Exploring The Molecular Mechanism Of Salvia Miltiorrhiza In The Treatment Of Deep Vein Thrombosis Based On Network Pharmacology,Molecular Docking,and Experimental Validation

Objective:Salvia miltiorrhiza,a traditional Chinese medicine,has been widely used in the treatment of cardiovascular diseases because of its ability to activate blood circulation,resolve blood stasis,anti-inflammatory and antioxidant effects.However,its mechanism of action in the treatment of DVT remains unclear.Oxidative stress damage to endothelial cells is the initiating factor of DVT.This thesis explores the effective components and molecular mechanisms of Salvia miltiorrhiza in the treatment of DVT through network pharmacological techniques,experimental validation,and provides a reliable basis for the clinical treatment of deep vein thrombosis.Methods:1.Network pharmacological analysis:（1）Using TCMSP database to search the active ingredients of Salvia miltiorrhiza and obtain the relevant action targets through Drug bank;（2）Using"Deep Vein Thrombosis"as the keyword,the disease-related genes were obtained from Gene Cards,OMIM,Phar GKB,TTD and Drug bank databases,and the target genes of Salvia miltiorrhiza for DVT were obtained by intersecting the target of Salvia with the DVT gene through the R language Venn package.（3）The target genes of Salvia miltiorrhiza for DVT were imported into the String website for protein interaction analysis,the core target genes were screened by the cyto NCA plug-in in Cytoscape 3.9.1 software,the network interactions of"Salvia miltiorrhiza-the active ingredient-target genes-DVT"were constructed by the Network Analyzer plug-in.（4）GO and KEGG enrichment analysis of target genes of Salvia miltiorrhiza for DVT using the R language cluster Profiler package;（5）Molecular docking of the core active ingredient Tanshinone IIA with key target genes using Auto Dock and analysis of the docking results.2.In vitro studies:（1）A model of oxidative damage was constructed using H₂O₂-induced HUVECs.The CCK8 method was used to detect the cell viability of HUVECs and observe the effects of different concentrations of H₂O₂and Tan IIA on the cell viability of HUVECs,and determine the final concentrations of H₂O₂and Tan IIA.HUVECs were grouped:The control group,H₂O₂group and H₂O₂+Tan IIA group,and incubated with dosing according to different groups.（2）The migration ability of HUVECs was observed by cell scratch assay,and the levels of MDA,SOD activity,and ROS were measured to observe the antioxidant effect of Tan IIA on H₂O₂-induced HUVECs,the pro-angiogenic ability of Tan IIA on H₂O₂-induced HUVECs was assessed by in vitro tube formation assay;TP53,AKT,and PTGS2protein expression levels were detected by WB.Results:1.The TCMSP database was used to identify 58 effective active ingredients of the Chinese herbal medicine Salvia miltiorrhiza and 117 corresponding action target genes.A total of 2247 genes related to deep vein thrombosis were screened from five databases,and 77 genes were obtained as the targets of Salvia miltiorrhiza for the treatment of deep vein thrombosis after the intersection.A network of 134 nodes and401 edges by using Cytoscape was constructed for the interactions between Salvia miltiorrhiza-the active ingredient-target genes-deep vein thrombosis,and the active ingredients were screened to include lignan,Tan IIA,salviaequinone d,dihydrodanshinolide,etc.GO enrichment analysis revealed that the biological functions of the target genes of Salvia miltiorrhiza in the treatment of deep vein thrombosis were mainly focused on the response to drugs,response to radiation,response to oxidative stress,etc.KEGG enrichment analysis showed that the target genes were mainly enriched in PI3K/AKT signalling pathway,chemotaxis-receptor activation,lipids and atherosclerosis.Molecular docking analysis confirmed that Tan IIA could form a good combination with the target protein through intermolecular forces.2.In the in vitro oxidative damage model of HUVECs,compared with the blank control group,the cellular ROS level and MDA content were increased（p<0.05）while SOD activity was decreased（p<0.05）in the H₂O₂group;compared with the H₂O₂group,the cellular ROS level and MDA content were decreased（p<0.05）,SOD activity was increased in the H₂O₂+Tan IIA group（p<0.05）.The scratch assay showed that the migration ability of HUVECs in the H₂O₂group was significantly reduced compared with the control group,however the pretreatment with Tan IIA could reduce the damage to endothelial cells by H₂O₂and promote their migration.The results of in vitro tube formation assay showed that compared with the blank control group,H₂O₂significantly reduced the tube formation of HUVECs,and Tan IIA pretreatment could promote the in vitro neovascularization of vascular endothelial cells after H₂O₂injury.The results of Western Blot experiments showed that compared with the control group,the expression levels of TP53 and PTGS2 in the induced group were significantly increased,the expression level of AKT was decreased.After pretreatment with Tan IIA,the levels of TP53 and PTGS2 were significantly decreased,the level of AKT was increased.Conclusion:Salvia miltiorrhiza can target and regulate various signaling pathways by acting on AKT and TP53 gene loci,thus having potential therapeutic effect on deep vein thrombosis.