Network Pharmacology-based Study On Action Mechanism Of YPF Powder Treating For Asthma

Objective:This study aimed to explore the action mechanism of YPF Powder treating for asthma by network pharmacology and perform further verification by cell experiment and animal experiment.Methods:1.Detailed steps for analyzing of the action mechanism of YPF Powder treating for asthma by network pharmacology were as follows:The chemical compounds of Astragalus membranaceus,atractylodes,Windproof which OB ≥30%or DL≥0.18 were obtained in TCMP Database.The compounds of the three drugs were combined and repeated compounds were deleted.The Corresponding target of each component was acquired in TCMSP database.The Corresponding gene name of each target protein was obtained in UniProt database and non-human target proteins were excluded.Finally,the target protein regulating by YPF Powder was acpuired.Asthma-related human genes were searched on NCBI Gene database.Then,the target genes of YPF Powder and asthma were mapped.The interaction protein of mapped gene was obtained through the String interaction database and the protein interaction network was constructed.Molecular functional analysis and biological process analysis of the interaction genes were carried out using Cytoscape’s BiNGO analysis tool,and then molecular function classification of hierarchical network and biological process classification of hierarchical network of the interaction genes were constructed.The pathway enrichment of the interaction protein was Used the DAVID database,and broad signaling pathways were eliminated.The first five pathways were extracted and the matching genes of the NOD-like receptor pathway which was ranked first were constructed the interaction network to further excavate the specific mechanism of Yupingfeng powder treating asthma.2.To explore the effect of YPFS on NLRP3 inflammasomes in Macrophages based on network pharmacology analysis results.Protocols were as follows:The inflammatory models of U937 cells were induced by LPS,and control group,model group(LPS,100 ng/ml)and YPFS group(YPFS,25 μg/ml)were set up.After CCK-8 assay was used to evaluate the viability of U937 cells.Cell suspensions with 1 ×106 cells were plated into 6-well cell culture plate with the volume of 2 mL/well.U937 cells were pre-treated with PMA(10 ng/ml)for 48 hours to induce macrophages.Then,cells were stimulated with LPS(100 ng/ml)for 48 h with or without the indicated concentration(25 μg/ml)of YPFS treatment for 2 h.The levels of IL-1β、TNF-α and IL-6 in cell serum were evaluated by ELISA.The mRNA and protein levels of IL-1β,NLRP3,Caspase-1 and ASC were assessed by real-time PCR and Western blot,respectively.3.The effect of YPF powder on NLRP3 inflammasome was further confirmed by animal experiment.Specific experimental steps are as follows:Asthma model was induced by OVA-sensitized Balb/c mice and all the mice was divided into 4 groups:normal group,dexamethasone group(1 mg/kg/d)and YPF powder group(13 g/kg/d),each group contains 10 mice.From the start of the atomization period,half an hour before each atomization,the YPF powder intragastric administration or dexamethasone injection was carried out for 12 days.The weight of the mice was recorded every 2 days and the general condition of the mice in the atomization period was observed.After treatment,the serum of mice was collected and the levels of IL-1β,TNF-a and IL-6 were detected by ELISA.The upper lobe of the left lung was collected and the histopathological changes of the lung tissue were observed by HE staining and the mucus secretion in the bronchus was observed by AB-PAS staining.The expression levels of IL-1β,NLRP3,Caspase-1 and ASC mRNA and protein on the upper lobe of the right lung were detected by Real-time PCR and Western Blot respectively.Results:1.Network pharmacology analysis results:A total of 372 human target proteins(31 of Astragalus membranaceus,39 of atractylodes and 120 of Windproof)which OB≥30%or DL≥0.18 and 793 asthma-related human genes were obtained.The in vivo reaction network that YPF powder against asthma was build through String database.Molecular function classification of hierarchical network and biological process classification of hierarchical network of the interaction genes were constructed using Cytoscape’s BiNGO analysis tool.It was found that YPF powder can treat asthma by regulating molecular functions including molecular transduction activity,enzyme moderator activity,binding,antioxidant activity and biological process including response to stimulate,immune system process,biological regulation,metabolic regulation,signal process.DAVID database pathway enrichment analysis showed that the top five pathways of YPF powder regulating asthma were NOD-like receptor signaling pathway,TNF signal pathway,PI3K-AKT signal pathway,HIF-1 signal pathway and NF-kB signal pathway.Using the String database to visualize the matching gene of the NOD-like receptor pathway showed that the NLRP3 inflammasome may play an important role in the process that YPF powder regulating the asthma.2.Ex vivo test results:Our results showed that YPFS didn’t exhibit obvious cytotoxicity at the concentration of 3.125,6.25 12.5 and 25 p.g/ml(P>0.05).Subsequently,25 μg/ml YPFS was used for the further experiments.We found that cytokine production in supernatant were all significantly increased in the LPS stimulation group compared to the control group(P<0.01).YPFS was able to interfere expressions of IL-1β、IL-6 and TNF-αcytokine production compared to the model group(P<0.01).Further,LPS stimulation significantly increased IL-1β,NLRP3,caspase-1 and ASC expression both at mRNA and protein level(P<0.01).YPFS significantly inhibit the mRNA and protein expression of NLRP3 inflammasome components as well as the IL-1β compared to model group(P<0.05 or P<0.01).3.Animal experiment results:changes in body weight showed that YPF powder can effectively slow down the weight loss rate of mice(P<0.01).The lung tissue inflammation score and mucus score showed that YPF powder significantly reduced inflammatory cell infiltration and mucus secretion in the mice(P<0.01).The results of ELISA showed that the levels of IL-1β,TNF-α and IL-6 in serum of asthmatic mice were prominently decreased(P<0.01).The results of Real-time PCR showed that the expression of IL-1β,NLRP3,Caspase-1 and ASC in lung tissue of mice was dramaticlly decreased(P<0.05 or P<0.01).The results of Western Blot showed that YPF powder could markedly reduce the expression of IL-1(3,NLRP3,Caspase-1,Pro-Caspase-1 and ASC protein(P<0.05 or P<0.01).Conclusions:1.Network pharmacology method prediction suggested that inflammasome may play an important role in the treatment of asthma.2.In vivo experiments confirmed that YPF powder have a certain therapeutic effect onOVA-induced asthma mice.3.In vitro and in vivo experiments confirmed that YPF powder may exert the therapeutic effect by downregulating the key protein of inflammasome including IL-1β,NLRP3,Caspase-1 and ASC.