Clinical Validation Of Platelet Differential MiRNA In Coronary Heart Disease With Blood Stasis Syndrome And Study Of Anti-platelet Mechanism Of Panax Notoginseng Saponins Based On MiR-223-3p

Platelet microRNAs(miRNAs)are currently a hot topic in coronary heart disease(CHD)research and can be used to detect and differentiate between disease and health states.Platelet miRNAs play an important role in regulating platelet gene expression and platelet function-related proteins,and are a key entry point for the treatment of thrombotic diseases.Panax notoginseng preparations(PNP),made from Panax notoginseng extract,have been widely used in the clinical treatment of thrombotic diseases such as CHD and ischemic stroke.Many clinical studies have been conducted to investigate the effects of PNP on platelet aggregation and coagulation function in patients with CHD or ischemic stroke.Currently,the main components of PNP in clinical application are Panax Notoginseng Saponins(PNS)or Panaxatriol Saponin(PTS),among which the most abundant types and dosage forms are PNS-based preparations.Pharmacological studies have confirmed that PNS can inhibit platelet activation and aggregation,but there is still a lack of research on whether it can regulate platelet function based on platelet differential miRNAs in CHD with blood stasis syndrome.In this thesis,randomized controlled studies on the effects of PNP on platelet aggregation and coagulation in patients with CHD or ischemic stroke were systematically evaluated;secondly,platelet differential miRNAs in CHD complicated with blood stasis syndrome,which were screened previously by second-generation sequencing technology in our group,were tested by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)to performed clinical validation.Then,based on network pharmacology and molecular docking technology,we proposed that PNS might base on miR-223-3p and then target P2RY12 to exert anti-platelet effects;finally,we tested the hypothesis by constructing platelet particle models in vitro.Study 1 Meta-analysis of the effects of Panax notoginseng preparations on platelet aggregation and coagulation in patients with coronary heart disease or ischemic strokeObjective:To observe the effects of PNP containing PNS or PTS on platelet aggregation and coagulation during adjuvant treatment of CHD or ischemic stroke.Methods:A total of 8 databases were searched for randomized controlled trials(RCTs)comparing PNP and aspirin(ASA)with ASA alone for the treatment of CHD or ischemic stroke.Subgroup analysis was performed according to saponin category.When statistical heterogeneity was significant,sensitivity analyses were performed by changing the effect model and adopting leave-one-out method.Publication bias was detected using funnel plots,Egger’s test,and Begg’s test.Results:Twenty RCTs involving 2216 patients were analyzed.Compared with ASA alone,PNP plus ASA had a stronger inhibitory effect on platelet aggregation rate(PAgR)[PNS,WMD=-6.10(-7.25,-4.95),P<0.00001;PTS,WMD=-3.53(-4.68,2.38),P<0.00001];PNS plus ASA better reduced fibrinogen(FIB)[WMD=-0.43(0.49,-0.36)]and D-dimer(DD)[WMD=-0.59(-0.67,-0.51),P<0.00001],while platelet count(PLT)(P=0.07)and prothrombin time(PT)(P=0.34)were not significantly different;PTS plus ASA better prolonged PT[WMD=1.90(1.47,2.32),P<0.00001]and prothrombin timebased international normalized ratio(PT-INR)[WMD=0.22(0.11,0.32),P<0.0001],whereas no significant difference in DD(P=0.1)and bleeding-related events(positive fecal occult blood,P=0.96,upper gastrointestinal bleeding,P=0.67,subcutaneous hemorrhage,P=0.51,bulbar conjunctival hemorrhage,P=0.51,and hematuria,P=0.58).There was no significant difference in gastrointestinal side effects(PNS,P=0.65;PTS,P=0.56)and urticaria(PNS,P=0.57;PTS,P=0.55)between PNP plus ASA and ASA alone.Conclusions:PNP combined with ASA may produce stronger antiplatelet aggregation and anticoagulation effects without increasing risk of bleeding risk,gastrointestinal side effects,and urticaria compared with ASA alone.Study 2 Clinical validation of platelet differential miRNA in coronary heart disease complicated with blood stasis syndromeObjective:To clinically validate miR-223-3p,miR-146a-5p,miR-126-3p,miR126-5p of platelet differential miRNAs screened by high-throughput sequencing in CHD complicated with blood stasis syndrome,and to explore the correlation between these four platelet miRNAs and the maximum platelet aggregation rate(MPAR)in patients with CHD.Methods:Ninety subjects were included in the study,including 30 with CHD complicated with blood stasis syndrome,30 with CHD complicated with non-blood stasis syndrome,and 30 healthy volunteers.The expression of platelet miR-223-3p,miR-146a-5p,miR-126-3p,miR-126-5p was detected by qRT-PCR in CHD complicated with blood stasis syndrome group,CHD complicated with non-blood stasis syndrome group and healthy control group.Pearson correlation test was used to analyze the correlation between miR-223-3p,miR-146a-5p,miR-126-3p,miR-126-5p and MPAR in patients with CHD.Results:There were no statistical differences in age and gender between the three groups of subjects.The relative expressions of platelet miR-223-3p,miR-146a-5p,miR-126-3p and miR-126-5p in the CHD complicated with blood stasis syndrome group were significantly higher than those in the healthy control group,and the relative expressions of platelet miR-223-3p,miR-146a-5p,miR-126-3p and miR-126-5p in the CHD complicated with non-blood stasis syndrome group were significantly higher than those in the healthy control group.The relative expressions of miR-223-3p,miR-146a5p,miR-126-3p,and miR-126-5p were not significantly different between the CHD complicated with blood stasis syndrome group and the CHD complicated with nonblood stasis syndrome group.The relative expression of platelet miR-223-3p,miR146a-5p,miR-126-3p,and miR-126-5p in patients with CHD were not significantly correlated with MPAR(r=0.038,P=0.77;r=0.139,P=0.29;r=0.047,P=0.723;r=0.076,P=0.565).Conclusion:(1)Platelet miR-223-3p,miR-146a-5p,miR-126-3p,and miR-1265p were differentially expressed between the CHD group and the healthy control group,and there was no significant difference in the expression of these four miRNAs between the CHD complicated with blood stasis syndrome group and the CHD complicated with non-blood stasis syndrome group.(2)The relative expression of platelet miR-223-3p,miR-146a-5p,miR-126-3p,miR-126-5p in patients with CHD may not be correlated with MPAR.Study 3 Exploring the antiplatelet mechanism of Panax Notoginseng Saponins based on network pharmacology and molecular dockingObjective:Based on network pharmacology and molecular docking techniques,combined with the pre-validated platelet differential miRNAs,we initially explored the target miRNAs,key targets and pathways for PNS to exert anti-platelet effects,so as to propose a mechanistic hypothesis and lay the foundation for subsequent experimental studies.Methods:The SwissTargetPrediction website was used to predict the targets of the main active components of PNS,the GenCLiP 3 database was used to obtain platelet function-related targets,and the Draw Venn Diagram online tool was used to draw Venn diagrams to obtain the intersection targets.The DAVID 6.8 database was applied for GO and KEGG enrichment analysis of the intersecting targets,and the most relevant pathways to the study topic were selected.The target genes of major components of PNS were intersected with genes of related pathways using Draw Venn Diagram online tool to obtain the key targets.Molecular docking of the key targets to the major components of PNS was performed using Autodock vina software and visualized and analyzed by PyMOL 2.4.0 software.The target miRNAs of the key targets were predicted by TargetScanHuman 7.1 database and combined with the pre-validated platelet differential miRNAs to identify the target miRNAs for the study.Results:There were 287 predicted targets for the 5 major active ingredients of PNS,6617 targets related to platelet function,and 250 intersecting targets for both.By GO and KEGG enrichment analysis,142 GO enrichment entries and 105 KEGG pathways were screened,focusing on platelet activation pathway and PI3K/AKT pathway according to the direction of the research topic.23 target targets were obtained by taking intersection of the main components of PNS and genes of platelet activation pathway,namely:P2RY12,ITGB1,SYK ROCK1,ROCK2,SRC,ITGB3,F2R,ITGA2B,PIK3CD,PIK3CB,F2,MAPK14,PIK3CG,PTGS1,PPP1CC,RAP1A,PIK3CA,TBXA2R,AKT2,AKT1,MAPK1,PRKACA.Using the KEGG bioinformatics database to predict the downstream target genes of P2RY12,the platelet activation pathway of the P2RY12/PIK3CA/AKT1 regulatory network was identified.Molecular docking showed that the major components of PNS have good affinity with the key targets P2RY12,PIK3CA and AKT1.TargetScanHuman 7.1 target gene prediction database suggested that miR-223-3p,a platelet differential miRNA validated in the clinical study section,and the key targets P2RY12,PIK3CA and AKT1 may have a direct target relationship,and the target miRNA was identified as miR-223-3p.Conclusion:PNS exerts antiplatelet effects that may be related to P2RY12 and downstream PIK3CA,AKT1 and miR-223-3p.PNS may regulate platelet function based on miR-223-3p targeting P2RY12 and downstream PIK3CA/AKT1 pathway.Study 4 Mechanism of miR-223-3p-based action of Panax Notoginseng Saponins targeting P2RY12 in regulating platelet functionObjective:To investigate the regulation of miR-223-3p on P2RY12 and downstream PIK3CA/AKT1 pathway and the mechanism of PNS regulating platelet function based on miR-223-3p targeting P2RY12.Methods:(1)In vitro culture of human megakaryocytic leukemia cells MEG-01,Phorbol 12-myristate 13-acetate(PMA)was used to induce maturation and differentiation.The expression of CD41/CD61 on the cell surface was measured by flow cytometry at different concentrations of PMA,and the morphological changes of the cells after induction were observed microscopically to screen the best induction concentration for the construction of platelet particle model.(2)Dual-luciferase reporter gene experiment:a dual luciferase reporter system containing P2RY12 3’-UTR wild type(Wide Type,WT)or mutant type(MUT)was constructed,and six groups were set up:P2RY12-3’UTR-WT+Control group,P2RY123’UTR-MUT+Control group,P2RY12-3’UTR-WT+miR-223-3p mimic group,P2RY12-3’UTR-MUT+miR-223-3p mimic group,P2RY12-3’UTR-WT+NC mimic group,and P2RY12-3’UTR-MUT+NC mimic group to verify the targeting regulation of miR-223-3p and P2RY12.(3)The regulation of P2RY12 and downstream PIK3CA/AKT1 pathway was investigated by transfecting miR-223-3p mimic or inhibitor and the corresponding negative control into platelet particle model,overexpressing and silencing miR-223-3p.Five groups were set up:Control group,NC mimic group,miR-223-3p mimic group,NC inhibitor group,and miR-223-3p inhibitor group.The expression of miR-223-3p,P2RY12,PIK3CA,AKT1 mRNA was detected by qRT-PCR,the expression of P2RY12 was detected by immunofluorescence,and the protein levels of P2RY12,PIK3CA,AKT1 were detected by Western Blot.(4)After screening the optimal dosing concentration and duration of PNS and Ticagrelor with CCK8,miR-223-3p mimic and corresponding negative control were transfected into platelet particle model,and Ticagrelor was used as a positive control drug to study the effect of PNS based on miR-223-3p targeting P2RY12 and downstream pathways regulating platelet function.Seven groups were set up:Control group,NC mimic group,miR-223-3p mimic group,NC mimic+Ticagrelor group,NC mimic+PNS group,miR-223-3p mimic+Ticagrelor group and miR-223-3p mimic+PNS group.qRT-PCR was performed to detect the expression of miR-223-3p,P2RY12,PIK3CA,AKT1 mRNA,immunofluorescence was performed to detect the expression of P2RY12,Western Blot to detect the protein levels of P2RY12,PIK3CA,AKT1.Results:(1)The surface CD41/CD61 positivity rate of cells differentiated by 10nM PMA induction for 3 days was significantly increased(P<0.0001),and the morphology of MEG-01 cells was changed compared with normal cells,with increased cell apposition,increased volume,poorly defined nuclei,irregular edges,coarse granular chromatin,cell vacuole formation,and some cells showing shuttle-shaped changes and extending pseudopods.(2)The relative activity of luciferase in the P2RY12-3’UTR-WT+miR-223-3p mimic group was significantly lower than that in the other five groups(P<0.0001).(3)miR-223-3p mimic and miR-223-3p inhibitor effectively up-and downregulated the relative expression levels of miR-223-3p mRNA;miR-223-3p mimic and miR-223-3p inhibitor effectively down-and up-regulated P2RY12,PIK3CA,AKT1 mRNA and protein levels,respectively.(4)Both PNS and Ticagrelor elevated the relative expression levels of miR-2233p mRNA;there was no significant difference in the effect of PNS and Ticagrelor to elevate the relative expression levels of miR-223-3p mRNA;both PNS and Ticagrelor caused a further increase in the relative expression levels of the up-regulated miR-2233p mRNA;there was no significant difference in the effect of PNS and Ticagrelor to further elevate the relative expression levels of the up-regulated miR-223-3p mRNA.Both PNS and Ticagrelor decreased the mRNA and protein levels of P2RY12,PIK3CA,and AKT1;there was no significant difference in the effect of PNS and Ticagrelor in downregulating the mRNA and protein levels of P2RY12,PIK3CA,and AKT1;both PNS and Ticagrelor caused a further decrease in the mRNA and protein levels of downregulated P2RY12,PIK3CA,and AKT1;there was no significant difference in the effect of both PNS and Ticagrelor to further decrease the mRNA and protein levels of down-regulated P2RY12,PIK3CA,AKT1.Conclusion:(1)Overexpression of miR-223-3p inhibits P2RY12 and downstream PIK3CA and AKT1 levels.(2)There was no significant difference between the effects of PNS and Ticagrelor in upregulating the mRNA level of miR-223-3p and downregulating the mRNA and protein levels of the target genes P2RY12 and downstream PIK3CA and AKT1.(3)Both PNS and Ticagrelor may regulate platelet function based on miR-223-3p targeting P2RY12 and the downstream PIK3CA/AKT1 pathway.