Study Of The Immunomodulatory Effects Of Formulas For Psoriasis By Network Pharmacology

ObjectiveThere are no exact records of psoriasis in Traditional Chinese medicine.None but "Baibi" is similar to psoriasis.Many formulas and herbs were used to treating this disease.It was very important to find out the commonness from those formulas for the study of mechanisms and effective components.It’s also very important for the clinical therapy and the new drug discovery for psoriasisPsoriasis was a chronic,inflammatory skin disease.Scientists found that immune cells in patients with psoriasis was increased evidently,which suggested that the pathogenesis of psoriasis was associating with immune function in 1970.Innate immunity in patients with psoriasis is abnormal,the skin lesions of immune cells(T cells,dendritic cells)was significantly increased.Therefore,Immunomodulatory effects of formulas for psoriasis will be researched in this paper.And we will try to carry out the in-depth study of traditional Chinese medicine for the treatment of psoriasis,reveal its modern connotation,and provide a foundation for the clinical treatment and drug development of psoriasis.Methods1.Prediction of the mechanisms of formulas for treating psoriasis by methods of computational pharmacology.63 formulas for psoriasis,including 129 herbs,were collected from dermatological journals for ten years.Small molecules were collected from Beilstein database,Chinese natural products database and 3D-molecular.structure database of Peking University.Dataset were imported in Discovery Studio(2.5.5 version,Accelrys).Then,4 chemical descriptors,including lipid water partition coefficient,hydrogen bond donor and hydrogen bond acceptor,were calculated for the analysis of drug-like.There are 10 key cell factors in the immune cascade network.Their 3D-structures were downloaded from PDB.And then were imported to Discovery Studio.The DS libdock module was employed to predict the interactions between small molecules in Chinese traditional medicine for treating psoriasis and target.The active sites were defined by the activity sequences which were reported in the literatures,and other indicators were using default parameters.Top 100 molecules with high Dockscore were defined as the potential active components.Formulas,herbs,target proteins,and results of virtual screening were imported into Cytoscape;a kind of software for network constructing and analysis,for the prediction of the mechanisms and effective components.2.Prediction of the interaction between the highest frequency 10 herbs and 6 therapy targets for psoriasis.Statistics of the frequency of herbs in the formulas,and constructs the small molecules dataset for the highest frequency 10 herbs.6 therapeutic targets were collected from Therapeutic Target Database.All the 6 target proteins had inhibitors in the structures.DS ligandfit was employed to carry out the half soft docking.Energy Grid was set as charm,Number of Monte Trials was set as 5000,Maximum Poses Retained was set as 1,Minimization Algorithm was set as Smart minimizer,Force field was set as charm,Iteretions was set as 1000,Gradient Tolerance was set as 0.001,all other parameters was set as defaults.The dockscores of inhibitors were set as threshold value.3.Study of the immunomodulatory effects and mechanisms of Yinxieling Tablets by network pharmacology.(1)Study of the immunomodulatory effects of Yinxieling Tablets by network pharmacology.① Virtual screening 886 small molecules were collected from 10herbs of Yinxieling Tablets.10 cytokines which were closely related to the pathogenesis of psoriasis all had known 3D-stricture.DS libdock was used to carry out virtual screening.② Construction and analysis of immune cascade network Immune cascade network was constructed by cytoscape,the key immune cells and cytokines of T cells immune were set as the nods and the relationship between them were set as the edges.And the network included 7 kinds of immune cells and 10 cytokines.8 topological parameters of network,such as degree,betweenness,closeness,et.al,were used to evaluate the essentiality of the nodes in the network.③ Prediction of the immunomodulatory effects of Yinxieling Tablets.Immunomodulatory effects and mechanisms of Yinxieling Tablets were prediction of by the results of computational virtual screening and network pharmacology.(2)Establishment and optimization of IL-23 induced psoriasis-like mouse models.Mice were cut back hair with electric clippers,20 μL IL-23(containing IL-23 antigen 500ng)was given to mice by hypodermic injection.Blank control group were injected with PBS.Mice received injection in 1,2,4 day.Mice were handled in 5 day.Optimization of conditions included mice specials,sexuality,and dose of IL-23.(3)Research on the intervention effects of Yinxieling tablets on IL-23 induced psoriasis-like mice.60 C57BL/6 mice were divided in to 6 groups randomly,including blank control group,Model group,positive control group,low dose group of Yinxieling tablets,middle dose group and high dose group of Yinxieling tablets,half of male and female.Mice were cut back hair with electric clippers,20 μL IL-23(containing IL-23 antigen 500ng)was given to mice by hypodermic injection.Blank control group were injected with PBS.Mice received injection in 1,2,4 day.Mice were handled in 5 day.Optimization of conditions included mice specials,sexuality,and dose of IL-23.4.Studies on the effective components of Yinxieling optimization formula(1)Prediction of the effective components of Yinxieling optimization formula The effective components of Yinxieling optimization formula were predicted of by virtual screening.Results of virtual screening were imported into cytoscape to construct the target-drug network.Topological parameters of network,such as degree,betweenness,closeness,et.al,were used to evaluate the essentiality of the targets in the network.And try to predict the most effective molecules and the bridge molecules.(2)Study on components which were been absorbed into the blood and the effective molecules.Adult male beagle dogs,12h fasting,take blank blood samples into anticoagulant tube.The dog was treated by gavage with Yinxieling optimization solution(9.6g/kg body weight).Drug-containing plasma were separated by centrifugation.And UPLC-Q-TOF/MS was used to analysis the small molecules in the blood.Active ingredients contrast in blood and forecast,forecast the active components in serum.Results1.Prediction of the mechanisms of formulas for treating psoriasis by methods of computational pharmacology.(1)Study of formulas and herbsThe highest frequency 10 herbs in turn:Rehmannia(Dihuang,44),Salvia miltiorrhiza(danshen,28),Rhizoma Smilacis Glabrae(Tufuling,26),Radix Paeoniae Rubra(Chishao,24),Lithospermum erythrorhizon(Zicao,22),Cortex Moutan Radicis(Mudanpi,21),Radix Angelicae Sinensis(Danggui,19),Caulis Spatholobi(Jixueteng,19),Lonicera Japonica(Jinyinhua,17),Rhizoma Imperatae(Baimaogen,16)。(2)Results of drug-like analysis There were 3835 drug-like molecules,and belonged to 106 herbs.(3)Results of virtual screening 772 molecules had interaction with 10 key cytokines.All these potential effective molecules belonged to 95 herbs.That means most formulas used in clinical had interaction with cytokines.Top 10 herbs which had most effective components in turn:Radix Ginseng Rubra(Hongshen,46)、Dioscorea opposita(Shanyao,32)、Radix Glycyrrhizae(Gancao,23)、Ixeris denticulata(Baijiangcao,22)、Rehmannia(Dihuang,22),Lonicera Japonica(Jinyinhua,21),Salvia miltiorrhiza(danshen,20),Herba Taraxaci(Pugongying,20)、Semen Persicae(Taoren,20),Cortex Mori(Sangbaipi,19).2.Prediction of the interaction between the highest frequency 10 herbs and 6 therapy targets for psoriasis.(1)Prediction of mechanisms10 herbs had interaction with 5 targets.Mechanism of herbs for psoriasis may include inhibiting the activity of Cathepsin S,Purine nucleoside phosphorglase,Mitogen-activated protein kinase 14,tumor necrosis factor alpha,and activating Peroxisome proliferators-activated receptor gamma.(2)Prediction of active components362 molecules had interaction with 5 targets.Rhizoma Smilacis Glabrae,Radix Angelicae Sinensis,Salvia miltiorrhiza and Cortex Moutan Radicis had the most effective components,Delphin from Salvia miltiorrhiza had interaction with 5 targets.It was also the most effective components for p38mapk.Peonoside from Cortex Moutan Radicis also had interaction with 5 targets.It was also the most effective components for PPAR-r.3.Study of the immunomodulatory effects and mechanisms of Yinxieling Tablets by network pharmacology.(1)Study of the immunomodulatory effects of Yinxieling Tablets by network pharmacology.① Virtual screening397 molecules had potential interaction with 8 targets.IL-17A had 260 potential effective components,and IFN-α had 192 potential effective components.IL-17A and IFN-α might be the primary targets for Yinxieling Tablets.IL-23 and IL-17F had no interaction with Yinxieling Tablets.② Study of immune cascade network by network pharmacologyAccording to the results of importance analysis,ranking the importance of cytokines:IL-23、IL-12、TNF-α、IFN-γ、IL-1 beta、IL-6、IL-17A、IL-17F、IL-22 and IFN-α.According to the results of target-drug network analysis,IL-17A and IFN-α were the most important nodes in the network.According to the results of target-drug network,IL-17A and IFN-α also were the most important nodes in the network,followed by IL-12 and IL-22.③ Prediction of the immunomodulatory effects of Yinxieling Tablets.Yinxieling Tablets took effects by the monarch drugs(Rehmannia glutinosaLibosch.,Angelica sinensis(Oliv.)Diels,Ligusticum chuanxiong Hort.),the ministerial drug(Arnebia euchroma(Royle)Johnst,),Smilax glabra Roxb.and Glycyrrhiza uralensis Fisch..IL-17A、IFN-α、IL-12 and IL-22 were the main targets.(2)Establishment and optimization of IL-23 induced psoriasis-like mousemodels.C57BL/6 mice model were successful。Incrassated white derma appeared in the fourth day.White derma easily brushed off.Red tissue could be seen after that white derma brushed off.Pathological observation suggested that lesions consisted with psoriasis.But,8 days later,there were no reaction byrepetitious stimulation.Pathological observation suggested that lesionspartly consisted with psoriasis for Kunming mice and NIH mice.8 days later,there were no reactions by repetitious stimulation.Molding effect was dose-dependent,middle and high dose groups,severity score and the positive rate were significantly higher than that of low dose group;mice model had gender differences,the positive rate and score of male mice were significantly higher than that of female mice.Therefore,combined with the model experimental results and literatures,we selected C57BL/6 for subsequent research,respectively at first,2,4 days and took subcutaneous injection of IL-23(500ng/20 μL)at 1,2,4 day.(3)Research on the intervention effects of Yinxieling tablets on IL-23 induced psoriasis-like mice.① Apparent symptomThe average score of the model group wasl.7 points,and the positive rate of skin lesions was 80%;The positive rate of skin lesions and the average score were 30%(0.6),40%(0.5),30%(0.2)for Yinxieling tablets low,middle and high dose groups;Positive control drug cyclosporine A was 30%(0.2 points).The results show that Yinxieling tablet every dose group and cyclosporine A group can significantly reduce the positive rate and score.And the Yinxieling tablet can prevent the psoriasis-like lesions induced by IL-23 antigen in mice.② Results of pathological examinationThe model group with cuticle thickening,dyskeratosis rare,typical Munro microabscess,thinning granular layer,acanthosis,thickening of the basal lamina or release of destruction in individual cases;dermal telangiectasia,congestion,extravasation of red blood cells,lymphocyte,neutrophil infiltration were also been seen.Lesions accorded with psoriasis.In the treatment groups with Yinxieling,lesions showed parallel changes in the model group,the effect of high dose group was significantly,and the granular layer increased significantly;anti-inflammatory effect of cyclosporine A group is obvious,but anti-keratosis effect is weak.③ The weight index of immune organCompared with the blank group,spleen index of middle dose group of Yinxieling tablet increased remarkably(P<0.01),and cyclosporine A group(P<0.05)also increased significantly;compared with the model group,the spleen weight index of middle dose group of Yinxieling tablet(P<0.01)increased significantly.And the spleen index of middle dose group of Yinxieling tablet were higher than the low dose and high dose group(P<0.05).Compared with the blank group,the thymus index of middle dose group of Yinxieling tablets(P<0.01)were significantly increased,and that of middle dose group Yinxieling tablet was higher than that of low dose group(P<0.05)and high dose group.These suggest that middle dose group of Yinxieling tablet can promote immune function better.Compared with the model group,thymus index of cyclosporine A group were significantly reduced(P<0.05).These suggest that cyclosporine A has obvious inhibitory effect.4.Studies on the effective components of Yinxieling optimization formula(1)Prediction of the effective components of Yinxieling optimization formula273 molecules from Yinxieling optimization formula had potential interaction with 8 targets.Ranked according to the importance of target,the first 5 targets are IL-17A,IFN-r,IL-22,IL-6 and IL-1β。IL-23 P19 subunit and IL-17F had no interaction with Yinxieling optimization formula.117 effective molecules from Glycyrrhiza uralensis Fisch.,47 molecules from Arnebia euchroma(Royle)Johnst.,36 molecules from Curcuma phaeocaulis Val.,32 molecules from Paeonia lactiflora Pall.,25 molecules from Smilax glabra Roxb.,13 molecules from Prunus mume(Sieh.)Sieb.et Zucc.,and 3 molecules from Chloranthus spicatus,there are 273 potential effective molecules in total.(2)Study on components which were been absorbed into the blood and the effective molecules.Comparing with the spectrograms of formula,herbs in vitro and in vivo,there are 34 components absorbed in blood directly those are identified from serum after lavage.Comparison of active components in the blood and predictive effective components,a total of 8 components were identified.Those are astilbin,glycyrrhizin,taxifolin,liquiritin apioside,liquiritin,liquiritigenin,isoliquiritigenin and rosmarinic acid.There were reported,6 components all had immunomodulatory or immunosuppressive effects.Conclusion1.Prediction of mechanism formulas for psoriasis by computer pharmacological methodThe mechanism and effective components of formulas on treating psoriasis were studied by computer method.Results show that most formulas had possible interaction with cytokines from immune cascade in the network.Top 10 herbs with highest frequency possibly inhibited the activities of cathepsin S,purine nucleoside phosphorylase,mitogen-activated protein kinase and tumor necrosis factor alpha activity,as well as activated PPAR-y.Thus they can achieve the purpose of cure psoriasis by playing anti-inflammatory and suppress role.2.Studies on the immunomodulatory effects and mechanism by network pharmacology.The predictive results by virtual screening and network pharmacology were according with that of animal experiments.Yinxieling Tablet had immunomodulatory effects.Low,middle and high dose of Yinxieling tablet groups can prevent the lesions induced by IL-23.but,the exact targets and mechanism need in-depth research.Yinxieling tablet took effect by immunomodulatory effect and correcting the balance of immune cascade network,while cyclosporine A took effect by immune inhibition effect.Those may lead to the slower onset,but lasting effect,less side effects of Yinxieling tablet.3.Studies on the effective components of Yinxieling optimization formula Comparison of active components in the blood and predictive effective components,a total of 8 components were identified.Those are astilbin,glycyrrhizin,taxifolin,liquiritin apioside,liquiritin,liquiritigenin,isoliquiritigenin and rosmarinic acid.