Ethanol extract of Liriodendron tulipifera leaves displays anti-inflammatory activity by suppressing the Syk/Src/NF-κB pathway

Ethnopharmacological relevance: Liriodendron tulipifera L. (family Magnoliaceae), native to North America, has traditionally been used to treat inflammation-related diseases, such as rheumatism, catarrh, stomachache, dysentery, or headache. Historical records also suggest its strong vermifuge activity against parasites. Aim of the study: Although L. tulipifera has been reported to exhibit notable anti-inflammatory activity, its underlying molecular mechanisms remain unclear. This study aimed to comprehensively evaluate the anti-inflammatory effects of an ethanol extract of L. tulipifera (Lt-EE) through in vitro and in vivo experiments and to elucidate its molecular mechanisms and target proteins. Materials and methods: RAW264.7 cells and HEK293T cells were used to analyze cellular activities and determine the molecular targets of Lt-EE. Griess reagent assays were used to evaluate the influence of Lt-EE on nitric oxide (NO) generation. In addition, western blotting was conducted to analyze the in vitro effects of Lt-EE at the protein level while real-time polymerase chain reaction (PCR) and reverse transcription PCR were used to assess the in vitro effects of Lt-EE at the transcript level. Luciferase reporter assays and cellular thermal shift assays (CETSAs) were employed to confirm the molecular target(s) of Lt-EE. To evaluate the in vivo effects, Lt-EE was administered at 50 and 150 mg/kg intraperitoneally in an LPS-induced peritonitis model, and at 100 and 150 mg/kg orally in an HCl/EtOH-induced acute gastritis mouse model. Results: Lt-EE was not cytotoxic but effectively suppressed the production of NO. mRNA expression levels of the pro-inflammatory cytokines iNOS, IL-6, and IL-1β were decreased by Lt-EE in a concentration-dependent manner. Moreover, the overall pathway of Lt-EE was revealed by western blotting. Through CETSA, the targets of Lt-EE were confirmed to be Syk and Src. Lt-EE significantly ameliorated inflammation in both LPS-induced peritonitis and HCl/EtOH-induced gastritis models. Conclusions: An ethanol extract of L. tulipifera leaves showed significant anti-inflammatory effects in vitro and in vivo by inhibiting Syk and Src and the NF-κB signaling pathway. These results indicate that Lt-EE is a potent anti-inflammatory agent.