The Study On The Modulatory Action And Molecular Mechahnism Of Pingchuan Granules On Asthmatic Eos Based On Notch And STAT Signaling Pathway

Objective:To evaluate the therapeutic effect of PingChuan Granules(PCG)treatment by using rat asthma model,to explore the target of PCG therapy,and to describe the molecular mechanism of PCG on asthma.To provide theoretical basis for clinical application of PCG.Methods:80 rats were randomly divided into 4 groups:control group,model group,dexamethasone(1 mg/kg)group,and PingChuan Granule(5.4 g/kg)group.Asthma model was constructed by inhalation of ovalbumin and aluminum hydroxide.The treatment intervention was performed in 5th after the last sensitization,once a day.On the day of challenge,treatment was given 1 hour before the challenge.Bronchial alveolar lavage fluid and lung tissue were collected after 24 hours in the last challenge,and relevant indicators were evaluated.In vivo experiments:1.HE staining was used to observe the histopathological changes in the lungs,and the ratios of WAi/Pi,WAm/Pi and N/Pi were quantitatively analyzed by pathological image analysis system.2.Ultrastructure of lung tissue was observed by transmission electron microscopy to evaluate the imaging changes.3.To evaluate EOS count in BALF and blood with automatic five-class blood analyzer.4.The expression of TIgE,OVA sIgE and EOS-related active substances ECP and Eotaxin in serum were detected by ELISA.5.The proteins contents of Notch 1,2,3,4,Jagged1,2,Delta 1,3,4 and Hes on Notch signaling pathway,STAT1,3,4,5,6 and its phosphoprotein on STAT signaling pathway were detected by Western blot in lung tissue.6.According to Western Blot,Real-Time PCR was used to detect the mRNA expression of Notch 1,2,4,Jagged 1,2,Deltal,3,Hes and P-STAT 1,3,4,5,6 in lung tissues.In vitro experiments:1.Separation of EOS in BALF by Percoll discontinuous density gradient separation,and identification of isolated cells by Wright-Giemsa staining.2.The proteins contents of Notch1,2,3,4,Jaggedl,2,Deltal,3,4 and Hes on Notch signaling pathway,STATl,3,4,5,6 and its phosphoprotein on STAT signaling pathway were detected by Western blot in EOS.3.According to Western Blot,Real-Time PCR was used to detect the mRNA expression of Notch 1,2,3,4,Jagged 1,Delta 1,3,4 and Hes and P-STAT 1,3,4,5,6 in EOS.Results:In vivo experiments:1.Lung histopathological observations showed that the vascular lumen of the asthma model group was narrow,with a large number of inflammatory cells infiltrating around the blood vessels and in the lung tissues,the neutrophils and eosinophils were predominant.The basement membrane was thickened,and the smooth muscle of the wall and connective tissue in the outer bronchial layer was extensive hyperplasia.Inflammatory infiltration in the dexamethasone group and PingChuan granule group was alleviated,and the effect is obvious.Image quantitative analysis showed that the ratios of WAi/Pi,WAm/Pi and N/Pi in the model group were significantly higher than control group(P<0.01).The treatment groups obviously reduced the ratios,and the difference was statistically significant(P<0.05,P<0.01).2.Ultrastructure of TEM scanning showed that the number of EOS cells in the model group increased significantly.The mitochondria swollen,hazy blur or even partial loss,vacuolar degeneration phenomenon,the number of lamellar bodies was significantly reduced,and emptying increased.Thebasement membrane thickened,chromatin accumulation.Dexamethasone and PingChuan Granules treatment group can reduce the degree of cell damage,with EOS cell number decreased,mitochondrial vacuolization,phlegm clearer,basement membrane is more complete.3.Blood analyzer analysis showed that the EOS counts in the BALF and blood of model group were significantly increased(P<0.01),and the number of EOS in each treatment intervention group was significantly decreased(P<0.01).4.The results of ELISA showed that the levels of TIgE and OVA sIgE levels in the serum and the EOS activation markers ECP and Eotaxin in the lung tissue on the model group were significantly increased(P<0.01).Compared with the model group,the treatment intervention groups were significantly reduce the content of the above indicators,the difference was significant(P<0.05,P<0.01).5.Western Blot and Real-Time PCR results showed that the protein and mRNA expression of Notch1,Notch2,Notch4 and Jagged1,Jagged2,Hes,P-STAT1,3,5,6 were significantly up-regulated in the model group.The protein and mRNA expressions of Deltal,Delta3 and P-STAT4 were obviously down-regulated in the model group,the difference was significant(P<0.01).Each treatment intervention group improved the expression of the above mRNA and protein in different degrees(P<0.05,P<0.01).In vitro experiments:1.EOS isolated from BALF was identified,and the result of Wright-Giemsa stained was positive,consistent with the morphological characteristics of EOS,so cells were identified as EOS.2.Western Blot results showed that the expression of Notch1,Notch2,Notch4 and Jagged1,Jagged2,Delta4,Hes,P-STAT1,3,5,and 6 were significantly up-regulated and Notch3,Deltal,Delta3 and P-STAT4 were significantly down-regulated in the model group,the difference was significant(P<0.01).Each treatment intervention group improved the expression of the above protein in different degrees(P<0.05,P<0.01).3.The results of Real-Time PCR showed that the mRNA expression of Notch1,Notch2,Notch4 and Jagged1,Delta4,Hes,P-STAT1,3,5,6 were significantly up-regulated in the model group,and the mRNA expression of Notch3,Delta1,Delta3 and P-STAT4 was obviously down-regulated.The difference was significant(P<0.01).Each treatment intervention group improved the expression of the above mRNA in different degrees(P<0.05,P<0.01).Conclusions:1.In the asthma model,the typical effector cells EOS and its active substances were highly expressed and could be used as biomarkers for the evaluation of anti-asthma efficacy.PingChuan Granules can significantly reduce the high expression of EOS.2.PingChuan Granules has a definite therapeutic effect on asthma,and the therapeutic effect is equal to that of dexamethasone.3.PingChuan Granules have the effect in inhibiting asthma,and its molecular mechanism is closely related to its inhibition of the activation of Notch and STAT signaling pathways.4.Pingchuan Granules had a regulatory effect on EOS in the asthma model,and its mechanism was closely related to the down-regulation of Notch and STAT signaling pathways.5.PingChuan Granules has a clear role in the treatment of asthma.The molecular mechanism is to inhibit the expression of Notch and STAT signaling pathways in EOS and reduce EOS content,thereby alleviating asthma symptoms.