| Objectives:1.By building a drug-active ingredient-target network based on network pharmacology,we investigated the targets and mechanisms of the combination of drugs commonly used by Professor Wang Sumei,Bombyx mori and Scorpion,in the treatment of TD,to provide theoretical basis for its prevention and treatment of tic disorders and a scientific reference for the promotion and application of antitwitch bug drugs.2.Using immunohistochemistry to detect the changes in the number of NF-κBp65 in the striatum of TD rats induced by IDPN,we clarify whether Bombyx mori and scorpion play a role in regulating neuroimmunity during the tic disorder treatment process.Methods:1.Network pharmacology research:①Drug target prediction:through Herb database search and literature collection as a supplement,we screen the effective active ingredients of Bombyx mori and Scorpion,and predict the target through SwissTargetPrediction database and PharmMapper database,and correct gene names through UniProt database;②Disease target prediction:potential targets related to refractory tic disorder were collected from online mendelian inheritance in man database,GeneCards database,TTD database,DisGeNet database,DRUGBANK database;③ The intersection targets of Bombyx mori-scorpion and refractory tic disorder were uploaded to the STRING11.5 database,and we draw the drug and disease PPI network;④Through the DAVID Bioinformatics Resources platform,the core targets of the Bombyx mori-scorpion drug combination treatment of refractory TD were analyzed by the means of GO annotation and KEGG pathway enrichment.2.Animal experiment research:25 male SD rats were randomly divided into 5 groups according to body weight,namely blank control group,model group,tiapride hydrochloride group,Chinese medicine group A(Jianpi Zhidong decoction group)and Chinese medicine group B(Jianpi Zhidong decoction+bombyx mori-scorpion group).There are 5 rats in each group.Except for the blank control group,the other 4 groups were intraperitoneally injected with IDPN(250 mg/kg/d)for 7 days to establish a TD rat model,and the Diamond method was used to determine whether the model was successfully established,and the stereotyped motor score and body weight were recorded.Continuous intragastric administration lasted for 4 weeks,in which the blank control group and the model group were given normal saline(10 mg/kg/d),and the drug groups were given tiapride hydrochloride suspension(21 mg/kg/d),Jianpi Zhidong decoction(16mg/kg/d),Jianpi Zhidong Decoction+Bombyx mori and Scorpion(16mg/kg/d)respectively,subsequently stereotyped movement score and weight measurement were performed again after the administration,and the number of NF-κBp65 positive cells and the mean optical density value of striatum of rats in each group were analyzed by immunohistochemistry.Results:1.Network pharmacological study:a total of 31 important active ingredients were screened from the Bombyx mori and Scorpion drug pair,including dihydroabietin benzoate,dibutyl phthalate,aurantiamide acetate,pheophorbide A,linoleic acid,Stearic acid methyl ester,and so on;1,403 effective targets for tic disorders,including 218 drug-disease intersection targets,such as ALB,AKT1,IL6,TNF,EGFR,IL1B,MAPK3,ESR1 and others;GO annotation analysis results suggest that the drug combination is mainly involved in signal transduction,chemical synaptic transmission,positive regulation of cell proliferation,postitive regulation MAPK cascade,lipid and atherosclerosis,5-hydroxytryptaminergic synapses,epidermal growth factor receptor tyrosine kinase inhibitor resistance and so on.The KEGG pathway enrichment analysis revealed that the drug combination may act mainly through Neuroactive ligand-receptor interactions,Calcium signaling pathway,MAPK signaling pathway,PI3K-Akt signaling pathway,cAMP signaling pathway,and AGE-RAGE signaling pathway.2.Animal experiments showed:①Body weight:there was a difference in body weight between the model group and the blank group after 7 days of modeling(P=0.017<0.05);after 4 weeks of gavage,there was a significant difference between the model group and the blank group(P=0.000<0.01),and there was a difference between the Chinese medicine group A and the model group(P=0.049<0.05),the comparison between the Chinese medicine group A and the Chinese medicine group B,the comparison between the Western medicine group and the model group,the Chinese medicine group A compared with Western medicine group,and Chinese medicine group B compared with Western medicine group,showed no statistical difference(P=1.000>0.05),and the difference between Chinese medicine group B and model group was not statistically significant(P=0.880>0.05).②Stereotyped movement score:after 7 days of modeling,except for the blank group,the stereotyped movement score of each rat in the four groups was greater than 1,which suggested successful modeling,and there was no statistical difference in the stereotyped movement score in the four groups(P=0.110>0.05);after 4 weeks of gavage,there was a statistical difference in the stereotyped movement score between the Chinese medicine group A and thiopride hydrochloride group and the model group(P=0.046<0.05).There was no statistically significant difference in stereotyped motor scores between Chinese medicine group B and model group(P=0.214>0.05),and no statistically significant difference in stereotyped motor scores between Chinese medicine group A and Chinese medicine group B,Chinese medicine group A and Western medicine group,and Chinese medicine group B and Western medicine group(P=1.000>0.05).③Number of NF-κBp65 positive cells and mean optical density values:the number of NF-κBp65 positive cells and mean optical density values of brain striatum of TD rats in each group were compared between two groups,and the results were not statistically significant(P=0.740>0.05,P=0.567>0.05).Conclusion:1.The Bombyx mori-Scorpion drug combination works on multiple components,multiple targets,and multiple pathways.Its mechanism may be that the core active components such as dihydroabietin benzoate,dibutyl phthalate,aurantiamide acetate,pheophorbide A,linoleic acid,Stearic acid methyl ester act on the cell membrane through the core targets such as ALB,AKT1,IL-6,TNF,EGFR,IL1B,MAPK3,ESR1 and so on.By participating in signal transduction,chemical synaptic transmission and other biological functions,it regulates Neuroactive ligand-receptor interaction,Calcium signal pathway,MAPK signal pathway,PI3K-Akt signal pathway and so on.2.NF-κBp65 was positively expressed in the striatum of IDPN-induced TD model rats,but compared with the blank group,the expression of NF-κBp65 in the striatum of model group rats had no statistically significant difference,which may be related to model factors,protein expression sites,experimental factors and so on,which provided a reference for further experiments.Meanwhile,at certain doses,the combination of Bombyx mori and Scorpion had no significant effect on the expression of NF-κBp65 protein in the striatum of TD rats induced by IDPN. |
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