Purpose:First of all,through the contrast of silver particles to verify white mange mixture of blood heat treatment the clinical curative effect and safety of patients with psoriasis;Then,application of metabonomics technology,analysis of serum metabolic characteristics of patients with psoriasis,the white mange mixture of blood heat treatment psoriasis before and after the change of serum metabolic state,to explore the diagnosis and treatment white mange mixture of blood heat and its possible mechanism of psoriasis;Again through the network pharmacology to study the effect of white mange mixture to treat psoriasis may targets and pathways;Finally,to carry out the white mange mixture intervention in patients with psoriasis and Ha Ca T cells in rats of experimental study on possible mechanism is verified.Material and method:1.A total of 66 patients with blood-heat psoriasis were collected from the dermatology department of the Affiliated Hospital of Liaoning University of Traditional Chinese Medicine.Firstly,the diagnosis and classification of the outpatients were identified.The patients to be included should meet the diagnostic criteria of Western medicine psoriasis and TCM syndrome,and the patients meeting the diagnosis criteria were screened according to the inclusion and exclusion criteria.Will be included in the psoriatic patients were randomly divided into two groups: treatment group in 33 people,give white mange mixture 50 ml,2times daily oral,joint card berth triol cream twice daily for external use;Positive drug control group 33 patients,Xiaoyin granules 5.25 g twice a day oral,combined with carpotriol cream twice a day for external use;After 3 months of treatment,subjects were followed up before treatment,30 days of treatment,60 days of treatment,and 90 days(at the end of treatment).The efficacy and safety of the two groups were evaluated.The main index was PASI score,and the secondary index was PGA score,efficacy index and TCM main clinical symptom Assessment Scale score.2.Application of metabonomics technology test and application of 11 patients with psoriasis metabolites white mange mixture of metabolites,after treatment and detection of metabolites of 10 cases of normal healthy people.Then,the difference of psoriatic patients and normal healthy people metabolites were analyzed,and its applications in patients with psoriasis white mange mixture after treatment differences metabolites were analyzed.Specific methods: The serum samples were first remelted and pretreated,and then the samples were detected by ultra-high performance liquid chromatography and high resolution mass spectrometry,and the data were quality-controlled and standardized,and the metabolite content was counted and the percentage of metabolite content histogram was drawn.Then,principal component analysis,partial least squares discriminant analysis and orthogonal partial least squares discriminant analysis were performed,ROC curve was drawn and the area under the AUC curve was calculated,and T-test volcano map was drawn,focusing on metabolites with p < 0.05 and FC absolute value greater than 2 in the volcano map.These metabolites were significantly different between groups.Pearson correlation heat map was used to observe whether the correlation of metabolites changed among different groups.To obtain the metabolites of filtered according to the VIP value and FC,the metabolites,namely characteristic differences of different metabolites pathway analysis,analysis of different metabolites of metabolic pathways,predict white mange mixture mechanism for the treatment of psoriasis.3.The network pharmacology: through TCMSP respectively by white mange mixture of salvia miltiorrhiza,bean root,radix isatidis,flos lonicerae,radix paeoniae rubra,mountain cogongrass rhizome,spreading hedyotis herb,cortex phellodendri as keywords retrieval and filtered according to the condition of effective components,obtain 8 TCM active ingredients.Then the active compounds of Rehmannia and scorpion and their targeting genes were obtained through BATMAN-TCM database.The protein target of drug acquisition was converted to Gene Symbol in the Uniprot database.The Gene Cards database was retrieved with the keyword "Psoriasis",and the obtained targets were imported into Excel for data processing.The Score value was set to be greater than the median,and the potential targets of psoriasis were obtained.Using microscopic letter online mapping tools build white mange mixture with psoriasis intersection targets Wayne figure.White mange mixture of active ingredient and the corresponding targets,disease targets import Cyto Scape3.8.2,build "Chinese medicine"-the active ingredient-gene targets-disease network diagram.The built-in tool of Cyto Scape3 was used to analyze network topological parameters of effective ingredients and targets,and to determine core targets and main active ingredients for drug efficacy based on network topological parameters such as connectivity,intermediation and compactness.The intersection target was submitted to STRING database to construct PPI network model,and imported into Cyto Scape3.8.2 for visualization analysis to obtain its degree value.The common targets of drugs and diseases were imported into DAVID database for KEGG pathway enrichment analysis and GO enrichment analysis.4.Experimental study: In the first part,HE staining showed that the epidermal layer was clear in the blank control group,there was no inflammatory cell infiltration in the dermis,and the structure of sebaceous glands and hair follicles was clear.And model control group see a lot of parakeratosis cutin cell,the western medicine control group and traditional Chinese medicine(TCM)low,medium and high dose group of skin by the thick and thin,cutin cell morphology.TUNEL test showed that compared with the blank control group,the number of apoptotic cells in the model control group was significantly reduced(p < 0.01),and the apoptotic cells in the western medicine control group and the low-dose,medium-dose and high-dose Chinese medicine groups were significantly increased(p < 0.01).The expression levels of HSP27,bax,bcl-2,p53 and caspase-3 in rat skin tissues were detected by western-blot.Compared with blank control group,the expression levels of HSP27 and bcl-2 in skin tissue of other groups were significantly increased(p < 0.01),and the expression levels of bax,p53 and caspase-3 in skin tissue of rats in high-dose,medium-dose and low-dose groups and western control group were significantly increased(p < 0.01).Compared with model control group,the expressions of HSP27 and bcl-2 in skin tissue of rats in high-dose,medium-dose and low-dose groups and western control group were significantly decreased(p< 0.01),while the expressions of bax,p53 and caspase-3 were significantly increased(p <0.01).Compared with blank control group,HSP27 in model control group increased significantly(p < 0.01).Compared with model control group,HSP27 in western medicine control group and Chinese medicine low,medium and high dose groups was significantly decreased(p < 0.01).In the second part,the expression of col-1,MMP-1,MMP-3 and TIMP-1 in the skin tissue of rats in each group was detected by western-blot.Compared with blank control group,the expression levels of col-1,MMP-1,MMP-3 and TIMP-1 in skin tissue of other groups were significantly increased(p < 0.01).Compared with model control group,the expression levels of col-1,MMP-1,MMP-3 and TIMP-1 in skin tissue of rats in high-dose,medium-dose and low-dose groups and western control group were significantly decreased(p < 0.01).The protein expression levels of col-1,MMP-1,MMP-3 and TIMP-1 in the skin tissue of rats in each group were detected by immunohistochemistry.Compared with blank control group,the expression levels of col-1,MMP-1,MMP-3 and TIMP-1 in skin tissue of other groups were significantly increased(p < 0.01).Compared with model control group,The expression levels of col-1,MMP-1,MMP-3 and TIMP-1 in skin tissue of rats in high-dose,medium-dose and low-dose groups and western control group were significantly decreased(p < 0.01).In the third part,the results of CCK-8 detection showed that compared with blank control group,the proliferative activity of other groups was significantly increased(p <0.01).Compared with model control group,cell proliferation activity in serum groups with different doses and HSP27 inhibitor intervention group was significantly decreased(p <0.05).Flow cytometry showed that compared with blank control group,apoptosis rate of model control group was significantly decreased(p < 0.01),and apoptosis rate of drug serum group and inhibitor intervention group were significantly increased(p < 0.01).Compared with the model control group,the percentage of apoptosis in the intervention groups with different doses of drug-containing serum and inhibitor was significantly increased(p < 0.01).The contents of col-1,MMP-1,MMP-3 and TIMP-1 in cell culture supernatant of other groups were significantly increased compared with blank control group by ELISA(p <0.01).Compared with the model control group,the contents of col-1,MMP-1,MMP-3 and TIMP-1 in the cell culture supernatant of each dose containing serum and inhibitor intervention group were significantly decreased(p < 0.01).western-blot analysis showed that compared with blank control group,the expression levels of HSP27 and bcl-2 in other groups were significantly increased(p < 0.01),while the expression levels of bax,p53 and caspase-3in drug-containing serum groups and inhibitor intervention groups were significantly increased(p < 0.01).The expression levels of bax,p53 and caspase-3 in model control group were significantly decreased(p < 0.01).Compared with model control group,the expression levels of HSP27 and bcl-2 in each dose containing serum and inhibitor intervention group were significantly decreased(p < 0.01),while the expression levels of bax,p53 and caspase-3were significantly increased(p < 0.01).Results:1.Clinical research: Participants included 66 patients,33 cases in Baidan mixture group(experimental group)and 33 cases in silver granule group(control group).During the study,there were 5 cases of total loss,3 cases in experimental group and 2 cases in control group.The efficacy of shedding cases was analyzed.In the control group,there were 18 males(54.5%)and 15 females(45.5%).In the treatment group,there were 16 males(48.5%)and 17females(51.5%).Chi-square test was used for gender difference between the two groups,and there was no statistical significance(P> 0.05).The mean age of the control group was49.55±10.36 years old,and that of the treatment group was 48.24±11.29 years old.Independent sample t test was used for age difference between the two groups,and there was no statistical significance(P> 0.05).The mean course of disease was 10.00(3.00,12.50)years in the control group and 9.00(1.00,12.00)years in the treatment group.Manwhitney U test was used to detect the difference in disease course between the two groups,and the result was not statistically significant(P> 0.05).The PASI score before intervention was 11.73±4.68 in the control group and 12.47±5.72 in the treatment group.Independent sample T-test showed no significant difference in PASI scores between the two groups before intervention(P> 0.05),indicating that baseline balance was comparable.In the control group,before intervention,skin color score was 4.74±1.41,skin burning sensation score was 4.29±1.41,itching degree score was 5.65±2.83,irritability score was 4.48±1.41 and TCM syndrome score was19.16±7.07.Before intervention,skin color score was 4.8±1.41,skin burning sensation score was 4.53±1.41,itching degree score was 6.0±2.83,irritability score was 4.90±3.54,TCM syndrome score was 20.23±9.19.According to independent sample t test,there was no statistical significance in TCM syndrome scores between the two groups before intervention(P> 0.05).The PASI score of the control group was 5.46±2.82 after intervention.The PASI score of the treatment group was 5.58±3.01 after intervention.By paired sample t test,PASI scores in both groups were significantly lower after intervention than before intervention(P<0.001).In the control group,after intervention,skin color score was 1.97±1.66,skin burning sensation score was 1.77±1.71,pruritus degree score was 3.84±4.95,irritability score was2.87±1.65 and TCM syndrome score was 10.45±5.54.After intervention,skin color score was2.27±2.03,skin burning sensation score was 1.97±1.87,itching degree score was 1.87±2.12,irritability score was 2.20±1.59 and TCM syndrome score was 7.93±5.96.According to the paired sample T-test,skin color score,skin burning sensation score,itching degree score,irritability score and overall TCM syndrome score in both groups were significantly lower after intervention than before intervention score(P< 0.001).The difference of PASI in the control group was 6.27±3.45,and that in the treatment group was 6.89±3.98.Independent sample t test showed that there was no significant difference in PASI scores between the two groups after intervention(P> 0.05).As for TCM syndrome evaluation between the two groups after intervention,according to independent sample t test,the differences in itching degree score(P < 0.001)and irritability score(P < 0.05)between the two groups after intervention were statistically significant,indicating that the improvement degree of the treatment group was better than the control group in the above two aspects.There were no significant differences in skin color score,skin burning sensation score and overall TCM syndrome score between the two groups after intervention(P> 0.05).In the control group,0 cases were cured,14 cases were effective,14 cases were effective,3 cases were ineffective,the effective rate was 45.2%,the total effective rate was 90.3%.In the treatment group,0 cases were cured,14 cases were effective,12 cases were effective,4 cases were ineffective,the effective rate was46.7%,the total effective rate was 86.7%.Fisher’s Chi-square accuracy test showed no statistically significant difference in efficacy between the two groups,and no statistically significant difference in recovery rate and total effective rate between the two groups after intervention.2.Metabolomics study: Compared with healthy people,psoriasis patients had 211 metabolites with P < 0.05,63 metabolites were obtained after screening according to VIP value and FC,which were potential biomarkers related to psoriasis.A total of 17 differential metabolites were screened in HMDB database.White mange mixture compared before and after treatment,P < 0.05 the metabolite of a total of 140,according to the values of VIP and FC metabolites21 after screening,for white mange mixture to treat psoriasis related potential biomarkers,in the communist party of China(HMDB database screening to differences metabolites 20.ROC curve analysis was performed on the differential metabolites of psoriasis patients and healthy people to evaluate their diagnostic performance,and the AUC value was calculated and the ROC curve analysis diagram was drawn.Among them,the AUC value of 31 compounds was greater than 0.7.White mange mixture before and after treatment of ROC curve analysis differences metabolites,22 compounds the AUC value > 0.7.The enrichment analysis of different metabolites between psoriasis patients and healthy people showed that 32 metabolic pathways were involved.The metabolic pathways were ranked according to P value,and there were 5 metabolic pathways with P < 0.05,including glycerol phospholipid metabolism,alanine aspartic acid and glutamic acid metabolism,histidine metabolism,caffeine metabolism,purine metabolism,etc.White mange mixture before and after treatment differences metabolites concentration analysis,the results involving 22 metabolic pathways,including P < 0.05 metabolic pathways have four,including: steroid hormone biosynthesis,pyrimidine metabolism,sheath lipid metabolism,starch and sucrose metabolism.3.Network Pharmacology:In TCMSP database can retrieve the white mange total 8 taste of traditional Chinese medicine,according to a set of OB and DL value filtering,active ingredients for 225,including spreading hedyotis herb seven,four cogongrass rhizome,radix isatidis,39,radix paeoniae rubra,29,65,salvia miltiorrhiza,cortex phellodendri 37,honeysuckle,23,21 mountain bean root,The Batman-TCM database was used to set the threshold value > 20,P < 0.05 as the screening standard bricks,and 16 active compounds were obtained by searching Rehmannia and scorpion,among which 4 were Rehmannia and 12 were scorpion.White mange mixture of eight kinds of traditional Chinese medicine(TCM)in225 active ingredients found in TCMSP database corresponding "Targetname",the predicted gene targets in Uniprot database converted into standard name,to weight for the 510 targets.Psoriasis related targets were screened out in Gene Cards,and the Score value was set to be greater than the median as potential targets for psoriasis,and 854 disease targets were obtained.White mange mixture of active ingredients,drugs and disease common target import Cytoscape 3.8.2 building network,a total of 744 nodes,6704 wire,By Cytoscape 3.8.2 built-in Networkanalyzer function analysis in the "white mange mixture party-active ingredients-drug targets-psoriasis" the characteristics of the network,found that the drug active ingredient quantity is red paeony root,salvia miltiorrhiza bean root,radix rehmanniae,mountain,The top 10 active ingredients were quercetin,β-sitosterol,luteolin,stigmasterol,kaempferol,gamma-aminobutyric acid,flaxanthin,tanshinone IIA,formononetin,cryptotanshinone.White mange mixture drugs with psoriasis intersection targets 121 genes into String,get the protein interaction network,further by topological parameters,gene screening in 51.White mange mixture drug targets 379 genes into String,get the protein interaction network diagram,is obtained by topological parameters selection,a total of 28 genes.121 common targets were imported into the DAVID database for GO function analysis,and 735 biological processes GO-BP,67 cellular components GO-CC,and 116 molecular functional Go-MF were obtained.The enrichment analysis results showed that:The biological functions mainly include inflammatory response,negative regulation of apoptosis and positive regulation of cell proliferation.Cell components mainly included macromolecular complex,cell membrane cave-like invagination,membrane raft,RNA polymerase II transcription factor complex,etc.Molecular functions mainly include enzyme binding,identical protein binding,protein binding,cytokine activity and so on.In DAVID database entry white mange mixture with psoriasis and 121 common targets,KEGG pathway enrichment analysis path,you get 154 sorted according to the Log10 P values,High ranking Pathways were Pathways in cancer,Lipid and atherosclerosis,AGE-RAGE signaling pathway in diabetic complications,and Fluid shearstress and atherosclerosis.4.Experimental study: In the first part,HE staining showed that the epidermal layer was clear in the blank control group,there was no inflammatory cell infiltration in the dermis,and the structure of sebaceous glands and hair follicles was clear.And model control group see a lot of parakeratosis cutin cell,the western medicine control group and traditional Chinese medicine(TCM)low,medium and high dose group of skin by the thick and thin,cutin cell morphology.TUNEL test showed that compared with the blank control group,the number of apoptotic cells in the model control group was significantly decreased(p < 0.01),while the number of apoptotic cells in the western medicine control group,low-dose Chinese medicine group,medium-dose Chinese medicine group and high-dose Chinese medicine group was significantly increased(p < 0.01).The expression levels of HSP27,bax,bcl-2,p53 and caspase-3 in rat skin tissues were detected by western-blot.Compared with blank control group,the expression levels of HSP27 and bcl-2 in skin tissue of other groups were significantly increased(p < 0.01),and the expression levels of bax,p53 and caspase-3 in skin tissue of rats in high-dose,medium-dose and low-dose groups and western control group were significantly increased(p < 0.01).Compared with model control group,the expressions of HSP27 and bcl-2 in skin tissue of rats in high-dose,medium-dose and low-dose groups and western control group were significantly decreased(p < 0.01),while the expressions of bax,p53 and caspase-3 were significantly increased(p < 0.01).Compared with blank control group,HSP27 expression was significantly increased in model control group(p < 0.01).Compared with model control group,HSP27 in western medicine control group,low dose group,medium dose group and high dose group was significantly decreased(p < 0.01).In the second part,the expression of col-1,MMP-1,MMP-3 and TIMP-1 in the skin tissue of rats in each group was detected by western-blot.Compared with blank control group,the expression levels of col-1,MMP-1,MMP-3 and TIMP-1 in skin tissue of other groups were significantly increased(p < 0.01).Compared with model control group,the expression levels of col-1,MMP-1,MMP-3 and TIMP-1 in skin tissue of rats in high-dose,medium-dose and low-dose groups and western control group were significantly decreased(p < 0.01).The protein expression levels of col-1,MMP-1,MMP-3 and TIMP-1 in the skin tissue of rats in each group were detected by immunohistochemistry.Compared with blank control group,the expression levels of col-1,MMP-1,MMP-3 and TIMP-1 in skin tissue of other groups were significantly increased(p < 0.01).Compared with model control group,The expression levels of col-1,MMP-1,MMP-3 and TIMP-1 in skin tissue of rats in high-dose,medium-dose and low-dose groups and western control group were significantly decreased(p < 0.01).In the third part,the proliferative activity of cells measured by CCK-8 showed that compared with blank control group,the proliferative activity of cells in other groups was significantly increased(p < 0.01).Compared with the model control group,the cell proliferation activity in the intervention group with different doses of drug-containing serum and HSP27 inhibitor was significantly decreased(p < 0.05).Flow cytometry showed that compared with blank control group,the percentage of apoptotic cells in model control group was significantly decreased(p< 0.01),and the percentage of apoptotic cells in each dose serum containing drug and inhibitor intervention group was significantly increased(p < 0.01).Compared with the model control group,the percentage of apoptosis in the intervention groups with different doses of drug-containing serum and inhibitor was significantly increased(p < 0.01).The contents of col-1,MMP-1,MMP-3 and TIMP-1 in cell culture supernatant of other groups were significantly increased compared with blank control group by ELISA(p < 0.01).Compared with the model control group,the contents of col-1,MMP-1,MMP-3 and TIMP-1 in the cell culture supernatant of each dose containing serum and inhibitor intervention group were significantly decreased(p < 0.01).western-blot analysis showed the expression levels of HSP27,bax,bcl-2,p53 and caspase-3.Compared with blank control group,the expression levels of HSP27 and bcl-2 in other groups were significantly increased(p < 0.01),and the expression levels of bax,p53 and caspase-3 in different doses of drug-containing serum and inhibitor intervention groups were significantly increased(p < 0.01).The expression levels of bax,p53 and caspase-3 in model control group were significantly decreased(p <0.01).Compared with model control group,the expression levels of HSP27 and bcl-2 were significantly decreased(p < 0.01),while the expression levels of bax,p53 and caspase-3 were significantly increased(p < 0.01).Conclusion:1.Clinical studies have confirmed that oral white mange mooring triol cream mixture combined external card can effectively improve blood heat syndrome clinical symptoms in patients with psoriasis,curative effect is distinct,good security,compare the silver particles,white mange mixture has more advantages in terms of improve the itch and fidgety.2.Patients with psoriasis obvious metabolic disorder and the differences between lipid metabolites as the main characteristics of white mange mixture in the treatment of psoriasis mechanism may be related to steroid hormone biosynthesis,pyrimidine metabolism,sheath lipid metabolism and so on many kinds of ways.3.White mange mixture has many components,treatment effect,the main active ingredient for beta sitosterol,quercetin,luteolin,stigmasterol,kaempferia galanga phenol,core targets including TNF,STAT3,PTGS2,AR,VEGFA,IL-1 b,TP53,MMP9,The main Pathways include Pathways in cancer,Lipid and atherosclerosis,AGE-RAGE signaling pathway in diabetic complications,and Fluid shearstress and atherosclerosis,etc.4.White mange mixture of psoriasis model rats and Ha Ca T cells have obvious intervention effect,can effectively improve the psoriasis rat epidermis layer thickening,cutin cell parakeratosis,subcutaneous tissue pathological phenomena such as inflammatory cells infiltration.White mange mixture that HSP27 after intervention,the BCL-2,col-1,MMP 1,the expression of MMP-3 and TIMP-1 decreased significantly,and make the bax,p53,caspase-3 a significant rise in expression,by promoting apoptosis,inhibition of proliferation and restore the extracellular matrix reconstruction treatment of psoriasis. |