| Objective:1.To summarize and excavate professor Zhao Ruihua’ s medication and core prescription for Adenomyosis dysmenorrhea.2.To analyze and verify the molecular mechanism and signal pathway of Dangui Xiaozheng decoction in treating Adenomyosis.Methods:1.Core prescription of Professor Zhao Ruihua for Adenomyosis dysmenorrhea based on data miningBy sifting through real world,clinically valid medical records,the data were incorporated into the prescriptions of Professor Zhao Ruihua for patients with effective Adenomyosis dysmenorrhea.After data normalization and collation,through data mining methods such as Drug frequency,Four Natures,Five Flavours,Channel Tropism and Drug Classification,Association rules,Cluster analysis and combining with clinical practice,the interview results of the expert herself are synthesized,get Professor Zha.o Ruihua’s medication and core prescription for Adenomyosis dysmenorrhea.2.To explore the molecular mechanism of Dangui Xiaozheng decoction in treating Adenomyosis based on Network pharmacology and animal experimentsTo explore the pharmacological mechanism of compound prescription of Chinese herbal compound by using the method of network pharmacology.Firstly,the active components of Dangui Xiaozheng decoction were searched and screened by TCMSP database,and the protein targets of these compounds were screened.The candidate genes were obtained from GeneCards,DisGeNET and other databases,and from the cross network of Adenomyosis and drug targets."Dangui Xiaozheng decoction-compound-target-Adenomyosis" network was constructed by Cytoscape,PPI network was constructed by STRING website,and GO and KEGG enrichment analysis was performed by Metascape,and visually express the result.Finally,the important signal pathways and related targets suggested by the research of network pharmacology were verif ied by animal experiments,and the material basis and mechanism of TCM compound prescription were expounded preliminarily.Results:1.Core prescription of Professor Zhao Ruihua for Adenomyosis dysmenorrhea based on data mining(1)General data results:A total of 802 prescriptions were obtained in 122 patients,with an average of 7 consultations.The mean age of the patients was 38.30±0.51 years.The mean duration of Adenomyosis was 32.55 ± 3.23 months,and the mean duration of dysmenorrhea was 78.15±8.42 months,which was significantly higher than that of Adenomyosis(P<0.01).There were 26(21.31%)patients with adenomyoma,40(32.79%)patients with menorrhagia,14(11.48%)patients with chronic pelvic pain,and 32(26.23%)patients with fertility requirements.(2)Drug frequency:The 802 prescriptions included in this study involved a total of 105 traditional Chinese medicines,with a cumulative frequency of 12,320.The maximum number of drug flavors included in a single prescription was 24,at least 9,with an average of(15.36±2.85)flavors.Of the drugs used more than 50%of the time,14 were,they are Poria,Cmnamomi Mmulus,Galli Gigerii Endothelium Corneum,Paeoniae Radix Alba,Coicis Semen,Atractmlodis Macrocephalae Rhizoma,Notoginseng Radix et Rhizoma,Glycyrrhiza uralensis,Codonopsis Radix,Fenugreek,Zingiber is Rhizoma Recens,Salvia Miltiorrhiza,Curcumae Longae Rhizoma and Bupleuri Radix.(3)Four Natures,Five Flavours,C hannel Tropism and Drug Classification:According to the statistics of Four Natures of traditional Chinese medicine,the drugs with warm and flat nature were used more frequently,and the drugs with warm and hot nature were much more than those with cold and cool nature.The statistical results of Five Flavours of traditional Chinese medicine showed that pungent,sweet and bitter drugs were used the most.The Channel Tropism of drugs mainly involves spleen channel,liver channel,lung channel,stomach channel and kidney channel.According to the classification of drug efficacy,it was found that the main herbs included in this study were tonic,antipyretic,activating blood and removing blood stasis,hemostatic,regulating qi and warming Li.(4)Drug association rules:In order of support,the main drugs involved in the drug relationship with support>85%were Poria,Cmnamomi Mmulus,Galli Gigerii Endothelium Corneum,Paeoniae Radix Alba,Notoginseng Radix et Rhizoma,Atractmlodis Macrocephalae Rhizoma,Coicis Semen,etc.Combined with Professor Zhao Ruihua ’s prescription writing habit,the commonly used drug combinations were Cmnamomi Mmulus-Paeoniae Radix Alba,Galli Gigerii Endothelium Corneum-Coicis Semen,PoriaAtractmlodis Macrocephalae Rhizoma,etc.(5)Drug cluster analysis:In this study,the high-frequency use of traditional Chinese medicine mainly clustered into 3 categories.The first category is Curcumae Rhizoma,Gleditsiae Spina,Salvia Miltiorrhiza,Notopterygii Rhizoma et Radix,Glycyrrhiza uralensis,Bupleuri Radix and Atractmlodis Ma.crocephalae Rhizoma.The second category is Zingiberis Rhizoma,Euodiae Fructus,Citri Reticulatae Pericarpium,Zingiberis Rhizoma Praeparatum,Radix astragali preparation,Galli Gigerii Endothelium Corneum,Coicis Semen,Not.oginseng Radix et Rhizoma,Curcumae Longae Rhizoma,Cyperi Rhizoma,Cmnamomi Mmulus,Paeoniae Radix Alba and Fenugreek.The third category is Poria,Atractmlodis Macrocephalae Rhizoma,Amomi Fructus,Pogostemonis Herba,Zingiberis Rhizoma Recens,Jujubae Fructus,Codonopsis Radix,Glycyrrhiza uralensis,Pinelliae Rhizoma and Rosae Chinensis Flos.(6)Core prescription analysis:the data mining results of 802 prescriptions combined with the tutor’s clinical prescription medication habits and expert interview results,the key prescriptions of Professor Zhao Ruihua in the treatment of dysmenorrhea in Adenomyosis were:Cyperi Rhizoma,Salvia.Miltiorrhiza,Cmnamomi Mmulus,Galli Gigerii Endothelium Corneum,Coicis Semen,Notoginseng Radix et Rhizoma,Paeoniae Radix Alba,Curcumae Longae Rhizoma,Zingiberis Rhizoma and Dioscoreae Rhizoma.Professor Zhao Ruihua named it Dangui Xiaozheng decoction.2.To explore the molecular mechanism of Dangui Xiaozheng decoction in treating Adenomyosis based on Network pharmacology and animal experiments(1)The active ingredients and targets:The effective components of each Traditional Chinese medicine were obtained,including 18 kinds of Cyperi Rhizoma,65 kinds of Salvia Miltiorrhiza,7 kinds of Cmnamomi Mmulus,24 kinds of Galli Gigerii Endothelium Corneum,9 kinds of Coicis Semen,8 kinds of Notoginseng Radix et Rhizoma,13 kinds of Paeoniae Radix Alba,3 kinds of Curcumae Longae Rhizoma,5 kinds of Zingiberis Rhizoma,16 kinds of Dioscoreae Rhizoma,including luteolin,sitosterol,beta sitosterol,stigmasterol and catechins.All the collected compounds were combined and weighed,and the compounds without corresponding action targets were deleted.Finally 126 potential effective compounds were obtained.A total of 554 targets were obtained by combining the corresponding targets of each drug component and deleting the repeated values.(2)Acquisition of drug targets:1309 and 154 disease-related targets were obtained in Genecards and DisGeNET respectively,and a total of 1353 disease targets were obtained by combining and removing repetitive values.The intersection of adenomyosis target genes and potential targets of Dangui Xiaozheng decoction compound was obtained by Venn diagram.183 common targets were obtained.(3)Network diagram construction of "TCM-compound-target disease":The 126 active components of adenomyosis and 183 common targets of drug disease were introduced into Cytoscape3.8.2 software,respectively,to construct a Network diagram of Dangui Xiaozheng decoction-compound-target-AM.The network consists of 320 nodes and 1380 edges.The top 7 compounds were quercetin,luteolin,β-sitosterol,stigmasterol,bile triene,kaoneferol and tanshinone IIA,with the corresponding degree values of 97,90,75,45,41,39,23,respectively.It was speculated that these compounds might be important active components of Dangui Xiaozheng Decoction in the treatment of AM.(4)PPI network diagram:183 common targets Input STRING website to construct PPI network.The result shows that there are a.total of 183 nodes,3964 edges,and the average degree value of network nodes is 43.32.In order of degree value,AKT1,TNF,IL6,TP53,EGFR,HIF1A and PTGS2 are the core proteins in the network,and there are many interrelationships,indicating that Dangui Xiaozheng decoction can play a therapeutic role through these proteins.(5)GO and KEGG enrichment analysis:GO enrichment analysis showed that a total of 2467 cell biological processes were enriched,including 2177 BP,105 CC,185 MF and 201 KEGG signal pathways.GO enrichment analysis is mainly related to biological processes such as cell membrane signal transduction,transcriptional regulatory factors,response to hormones,response to decreased oxygen levels,response to steroid hormones,inflammatory response,reproductive structure development and so on.KEGG enrichment analysis mainly involved cancer signaling pathway,HIF-1 signaling pathway,MAPK signaling pathway,chalasin signaling pathway,NF-Kappa B signaling pathway,cAMP signaling pathway,and necrotic apoptosis,suggesting that Dangui Xiaozheng decoction may play a role through regulating multiple signaling pathways.(6)Animal experimental results:①HE staining and endometrial infiltration depth of mice uterus:the infiltration degree of mice in each group was different.Comparing the infiltration depth of mice in each group,the results showed that the number of grade 2 and grade 3 infiltration of AM mice uterus after TCM treatment(2/8,1/8)was less than that of model group(4/8,3/8),and the difference was statistically significant(P<0.01).②The expressions of HIF-1 alpha,VEGFA and COX2 in mice uterus:Compared with blank group,the expressions of HIF-1 alpha and COX2 were increased in model group(P<0.05),while VEGFA expression was increased,but there was no significant difference(P>0.05).After Dangui Xiaozheng decoction treatment,the expressions of HIF-1 alpha,VEGFA and COX2 in the TCM treatment group were decreased compared with the model group,and the differences were statistically significant(P<0.05).Conclusion:1.For the treatment of adenomyosis dysmenorrhea,Professor Zhao Ruihua uses simple prescription and gentle medication.She often uses warm and hot drugs,mainly pungent,sweet and bitter drugs.The commonly used drug combinations were Cassia twig-Paeoniae alba,Endothelium corneum-Coix seed,Poria-Rhizoma atractylodis macrocephalae and so on.The core prescription is Dangui Xiaozheng decoction:Rhizome rhizome,Radix salviae miltiorrhizae,Cassia twig,Endothelium corneum,Coix seed,Panax notoginseng powder,Radix paeoniae alba,Turmeric,Rhizoma zingiberis and Rhizoma Dioscoreae.2.The effective components of Dangui Xiaozheng decoction in the treatment of adenomyosis include quercetin,luteolin,β-sitosterol,stigmasterol,catechin,kaherol,tan shone IIA,etc.The potential targets are AKT1,TNF,IL6,TP53,EGFR,HIF1A,PTGS2,etc.KEGG results and animal experiments proved that Dangui Xiaozheng decoction may play a role in the treatment of adenomyosis by mediating HIF-1 signaling pathway to improve the hypoxic microenvironment of AM lesions,inhibiting angiogenesis and prostagranin production. |
