Cimifugin suppresses type 2 airway inflammation by binding to SPR and regulating its protein expression in a non-enzymatic manner

Cimifugin is one of the main bioactive components of Yu-Ping-Feng-San, a well-known traditional Chinese medicine, which can effectively relieve Allergic asthma (AA) and atopic dermatitis and reduce recurrence in clinic. However, the underlying mechanism of cimifugin on AA is still unknown. In the present study, we aimed to investigate the effect and mechanism of cimifugin on AA. In vivo and in vitro experimental studies were performed. The effect of cimifugin on AA was demonstrated in vivo and in vitro. Sepiapterin reductase (SPR) was predicted as the most potent target of cimifugin in treating AA by reverse docking. Molecular docking and microscale thermophoresis (MST) were used to analyze the direct binding between cimifugin and SPR. Overexpression and interference of SPR were performed to verify whether targeting SPR is a key step of cimifugin in the treatment of AA. QM385, an inhibitor of SPR, was administrated in vivo and in vitro to evaluate the role of SPR in AA. Further, HPLC and cell-free direct hSPR enzyme activity assay were performed to research whether cimifugin regulated SPR by influencing the enzyme activity. Simultaneously, the inhibitors of protein degradation were used in vitro to explore the mechanism of cimifugin on SPR. We found cimifugin effectively alleviated AA by reducing airway hyperresponsiveness, inhibiting type 2 cytokines-mediated airway inflammation, and restoring the expression of epithelial barrier proteins. Molecular docking predicted the direct binding ability of cimifugin to SPR, which was further verified by MST. Notably, the therapeutic effect of cimifugin on AA was dampened with SPR interfering, in contrast, the phenotypic features of AA were significantly alleviated with QM385 application both in vivo and in vitro. Interestingly, cimifugin showed no effect on the enzyme activity of SPR, as the level of its substrate sepiapterin was not affected with cimifugin treatment by cell-free enzyme activity assay. Furthermore, we found cimifugin could reduce SPR protein expression without affecting its mRNA expression probably through autophagosome pathway. To our knowledge, we're reporting for the first time that cimifugin can suppresses type 2 airway inflammation to alleviate AA by directly binding to SPR and regulating its protein expression in a non-enzymatic manner. [Display omitted]