| Objective:1.To observe the clinical efficacy of AST in the treatment of BA airway inflammation and the correlation analysis of type Ⅱ innate lymphoid cells(ILC2s%)in peripheral blood of BA patients;2.Through HPLC,UPLC-Q-TOF-MS,network pharmacology prediction and analysis of AST drug components and action targets,as well as animal experiment verification,to explore the mechanism of AST in the treatment of BA.Methods:1.A total of 70 patients diagnosed with BA from the Acupuncture and Moxibustion Rehabilitation Department and Respiratory Department of Jiangsu Province Hospital of Chinese Medicine in 2020-2021 were included in this study.The patients were divided into Xinbike group(n=35)and Xinbike+AST group(n=35).Xinbike group was given budesonide and formoterol fumarate powder for inhalation,Xinbike+AST group was given AST on the basis of budesonide and formoterol fumarate powder for inhalation,the total course of treatment was 6 weeks,at the end of the treatment,2 cases dropped out in Xinbike+AST group and 1 cases dropped out in Xinbike group.(1)Before treatment and at the end of treatment,34 cases of BA in the Xinbike group and 33 cases of BA in the Xinbike+AST group were compared in terms of pulmonary function related indicators and Asthma Control Test(ACT);(2)According to the patients’ wishes,BA patients in 2021 were divided into 14 cases of Xinbike group and 14 cases of Xinbike+AST group,flow cytometry was used to analyze the peripheral blood ILC2s%level.(1)The correlation between peripheral blood ILC2s%and clinical data of BA patients before treatment was analyzed;(2)Before treatment and at the end of treatment,the absolute values of peripheral blood eosinophils(EOS),exhaled nitric oxide(FeNO),pulmonary function-related indicators,BA control level,BA Control questionnaire-7(ACQ-7)score and peripheral blood ILC2s%were analyzed;2.Traditional Chinese Medicine Systems Pharmacology Data Base and Analysis Platform TCMSP)searched for the five most important components of AST drug prescription:Mustard seed,Corydoid Yanhusuo,Gansui,Asarum and Ginger,selected their chemical components,and established a self-built database.HPLC was used to identify the chemical components of AST core components as quality control.The chemical components of AST drugs were detected by UPLC-Q-TOF-MS,and analyzed in the self-built database.TCMSP and Swiss Target Prediction were used to screen the chemical components of AST drug.Meanwhile,targets of BA airway inflammation,airway hyperresponse and airway remodeling were collected in GeneCards.The core target is obtained by the intersection of drug action target and BA mechanism target,and PPI,GO and KEGG signal pathway are analyzed by STRING and DAVID.Cytoscape3.7.2 software is used to draw the network of "TCM-chemical composition-target-disease" and perform visualization analysis.To sum up,the possible mechanism and signaling pathway of AST treatment of BA were speculated;3.The mouse model of BA induced by House dust mite(HDM)was established,and the mechanism and signal pathway of AST in treating BA were verified by lung histopathology,lung function detection,Western blot(WB)and Flow cytometry.Results:1.(1)Comparing the clinical efficacy of 34 cases of Xinbike group and 33 cases of Xinbike+AST group:①Before and after treatment:1)in the Xinbike group ACT score improved(P<0.05),but had no significant improvement in several indexes of pulmonary function(P>0.05);2)in Xinbike+AST group ACT score(P<0.001)significantly decreased,and FEV1%(P<0.05)and MMEF75/25(P<0.05)significantly improved.②At the end of the treatment,the comparison between the two groups showed that the AST could improve the FVC%(P<0.05)and the ACT score(P<0.01)in BA patients on the basis of the treatment of the Xinbike,with statistical significance;(2)Comparing the clinical efficacy of 14 cases of Xinbike group and 14 cases of Xinbike+AST group:①ILC2s%in peripheral blood was positively correlated with the absolute value of EOS(r=-0.706,P<0.001)and FeNO(r=-0.971,P<0.001);②Before and after treatment,the two groups were compared within the group.It was found that 1)in the Xinbike group,the FeNO level decreased(P<0.05),the ACQ-7 score improved(P<0.01),and the levels of ILC2s%(P<0.01)in peripheral blood decreased,but there was no significant improvement in BA control level and lung function(P>0.05);2)In Xinbike+AST group,the control level of BA patients increased(P<0.05),the FeNO level decreased(P<0.05),the ACQ-7 score improved(P<0.05),and the levels of ILC2s%(P<0.01)in peripheral blood decreased;③Compared between the two groups at the end of treatment,the BA control level of the Xinbike+ AST group was better than that of the Xinbike group(P<0.05),but there was no significant difference in other clinical indicators and peripheral blood ILC2s%(P>0.05);2.HPLC analysis showed that sinapine thiocyanate,tetrahydropalmatine,asarcin,6-gingerol and euphol could be used as the main chemical components for AST drug quality control.UPLC-Q-TOF-MS was combined with self-built database analysis to obtain 41 chemical components in AST drug composition,and 162 potential targets were obtained by screening of drug and BA mechanism action targets,and the first 53 were selected as key targets according to Degree value.The network analysis of "TCM-chemical components-target-disease" was used to obtain the key chemical components such as Salutaridine,erucinic acid,dihydrocapsaicin,6-gingerol,and the key targets such as SRC and PIK3CA.KEGG analysis showed that the signaling pathways of AST in BA treatment may mainly revolve around PI3K-AKT,ErbB,Sphingolipid,etc;3.(1)Lung function:Compared with normal group,Penh level in model group was significantly increased,with statistical difference(P<0.001),while Penh level in AST group was not significantly different(P>0.05).Compared with model group,Penh level of AST group was significantly decreased,with statistical difference(P<0.01).(2)Related protein expression:Compared with normal group,the expression of P-Akt and PI3K protein in lung tissues of mice in model group was significantly increased,with statistical significance(P<0.001),and the expression of PTEN protein was significantly down-regulated,with statistical significance(P<0.001),while the expression of AKT protein was not significantly different(P>0.05).Compared with model group,the expression of P-Akt and PI3K protein in AST group was significantly down-regulated(P<0.01),the expression of PTEN protein was significantly increased(P<0.001),and there was no significant difference in the expression of AKT protein(P>0.05).(3)ILC2s:Compared with the normal group,the level of ILC2s in the lung tissues of mice in the model group was significantly increased,with statistical significance(P<0.001);Compared with the model group,the level of ILC2s in lung tissues of mice in AST group was significantly decreased,with statistical significance(P<0.01).Conclusion:1.(1)There was a positive correlation between the ILC2s%in peripheral blood of BA patients and the absolute value of FeNO and EOS in peripheral blood,suggesting that there was a close relationship between ILC2s%and BA airway inflammation;(2)AST combined with Xinbike and Xinbike treatment can effectively improve FeNO,peripheral blood ILC2s%and ACQ-7 score in BA patients;(3)AST combined with Xinbike is superior to Xinbike alone in improving lung function,improving ACT score and BA control level;2.HPLC analysis showed that sinapine thiocyanate,tetrahydropalmatine,asarcin,6-gingerol and euphol can be used as the main chemical components for AST drug quality control;3.UPLC-Q-TOF-MS combined with network pharmacological showed salutaridine,erucinic acid,dihydrocapsaicin,6-gingerol may be the key chemical components of AST in the treatment of BA,and were mainly derived from white mustard seed,gansui and ginger;SRC,PIK3CA were key targets;The signaling pathways of AST treatment on BA may mainly revolve around PI3K-AKT;4.The results were verified by animal experiments:the mechanism of AST therapy on BA may be through downregulation of PI3K-AKT signaling pathway and inhibition of ILC2s differentiation. |
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