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, Tgf beta, 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-kappa B p65, while also inhibiting the nuclear translocation of p65, leading to the suppression of transcription of pro -inflammatory genes initiated by NF-kappa B. Collectively, our data identified and quantified the key chemicals of EW and provide insights into the optimization of the herbal composition for neuroprotection.

Automated summary

Ethnomedicine Eerdun Wurile (EW) can significantly promote poststroke neuro-recovery through modulation of microglia polarization. Alantolactone (Ala) and dehydrodiisoeugenol (Deh) were identified as the active anti-inflammatory components of EW. They downregulated the expression of pro-inflammatory genes and upregulated the expression of anti-inflammatory genes in microglia. Furthermore, they enhanced the proliferation of N2a cells and promoted neurite outgrowth through the upregulation of specific genes. The mechanism of action involves the downregulation of NF-kappa B p65 expression and inhibition of its nuclear translocation.