| Panax notoginseng is a common Chinese herbal medicine,which has the function of both promoting blood circulation as well as stopping bleeding.Its main bioactive ingredient is panax notoginseng saponins(PNS).At present,PNS has been widely used for the treatment of many cardiovascular diseases,especially for patients with refractory coronary heart disease(CHD)with blood stasis syndrome(BSS),such as antiplatelet drug resistance,secondary nitroglycerin failure,and chest tightness or pain after coronary artery bypass grafting.However,PNS contains dozens of saponin ingredients,numerous targets and complex mechanisms,which limit the application and therapeutic effect of PNS in clinical practice to a certain extent.In addition,previous mechanism researches about PNS in the treatment of CHD patients with BSS were scattered and single;it’s thereby difficult to form a clear and systematic regulatory network.As a result,this study applied methods of network pharmacology,clinical verification and cytological research,in order to systematically reveal the multi-target and multi-level network mechanism of PNS in the treatment of CHD patients with BSS from the transcriptomics level.It would provide objective evidences for improved application of PNS in clinical practice,and help to promote the efficacy in treating CHD.ObjectiveTo systematically reveal the multi-target and multi-level mechanism of PNS in the treatment of CHD patients with BSS from the level of IncRNA-miRNA-mRNA regulatory network,by the methods of network pharmacology,clinical verification and cytological function research.MethodsPart one:network pharmacology-based prediction of target and lncRNA--miRNA-mRNA network in the treatment of PNS for CHD patients with BSSAccording to the "Pharmacopoeia of the People’s Republic of China(2015 version)",PNS mainly contains 5 bioactive ingredients:panax notoginsenoside R1,ginsenoside Rg1,ginsenoside Re,ginsenoside Rbl and ginsenoside Rd.Firstly,the PharmMapper database was used to predict the targets of each ingredient by reverse pharmacophore matching methods.The top 300 target genes that match each ingredient of PNS were separately predicted,and all genes were then uniformed and deduplicated.Meanwhile,target genes related to human CHD were searched and screened in NCBI GEO database.GEO2R was used to analyze the differentially expressed genes in 393 patients with CHD and non-CHD in the GSE20686 geneset.Among those genes,targets with the fold change≥1.5 and P<0.05 were selected.15 cases of CHD patients with BSS,CHD patients with non-BSS and healthy persons(5 in each group)were analyzed and used to predict the targets of CHD patients with BSS,based on the high-throughput sequencing data from our team’s previous work.Secondly,all the predicted genes of PNS,CHD and CHD patients with BSS were uniformed.VENN website was then used to analyze the intersection genes of the above three groups.Besides,GeneCards,GeneMANIA,and WebGestalt GSAT databases were applied to analyze the GO functions,co-expression network,and KEGG signaling pathways of the predicted target.Lastly,according to the functions and main signal pathways of predicted target,accompanied with the lncRNA-miRNA-mRNA regulatory network of CHD patients with BSS constructed earlier by our team,the regulatory network of PNS for CHD patients with BSS was further predicted.Part two:clinical verification of the predicted target and lncRNA-miRNA--mRNA network of PNS in the treatment of CHD patients with BSS80 unstable angina pectoris(UA)patients with BSS were registered and randomly divided into test group and control group(40 cases in each group)in a randomized double-blind placebo-controlled trial.The test group was given basic western medicine treatment+Xuesaitong soft capsule(main component is PNS,hereinafter referred to as PNS);the control group was given basic western medicine treatment+PNS placebo.The whole course of the trial was 4 weeks.Baseline indicators such as gender,age,smoking,drinking,body mass index(BMI),risk factors,medication and revascularization were all observed,in order to evaluate the balance between two groups.Moreover,the primary and secondary efficacy indicators were compared to evaluate the clinical effectiveness of PNS in the treatment of UA patients with BSS.The primary indicators involved angina pectoris effect and the efficacy of traditional Chinese medicine(TCM)syndrome,which were obtained by analyzing scores of the Seattle angina questionnaire(SAQ)and the evaluation questionnaire for CHD patients with BSS(BSSQ)respectively.The secondary indicator was the efficacy of single TCM symptoms and signs,which were obtained by analyzing the manifestations before and after treatment.Furthermore,safety indicators involving complete blood count,liver function,renal function,blood glucose,blood lipids,myocardial enzymes,coagulation and electrocardiogram were analyzed before and after treatment,in order to evaluate the clinical safety of PNS.Notably,to verify the target genes and IncRNA-miRNA-mRNA regulatory network that were predicted in part one,the plasma expressions of target and its network before and after treatment in the two groups were detected by quantitative real time polymerase chain reaction(qRT-PCR);and the values of 2-ΔΔCt and fold change were calculated.Part three:mechanical research of lncRNA-miRNA-mRNA regulatory network induced by PNS based on RNA interference technologyOxidative stress injury of coronary vascular endothelium was molded by hydrogen peroxide(H2O2)in human umbilical vein endothelial cells.MiR-3656 inhibitor and miR-3656 mimics were prepared by synthesized siRNA;and IncR CTB-114C7.4 overexpression and IncR CTB-114C7.4 knockdown were prepared by lentiviral shRNA.Meanwhile,the molding concentration of H2O2,and the optimal treatment time and concentration of PNS were screened by MTT.After accomplishment of the above preliminary works,the experiment was divided into 7 groups:control group,H2O2 model group,H2O2+PNS treatment group,H2O2+1ncR CTB-114C7.4 overexpression group,H2O2+miR-3656 inhibitor group,H2O2+PNS+lncR CTB-114C7.4 knockdown group,H2O2+PNS+miR-3656 mimics group.The model group was compared with the control group,PNS treatment group.lncR CTB-114C7.4 overexpression group and miR-3656 inhibitor group respectively.Similarly,the PNS treatment group was compared with the lncR CTB-114C7.4 knockdown group and miR-3656 mimics group as well.The expressions of all RNA in the network were detected by qRT-PCR,and the values of 2-ΔΔCt and fold change were also calculated to analyze the initiator and specific mode of the network.Besides,BCL2A1 and Beclinl proteins were detected by Western Blot.Their differential expressions among groups were compared by calculating gray ratio;and the consistence between protein and mRNA was checked.Furthermore,apoptosis,autophagosomes and autophagic flow in all experimental groups were analyzed by flow cytometry and transmission electron microscopy,combined with detecting the changeable expression of LC3-Ⅱ protein under the condition of lysosomal inhibitor.Therefore,regulatory effects of the network on apoptosis and autophagy were determined.ResultsPart one:network pharmacology-based prediction of target and lncRNA--miRNA-mRNA network in the treatment of PNS for CHD patients with BSS1.656 target genes of PNS were predicted by the PharmMapper database.225 target genes of CHD were analyzed in the GSE20686 geneset.221 target genes of CHD patients with BSS were obtained from our team’s previous high-throughput sequencing data.2.Intersection of the above three groups were compared and analyzed;thereby a shared gene BCL2A1 was received.GO function and KEGG pathway of BCL2A1 and its co-expression network were further analyzed.It’s revealed that BCL2A1 could regulate protein binding,and was closely related to the signaling pathways of apoptosis and autophagy.3.BCL2A1 could act as a downstream factor of NR4A1,thus co-participating in apoptosis signaling pathway.Moreover,NR4A1 was involved in the network of lncR CTB114C7.4-miR3656-NR4A1,which was the potential mechanism of CHD patients with BSS and was also relevant to apoptosis.Therefore,BCL2A1 may be the downstream target of lncR CTB114C7.4-miR36564.BCL2A1 protein could bind to Beclin1 protein,thus affecting the function of Beclinl and finally mediating the process of autophagy.The above predicted results have shown that PNS may target BCL2A1 by regulating IncR CTB114C7.4-miR3656-BCL2A1/Beclin1 network,and then take part in apoptosis or autophagy,thus exerting protective effect on CHD patients with BSS.Part two:clinical verification of the predicted target and lncRNA-miRNA--mRNA network of PNS in the treatment of CHD patients with BSS1.There were no statistical differences between the test group and the control group regarding gender,age,smoking,drinking,BMI,risk factors,medication and revascularization,which indicated that the baseline was balanced.2.In terms of the angina efficacy,PNS could reduce the frequency of angina attack and improve the steady state of angina.3.In terms of nitroglycerin cut-off rate,PNS could reduce the usage of nitroglycerin.4.In terms of the therapeutic effect on TCM syndrome,PNS could reduce the BSSQ score of CHD patients with BSS.Besides,it also increased the reduction rate from 6.63%to 34.44%.5.In terms of the therapeutic effect on symptoms and signs,PNS could significantly improve the chest pain and tightness,accompanied with effectiveness of 80.65%and 76.32%respectively6.In terms of safety indicators,PNS had no impact on complete blood count,renal function,coagulation and electrocardiogram.Interestingly,it could improve the levels of blood glucose,AST,ALT,LDL,CK and CK-MB,indicating that PNS was safe in clinical practice.7.In terms of the target genes and regulatory network,PNS could upregulate the plasma expressions of IncR CTB-114C7.4 and BCL2A1,while downregulate miR-3656 and Beclinl level in the test group.As a result,it’s confirmed that PNS was effect and safe for the treatment of CHD patients with BSS.More importantly,the trial verified the predicted target and network of PNS,which farther suggested that there may exist a specific mode of IncR CTB114C7.4↑-miR3656↓-BCL2A1↑/Beclin1↓.Part three:mechanical research of IncRNA-miRNA-mRNA regulatory network induced by PNS based on RNA interference technology1.It’s found that lncR CTB-114C7.4 was the direct target of PNS and the initiator of the network.2.Specific regulatory mode of the network was lncR CTB114C7.4↑-miR3656↓-BC--L2A1↑.Regrettably,Beclinl mRNA did not participate in the network directly3.The expression level of BCL2A1 protein was consistent with its corresponding mRNA in each group,which indicated that PNS promoted BCL2A1 protein by activating the above network.Moreover,analysis about the expressions of BCL2A1 and Beclinl proteins illustrated that they maintained a relatively fixed negative correlation,indicating interactions between them.4.In terms of apoptosis,H2O2 promoted the process of apoptosis in model group,especially the early phase of apoptosis;however,groups of PNS treatment,lncR CTB-114C7.4 overexpression and miR-3656 inhibitor could suppress early and total apoptosis induced by H2O2.Furthermore,lncR CTB-114C7.4 knockdown or miR-3656 mimics on the basis of PNS could reverse the protective effects of PNS on early and total apoptosis.5.In terms of autophagy,H2O2 could accelerate autophagosomes formation and the synthesis of autophagy-related membranes,accompanied with a tendency of increasing autophagy flow in the model group.On the contrary,PNS treatment group and lncR CTB-114C7.4 overexpression group could not only reduce the synthesis of autophagy-related membranes,but also inhibit autophagosome formation and autophagic flow.Notably,lncR CTB-114C7.4 knockdown group could promote autophagosomes formation and then reactivated the process of autophagy.The above experimental results showed that PNS could initiate the expression of lncR CTB-114C7.4,and thereby activated the lncR CTB114C7.4↑-miR3656↓-BCL2--A1↑ regulatory network.It then affected the protein expressions of BCL2A1 and Beclin1,and thus inhibited the apoptosis and autophagy of vascular endothelial cells.Conclusion1.It’s indicated from this study that BCL2A1 was the predicted target gene for PNS in the treatment of CHD patients with BSS.The regulatory network may be lncR CTB114C7.4-miR3656-BCL2A1/Beclin1.2.PNS was demonstrated to be effective in the treatment of CHD patients with BSS.Based on the clinical effectiveness,it’s further verified that PNS could regulate the expressions of predicted target gene and network.3.Initiator of the network regulated by PNS was lncR CTB-114C7.4 Specific regulatory mode of the network was lncR CTB114C7.4↑-miR3656↓-BCL2A--1↑.The cytological function induced by the network was that it inhibited the processes of apoptosis and autophagy in vascular endothelial cells by up-regulating the expression of BCL2A1 protein and down-regulating Beclinl protein level,thus playing a protective role in the treatment of CHD patients with BSS. |
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