Mahonia bealei (Fort.) Carr. Leaf extract modulates the TLR2/MyD88/NF-κB signaling pathway to inhibit PGN-induced inflammation in RAW264.7 cells

Ethnopharmacological relevance: Mahonia bealei (Fort.) Carr. (M. bealei) is a traditional Chinese medicinal plant and one of the herbs used by the Hmong people. It has been recorded in the most recent editions of the Chinese Pharmacopoeia as well as in Hmong medicinal works such as Hmong Pharmacopoeia, for its ability to clear away heat, dry up dampness, and to remove fire and toxins. Currently, the plant is widely cultivated in China, Japan, Mexico, the United States, and Europe, and it appears to be naturalized in the eastern United States. Our earlier research demonstrated that M. bealei leaf extract (MBE) effectively reduced inflammation in xylene-induced ear swelling in mice and cotton ball granuloma in rats. The unclear specific anti-inflammatory mechanism necessitated further investigation in this study. Aim of the study: The aim of this study was to investigate the mechanism of anti-inflammatory effects of MBE on PGN-stimulated RAW264.7 macrophages through the TLR2/MyD88/NF-kappa B signaling pathway. Methods: Firstly, M. bealei leaf extract (MBE) was obtained by ultrasonic synergistic high-speed homogenization extraction. The main active components in MBE were determined by UPLC-Q-TOF-MS/MS and the interaction of the main components with TLR2 was verified by molecular docking technique. Then, PGN-induced RAW264.7 cells were used to establish an inflammation model, and then the administration concentration of MBE and the modeling concentration of PGN were determined by the CCK-8 method, and the protective effect of MBE on PGNinduced cellular inflammation was evaluated. Meanwhile, the effect of MBE on the morphology of RAW264.7 cells was observed by scanning electron microscopy. The effect of MBE on the focal death of RAW264.7 cells was determined by flow cytometry. Subsequently, NO levels were measured using the Griess method, while PGE2, TNF-alpha, IL-1 beta, IL-6, and IL-10 concentrations were assessed via ELISA. ROS levels were determined by fluorescent staining in each group of cells. The expression levels of TLR2, MyD88, I kappa B, IKK, and NF-kappa B in the TLR2/MyD88/ NF-kappa B pathway were analyzed using RT-qPCR for mRNA and Western blot for protein. Results: Nineteen major components were detected from MBE by UPLC-Q-TOF-MS/MS, and 10 of them with higher relative abundance were selected for molecular docking with TLR2, respectively, and all of them showed low binding energies and good stability. MBE was shown to be effective in ameliorating the PGN-induced inflammatory condition of RAW264.7 cells, as demonstrated by the CCK-8 method, scanning electron microscopy, and flow cytometry. MBE effectively suppressed the release of inflammatory cytokines NO, PGE2, TNF-alpha, IL-1 beta, and IL-6, while enhancing IL-10 production. The RT-qPCR and Western Blot analyses demonstrated that MBE could decreased the mRNA and protein levels of TLR2 and MyD88, suppressed the phosphorylation of I kappa B, NF-kappa B, and IKK, and consequently inhibited the TLR2/MyD88/NF-kappa B signaling pathway. Conclusion: The present study showed that MBE effectively inhibited PGN-induced inflammation and ameliorated cell injury in RAW264.7 cells. This may be attributed to its inhibition of the TLR/MyD88/NF-kappa B pathway, which further regulates the release of downstream related inflammatory factors and exerts anti-inflammatory effects. These findings also suggest the potential of M. bealei as a natural drug for the treatment of inflammation-related diseases.