To Explore The Biological Markers Of Psoriasis Vulgaris Based On Metabonomics And To Study The Effects Of Drugs On Markers

Research background: Psoriasis vulgaris is a kind of common and easy to relapse chronic inflammatory skin disease.At present,the clinical diagnosis of this disease is mainly based on the clinical manifestations,lesion location and histopathological changes.However,histopathological examination has a certain trauma,the specificity and sensitivity of routine laboratory examination indexes for psoriasis vulgaris are low,the clinical value and significance are limited,so there is still a lack of specific diagnostic criteria for psoriasis vulgaris.in addition,the causes of the disease are complex,the pathogenesis is not completely clear and the patients are often accompanied by a variety of endogenous metabolic disorders.Metabolomics is a technique to study the metabolic network of the body by investigating the metabolite spectrum and its dynamic changes of living organism is stimulated or disturbed(such as genic mutation or pathophysiological status).In recent years,it has been widely used in many fields,such as disease diagnosis,pathogenesis research,individualized administration and drug development.Therefore,the study of endogenous metabolites of patients with psoriasis vulgaris by applying the metabonomics technology combined with multivariate statistical analysis,the obtained biomarkers will be helpful for the diagnosis of the disease,the further study of the pathogenesis and the formulation of the individualized dosage regimen.Methotrexate(MTX)and tripterygium glycosides are commonly used drugs for the treatment of psoriasis,and mainly play a role through immunosuppression,but its side effects are obvious and individual differences are large.Psoriasis vulgaris is a disease that endogenous metabolites are disordered,therefore,the role of MTX and tripterygium glycosides in the treatment of psoriasis can be studied from the perspective of metabolites by observing the effects of drugs on the biomarkers.Research purpose: 1.Aiming to find the potential biomarkers of psoriasis vulgaris based on metabonomics.2.Aiming to investigate the effects of MTX and tripterygium glycosides on these biomarkers that matching with the patients in psoriasis model of mice.Research method: 1.Sample collection and processingPatients with psoriasis vulgaris who were treated in the Department of Dermatology of the First Affiliated Hospital of Jinan University and met the inclusion criteria were selected as the case group,the healthy volunteers in hospital examination center were selected as control group.Blood samples were collected fasting from the patients and the healthy volunteers in the morning and placed into 5 m L EDTA-K2 anticoagulant tube,The samples were stand for 2 hours after mixing 5~6 times by upside down gently and then centrifuged(4000 r/min,15 min,4?),the supernatants were stored at-80 °C until analysis.2.Plasma metabonomics analysis based on ~1H-NMR and UPLC/ Q-TOF MS Nuclear magnetic resonance ~1H spectroscopy(~1H-NMR)and ultra-performance liquid chromatography/quadrupole time of flight mass spectrometry(UPLC/Q-TOF MS)techniques were used to analyze the plasma metabolomic profiling between the patients with psoriasis vulgaris and the healthy volunteers,respectively.principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA)were used for pattern recognition,these variables of variable importance projection(VIP)>1 in the OPLS-DA model were selected for independent sample t test,P <0.05 is the critical value,so as to screen out the potential biomarkers and analyze the related metabolic pathways.The results of the two methods were compared to find out the similarities and differences in the biomarkers.3.To establish the psoriasis model of mice by IMQ induced and to find the biomarkers that matching with the patients in the model mice 24 female BALB/c mice were used as the experimental subjects,the mice were anesthetized with 4% chloral hydrate(0.1ml/10g)after feeding for 3 days and its back hair were removed,forming an exposed area about 2 cm x 3 cm.The mice were randomly divided into 2 groups,namely the model group and the blank group,and 12 mice per each group.The model group were coated with 5% IMQ cream 62.5mg on the back of mice,the blank group were coated with the same amount of vaseline.At the same time,the two groups were treated with normal saline(0.2m L/t/d)for successively 7 days.The mice were killed at the eighth day,whether there is psoriasis-like lesions can be evaluated by naked eyes and pathological examination.Next,UPLC/Q-TOF MS technique was used to analyze the plasma metabolomic profiling between the blank mice and the model mice,aiming to find the potential biomarkers that matching with the patients with psoriasis vulgaris in the model mice.4.The effects of MTX and tripterygium glycosides on the lesion location and the matching markers of mice psoriasis 24 female BALB/c mice were used as the experimental subjects,the mice were anesthetized with 4% chloral hydrate(0.1ml/10g)after feeding for 3 days and its back hair were removed,forming an exposed area about 2 cm x 3 cm.The mice were randomly divided into 2 groups,namely the MTX group and the tripterygium glycosides group,and 12 mice per each group.The MTX group were coated with 5% IMQ cream 62.5mg on the back of mice,at the same time,its were treated with MTX(1mg/kg/d)for successively 7 days.The tripterygium glycosides group were coated with 5% IMQ cream 62.5mg on the back of mice,at the same time,its were treated with tripterygium glycosides(10mg/kg/d)for successively 7 days.The mice were killed at the eighth day,whether skin lesions has improved can be preliminarily judged by naked eyes.Next,UPLC/Q-TOF MS technique was used to investigate the effects of MTX and tripterygium glycosides on these biomarkers that matching with the patients with psoriasis vulgaris in the model group.Research results: 1.Plasma metabonomics analysis based on ~1H-NMR and UPLC/ Q-TOF MS ~1H-NMR and UPLC/Q-TOF MS techniques were used to analyze the plasma metabolomic profiling between the patients with psoriasis vulgaris and the healthy volunteers,respectively.The results showed that the two groups could be clearly distinguished,and there were significant differences in the levels of endogenous metabolites between the two groups.21 differential metabolites were identified by ~1H-NMR technique combined with multivariate statistical analysis,namely very low density lipoprotein(VLDL),low density lipoprotein(LDL),lipids,leucine,isoleucine,valine,?-hydroxybutyrate,lactate,alanine,arginine,N-acetylglycoproteins,acetoacetate,glutamate,pyruvate,glutamine,citrate,creatine,creatinine,glucose,aspartate and phenylalanine.19 differential metabolites were identified by UPLC/Q-TOF MS technique combined with multivariate statistical analysis,namely threonine,leucine,phenylalanine,tryptophan,palmitamide,linoleic amide,oleamide,stearamide,cis-11-eicosenamide,Trans-13-Docosenamide,uric acid,Lysophosphatidylcholine(Lyso PC)(16:0),Lyso PC(18:3),Lyso PC(18:2),Lyso PC(18:1),Lyso PC(18:0),oleic acid,arachidonic acid and N-linoleoyl taurine.It can be concluded from above results that the two metabolomics techniques have goodclustering effect,but most of the differential metabolites obtained by the two methods were different.2.To establish the psoriasis model of mice by IMQ induced and to find the biomarkers that matching with the patients in the model mice It can be seen by naked eyes at the eighth day that the dorsal skin of the blank mice were smooth,but the model mice were covered with scales on the erythema,and the skin thickening serious.It can be seen by pathological examination that the epidermis of the blank mice was very thin and the cells were morphologically normal,but the model mice has many histopathologic changes,such as hyperkeratosis,parakeratosis,acanthosis cell layer thickening and a large number of inflammatory cell infiltration,this is similar to psoriasis-like lesions.the preliminary results showed that the model was successful.Next,UPLC/Q-TOF MS technique was used to analyze the plasma metabolomic profiling between the blank mice and the model mice,we could found that threonine,phenylalanine,tryptophan,palmitamide,oleamide,oleic acid,stearamide,Trans-13-Docosenamide,Lyso PC(16:0),Lyso PC(18:3),Lyso PC(18:2)and Lyso PC(18:0)are the potential biomarkers that matching with the patients with psoriasis vulgaris in the model mice.It can be concluded from above results that there were some similarities in biomarkers between the model mice and the patients with psoriasis vulgaris.3.The effects of MTX and tripterygium glycosides on the lesion location and the matching markers of mice psoriasis It can be seen by naked eyes at the eighth day that the dorsal skin of the MTX group and the tripterygium glycosides group had an improvement in erythema,scales and thickening than that of the model group,the results showed that the drugs can significantly improve the skin lesions of psoriatic mice.UPLC/Q-TOF MS technique combined with multivariate statistical analysis were used to study the endogenous metabolites of the blank group,the model group,the MTX group and the tripterygium glycosides group,The results showed that the drug therapy group and the model group can be clearly distinguished,and there was a tendency to shift to the blank group.In these biomarkers that matching with the patients in the model mice,the levels of phenylalanine,Lyso PC(16:0),Lyso PC(18:2)and Lyso PC(18:0)in the MTX group were significantly lower than that of the model group,and there were significant difference between the MTX group and the model group(P?0.05),butthere were no significant difference between the MTX group and the blank group(P?0.05);the levels of phenylalanine,tryptophan,Lyso PC(16:0),Lyso PC(18:2)and Lyso PC(18:0)in the tripterygium glycosides group were significantly lower than that of the model group,and there were significant difference between the tripterygium glycosides group and the model group(P ? 0.05),but there were no significant difference between the tripterygium glycosides group and the blank group(P?0.05).It could be concluded from above results that the therapeutic effects of MTX and tripterygium glycosides may be related to the level of amino acids and LPCs.Research conclusion: ~1H-NMR and UPLC/Q-TOF MS techniques as the two common methods in metabonomics research,there is a certain complementarity in biomarker discovery.In this study,~1H-NMR and UPLC/Q-TOF MS techniques were used to study the endogenous metabolites between the patients with psoriasis vulgaris and the healthy volunteers,the results showed that the two methods have a good clustering effect.The biomarkers were identified by metabonomics techniques combined with multivariate statistical analysis will be helpful for the diagnosis of the disease,the further study of the pathogenesis and the formulation of the individualized dosage regimen.The psoriasis-like lesions of mice had been successfully induced by IMQ,and when exploring the effects of MTX and tripterygium glycosides on these markers that matching with the patients in the model mice,It was found that the therapeutic effects of MTX and tripterygium glycosides may be related to the level of amino acids and LPCs.This will help to establish the pharmacodynamics indicators,so as to provide the basis for the adjustment of drug dosage and the individualized administration.