Analysis and identification of key anti-inflammatory molecules in Eerdun Wurile and exploration of their mechanism of action in microglia

Ethnomedicine Eerdun Wurile (EW) can significantly promote poststroke neuro-recovery through modulation of microglia polarization. Fraction 4–6 (F4-6) isolated from EW via serial fractionation inhibits the expression of pro-inflammatory genes in LPS stimulated microglia. However, the key active molecules of F4-6 have not been identified. Herein, we identified alantolactone (Ala) and dehydrodiisoeugenol (Deh) as the active anti-inflammatory components of F4-6 by UPLC-qTof MS analysis. We confirmed that, F4-6, Ala, Deh and mixture of Ala and Deh (Mix) downregulate the expression of several pro-inflammatory genes including Ccl2 , Cox2 and Il6 in LPS-treated microglia in a similar pattern. At the same time upregulate the expression of anti-inflammatory genes including Hmox1 , Tgfβ , Igf1 and Creb1 . Moreover, the conditioned culture media obtained from F4-6 treated microglia significantly enhanced proliferation of N2a cells, and promoted neurite outgrowth possibly through upregulation of Nefh and Dlg4 . Mechanistically, F4-6 strongly downregulated the expression of NF-κB p65, while also inhibiting the nuclear translocation of p65, leading to the suppression of transcription of pro-inflammatory genes initiated by NF-κB. Collectively, our data identified and quantified the key chemicals of EW and provide insights into the optimization of the herbal composition for neuroprotection.