Paeoniflorin inhibits APEC-induced inflammation in HD11 cells through the NF-κB signaling pathway by activating CB₂R

Avian pathogenic Escherichia coli (APEC) is a bacterial pathogen that threatens poultry reproduction by inciting systemic inflammation and leading to chicken colibacillosis. The endocannabinoid system (ECS) is an immunomodulator system that regulates inflammatory responses. In this study, we aimed to investigate the anti-inflammatory effect of paeoniflorin on APEC-infected HD11 cells and its underlying mechanism. The results showed that paeoniflorin significantly reduced the expression levels of pro-inflammatory factors (IL-1β, IL-6, TNF-α), M1-type macrophage-associated markers (IL-12, iNOS, CD86), and chemokines (CXCL8, CXCL12, CCL1, CCL5, CCL17). Additionally, paeoniflorin significantly reduced the expression of MAGL and restored that of DAGL and CB₂R, thereby activating the ECS. Furthermore, we found that paeoniflorin and CB₂R exhibited stable conformations through molecular docking and molecular dynamics simulations. The addition of the CB₂R inhibitor AM630 notably diminished paeoniflorin's inhibitory effects on the phosphorylation levels of proteins in the NF-κB signaling pathway and on inflammatory responses. These results indicate that the anti-inflammatory effects of paeoniflorin on APEC-induced HD11 cells may be mediated by the inhibition of the NF-κB signaling pathway through the activation of CB₂R. This work has the potential to provide a new agent for the control and prevention of chicken colibacillosis, as well as contribute to modern research in traditional Chinese medicine.