| BackgroundPlaque psoriasis,known as psoriasis vulgaris,has a complex pathogenesis.Although there are many types of treatment methods,there is a lack of long-term effective methods.According to the previous research results of the research team,it was found that:① the initial cross-sectional survey showed that patients with plaque psoriasis had abnormal fat metabolism;② The results of serum metabolomics in patients with psoriasis suggest that there is an imbalance in sphingolipid metabolism in the serum,with abnormal expression of ceramide(Cer)and sphingomyelin(SM)③The proteomic results suggest that the expression of proteases SPTLC2 and SPHK,which are closely related to the production of Cer and sphingosine 1-phosphate(S1P),is abnormal in the skin lesions of psoriatic mice.Based on literature research,it is suggested that the core of sphingolipids metabolism is two biologically active sphingolipids,Cer and SIP,which can transform each other and participate in various metabolic activities of skin cells.Cer can inhibit programmed apoptosis of keratinocytes,making them unable to differentiate normally,leading to excessive proliferation;Secondly,Cer is an important component of the skin barrier and has a good water locking ability.And S1P can participate in NF-κB signal pathway transduction affects the migration and distribution of lymphocytes.Therefore,plaque psoriasis is closely related to imbalance in sphingolipids metabolism.Kai-Xuan-Jie-Du-Chu-Shi formula(KXJD)is a long-term clinical experience prescription used by the Dermatology Department of Guang’anmen Hospital,Chinese Academy of Chinese Medicine.It is proposed by the mentor,Director Song Ping,on the basis of inheriting the experience of well-known traditional Chinese medicine and combining a large number of clinical practices.This prescription is composed of eight herbs,including Guizhi(Cinnamomi Ramulus),Mahuang(Ephedra Herba),Huanglian(Coptidis Chinensis Franch),Qinghao(Artemisia Annua L.),Baihuasheshecao(Hedyotis Diffusa Willd),Huzhang(Polygonum Cuspidatum Seib.Et Zucc),Qingdai(Indigo Naturalis)and Longdancao(Adenophora capillaris).KXJD formula can not only dispel pathogenic wind-cold-dampness of skin and muscles through diaphoresis but cool the blood and relieve toxins.Previous studies have suggested that KXJD formula has a certain regulatory effect on lipid metabolism in psoriasis.Therefore,this study proposes a hypothesis that the KXJD formula can ultimately improve the skin lesions of plaque psoriasis by regulating the metabolism of sphingolipids,improving the skin barrier function,inhibiting epidermal cell proliferation and inflammatory signal pathways,and affecting the distribution of immune cells.ObjectiveThis study first used a mouse model of psoriasis as the research object to clarify the efficacy of KXJD formula on plaque psoriasis;Secondly,targeted lipomics detection methods was carried out to identify whether there was abnormal sphingolipids metabolism in plaque psoriasis and the specific regulation mechanism of KXJD on sphingolipids metabolism.Network pharmacology combined with molecular docking to predict the potential targets involved in regulating sphingolipids metabolism;Finally,using the mouse model and the HaCat cell model of psoriasis as research objects,and the functional antagonists of the S1P signaling pathway of sphingolipids metabolites as controls,to explore the mechanism of KXJD formula and its active components in regulating sphingolipids metabolism in plaque psoriasis.Methods1.Effect and safety evaluation of KXJD formula on the skin lesions of imiquimod induced psoriasis model mice60 SPF male C57 mice were randomly divided into 5 groups,namely blank group,model group,KXJD-M group,KXJD-H group,and FTY720 group.Except for the blank group,the other groups were treated with external coating of 62.5 mg of imiquimod to establish a psoriasis mouse model.After 5 days of intervention,samples of spleen,skin,thymus,and serum were taken on the 6th day.Record the daily weight of mice,collect the skin lesion image every day,and conduct PASI score;Thymus and spleen index were measured;Shoot the hyperplasia of dermal capillaries;Based on the above results,we evaluate the improvement and safety of KXJD formula on the symptoms and signs of skin lesions in mice with psoriasis,and screen the concentration of KXJD with good efficacy.Subsequently,HE staining was used to measure the thickness of the epidermis,and immunohistochemical method was used to detect the percentage of PCNA+cells in the skin lesions to evaluate the epidermal hyperplasia;WB method was used to detect the content of Claudin-1 and Occludin proteins in the skin lesions to evaluate the skin barrier function;Detection of NF-κB signal pathway by WB method to assess inflammatory signaling pathways;WB method was used to detect the expression of CD3 in skin lesions and spleen to assess the migration and distribution of lymphocytes;Elisa method was used to detect the content of IL-23 and IL-17 to assess the degree of inflammation of the disease;At the same time,liver and kidney tissues were stained with HE,and serum biochemical indicators ALT,AST,and BUN were detected by Elisa method to evaluate the drug safety of KXJD formula.2.Targeted lipidomics detection of sphingolipid metabolites in the skin lesions of imiquimod-induced psoriasis-like mice treated with KXJD formulaIn Experiment 1,10 skin lesions of mice in the healthy group,the psoriasis model group,and the high-dose KXJD formula were collected,and the content of sphingolipids metabolites in the skin lesions,including Cer,SIP,Sph,SM,GluCer,LacCer,and GalCer,was detected and analyzed by targeted detection using liquid chromatography and mass spectrometry.3.Prediction of potential targets of KXJD formula for the treatment of plaque psoriasis based on network pharmacology and molecular dockingUsing network pharmacology combined with molecular docking technology to predict the potential targets of KXJD formula in the treatment of plaque psoriasis.Firstly,TCMSP and TCMIP online databases of traditional Chinese medicine were used to screen active ingredients to obtain active targets;Then,five disease databases(GeneCards,OMIM,TTD,DisGeNET,and DrugBank)were used to establish targets for plaque psoriasis and obtain cross targets for drugs and diseases.Using the obtained intersection targets,protein-protein interaction maps were established,gene function analysis and pathway enrichment analysis could be carried out.At the same time,a drug-component-target network diagram was constructed,and core active components are selected based on topological parameters.Finally,molecular docking technology is used to dock core components with target proteins.4.KXJD formula participates in the regulation of sphingolipids metabolism in the skin lesions of imiquimod-induced psoriasis-like miceSelect the skin lesions of the healthy group,the model group,the high-dose group of KXJD formula,and the FTY720 group in Experiment 1.Immunohistochemical method was used to detect the content of S1P in skin lesions;Immunofluorescence assay was used to detect the content of Cer in the skin lesions;WB method was used to detect the content of protease SPHK,SPTLC2,and ASAH1 related to the production of Cer and SIP;The mRNA levels of five S1P receptors were detected by q-PCR method to comprehensively evaluate the specific regulatory mechanism of KXJD formula on sphingolipids metabolism.5.KXJD formula participates in the regulation of sphingolipids metabolism in the HaCat cell model of psoriasisA psoriasis cell model was established by using three inflammatory factors(IL-17,IL-22,and TNF-a,1:1:1,each 40ng/ml)to stimulate HaCat cells for 48h.Prepare rat serum containing drugs,determine the intervention concentration of the serum containing drugs,kaempferol and FTY720 using MTT method.Subsequently,WB method was used to detect the expression of intercellular junction protein and NF-κBsignal related proteins,cell cycle detection by flow cytometry,and inflammatory factors detection by q-PCR to evaluate the improvement effect of KXJD formula on HaCat cell model.Finally,the regulatory mechanism of KXJD formula on sphingolipids metabolism was evaluated by immunofluorescence assay to detect the content of S1P and Cer,WB assay to detect the content of sphingolipids metabolism related proteins SPHK,SPTLC2,and ASAH1,and q-PCR assay to detect the mRNA expression level of S1P receptor.Results1.Effect and safety evaluation of KXJD formula on the skin lesions of imiquimod induced psoriasis model mice(1)Compared with the blank group,KXJD formula can alleviate the weight loss of psoriasis mice(P<0.05);(2)Compared with the model group,KXJD formula can improve the erythema,scale,infiltration,and PASI scores of psoriatic mice(P<0.01);(3)Compared with the model group,KXJD formula can improve the organ index(P<0.01)and capillary proliferation in mice with psoriasis;(4)Compared with the model group,KXJD formula can reduce the epidermal thickness and the number of PCNA positive cells(P<0.01);(5)Compared with the model group,KXJD formula can increase the content of Claudin-1 and Occludin protein in psoriatic mice(P<0.01);(6)Compared with the model group,KXJD formula can reduce p-NFκB and p-IκBa in mice with psoriasis(P<0.01);(7)Compared with the model group,KXJD formula can reduce the content of CD3 in the skin lesions and spleen of psoriatic mice;(8)Compared with the model group,KXJD formula can reduce the content of IL-17 and IL-23 in serum of mice with psoriasis(P<0.05);(9)HE staining of liver and kidney tissues showed no significant pathological changes among the groups,and there was no significant difference in ALT,AST,and BUN.2.Targeted lipidomics detection of sphingolipid metabolites in the skin lesions of imiquimod-induced psoriasis-like mice treated with KXJD formulaFor Cer,compared with the healthy group mice,the total content of Cer in the model group mice increased(P<0.05).Among them,the content of 55 Cers in the skin lesions of the model group mice increased(P<0.05),the content of 15 Cers decreased(P<0.05),and the content of 12 Cers did not change significantly(P>0.05).Compared with the model group,after the intervention of KXJD formula,the content of 11 Cers decreased(P<0.05),while the content of 3 Cers increased.For Sph and S1P,compared with the healthy group mice,the content of Sph and SIP in the skin lesions of the model group mice increased(P<0.05).Compared with the model group,after the intervention of KXJD formula,the content of two types of Sph decreased(P<0.05)and the content of S1P decreased(P<0.05).For SM,compared with the healthy group mice,there was no significant increase or decrease in the total content of SM in the skin lesions of the model group mice(P>0.05).Among them,the content of 5 types of SM in the skin lesions of the model group mice increased(P<0.05),the content of 11 types of SM decreased(P<0.05),and the content of 8 types of SM showed little change(P>0.05).Compared with the model group,after the intervention of KXJD formula,there were 3 types of SM levels increased(P<0.05)and 2 types of SM levels decreased(P<0.05).Compared with the healthy group mice,the total content of GluCer,GalCer,and LacCer in the skin lesions of the model group mice showed a significant increase(P<0.05).For GluCer,23 of them showed an increase in content(P<0.05),while 1 showed little change(P>0.05);For GalCer,19 contents significantly increased(P<0.05),while 3 contents showed little change(P>0.05);For LacCer,12 contents significantly increased(P<0.05),while 4 contents showed little change(P>0.05).Compared with the model group,KXJD formula can downregulate the expression of some glycosphingolipids(P<0.05).Among them,14 species are GluCer and 5 species are GalCer.3.Prediction of potential targets of KXJD formula for the treatment of plaque psoriasis based on network pharmacology and molecular dockingA total of 69 active ingredients were selected from traditional Chinese medicine prescriptions,with a total of 266 potential targets;A total of 1201 targets were searched for diseases.There were 108 cross targets in total.The top three targets in the constructed protein-protein interaction map are TNF,Aktl,and TP53.In GO analysis,the biological processes involved include gene expression,drug response,inflammatory response,response to hypoxia/exogenous stimulation/lipopolysaccharide,and positive regulation of apoptosis;The cellular components involved in intervention are mainly related to cytosol,cytoplasm,extracellular space,extracellular secretions,macromolecular complexes,chromosomes,membrane rafts,etc;The molecular functions of intervention mainly include protein binding,enzyme binding,identical protein binding,cytokine binding,transcription cofactor binding,and heme binding.The pathway enrichment analysis showed that the most relevant signaling pathways were lipid and atherosclerosis signaling pathways.In the constructed active componenttarget protein-pathway network,the top three core components are quercetin,luteolin,and kaempferol.Molecular docking was performed between top 3 core componnets and S1PRs which was significant for lipid and atherosclerosis signaling pathways.The docking results show that the affinity of core components to target proteins is strong,suggesting that S1PRs may be potential intervention targets for KXJD formula.4.KXJD formula participates in the regulation of sphingolipids metabolism in the skin lesions of imiquimod-induced psoriasis-like mice(1)Compared with the model group,KXJD formula can reduce the content of S1P in psoriatic skin lesions and increase the content of Cer in psoriatic mouse skin lesions(P<0.05);(2)Compared with the model group,KXJD formula can reduce the content of sphingolipids metabolism regulating enzymes,including SPHK1/2,SPTLC2,and ASAH1 in psoriatic lesions(P<0.05);(3)Compared with the model group,KXJD formula can reduce the mRNA level of S1PR1-5 in the skin lesions of psoriatic mice.5.KXJD formula participates in the regulation of sphingolipids metabolism in the HaCat cell model of psoriasis(1)Compared with the model group,the serum containing KXJD can inhibit the proliferation of psoriasis cell model(P<0.01);(2)Compared with the model group,the drug-containing serum can increase the content of tight junction protein in the psoriasis cell model(P<0.01);(3)Compared with the model group,the serum containing KXJD can reduce the number of G0/G1 phase cells and increase the number of S phase cells in the psoriasis cell model(P<0.01);(4)Compared with the model group,the serum containing KXJD can reduce p-NFκB and p-IκBα in psoriasis cell model(P<0.01);(5)Compared with the model group,the serum containing KXJD can reduce the mRNA level of IL-6,IL-8,and TNFα in psoriasis cell models(P<0.05);(6)Compared with the model group,the serum containing KXJD can reduce the content of S1P and increase the content of Cer in the psoriasis cell model;(7)Compared with the model group,the serum containing KXJD can reduce the levels of ASAH1,SPTLC2,and SPHK1 proteins in the psoriasis cell model(P<0.01);(8)Compared with the model group,the serum containing KXJD can reduce the mRNA level of S1PR2 in the psoriasis cell model(P<0.05).Conclusion1.KXJD formula can significantly improve local skin lesions in psoriasis model mice,including repairing skin barrier,inhibiting epidermal proliferation,and inhibiting NF-κB Inflammatory signaling pathway,reduces inflammatory factors,and affects lymphocyte distribution.2.KXJD formula has good safety.3.There is an imbalance in sphingolipids metabolism in plaque psoriasis,KXJD formula has effects on sphingolipid metabolites.4.KXJD formula can participate in the regulation of related processes of sphingolipids metabolism by interfering with the levels of related sphingolipids metabolites(Cer,S1P).5.The potential mechanism of KXJD formula for improving skin lesions in plaque psoriasis may be related to regulating processes related to sphingolipids metabolism... |
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