Potential Mechanism Of Activating T Lymphocytes By Compounds In Danshen Based On Network Pharmacology

Objective:To screen the pharmacokinetic characteristics of compounds in Salvia miltiorrhiza based on network pharmacology,and explore the relationship between their targets and T cell signaling pathways for compounds with qualified pharmacokinetic characteristics.Multiple experimental methods were used to verfy the effect and the mechanism of the selected compounds on T lymphocytes.Provide new ideas for the development and application of compounds in Chinese medicine.Methods:1.Using network pharmacology to predict compounds in Salvia miltiorrhiza that may act on T lymphocytes and the underlying mechanism of action.（1）Using Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform（TCMSP）screened compounds in Salvia miltiorrhiza which satisfied oral bioavailability（OB）>50%,Caco-2 permeability（Caco-2）>0.4 and drug-like properties（DL）>0.2.（2）Using the Swiss TargetPrediction platform to find all the targets of small molecule compounds that meet the pharmacokinetic characteristics.（3）Input the acquired targets into the STRING platform to screen the compounds acting on the TCR signaling pathway and obtained the interaction relationship between the corresponding proteins of the targets,which were convenient to find the pathway enrichment information corresponding to the interacting targets.（4）Using the STRING database and running R and Cytoscape software to sort out the functions of the four compound interaction targets enriched in the TCR signaling pathway.Enrichment analysis of Kyoto Encyclopedia of Genes and Genomes（KEGG）in Cytoscape software to screen the targets of 4 compounds enriched on TCR signaling pathway.（5）Using Schr?dinger Suite software for molecular docking,analyzing the affinity of compounds and key targets,and obtain the possibility of interaction between small molecular compounds and corresponding targets;using the plug-in CentiScaPe in CytoScape software to analyze the key targets;（6）Screen the mechanism of Salvia miltiorrhiza on TCR signal pathway using BATMAN-TCM platform.2.Cryptotanshinone in Salvia miltiorrhiza was selected to verify the network pharmacology results.（1）The CCK-8 method was used to detect the effect of cryptotanshinone on the viability of Jurkat and HUT-78,and to detect the effect of different drug concentrations(1,2,4μmolμmol·L^-1)and action time（0,1,2,3 d）on Jurkat and HUT-78,respectively.（2）Flow cytometry was used to detect the effect of cryptotanshinone on Jurkat and HUT-78 about activation and apoptosis.Results:1.Analysis results of network pharmacologyAccording to the screening data,there are 10 kinds of compounds in Salvia miltiorrhiza which meet the pharmacokinetic characteristics of OB>50%,Caco-2>0.4,and DL>0.2 which are Danshenol B,Tanshinaldehyde,（6S）-6-（hydroxymethyl）-1,6-dimethyl-8,9-dihydro-7H-naphtho[8,7-g]benzofuran-10,11-Dione（（6S）-6-（hydroxymethyl）-1,6-dimethyl-8,9-dihydro-7H-naphtho[8,7-g]benzofuran-10,11-dione）,miltiononeⅡ,formyltanshinone,przewaquinone C,cryptotanshinone,isocryptotanshinone,danshenspiroketallactone and epidanshenspiroketallactone.After testing,the number of 10 compounds’targets（correlation>0）were 100,75,100,55,32,96,56,21,35,54,respectively.GO enrichment analysis of the four compounds interaction targets were involved in the immune process.KEGG found that four compounds act on the target of TCR signaling pathway.Molecular docking was treated by Schr?dinger Suite software.（6S）-6-（hydroxymethyl）-1,6-dimethyl-8,9-dihydro-7H-naphtho[8,7-g]benzofuran-10,11-dione,przewaquinone C and cryptotanshinone docked with the target PIK3CA and their molecule score<-5kcal·mol^-1.It indicated that these compounds tightly bound to PIK3CA,and acted on PIK3CA to activate T cells.Through CentiScaPe topology analysis and evaluation of intermediary centrality and degree,it was found that the four compounds corresponded to their respective targets with the best binding and the target corresponding to the TCR signaling pathway was cryptotanshinone.The key target of its action was PIK3CA,intermediary centrality and the degree scores are 2157.37 and 24 respectively.At the same time,the screening of BATMAN-TCM showed that the activation of TCR by Salvia miltiorrhiza was through the PI3K/AKT signaling pathway.It showed that Salvia miltiorrhiza contains some compounds that could activate T lymphocytes,and it activated T lymphocytes by acting on PI3K.2.Results of in vivo and in vitro verification experimentsCompared with the control group(the concentration of cryptotanshinone was0μmol·L^-1),Jurkat and HUT-78 cells proliferated significantly at different concentrations（P<0.01）at 2d,3d and showed concentration-dependent,the difference was statistical significance.After 1d of treatment with cryptotanshinone,Jurkat cells didn’t proliferate significantly,and only high concentrations(4μmol·L^-1)in HUT-78 cells proliferated significantly（P<0.05）.At the same time,compared with the control group,the expression of CD69 in Jurkat and HUT-78 cells increased at each concentration in the experimental group.Compared with the control group,the number of apoptosis of Jurkat and HUT-78cells didn’t increase under each concentration treatment in the experimental group.Conclusion:1.Among 10 compounds met the TCMSP screening conditions in Salvia miltiorrhiza,4 compounds could act on the TCR signaling pathway,namely（6S）-6-（hydroxymethyl）-1,6-dimethyl-8,9-dihydro 7H-naphtho[8,7-g]benzofuran-10,11-dione,przewaquinone c,cryptotanshinone and danshenspiroketallactone.2.Molecular docking found that（6S）-6-（hydroxymethyl）-1,6-dimethyl-8,9-dihydro-7H-naphtho[8,7-g]benzofuran-10,11-diKetone,przewaquinone C,cryptotanshinone were tightly bound to PIK3CA(score<-5kcal·mol^-1),while danshenspiroketallactone didn’t bind well to PIK3R1(score was-3.61kcal·mol^-1>-5kcal·mol^-1).Topological analysis showed that cryptotanshinone preferentially bound to PIK3CA.It showed that（6S）-6-（hydroxymethyl）-1,6-dimethyl-8,9-dihydro-7H-naphtho[8,7-g]benzofuran-10,11-dione,przewaquinone c and cryptotanshinone could activate T cells,and the effect of cryptotanshinone may be better.The results of BATMAN-TCM platform supported the above detection.3.Low,medium and high concentration(1,2,4μmol·L^-1)cryptotanshinone treatment for 2d,3d could obviously promote the proliferation of Jurkat and HUT-78 cells（P<0.01）.And at the same time,low,medium and high concentrations(1,2,4μmol·L^-1) cryptotanshinone might stimulate and activate Jurkat and HUT-78 cells,but it didn’t affect the apoptosis of Jurkat and HUT-78 cells.