Apelin-13 Attenuates Lipopolysaccharide-Induced Inflammatory Responses and Acute Lung Injury by Regulating PFKFB3-Driven Glycolysis Induced by NOX4-Dependent ROS

Purpose: Acute lung injury (ALI) is a life-threatening condition with limited therapeutic options. Macrophage inflammation plays a key role in the development of ALI. Abnormal glycolysis of macrophages contributes to the inflammatory response. However, the role of macrophage glycolysis in ALI still requires investigation. Apelin-13 has been shown to protect against ALI, whereas the underlying mechanisms remain unclear. In this study, we explored the effect of apelin-13 on lipopolysaccharide (LPS)-induced inflammation and ALI via regulation of glycolysis by modulating redox homeostasis in macrophages. Methods: Serums from 34 patients with sepsis and 13 healthy volunteers were analyzed. In vivo, the protective effect of apelin-13 against LPS-induced ALI was evaluated using a mouse model of LPS-induced ALI. In vitro, mouse bone marrow macrophages (BMDMs) were pretreated with the antioxidant, NADPH oxidase (NOX) 4 (NOX4) small-interfering RNA (siRNA), the 6-phosphofructo-2 -kinase/fructose- 2,6-biphosphatase 3 (PFKFB3) siRNA, or the PFKFB3 overexpression plasmid before exposure to LPS. Results: Serum apelin-13 levels were significantly elevated in patients with sepsis and sepsis-associated acute respiratory distress syndrome (ARDS) ( P < 0.0001). In vivo, apelin-13 suppressed LPS-induced ALI and inflammatory cytokine production ( P < 0.05). Furthermore, apelin-13 reduced hydrogen peroxide (H 2 O 2 ) content, NOX4 protein levels, and glycolysis. In vitro, LPS stimulation elevated NOX4 protein levels and reactive oxygen species (ROS) production ( P < 0.05). These changes resulted in the accumulation of glycolysis in BMDMs. Treatment with antioxidant or NOX4 siRNA inhibited LPS-induced glycolysis and inflammatory cytokine production ( P < 0.05). Moreover, in vitro experiments revealed that PFKFB3 regulates the release of pro-inflammatory cytokines by modulating glycolysis. In contrast, the action of apelin-13 opposed the effects of LPS. Conclusion: In conclusion, apelin-13 protects against LPS-induced inflammatory responses and ALI by regulating PFKFB3-driven glycolysis induced by NOX4-dependent ROS. Keywords: acute lung injury, inflammation, mice, apelin-13, NADPH oxidase 4, glycolysis, PFKFB3