Investigation of anti-asthmatic constituents and mechanisms of Cimicifugae Rhizoma based on LC-MS analysis, network pharmacology and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Cimicifugae Rhizoma was used for the treatment of asthma in traditional Chinese medicine. The triterpenoid partition of Cimicifugae Rhizoma, named as 'Ximingting', is commercially used for perimenopausal syndrome. However, the anti-asthma constituents and mechanism of Cimicifugae Rhizoma, as well as the therapeutic effect of 'Ximingting' on asthma remain unknown.

AIM OF THE STUDY: This study aims to illustrate anti-asthma constituents of Cimicifugae Rhizoma and 'Ximingting', and explore the underlying molecular mechanisms.

MATERIALS AND METHODS: The ethyl acetate fraction of Cimicifugae Rhizoma extract (EAEC) was prepared accroding to the manufacturing process of 'Ximingting'. The chemical composition of EAEC were analyzed by UPLC-MS/MS. An ovalbumin (OVA)-induced asthma mouse model was used for evaluating the bioassay in vivo. Network pharmacology was adopted for predicting anti-asthmatic targets/pathways, validated by molecular docking, ELISA, Western blot, qRT-PCR, immunofluorescence, and flow cytometry.

RESULTS: Fifty-eight constituents (mainly triterpenoids) were identified in EAEC. EAEC significantly attenuated OVA-induced airway inflammatory infiltration, reduced inflammatory cytokines, and restored Th1/Th2 balance in mice. Network pharmacology indicated that anti-asthmatic effect of EAEC was related to inflammation, oxidative stress, and T-cell differentiation. Further experiments demonstrated that EAEC activated Keap1-Nrf2 signaling to enhance antioxidant capacity, and inhibited STAT6 phosphorylation and GATA3 expression, thereby blocking CD4+ T cell differentiation into Th2 cells.

CONCLUSION: Triterpenoids in Cimicifugae Rhizoma exert anti-asthmatic effects by activating Keap1-Nrf2 pathway against oxidative stress and regulating STAT6/GATA3 pathway to balance immunity. These findings reveal the anti-asthmatic mechanism of Cimicifugae Rhizoma and suggest the potential of 'Ximingting' for further anti-asthmatic investigation.