Background: Sepsis-induced intestinal injury plays a pivotal role in the progression of sepsis and multiple organ dysfunction syndrome (MODS). This study aimed to investigate the protective effects of Esketamine (ESK) on sepsis-induced intestinal injury, focusing on the modulation of inflammation, oxidative stress, and macrophage polarization via the Toll-like receptor (TLR)4/NF-κB signaling pathway. Additionally, in vitro studies were conducted to further elucidate the molecular mechanisms underlying ESK's effects on macrophage polarization.

Methods: A rat model of sepsis was established using the cecal ligation and puncture (CLP) method. ESK was administered at doses of 30 mg/kg and 60 mg/kg. Serum levels of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) were measured using ELISA, and histopathological analyses were performed on intestinal tissues to evaluate injury. TLR4/NF-κB pathway markers were assessed using Western blotting and qPCR analyses. In vitro, RAW 264.7 macrophages were treated with lipopolysaccharide (LPS) to induce sepsis-like conditions, followed by ESK treatment. Immunofluorescence staining and flow cytometry were used to evaluate macrophage polarization, focusing on M1 markers (CD86, iNOS) and M2 markers (Arg1, CD206).

Results: ESK treatment significantly ameliorated sepsis-induced intestinal injury in vivo. It reduced serum levels of pro-inflammatory cytokines while increasing the anti-inflammatory cytokine IL-10 compared to the CLP rats (p < 0.05). ESK also promoted M2 macrophage polarization (increased Arg1 and CD206 expression) and inhibited M1 macrophage polarization (decreased CD86 and iNOS expression) compared to the CLP rats (p < 0.05). In vitro, ESK demonstrated similar effects in LPS-stimulated RAW 264.7 cells, modulating macrophage polarization by inhibiting the TLR4/NF-κB signaling pathway compared to the LPS-stimulated cells (p < 0.05). Co-treatment with a TLR4 agonist abrogated these effects, confirming the involvement of TLR4 in the mechanism of action of ESK.

Conclusion: ESK protects against sepsis-induced intestinal injury by modulating macrophage polarization and suppressing inflammation through the TLR4/NF-κB signaling pathway. Both in vivo and in vitro studies demonstrated ESK's ability to shift macrophages towards an anti-inflammatory M2 phenotype, highlighting its potential as a therapeutic agent for sepsis management. This study first investigated the effects of ESK on sepsis-induced intestinal damage and the potential molecular mechanisms involved.