Research The Effect And Mechanism Of Cimifugin On Alleviating Asthma

Allergic asthma is a chronic airway disease characterized by airway hyperresponsiveness,wheezing,coughing and dyspnea.Allergic asthma inflicts more than 350 million people worldwide in the 21st century,which is an urgent problem to be solved.Drugs commonly used clinical for the treatment of asthma,such as glucocorticoids,β2 receptor agonists,leukotriene modulators,sustained-release theophylline,etc.,usually take effect only in the elicitation period,and can not reduce the recurrence of asthma,meanwhile the adverse reactions are serious.Therefore,it is urgent to find effective drugs to treat allergic asthma.Yu-ping-feng-san plays an important role in alleviating allergic asthma and allergic dermatitis,which is widely used for allergic disorders.Previous studies have found that cimifugin,one of the main components of Yu-ping-feng-san,can ingest into the blood and relieve allergic dermatitis effectively.However,its effect on allergic asthma is still unclear.In addition,the reverse molecular docking was performed to predict the targets of cimifugin.The score of Sepiapterin reductase(SPR)was the highest.It has been reported that the inflammatory response mediated by T cells and eosinophils was significantly reduced after the inhibition of SPR.In this case,does SPR play an important role in allergic asthma?Does cimifugin have a regulatory effect on SPR to relieve asthma?Is there a regulatory effect of cimifugin on SPR in relieving asthma?In this study,we explored the effect and mechanism of cimifugin against allergic asthma as follows:1.First of all,to investigate the effect of cimifugin on airway responsiveness,cimifugin(25mg/kg)was administrated in sensitization phase,elicitation phase and the whole course of the mice with asthma induced by house dust mite(HDM).Different concentrations of cimifugin(6.25mg/kg,12.5mg/kg)were given during the elicitation period to investigate the effects of cimifugin on inflammatory cell infiltration and type 2 cytokine expression in asthmatic mice.HBE135-E6E7 cells were stimulated with poly(I:C)and TNF-α to establish cell model in vitro.Different concentrations of cimifugin were given 6 hours before stimulation to investigate the effect of cimifugin on the expression of epithelial derived alarm molecules and tight junctions.The results showed that cimifugin could effectively alleviate the airway hyperresponsiveness in mice at all stages,and the effect of cimifugin in the elicitation phase was most obvious.Cimifugin could reduce the number of eosinophils in blood,decrease the level of IgE in serum,and decrease the expression of IL-5 and IL-13 in bronchoalveolar lavage fluid.HE staining also showed that cimifugin could significantly reduce the thickening of airway and the infiltration of inflammatory cells.Cimifugin could reduce the levels of TSLP and IL-33 in supernatant while restore the expression of occludin.These results suggest that cimifugin can relieve allergic asthma.2.We evaluated the important role of SPR in asthma.To verify the effect of SPR in asthma,QM385,an inhibitor of SPR,was given in different phases of the HDM-induced asthma model.According to the GEO database,we found that the expression profiles(GSE64913,GSE63142,GSE43696,GSE41649)of bronchial epithelial cells from healthy subjects and asthma patients obtained by bronchial brush technology.We defined them in new groups to explore the expression of SPR in epithelial cells.HBE135-E6E7 cells were stimulated with poly(I:C)and TNF-α.The protein and mRNA expression of SPR were detected by Western blot and PCR.Furthermore,HBE135-E6E7 was co-stimulated with 20ng/mL TNF-α and 100μg/mL Poly(I:C)for 24h after being pretreated with QM385(5μM).The expression of TSLP and IL-33 in the supernatant was detected by ELISA and the expression of tight junctions was detected by western blot.The results showed that the expression of SPR protein was significantly increased in the model group.After administration of QM385,the number of eosinophils in blood,the level of IgE in plasma and the levels of IL-4,IL-5 and IL-13 in bronchoalveolar lavage fluid were significantly reduced.The results of HE staining also showed that QM385 could significantly reduce the airway thickening and infiltration of inflammatory cells.GEO showed that SPR was expressed in epithelial cells,but there was no significant change in the gene expression level between healthy people and asthma patients.The expression of SPR protein in epithelial cells was up-regulated by stimulation of poly(I:C)and TNF-α,but the mRNA level did not change significantly.The expression of TSLP and IL-33 in the supernatant was significantly downregulated after inhibition of SPR while the expression of occludin was restored.Above results show that SPR plays a key role in allergic asthma,and the use of SPR inhibitor can alleviate the symptoms of asthma.3.Finally,we tend to explore the mechanism of cimifugin regulating SPR.Schrodinger and MicroScale Thermophoresis(MST)were used to predict or verify the binding of cimifugin with SPR.High-pressure liquid chromatography(HPLC)and Cell-Free Direct hSPR Enzyme Activity Assay were used to detect whether cimifugin could inhibit SPR activity.Furthermore,HBE135-E6E7 was co-stimulated with 20ng/mL TNF-α and 100μg/mL Poly(I:C)for 24h after being pretreated with cimifugin(1μM,10μM)and QM385(5μM).The expression of TSLP and IL-33 in the supernatant was detected by ELISA and the expression of SPR was detected by western blot to explore whether cimifugin affect SPR protein expression.The results showed that,1)There was a direct binding between cimifugin and SPR.The score of cimifugin with SPR was close to that of QM385 with SPR when predicted by Schrodinger,which indicated that there was a direct binding between cimifugin with SPR.Meanwhile,the Kd value of cimifugin with SPR was close to that of QM385,which proved that there was a direct binding between cimifugin with SPR.2)Cimifugin might play an important role through SPR.After co-stimulation,the expression of TSLP and IL-33 were down-regulated when HBE135-E6E7 was interfered with siSPR and treated with cimifugin at the same time.There was no significant change compared with the administration of cimifugin alone or siSPR alone.In addition,the level of TSLP and IL-33 was further up-regulated when HBE135-E6E7 was transfected with SPR overexpression plasmid,compared with the costimulation group.Administration of cimifugin could significantly reduce the level of TSLP and IL-33,suggesting that cimifugin might exert its effect through SPR.3)Cimifugin did not inhibit the enzymatic activity of SPR.HPLC showed that the expression of sepiapterin did not increase in cimifugin treated group.Cell-Free Direct hSPR Enzyme Activity Assay showed the inhibitory rate of cimifugin on SPR enzyme activity was close to zero.4)Cimifugin might down-regulated the expression of SPR.Cimifugn,given in elicitation phase,reduced the expression of SPR in lung of HDM-induced asthma model.Meanwhile,cimifugin reduced SPR expression in HBE135-E6E7.These results suggested that cimifugin might play an important role by directly binding with SPR.However,cimifugin did not inhibit the enzymatic activity of SPR.Therefore,it might work in a nonenzymatic manner.Conclusion:1.Cimifugin can effectively relieve the symptoms of allergic asthma.2.SPR plays a key role in asthma.3.Cimifugin might exert its effect by directly binding with SPR,and its mechanism might be related to the downregulation of SPR expression.