Esculetin rebalances <scp>M1</scp>/<scp>M2</scp> macrophage polarization to treat sepsis‐induced acute lung injury through regulating metabolic reprogramming

Abstract

Sepsis-induced acute lung injury (SALI) is characterized by a high incidence and mortality rate, which has caused a serious medical burden. The pharmacological effects of esculetin (ELT), such as antibacterial and anti-inflammatory actions, have been widely confirmed. However, the therapeutic effects and mechanisms of ELT on SALI still need to be further clarified. In this study, we first evaluated the therapeutic potential of ELT on a caecal ligation and puncture (CLP) induced septic rat model, particularly in the treatment of acute lung injury. Afterwards, we explored the effect of ELT on macrophage polarization in vivo and in vitro. Then, we investigated the anti-inflammatory mechanism of ELT based on modulating the metabolic reprogramming of macrophage (the effect on glycolysis in M1, and the effect on fatty acid β-oxidation in M2). In addition, macrophage metabolic inhibitors (glycolysis inhibitor: 2-DG, and fatty acid β-oxidation inhibitor: etomoxir) were used to verify the regulatory effect of ELT on macrophage metabolic reprogramming. Our results proved that ELT intervention could effectively improve the survival rate of SALI rats and ameliorate pathological injury. Next, we found that ELT intervention inhibited M1 polarization and promoted M2 polarization of macrophages in vivo and in vitro, including the downregulation of M1-related markers (CD86, iNOS), the decrease of pro-inflammatory factors (nitric oxide, IL-1β, IL-6, and TNF-α), the upregulation of M2-related markers (CD206, ARG-1), the increase of immunomodulatory factors (IL-4 and IL-10). Subsequently, seahorse analysis showed that ELT intervention inhibited the glycolytic capacity in M1, and promoted the ability of fatty acid β-oxidation in M2. Besides, ELT intervention inhibited the level of glycolysis product (lactic acid), and the expression of glycolysis-related genes (Glut1, Hk2, Pfkfb1, Pkm and Ldha) and promoted the expression of fatty acid β-oxidation related genes (Cpt1a, Cpt2, Acox1). In addition, we found that the inhibitory effect of ELT on M1 polarization was comparable to that of 2-DG, while intervention with etomoxir abolished the promoting effect of ELT on M2 polarization. ELT inhibited the inflammatory response in SALI by correcting macrophage polarization (inhibiting M1 and promoting M2). The mechanism of ELT on macrophage polarization was associated with regulating metabolic reprogramming (inhibiting glycolysis in M1 and promoting fatty acid β-oxidation in M2).