| Objective:Our research group has verified the efficacy of Xiaoqinglong Decoction in treating AR through animal or clinical experiments in the previous study,and explored the mechanism of efficacy.It has been found that Xiaoqinglong Decoction can restore the imbalance of Th1/Th2;the catechins in Xiaoqinglong Decoction can play an anti-inflammatory role;the metabolomics study of mice provides the metabolic basis for the therapeutic effect of Xiaoqinglong Decoction...These findings provide the research foundation for the mechanism of Xiaoqinglong Decoction on allergic rhinitis.However,it has not been verified in humans whether the metabolic differences caused by Xiaoqinglong Decoction in mice with allergic rhinitis also occur in humans.Additionally,metabolomics is a high-throughput test method that requires confirmation by other studies to validate its ability to analyze differential changes and identify associated influence pathways,This study aimed to collect nasal lavage fluid and blood samples from patients treated with Xiaoqinglong Decoction and from healthy subjects,evaluate symptom improvement,and carry out correlation analyses using network pharmacology and metabolomics results verified by mass spectrometry.The goal was to provide a mechanism for the therapeutic effect of Xiaoqinglong Decoction on allergic rhinitis at the gene-protein-metabolite levels.Method:1.Analysis of the therapeutic effect of Xiaoqinglong decoction on AR patients20 patients with allergic rhinitis and 10 healthy subjects were recruited from the clinic.All patients were tested for allergens at the time of enrollment and did not take any other drugs within 2 weeks before administration.The treatment duration was 2 weeks.The symptoms of patients were evaluated with visual analog scale(VAS),total nasal symptom score(TNSS),and mini rhinoconjunctivitis quality of life questionnaire(Mini RQLQ)before and after treatment;The serum IL-13 levels of patients and healthy subjects were measured by ELISA to evaluate the inflammatory status of patients.The curative effect of Xiaoqinglong Decoction on allergic rhinitis was preliminarily confirmed.2.Blood metabolomics analysis of patients with allergic rhinitis treated with Xiaoqinglong DecoctionWith liquid chromatography-mass spectrometry(LC-MS),the serum samples of patients before and after treatment and healthy subjects were analyzed for metabolites,and the reliability of data was ensured through quality control experiments.The distribution of the total metabolic difference of the sample was displayed through principal component analysis,and the model transformation of Xiaoqinglong decoction group-patient group and patient group-health group was performed through partial least squares discriminant analysis,so as to more intuitively display the differences between its metabolic groups and obtain the serum differential metabolites.The metabolic pathway enrichment analysis of these differential metabolites was carried out to explain the action pathway of Xiaoqinglong Decoction in blood.3.Metabolic analysis of nasal lavage fluid in patients with allergic rhinitis treated with Xiaoqinglong DecoctionBy using LC-MS technology the metabolites of nasal lavage fluid samples of patients before and after treatment and healthy subjects were analyzed,and data reliability were ensured by quality control.The distribution of the total metabolic difference of the sample was displayed through principal component analysis,and the model transformation of Xiaoqinglong decoction group-patient group and patient group-health group was performed through partial least squares discriminant analysis,so as to more intuitively display the differences between the metabolic groups of nasal lavage fluid and obtain the differential metabolites of nasal lavage fluid.The metabolic pathway enrichment analysis of these different metabolites was carried out to explain the functional pathway of Xiaoqinglong Decoction in the local nasal cavity.4.The combined study of network pharmacology and metabolomics of Xiaoqinglong Decoction in treating allergic rhinitis patientsWe collected the active ingredients of Xiaoqinglong Decoction through the public database,and compared them with the mass spectrum analysis results of Xiaoqinglong Decoction in the previous study of our research group.It’s to obtain the verified effective ingredients of Xiaoqinglong Decoction.According to the extracted components,the corresponding treatment targets were obtained,and the target set of Xiaoqinglong Decoction for AR treatment was obtained by screening the relevant targets of allergic rhinitis in the’database.Conduct pathway enrichment analysis and network construction for the target.Result:1.Effect of Xiaoqinglong Decoction on patients with allergic rhinitis(1)There were no statistical differences between the age and gender of patients and healthy subjects,so comparative statistical analysis of their indicators could be carried out.(2)The total scores of VAS,mini-RQLQ and TNSS were significantly decreased after two weeks of treatment.This showed that Xiaoqinglong Decoction that improve the symptoms of patients and improve the quality of life.The patients’ nasal congestion,runny nose,sneezing and itching were statistically analyzed and significantly improved after two weeks.It showed that Xiaoqinglong Decoction didn’t only aim at some single symptom,but also improve all common allergic rhinitis symptoms.Through the statistical analysis of IL-13 in patients before and after treatment and in healthy subjects by ELISA,it was found that the expression of IL-13 in patients was increased compared with that in healthy groups,while Xiaoqinglong Decoction could reduce the level of IL-13 in patients.2.Analysis of serum metabolomics of patients with allergic rhinitis treated withXiaoqinglong Decoction(1)Obtained the total ion chromatography through liquid chromatography-mass spectrometry.(2)The quantitative values of metabolites from multiple QC samples were used for quality control.The Pearson correlation coefficient between QC samples was compared,and the sample correlation was close to 1.This showed that there was a certain system stability in the experimental detection,and the quality of data was guaranteed.(3)Metabolite identification and principal component analysis(PCA)According to the results of the ion diagram,software analysis was used to identify different metabolites and carry out relative quantitative analysis.Principal component analysis was carried out for the patient group,Xiaoqinglong decoction group,and health group,and its multivariables were converted to display in the twodimensional diagram.Its position difference represented the difference of body metabolites.In the positive and negative ion mode,there were some differences among the three groups.(4)Comparative analysis between groups by Discriminant Analysis of Partial Least Squares(PLS-DA)In order to better compare the differences between groups and obtain the different metabolites between groups,PLS-DA was used to analyze the metabolites of Xiaoqinglong Decoction group,AR group and healthy group under the positive and negative ion mode.The PLS-DA model was sorted and verified,and it showed that R2>Q2,and the intercept between Q2 regression line and Y-axis is negative.It showed that the model is reliable and not over-fitting.Based on this,the VIP values of different metabolites were obtained,and the different metabolites were screened according to the criteria of VIP>1.0,FC>1.2 or FC<0.833 and P-value<0.05.Compared with allergic rhinitis group,Xiaoqinglong decoction group obtained differential metabolites,including 22 negative ion modes and 62 positive ion modes.Compared the differential metabolite of Xiaoqinglong Decoction group VS AR group with that of AR group VS healthy group,and removed the drug and chemical metabolites.In the two comparisons,the common differential metabolite with a callback trend(the expression trend of drug-AR group is opposite to that of AR group-healthy group)was undecylic acid,with FC values of 0.709 and 1.424,respectively.(5)Differential pathway enrichment analysisThe enrichment of differential metabolites in Xiaoqinglong Decoction group and AR group was compared:the enrichment pathway of differential metabolites in negative ion mode was D-arginine and D-ornithine metabolism,taste conduction,tyrosine metabolism,glutathione metabolism,arginine and proline metabolism,ABC transport protein,2-oxycarboxylic acid metabolism,steroid hormone biosynthesis,arginine biosynthesis,phenylalanine metabolism,amino acid biosynthesis;Among them,the positive ion mode differential metabolite enrichment pathway is oxidative phosphorylation,retinol metabolism,inflammatory mediator regulation of TRP channel,purine metabolism,tyrosine metabolism,riboflavin metabolism,drug metabolism-cytochrome P450,cAMP signal pathway,gap junction,synaptic perivesicular phase,trypanosomiasis,tryptophan metabolism,vitamin digestion and absorption,arachidonic acid metabolism,Linolenic acid metabolism,neuro-active ligand receptor interaction,serotonin synapse,taste conduction,caffeine metabolism,phenylalanine,tyrosine and tryptophan biosynthesis,2-oxo-carboxylic acid metabolism,mineral absorption,phenylalanine metabolism,amino acid biosynthesis,ABC transport protein.Compared the enrichment pathway of Xiaoqinglong decoction group-AR group with that of AR group-health group,removed the drug or chemical material enrichment pathway,and the common enrichment pathway is steroid hormone biosynthesis,arachidonic acid metabolism,and serotonin synapse.3.Metabolic analysis of nasal lavage fluid in patients with allergic rhinitis treated with Xiaoqinglong Decoction(1)Obtained the total ion flow chromatogram through liquid chromatographymass spectrometry.(2)The quantitative values of metabolites from multiple QC samples were used for quality control.The Pearson correlation coefficient between QC samples was compared,and the sample correlation was close to 1.This showed that there was a certain system stability in the experimental detection,and the quality of data was guaranteed.(3)Metabolite identification and principal component analysis(PCA)According to the results of the ion diagram,software analysis was used to identify different metabolites and carry out relative quantitative analysis.The principal component analysis was carried out for the nasal lavage fluid of the patient group,the Xiaoqinglong decoction group and the healthy group,and its multivariable was converted to displayed in the two-dimensional diagram,and its position difference represented the difference of the body metabolites.In the positive and negative ion mode,there were some differences among the three groups.(4)Comparative analysis results between groups of Discriminant Analysis of Partial Least Squares(PLS-D A)In order to compare the differences between groups and obtain the different metabolites between groups,PLS-DA was used to analyze the metabolites of nasal lavage fluid in Xiaoqinglong Decoction group,AR group and healthy group under positive and negative ion mode.The PLS-DA model was sorted and verified,and it showed that R2>Q2,and the intercept between Q2 regression line and Y-axis is negative.It showd that the model is reliable and not over-fitting.Based on this,the VIP values of different metabolites were obtained,and the different metabolites were screened according to the criteria of VIP>1.0,FC>1.2 or FC<0.833 and P-value<0.05.Compared with allergic rhinitis group,Xiaoqinglong decoction group obtained differential metabolites,including 11 negative ion modes and 9 positive ion modes.Compared the differential metabolites of nasal lavage fluid in Xiaoqinglong decoction group VS AR group with those in AR group VS healthy group,and removed the drug and chemical metabolites.In the two comparisons,the differential metabolites that appear together and have a callback trend(the contrast expression trend of drug-AR group is contrary to that of AR-health group)were trimethyladipic acid(3-methyladipic acid),with FC values of 1.277 and 0.570,respectively.Compared the differential metabolites of nasal lavage solution Xiaoqinglong decoction group VS AR group with those of serum Xiaoqinglong decoction group VS AR group.The common differential metabolites were(±)18 hydroxyeicosapentaenoic acid,with FC values of 2.549 and 3.598 respectively.(5)Differential pathway enrichment analysisThe enrichment of differential metabolites in Xiaoqinglong decoction group and AR group was compared:the enrichment pathway of differential metabolites in negative ion mode was anti-folate resistance,phenylalanine and tyrosine and tryptophan biosynthesis,and pyrimidine metabolism;Among them,the positive ion mode differential metabolite enrichment pathway was arginine and proline metabolism β-Alanine metabolism,glutathione metabolism,bile secretion,metabolic pathway.Compared the enrichment pathway of Xiaoqinglong decoction group-AR group with that of AR group-health group.The common enrichment pathway was antifolate resistance,phenylalanine and tyrosine and tryptophan,pyrimidine metabolism,arginine and proline metabolism β-Alanine and glutathione metabolism,metabolic pathway.Compared the enrichment pathway of nasal lavage solution Xiaoqinglong decoction group vs AR group with the enrichment pathway of serum Xiaoqinglong decoction group vs AR group.The common enrichment pathway was phenylalanine,tyrosine and tryptophan biosynthesis,arginine and proline metabolism,and glutathione metabolism.4.The combined study of network pharmacology and metabonomics of Xiaoqinglong Decoction in the treatment of allergic rhinitis(1)Obtained the active ingredients of Xiaoqinglong Decoction from the public database,and checked them with the mass spectrometry analysis results in the previous study to gain the 18 active ingredients of Xiaoqinglong Decoction.(2)130 kinds of action targets of 18 ingredients were collected from the database,and AR related targets were obtained through OMID,TTD and GeneCards database.By the combination of them,the target ESR2,TNF,IFNG,IL6,IL1B,VCAM1,ALOX5,IL2,CCL2,MPO,SELE,CYP3A4 of Xiaoqinglong Decoction for AR treatment were obtained.(3)Conducted gene ontology analysis and gene pathway analysis on the target,and made the target connection into protein-related association network diagram.The gene ontology,enrichment pathway and the relationship between the treatment targets of Xiaoqinglong Decoction were obtained.Among them,interleukin-6,tumor necrosis factor and interleukin-1 were the most closely related to other targets.(4)The correlation of genes,metabolites and pathways was shown in the network diagram.By the combination of the metabonomics content and the results of network pharmacology,we found different metabolites and their related targets through different methods,and displayed their connection and action pathways-TRP pathway,serotonin synaptic pathway,arachidonic acid pathway,and linoleic acid metabolism pathway.Conclusion:1.Xiaoqinglong Tang could effectively improve the symptoms and life quality of patients with lung qi deficiency cold type allergic rhinitis,and have good safety.2.After the intervention of Xiaoqinglong Tang,the serum reflects metabolic differences,with the core differential metabolite-undecanonic acid closely related to allergic rhinitis.Xiaoqinglong Decoction could interfere with the accumulation of metabolites by arachidonic acid metabolism Pathways,α-linolenic acid metabolism and etc.The co-enrichment pathways of Xiaoqinglong Decoction and AR differential metabolites include steroid hormone biosynthesis,arachidonic acid metabolism,and serotonin synapses.3.After the intervention of Xiaoqinglong Decoction,the nasal lavage fluid reflected metabolic differences.Trimethyladipic acid was the core differential metabolite closely related to allergic rhinitis;The differential metabolite of Xiaoqinglong Decoction in serum and nasal lavage fluid was(±)18 hydroxyeicosapentaenoic acid.The common Xiaoqinglong Decoction enrichment pathways in serum and NLF are phenylalanine,tyrosine,and tryptophan biosynthesis,arginine and proline metabolism,and glutathione metabolism.4.Network pharmacology research starts from the active ingredients of Xiaoqinglong Tang,which have 12 targets related to AR.The core targets are interleukin-6,tumor necrosis factor,and interleukin-1 β.The core components are quercetin,kaempferol,and luteolin.By correlating the metabonomic results of Xiaoqinglong Decoction with the results of network pharmacology,it was found that the TRP pathway,serotonin synaptic pathway,arachidonic acid pathway,and linoleic acid metabolism pathway are closely related to AR.These pathways and the detected targets may be the effective mechanisms of Xiaoqinglong Decoction in treating AR patients. |
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