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, Tgfß, 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.

Bioactive components of ethnomedicine Eerdun Wurile regulate the transcription of pro-inflammatory cytokines in microglia.

ETHNOPHARMACOLOGICAL RELEVANCE: The traditional Mongolian medicine Eerdun Wurile (EW) has remarkable neural recovery effect, and has been playing a key role in the clinical treatment of neurological disorders including ischemic stroke in Inner Mongolia Autonomous Region of China. The preliminary pharmacological studies in animal suggested that EW regulates the expression of trophic factors in brain lesion and may also balance the polarization of activated microglia (Gaowa et al., 2018). AIM OF THE STUDY: The pool of leading bioactive chemicals underlying the therapeutic effects of EW has not been identified. Therefore, the mechanism of action of EW is poorly understood. This study was aimed to identify the major group of compounds that contribute to the inhibition of neuroinflammation during stroke recovery through regulation of microglia polarization. MATERIALS AND METHODS: The extracts of EW in different solvents were evaluated for their inhibitory ability of cytokine (IP-10) expression in LPS stimulated BV2 cells. The most effective extract (of petroleum ether extract) was further separated to 18 fractionations on a semi-preparative HPLC column, which were assess for the IP-10 down-regulation efficiency by RT-qPCR. The potent isolate was further fractionated in 12 fractions, which showed fewer peaks. The fraction 6 from this isolates, which remarkably down-regulates cytokines expression including IP-10, TNFα and IL-1ß, was analyzed on UPLC-qTOF MS. The key chemicals were measured for their cytokine inhibition in BV2 cells and mouse primary microglia. RESULTS: After two consecutive fractionating by preparative HPLC, petroleum ether extraction of EW gave 12 fractions with relatively distinctive chromatograms. A particular fraction (fraction 6) preserved the inhibitory effects on expression of pro-inflammatory cytokines including IP-10, TNFα, IL-1ß and iNOS. The result of UPLC-qTOF MS analysis showed that the fraction contains 21 chemicals including costunolide, alantolactone, myristicin and linolenic acid, which significantly down-regulate the expression of key pro-inflammatory cytokines in LPS stimulated BV2 cells as well as mouse primary microglia. CONCLUSION: Collectively our data suggest that the bioactive chemical pool which is responsible for the therapeutic effects of EW can be extracted in petroleum ether, and fractionated to a relatively small multiple components. Such components include known anti-inflammatory chemicals, which may contribute to the possible microglia polarization in brain lesion during the recovery of ischemic stroke.