Study On The Effect Of Qingfei Huatan Decoction On The Metaplasia Of Clara Cells To Goblet Cells In Asthmatic Mice Based On Notch Signaling Pathway

ObjectivesTo explore the mechanism of Qingfei Huatan Decoction in the treatment of asthmatic mice;to prove that Qingfei Huatan Decoction can treat asthma by inhibiting the metaplasia of Clara cells to goblet cells and inhibiting the hypersecretion of mucus.MethodsIn experiment 1:60 C57BL/6J mice were randomly divided into Control group,model group,Qingfei Huatan Decoction 10.5,21.0,42.0 g/kg groups and ambroxol hydrochloride group,10 mice in each group.On the 1st day and 8th day,except for the Control group,mice were intraperitoneally injected with 0.5 mL of OVA suspension(containing 100 μg OVA,200 μg aluminum hydroxide),nebulized on the 15th-21st days,and administered 1 hour after the end of the nebulization,and the last administration 24 hours after the drug,some mice were tested by small animals pulmonary function analyzer to detect the Penh of the mice with lung function;blood was collected,and blood routine was performed;some mice were killed immediately,and lung tissue was collected for observation by hematoxylin-eosin(HE)staining.The degree of inflammation and pathological changes in mouse lung tissue,Masson staining was used to detect airway remodeling,AB-PAS staining was used to detect the distribution of airway mucus,and Real time RT-qPCR was used to detect Clara cell secretory protein(Ccsp)and Muc5ac mRNA expression in mice lung tissue.Western blot was used to detect CCSP and MUC5AC protein expression in mice lung tissue.In experiment 2:90 C57BL/6J mice were randomly divided into Control group,DAPT group,VPA group,OVA group,OVA+DAPT group,OVA+VPA group,Qingfei Huatan Decoction group,OVA+DAPT+Qingfei Huatan Decoction group and OVA+VPA+Qingfei Huatan Decoction group,10 mice in each group.On the 1st and 8th day,except the Control group,DAPT group and VPA group,mice were intraperitoneally injected with 0.5 mL OVA suspension(containing 100 μg OVA,200μg aluminum hydroxide),and from the 9th day to the 15th days,the DAPT group,OVA+DAPT group and OVA+DAPT+Qingfei Huatan Decoction group were intraperitoneally injected with DAPT.VPA group,OVA+VPA group and OVA+VPA+Qingfei Huatan Decoction group were intraperitoneally injected with VPA.The mice were administered 1 hour after the end of each nebulization,and 24 hours after the last administration,the mice were sacrificed,and the lung tissue was collected,and HE staining was performed to observe the inflammation degree and pathological changes of the mice lung tissue.Hes1,Ccsp and Muc5ac mRNA expression,Western blot detection of Notch1,Hes1,CCSP and MUC5AC protein expression in mice lung tissue.ResultsIn experiment 1:Compared with the Control group,the enhanced Pause in the model group was significantly increased;the content of eosinophils in the blood was significantly increased;the lung tissue morphology and alveolar wall were thickened;the collagen deposition content was significantly increased;The mucus content in the distal airway increased;the expression of Muc5ac mRNA in lung tissue was significantly increased,and the expression of Ccsp mRNA was significantly decreased;the expression of MUC5AC protein in lung tissue was significantly up-regulated,and the expression of CCSP protein was significantly down-regulated.Compared with the model group,the enhanced Pause of the mice in each administration group was significantly reduced;the content of eosinophils in the blood of Qingfei Huatan Decoction 21.0 and 42.0 g/kg was significantly reduced;the lung tissue morphology and alveolar wall became thinner;Qingfei Huatan Decoction 10.5,21.0,42.0 g/kg significantly decreased collagen deposition content;Qingfei Huatan Decoction 10.5,21.0,42.0 g/kg distal airway mucus content decreased;Qingfei Huatan Decoction 21.0,42.0 g/kg The expression of Muc5ac mRNA in lung tissue was significantly decreased,and the expression of Ccsp mRNA in Qingfei Huatan Decoction 10.5,21.0,and 42.0 g/kg was significantly increased;Qingfei Huatan Decoction 42.0 g/kg CCSP protein expression was significantly down-regulated.In experiment 2:Compared with Control group,mRNA expression of Muc5ac,Notch1 and Hes1 in OVA group were significantly increased,and Ccsp mRNA expression was significantly decreased.The expression of MUC5AC,Notch1 and Hes1 protein was significantly up-regulated,while the expression of CCSP protein was significantly down-regulated.Alveolar wall became thicker and the number of inflammatory cells increased significantly.Compared with OVA group,mRNA expression of Notch1,Hes1 and Muc5ac in OVA+DAPT group were significantly decreased,and Ccsp mRNA expression was significantly increased.The protein expression of Notchl,Hes1 and MUC5AC was significantly down-regulated,and the protein expression of CCSP was significantly up-regulated.The alveolar wall became thinner and the number of inflammatory cells decreased significantly.The mRNA expression of Notch1 and Hes1 in OVA+VPA group was significantly increased,and the mRNA expression of Ccsp was significantly decreased.The protein contents of MUC5AC,Notch1 and Hes1 were significantly up-regulated,while the protein content of CCSP was significantly down-regulated.Alveolar wall became thicker and the number of inflammatory cells increased significantly.Compared with OVA+Qingfehuatan Decoction group,the expression of Notch1 mRNA in OVA+DAPT+Qingfehuatan Decoction group was significantly decreased,and the expression of Ccsp mRNA was significantly increased.The protein content of Hes1 was significantly down-regulated.The alveolar wall became thinner and the number of inflammatory cells decreased significantly.In OVA+VPA+Qingfei Huatan Decoction group,the mRNA expression of Muc5ac,Notch1 and Hes1 were significantly increased,while the mRNA expression of Ccsp were significantly decreased.The protein contents of MUC5AC,Notchl and Hes1 were significantly up-regulated,while the protein content of CCSP was significantly down-regulated.Alveolar wall became thicker and the number of inflammatory cells increased significantly.ConclusionQingfei Huatan Decoction may alleviate asthma by inhibiting Notchl signaling pathway,inhibiting Clara cells to goblet cells metaplasia and reducing mucus hypersecretion.