The Effect And Mechanism Of Yangyin Bufei Prescription On Relieving Cough And Asthma And Expectoration By CAMP/PKA/AQP1 Signalling Pathway

Objective:Under the guidance of traditional Chinese medicine theory,the effects and molecular mechanisms of Yangyin bufei prescription(YYBFP)in cough,asthma and expectorant were discussed based on the analysis of cAMP/PKA/AQP1 signaling pathway.The grey relation analysis was used to elaborate the pharmacodynamic substance basis of YYBFP for cough,asthma and expectorant,and provide scientific basis for its clinical application.Methods:1.Single factor experiment and L9(3~4)orthogonal experiment were used to optimize the extraction process of YYBFP.The quality standard of YYBFP was established by HPLC and the characteristic map of YYBFP was established.2.YYBFP Chinese traditional medicine active ingredients and corresponding target gene information were screened,the target genes related to lung dryness and body fluid injury were collected,the active constituents-target-disease(C-T-D)network was constructed,the target protein interaction(PPI)network map was constructed,and GO enrichment analysis and KEGG pathway analysis were performed.Gene mapping predicted the expression of related genes in different organs and carried out molecular simulation of core components and key target proteins to predict the binding energy of major core components and key target proteins,identify critical pathways and targets for subsequent validation.3.The state of mice was observed and recorded,and the growth curve was drawn;The antitussive and antiasthmatic-relieving effects of YYBFP were verified by ammonia induced cough experiment and histamine phosphate induced asthma experiment;The effect of YYBFP on promoting phenol-red excretion was verified by phenol-red excretion experiment.The pathological conditions of trachea and lung were observed by HE staining;The coefficients of lung and spleen organs were measured;The immunoregulatory effects of YYBFP on mice were detected by NK killing and eluviation experiments;The levels of iNOS and IFN-γin serum of mice were detected by Elisa kit,and the anti-inflammatory and vasodilatory effects of YYBFP were observed;The contents of SOD and MDA in serum of mice were detected by the kit,and the antioxidant effect of YYBFP was observed;The effect of YYBFP on the expression of MMP-9 in mouse lung was detected by immunohistochemistry;The protein expression levels of cAMP/PKA/AQP1 signaling pathway were detected by Western blot analysis,which confirmed the efficacy of YYBFP in promoting water and liquid transport in the body;Based on grey relation analysis,the pharmacodynamic material basis of YYBFP was studied,the spectral effect relationship between the active ingredients in YYBFP and serum biochemical indexes was established,and the pharmacodynamic material basis of YYBFP for relieving cough,relieving asthma and removing phlegm was explored.Results:1.Through single factor experiment and L9(3~4)orthogonal experiment,the optimal extraction process of YYBFP was determined as the solid-liquid ratio of 1:10,extraction time of 1.5 h,and extraction for 3 times.The quality standard of YYBFP is established,and the feature map of YYBFP is constructed.2.There were 22 potentially active ingredients in YYBFP,involving 436 gene targets.According to the results of square solution and network pharmacology,5-HMF,chlorogenic acid,adenosine and rutin were finally selected as the characteristic components of YYBFP,and the cAMP/PKA/AQP1 signaling pathway predicted by KEGG enrichment analysis was selected as the key pathway for subsequent verification.Gene localization and molecular simulation predicted that all components of YYBFP had strong binding ability with key proteins,and SOD and iNOS were selected as the key targets for subsequent verification,and MDA and IFN-γwere selected as the key indicators for subsequent verification.3.In pharmacodynamic experiments,compared with Model,YYBFP could prolong the cough latency of mice and reduce the number of coughs within 3 min(P<0.05,P<0.05);The incubation period was prolonged,the number of wheezing within 6 min was decreased(P<0.05,P<0.05),and the amount of phenol red excretion in respiratory tract was increased(P<0.05);The tissue morphology of trachea and lung of model mice was improved;The killing rate of NK cells in spleen of mice was increased(P<0.05),the content of IFN-γin serum of mice was increased(P<0.05),and the content of iNOS was decreased(P<0.05).The content of SOD in serum of mice was increased and MDA content was decreased(P<0.05,P<0.05),which could relieve cough by correcting the disturbance of oxidation balance system in vivo.Decreased the expression of MMP-9 in lung tissue of model mice(P<0.05),alleviated the alveolar injury of mice,and played a role in relieving cough and asthma.YYBFP could promote the expression of PKA and AQP1 proteins(P<0.05),increase the expression of cAMP in serum of mice(P<0.001),and improve the body’s water transport through cAMP/PKA/AQP1 signaling pathway,thus playing an expectory role.YYBFP was proved to be effective in relieving cough,relieving asthma and removing phlegm,and the correlation between characteristic components and serum biochemical indexes was obtained by grey relational analysis,among which the correlation degrees of adenosine with SOD,MDA and IFN-γwere 0.758,0.679 and 0.725,respectively.The correlation degree of chlorogenic acid and 5-HMF with iNOS was 0.640 and 0.621,respectively,indicating that YYBFP has regulatory effect on its corresponding key targets.Conclusion:The effects and mechanism of YYBFP on relieving cough,relieving asthma and expelling phlegm were studied in this paper.The effect of YYBFP on relieving cough,relieving asthma and expelling phlegm was confirmed.Secondly,the effect of YYBFP on lung dryness and body fluid injury was studied by network pharmacology and molecular simulation theory.At the same time,the quality standard of YYBFP is established,and the feature map of YYBFP is constructed.The grey relational analysis method was used to construct the spectral relationship between the active ingredients in YYBFP and serum biochemical indexes,and the basis of YYBFP for cough,asthma and expectorant was explored.Finally,the action mechanism of YYBFP in relieving cough,relieving asthma and expelling phlegm was discussed,which proved that YYBFP can play a synergistic role through multi-components and multi-targets,providing scientific reference for its clinical application in traditional Chinese medicine.