen species (ROS) level was measured by flow cytometry and immunofluorescence techniques. The protein and mRNA levels of GPx4 and ACSL4 were also detected. The relationship between sirtuin 6 (SIRT6) and GPx4 was clarified by using SIRT6 inhibitor and activator, as well as in combination with sh-GPx4.

Results

Hyperoxia exacerbated lung injury in rats subjected to cecal ligation and puncture (CLP). Hyperoxia also intensified damage to alveolar epithelial cells (AECs) in a lipopolysaccharide (LPS) model. However, NMN ameliorated these detrimental effects. Furthermore, LPS + Hyperoxia treatment significantly upregulated Fe²⁺, MDA, ROS, and ACSL4 levels, exacerbating oxidative damage. Also, LPS + Hyperoxia treatment downregulated GSH and GPx4 levels, thereby reducing antioxidant capacity. Additionally, Erastin, a ferroptosis promoter, further intensified oxidative stress damage and inflammatory response. However, ferroptosis inhibitor Fer-1 alleviated this damage. Similarly, NMN inhibited ferroptosis in hyperoxia-aggravated septic lung injury. Co-treatment with NMN and sh-GPx4 reversed the protective effect of NMN against LPS-stimulated injury exacerbated by hyperoxia in AECs. NMN supplementation increased SIRT6 expression in AECs. SIRT6 inhibition decreased GPx4 expression and raised ferroptosis markers, while SIRT6 activation had opposite effects. Co-treatment with SIRT6 activator and sh-GPx4 reversed the inhibitory effect on ferroptosis.

Conclusion

Hyperoxia aggravates septic lung injury by inducing ferroptosis of AECs. NMN can mitigate hyperoxia-aggravated septic lung injury by up-regulating GPx4 through increasing SIRT6 and inhibiting ferroptosis of AECs.