| Objectives1.The network pharmacology method was used to search the active components of Gu-Ben-Qu-Shi-Hua-Yu decoction and predict the targets and key signaling pathways that were activated in the treatment of psoriasis.Then imiquimod(IMQ)-induced mouse psoriasis-like dermatitis model and serum samples from patients with psoriasis were used for the initialvalidation.2.Serum metabolic profiles were performed by high throughput ultra performance liquid chromatography/quadrupole time of flight mass spectrometry(UPLC-Q-TOFMS).To reveal possible mechanisms of Gu-Ben-Qu-Shi-Hua-Yu decoction for treating psoriasis from metabonomics,We analysised the serum metabolomics characteristics of psoriasis and the changes of serum metabolism before and after treatmented with Gu-Ben-Qu-Shi-Hua-Yu decoction.Methods1.Network PharmacologyThe effective components of Gu-Ben-Qu-Shi-Hua-Yu decoction were screened in the database.Corresponding action targets were extracted and intersected with the related targets of psoriasis.Constructed and analysed the network diagrams of Gu-Ben-Qu-Shi-Hua-Yu decoction-the active component-action target by Cytoscape software.The degree values of drug,active ingredient and action target in the network graph were obtained respectively.Constructed protein-protein interaction(PPI)networks by string website.The key targets of Gu-Ben-Qu-Shi-Hua-Yu decoction in the treatment of psoriasis were screened by CytoNCA plug-in.Then,the function annotation and signaling pathways of key targets were performed by GO and KEGG enrichment analysis using R software.Next,we preliminarily validated the targets predicted by network pharmacology.24 male BALB/C mice were randomized divided into Gu-Ben-Qu-Shi-Hua-Yu decoction,the control group and the normal group.Then the control and model groups were gavaged with saline,while the treatment groups were gavaged with Gu-Ben-Qu-Shi-Hua-Yu decoction.4 days after administration,the model group and the Gu-Ben-Qu-Shi-Hua-Yu decoction group were smeared with 5%imiquimod ointment six days in a row.Stop making the model on the 7th day,recorded the PASI score and collect blood and skin.The levels of TNF-α,IL-1β and IL-6 in serum were detected by ELISA.30 patients with psoriasis who admitting the dermatology clinic of Guangdong Provincial Hospital of traditional Chinese Medicine from June 2020 to December 2020 were included in this study,and 30 healthy volunteers were enrolled.Psoriasis patients oral Gu-Ben-Qu-Shi-Hua-Yu decoction for 12 weeks,and venous blood samples were taken at week-0 and week-12.ELISA was performed to detect IL-17A,IL-6,TNF-α,IL-1β,IL-23 and IL-10 secretion.2.MetabolomicsThe metabonomics was studied by UPLC-Q-TOFMS,and the serum sample data were collected by AnalystTF1.5 software.These data are preprocessed by Markerview software,and then multidimensional analysis was carried out by SIMCA14.1 software.Serum differential metabolites of different groups which satisfying VIP≥ 1.0 in OPLS-DA mode and multiple of difference(Fold change,FC)≥ 1.2 or ≤0.8,and P<0.05 at the same time were screened.According to the M/Z of the candidate biomarkers,obtained their further informations by searching the HMDB database.The identified metabolites were submitted to MetaboAnalyst5.0 for further analysis of the metabolic pathway of differential metabolites.Results1.Results of network pharmacology researchThere were 78 active ingredients in Gu-Ben-Qu-Shi-Hua-Yu decoction,including 15 in Poriacocos,29 in Radix Paeoniae Rubra,3 in RhizomaCurcumae,2 in Radix Rehmanniae,8 in Wumei,10 in RhizomaAtractylodis,7 in Atractylodesmacrocephala and 20 in Radix Astragali.According to the active components,a total of 233 targets were predicted and 1563 psoriatic targets were retrieved.107 overlapping genes were identified by Venn diagram.It was found that the corresponding active ingredient in higher quantities were Paeonia lactiflora,Paeoniae Radix,Rehmanniae Radix Praeparata,Smilacis Glabrae Rhixoma、HedysarumMultijugum Maxim.The top rank active ingredients were Quercetin,kaempferol,beta-sitosterol and Stigmasterol,while the top rank targets were TNF-α,IL-6,IL-17A and IL-10.Through Cytonca plug-in,35 key action targets including IL-6,TNF-α,TP53,MAPK8 and IL-17A were found.Through GO enrichment analysis and KEGG signaling pathway enrichment analysis,it was predicted that Gu-Ben-Qu-Shi-Hua-Yu decoction could treat psoriasis through IL-17 signaling pathway,TNF signaling pathway and arachidonic acid metabolism pathway,involving cell membrane,nucleus and a variety of cell microstructure,and is related to cytokines,growth factors,a variety of enzymes and protein functions.The contents of TNF-α,IL-17A and IL-6 in serum of imiquimod-induced mice were significantly increased.The levels of TNF α,IL-6 and IL-17A induced by imiquimod were significantly decreased after treatment with Gu-Ben-Qu-Shi-Hua-Yu decoction.Compared with healthy controls,the serum levels of IL-1β,IL-6,IL-17A,TNF-α and IL-23 in patients with psoriasis were significantly higher,while the level of IL-10 was lower.After treatment with Gu-Ben-Qu-Shi-Hua-Yu decoction,the contents of IL-1β,IL-6,IL-17A,TNF-α and IL-23 were significantly decreased,and the concentration of IL-10 was increased.2.serum metabolomics studyThere were 180 metabolites of psoriasis before treatment compared with the healthy control group(P<0.05).A total of 98 were screened according to VIP and FC,which are potential biomarkers associated with psoriasis.A total of 142 metabolites of psoriasis were screened before and after treatment,and 88 were screened according to VIP value and FC.Those two groups of data do Venn diagram,the overlapping part were a total of 55.Based on the further analysis of the serum metabolism of patients with PASI improvement rate≥50%,a total of 86 biomarkers were identified,of which 35 were consistent with the metabolites of all psoriasis before and after treatment.So these 35 metabolites were selected as possible differential metabolites of Gu-Ben-Qu-Shi-Hua-Yu decoction in the treatment of psoriasis.Which involving 22 metabolic pathways.The metabolites of 5-dihydroxyprostaglandin F1a,6-ketoprostaglandin F1a,leukotriene D4(LTD4)and leukotriene E4(LTE4),and metabolic pathways such as arachidonic acid metabolism,glycerol phospholipid metabolism and sphingomyelin metabolism are closely related to psoriasis.There were a total of 16 metabolites in the effective group before and after treatment,while there were no differences in the ineffective group,and 11 of them may be related to psoriasis.ROC curve analysis showed that 5 of the 7 metabolites with AUC value greater than 0.7 were statistically significant before treatment between the healthy group and the effective group,while the comparison between the healthy group and the ineffective group was not statistically significant before treatment.Conclusion1.The network pharmacology method was used to predict the composition of Gu-Ben-Qu-Shi-Hua-Yu decoction.Experimental results showed that this formula can reduce the level of related inflammatory factors in serum of mice and patients.2.The method of metabonomics was used to analyze the serum metabolic status of patients with psoriasis.It was preliminarily found that Gu-Ben-Qu-Shi-Hua-Yu decoction could improve the serum metabolic status of patients to a certain extent. |
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