Regularity Of Prescriptions Of ALI And The Therapeutic Mechanism Of Glycyrrhetinic Acid On ALI Based On Clinical Data

There is no exact disease name about acute lung injury(ALI)in traditional Chinese medicine.According to its clinical manifestations,it belongs to the category of "violent asthma".It can be expressed as heat toxin,phlegm-heat,phlegm-damp,blood stasis syndromes in the early stage,while the latter is mainly based on lung,spleen and kidney deficiency syndromes.At present,western medicine mainly focuses on symptomatic supportive therapy.A large number of clinical and experimental studies have shown that Chinese medicine is efficient in the treatment of ALI.This study will analyze and discuss the rules and mechanism of Chinese medicine in the treatment of ALI.Objectives:A retrospective study on prescriptions of traditional Chinese medicine(TCM)for ALI was conducted to explore the high-frequency medicines and the regularity of TCM prescriptions through data mining.The network pharmacology method was used to analyze the main active components and mechanism of ALI for the treatment of licorice.Exploring the therapeutic effect and the mechanism of NLRP3 inflammasome of the main active component of Glycyrrhetinic acid(GA)of licorice in ALI by establishing lipopolysaccharide(LPS)-induced ALI mouse model and macrophage model.Methods:1.Clinical studyStudy the medication rules of ALI based on data mining technology(1)Establish a prescription databaseThrough the hospital medical record retrieval system of the hospital case,the words "acute lung injury","acute respiratory distress syndrome" and"athma" were searched who were hospitalized in the first affiliated hospital of guangzhou university of traditional Chinese medicine from January 1,2010 to December 31,2018.The basic data of Chinese and western medicine diagnosis and Chinese medical composition,record detailed information of the Chinese medicine,such as the type of syndrome of clinical usage and dosage.Building a database with these data,we selected a total of 217 eligible prescriptions of patients with basic information(name,age,the TCM and western medicine in the diagnosis,the TCM syndrome types and medicine composition)and established information database with Excel software.(2)Data entry and verificationThe study calibrated the names of TCM according to the book "Chinese Pharmacy" to ensure the uniformity and integrity of the data.Due to the different processing methods of TCM,we defined the name according to the teaching material,such as wine cellar and hawthorn are called "Hawthorn",frying atractylodes and coke white surgery correspond to the "Atractylodes".The standardized Chinese medicine prescriptions were input into the database of TCMISS,the data would be reviewed by a special person to avoid errors and underrecording,so as to ensure the reliability of the data.(3)Data mining analysisThe statistical report module in the platform of prescriptions database was used to analyze the property and flavor,meridian distribution,medication frequency and prescription rules.Meanwhile,data mining was conducted based on entropy clustering and improved mutual information method to analyze the medication rules and high-frequency drugs of TCM prescriptions,to explore the potential new prescriptions for treating ALI,and to analyze the compatibility rules of high-frequency drug prescriptions deeply.At last,we drew the drug combination network diagrams.2.To explore the main active components and mechanism of licorice in ALI based on network pharmacologyThe Chinese medicine system pharmacology analysis platform(TCMSP)was used to extract all the chemical components of licorice.The oral availability degree(OB)?40%and the drug-likeness(DL)?0.5 were set as the screening conditions to obtain the main active ingredients.We combined TCMSP with comparative genomic database(CTD)to extract the targets of the main active components of licorice.Using CTD online analysis platform to excavate the targets of ALI,and set the inference score 90 and reference count?110 as the screening condition.Finally,20 gene targets were obtained,which were imported into the STRING(https://string-db.org/)database.We constructed a protein-protein interaction(PPI)network diagram by Cytoscape3.7.1 software.After constructed a visualization network diagram of the main active ingredient-targets-disease of licorice,we combined with MCODE plug-in for clustering analysis to obtain the interaction relationship between.targets of active ingredients and disease targets.18 key gene targets of Gene Ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)functional analysis were performed according to the Cytoscape3.7.1 software and DAVID6.8 database(https://david.ncifcrf.gov/tools.jsp).We input the key target genes in the DAVID 6.8 database,and restricted the species to“Homo Sapiens”for search,then extracted the analysis results of Go and KEGG.The potential gene targets were mapped to the KEGG signal pathway and the relationship between targets and signal pathway were shown according to the KEGG database.3.Experimental study(1)The effect of GA on LPS-induced ALI miceWe established a mouse model of ALI induced by LPS.C57 mice were randomly divided into 6 groups,including normal control group,LPS group,dexamethasone group,GA low,medium,high(10,20,40 mg/kg)dose group.After 7 days of continuous gavage in the drug-administered group,the model group and each drug-administered group were anesthetized with chloral hydrate,and the mice were modeled by performing tracheotomy and aspirating LPS.After 24 hours,the vital signs and general conditions of the mice were observed,and the dry and wet specific gravity(W/D)of the lung tissues was measured to evaluate the pulmonary edema.The paraffin sections of the lung tissues were prepared and the lung histopathological scores were taken after HE staining.We detected the content of IL-1? in the serum of mice by Elisa.After frozen sections of lung tissue were prepared,the positive expression rate of IL-1?and F4/80 proteins in lung tissue of mice were detected by IHC.Then we detected the co-localization of F4/80 and the NLRP3 inflammasome complex proteins of NLRP3 and Caspase-1.(2)The mechanism of GA regulates NLRP3 inflammasome in macrophagesMacrophage Raw264.7 was selected for cell experiments.After normal cell culture and passage,cells in the logarithmic growth phase were taken for experiments.The effect of different concentrations of GA on cell proliferation and cytotoxicity was observed by a real-time dynamic cell monitor(Incucyte),and suitable concentrations of GA was screened.Macrophages were divided into five groups,including normal control group,LPS group,and GA low,medium,and high dose groups(5,10,20 ?M).Cells in each drug-administered group were incubated with the corresponding concentration of GA for 24 h.According to the literature and experimental studies,LPS group and GA group were stimulated with LPS at a concentration of 1?g/mL for 24 h,and samples were collected for experiments.NO assay kit was used to detect the content of NO in macrophages.The changes of ROS in each group were detected by Incucyte and flow cytometry(FCM),and IL-1? in supernatant was detected by ELISA.The expression of PI3K,AKT,iNOS,TXNIP,TLR4 and NLRP3 inflammasome complexes of NLRP3,ASC,Caspase-1 proteins were detected by WB.The transcriptional level of NLRP3,Caspase-1,ASC and IL-1?mRNA in the cells was detected by RT-PCR.The colocalization of NLRP3/Caspase-1 and NLRP3/ASC proteins were detected by IF.After we transfected the macrophages with NLRP3 shRNA by using Lipo3000,the silencing effect of NLRP3 and the proteins expression of iNOS and Caspase-1 were detected by WB.Results:1.Clinical StudyThe regularity of TCM in ALI(1)Basic information:a total of 217 Chinese medicine prescriptions were selected in this study.The patients were aged between 20 and 80 years old,with an average age of 54.16±14.31 years,and more men than women.(2)The analysis of the distribution of TCM syndromes:it was found that phlegm-heat obstructing lung syndrome was the most common type,accounting for 24.8%.Then the lung phlegm turbidity resistance and Qi-stagnation and blood stasis,accounting for 13.7%,12.4%.Next syndromes were zheng-asthenic asthma,yang-asthenic water flood,phlegm-stasis obstruction.The deficiency of lung-kidney,lung-spleen and qi-yin accounted for less.Spleen and kidney deficiency syndrome accounted for the least,accounting for 1.3%.(3)The analysis of the four-gas and five-flavor properties of TCM:the drugs used was mainly warm,then the cold and flatness and the least was heat.The proportion of warm and cold drugs was 36.9%and 34.7%respectively,and the hot drug only accounted for 0.8%.The taste of the drug was mainly sweet,bitter and simba,accounting for 33.8%,33.1%and 28.3%respectively.The sour was less and the salty taste was the least.The most common use of the drug was the lung,accounting for 24.1%,the least was the trifocal and pericardium.(4)Frequency of drug:the frequency statistics showed that the most frequently used was licorice,followed by pinellia,poria,fritillaria.(5)The association rule analysis:the support was set to 10%and the confidence was set to 0.9,then 31 drug combinations were obtained.When the support was set to 20%,we obtained the drug combination with the highest degree of support of liquorice-pinellia.(6)Entropy clustering analysis:the correlation coefficient of the drug was set to 5,and the penalty degree was set to 2.Five new therapeutic combinations of ALI were finally obtained after the new combinations were extracted.(7)Compatibility analysis of licorice:the compatibility analysis was carried out among licorice-pinellia,licorice-poria,licorice-fritillaria.The support degree was set as 20%,and the confidence degree was set as 0.9.At last,we obtained the the compatibility regularity network diagram of licorice-pinellia,licorice-poria,licorice-fritillaria.2.The main active components and mechanism of licorice in treating ALI(1)Collection and screening of active ingredients:based on the sample size,data complexity and comprehensive literature considerations,24 active ingredients were screened by 0B?40%and DL?0.5.These chemical components had good oral availability and drug-like properties.After CTD predicted and analyzed 24 active components,four active components with polygenic targets were obtained,including glycyrrhetinic acid,liquiritin,glycyrol and inermine.(2)The main active ingredient-target network of licorice:the PPI network chart showed that the degree of CPCP3 and MAPK3 was the highest(degree=19),followed by TNF and MAPK1(degree=18),and the values of IL-1? and NLRP3 were 17 and 14,respectively.Each active ingredient of licorice exerted an average of 52 targets,and each target protein was connected to an average of 2.14 compounds.The degree of GA had the largest(88 nodes),which was the central node in the network.Then,the degree of liquiritin,glycyrol and inermine were respectively 6,5 and 5.(3)Functional analysis results of GO:the results showed that the biological process(BP)was significantly enriched in the peripheral sensory neurons Fc-epsilon receptor signaling pathway,DNA template transcriptional regulation,drug response,apoptosis process and aging,signal transduction and so on.The cellular component(CC)was significantly enriched in extracellular regions,nuclear structures,Golgi apparatus,cell membranes,protein complexes.Molecular function(MF)was significantly enriched in protein and enzyme binding,homodimerization activity,transcription factor binding,DNA binding.(4)Pathway enrichment analysis of KEGG:after repeated screening and elimination,we obtained 6 correlation signaling pathways,including IL-17,TNF,C-type lectin receptor,NOD-like receptor,Toll-like receptor and PI3K/AKT signaling pathways.1.Experimental Study(1)The protective effect of GA on ALI in mice?Different concentrations of GA could decrease lung dry and wet specific gravity index in mice with ALI and reduce the edema of lung tissues.The results of pathological section suggested that GA could reduce the infiltration of inflammatory cells and decrease the pathological score of lung tissues in mice.? Different concentrations of GA could suppress the expression of IL-1?protein in mice serum and lung tissues.Meanwhile,GA could decrease the expression level of F4/80 protein in lung tissues.? Different concentrations of GA could suppress the expression of NLRP3 and Caspase-1 proteins in mice lung tissues and decrease the formation of NLRP3 inflammasome in macrophages.Thus,GA could protect the mice lung tissues.?The efficacy of GA on ALI was concentration-dependent,and the higher the concentration of GA,the better the efficacy.(2)The mechanism of GA regulation of NLRP3 inflammasome in macrophages? Effect of GA on NO and IL-1? in macrophages:compared with the normal group,the content of NO and IL-1? in model group increased significantly.Compared with the model group,GA groups reduced the production of NO and IL-1?in macrophages.? Effect of GA on expression of NLRP3 inflammasome associated proteins and transcription levels in macrophages:compared with the normal group,the expression of iNOS,TLR4,NLRP3,caspase-1 and ASC protein was increased,TXNIP was decreased,and the transcription levels of NLRP3,Caspase-1,ASC and IL-1?were increased in the model group.Compared with the model group,GA groups inhibited the expression of iNOS,NLRP3,caspase-1 and TLR4 proteins,but had no significant effect on ASC and TXNIP.GA groups could reduce the transcription levels of NLRP3,Caspase-1 and IL-1?,but had no significant effect on ASC.? Effect of GA on the colocalization of NLRP3 inflammasome complexes in macrophages:after LPS stimulation,the protein expression of NLRP3,Caspase-1 and ASC in macrophages increased,and the colocalization area increased significantly,indicating the formation of NLRP3 inflammasome complexes.GA groups could reduce the colocalization area of NLRP3 inflammasome and inhibit the formation of NLRP3 inflammasome.?Effect of GA on ROS and calcium influx in macrophages:after LPS stimulation,GA groups could reduce the fluorescence intensity of ROS and intracellular Ca2+ in macrophages,and reduce ROS production and Ca2+ influx.? Effect of GA on the phosphorylation of PI3K/AKT in macrophages:after LPS stimulation,the phosphorylation level of PI3K/AKT was increased,and GA could inhibit the phosphorylation of PI3K/AKT,but there was no significant difference between different concentrations of GA.?Effect of transfection with NLRP3-shRNA on proteins related to NLRP3 inflammasome pathway in macrophages:after LPS stimulated the non-transfection group and the transfection group respectively,the results showed that the protein expressions of Caspase-1 and iNOS in the non-transfection group were significantly increased,while the expressions of Caspase-1 and iNOS proteins in the transfection group were not significantly changed.GA had no significant effect on the expressions of Caspase-1 and iNOS proteins in the transfection group.Conclusions:1.Clinical Study(1)The most common TCM syndromes in ALI inpatients were phlegm-heat obstructing lung,followed by phlegm-phlegm obstructing lung,qi stagnation and blood stasis,and positive deficiency and asthma.(2)Licorice is the most frequency traditional Chinese medicine in the treatment of ALI,followed by pinellia,poria,fritillaria.(3)According to the analysis of association rules,licorice-pinellia is the drug combination with the highest degree of association in the treatment of ALI.(4)The new prescriptions are applicable to the syndromes of phlegm-dampness,phlegm-heat,qi stagnation and blood stasis syndrome,and so on.2.The network pharmacology study(1)There are 4 effective active ingredients with multi-target effect in licorice,and 18 key targets of licorice on ALI are obtained.Glycyrrhetinic acid has the most gene targets,which may be the main active ingredient of licorice in treating ALI.(2)There are 6 signaling pathways for treating ALI with licorice,including IL-17 signaling pathway,TNF signaling pathway,C-type lectin receptor signaling pathway,NOD-like receptor signaling pathway,Toll-like receptor signaling pathway and PI3K/AKT signaling pathway.3.Experimental Study(1)Different concentrations of GA can reduce the inflammatory response in the early stage of LPS-induced ALI mice,reduce the pathological damage of the lung tissue of mice,and have protective effect on the lung tissue of ALI mice,which is related to the inhibition of the formation of NLRP3 inflammasome complexes by GA in macrophages.(2)NLRP3 is the key protein that GA regulates inflammatory response of macrophages,and NLRs signal pathway is the potential signaling pathway for GA to treat ALI.GA can inhibit the phosphorylation of PI3K/AKT proteins by reducing the ROS produced by macrophages after LPS stimulation,thereby inhibiting the formation and activation of NLRP3 inflammasome,thereby reducing the production of downstream inflammatory mediators IL-1? and NO,and finally reducing the inflammatory response of macrophages and playing an anti-inflammatory role.