Metabolomic Study On The Intervention Of Yiqi Liangxue Shengji Recipe On Vascular Injury And Repair In Rat

Background:Percutaneous coronary intervention(PCI)is one of the important treatment methods for coronary heart disease,which significantly benefits patients with coronary heart disease.In recent years,with the promotion of the concept of "intervention without implantation",PCI technology has become more mature.However,interventional treatments such as stents and drug balloons are invasive procedures that inevitably damage the vascular endothelium,leading to problems such as restenosis and long-term vascular lumen loss caused by poor endothelial repair.The repair process of endothelial injury involves multiple pathophysiological processes,including inflammation,oxidative stress,platelet aggregation,proliferation and migration of vascular smooth muscle cells(VSMC),and repair of vascular endothelial cells(VEC).Although significant progress has been made in the research and development of anti platelet aggregation and lipid regulating drugs in modern medicine,endothelial damage repair is a complex pathological change,and there is still a residual risk of poor repair after intensive treatment with secondary preventive drugs.Traditional Chinese medicine(TCM)has achieved certain effects in the intervention of promoting injury repair after PCI.Previous studies have shown that Yiqi Liangxue Shengji formula(YQLXSJ)can improve clinical symptoms,improve quality of life,reduce the occurrence of MACE in patients after PCI,and has the effects of reducing inflammatory reactions,inhibiting VSMC proliferation,and promoting VEC repair.However,the ingredients of this prescription are not clear enough and the relevant research is not in-depth enough.The connotation of its mechanism needs to be further clarified.In recent years,metabolomics has been widely used in the study of TCM prescriptions,helping to characterize the pharmacodynamic material basis of TCM and explain its pharmacological mechanism.Therefore,this study constructed a balloon injury model of the abdominal aorta in rats,and used metabonomics to explain the mechanism of YQLXSJ in the repair of vascular injury after PCI.Objective:1.To evaluate the efficacy of YQLXSJ on vascular repair after balloon injury in rats.2.To explore the metabolic markers and metabolic pathways of YQLXSJ for intervention in vascular injury repair.3.To characterize the pharmaceutical ingredients of YQLXSJ,integrate network pharmacology to obtain the core targets of YQLXSJ for intervention in vascular injury repair after PCI,construct a metabolic interaction network,and reveal the metabolic regulation mechanism.Methods:1.The model of balloon-injured abdominal aorta was established in SD rats.A total of 40 rats were divided into the sham,model,YQLXSJ,and positive(atorvastatin calcium tablets)groups.Abdominal aortic ultrasound was used to evaluate vascular function,including intima-media thickness(IMT),peak systolic velocity(PSV),end diastolic velocity(EDV)and resistance index(RI).The morphological changes of blood vessel were observed by H&E staining,and the intimal thickness(IT),medial thickness(MT),and the ratio(IT/MT)were measured for quantitative analysis.Immunofluorescence was used to observe the distribution of VEC and VSMC in vascular tissue.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of serum inflammatory factor C-reactive protein(CRP)and nitric oxide(NO).2.Based on UPLC-MS/MS technology,the rat serum was used for metabolomics.The data were processed by PCA and OPLS-DA using multivariate statistical analysis methods.The differential metabolites were screened and identified according to the criteria of VIP>1,P<0.05,and FC>1.2 or FC<0.8.After comparison between groups,the metabolic markers of YQLXSJ for intervention in the repair of vascular injury were obtained.MetaboAnalyst 5.0 software was used to analyze the metabolite clustering and metabolic pathway.3.Based on UPLC-MS/MS technology,the chemical components of YQLXSJ were analyzed.The targets of active ingredients were searched in the TCMSP and SwissTargetPrediction,the targets of disease were searched in the GeneCards and OMIM with the keyword ’in-tent restenosis’,and all targets were unified by Uniprot.Cytoscape was used to build the ’component-disease-target’ network,and the top 5 key components of YQLXSJ were obtained according to the degree value in the network.The metabolite targets were collected in HMDB.The core targets were obtained after intersection of component targets,disease targets and metabolite targets,which were verified by molecular docking and ELISA.The key pathways were obtained via KEGG PATHWAY.The ’target-pathway-metabolite’network was constructed by Cytascape and the ’metabolic interaction network’ was drawn to show the relationship among the core targets,metabolic pathways and metabolites.Results:1.There was no statistically significant difference in the body weight of rats in each group at four weeks(P>0.05).The results of ultrasound:Compared with the sham group,IMT,PSV,RI were significantly increased and EDV was decreased in the model group(P<0.01).Compared with the model group,IMT,PSV,RI were decreased and EDV was increased in the YQLXSJ and positive groups(P<0.01).The results of H&E staining:In the model group,the neointima and media were thickened,but intimal thickening was more significant.A large amount of VSMC proliferation and disordered cell arrangement were observed in the neointima.Both YQLXSJ and atorvastatin attenuated the degree of intimal hyperplasia and proliferation of VSMC.Compared with the sham group,IT,MT,and IT/MT were obviously increased in the model group(P<0.01).Compared with the model group,IT,MT,and IT/MT were decreased in the YQLXSJ and positive groups(P<0.05).The results of immunofluorescence:In the model group,the vWF fluorescence intensity was the weakest,but the intensity of α-SMA was the strongest.Compared with the model group,both the fluorescence intensity of the YQLXSJ and positive groups had corresponding callbacks.The results of ELISA:Compared with the sham group,the level of NO was significantly decreased and the level of CRP was increased in the model group(P<0.01).Compared with the model group,the content of NO was improved and the level of CRP was reduced(P<0.05).2.The metabolic profile of the model group was significantly different from that of the sham group and YQLXSJ group,but the metabolic profile of YQLXSJ group was close to the sham group.After OPLS-DA analysis,a total of 49 metabolic markers were obtained,including citrulline,spermidine,ornithine,glutamine,glutathione,PC(15:0/15:0),LysoPC(16:1/0:0),pyruvate,succinic acid,α-Ketoglutaric acid,etc.Six important metabolic pathways were obtained,including Arginine biosynthesis,Alanine,aspartate and glutamate metabolism,Citrate cycle,Glutathione metabolism,Arginine and proline metabolism,and Glycerophospholipid metabolism.3.A total of 95 chemical components were identified in YQLXSJ,of which quercetin,luteolin,fisetin,kaempferol,isorhamnetin and tanshinone Ⅱa are the key components.After integrating metabolomics and network pharmacology,5 core targets were obtained:NOS2,NOS3,ODC1,VEGFA and PLA2G2A,5 key pathways were Arginine and proline metabolism,Arginine biosynthesis,Glutathione metabolism,Glycerophospholipid metabolism and HIF-1 signal pathway,and 14 key metabolites were screened.The results of molecular docking:the best binding energies of key components and core targets were less than-5 kcal/mol.The results of ELISA:Compared with the sham group,the levels of NOS2,VEGFA,ODC1 and PLA2G2A increased significantly in the model group(P<0.01),while the level of NOS3 decreased significantly(P<0.01).Compared with the model group,the levels of NOS2,VEGFA,ODC1 and PLA2G2A decreased in the YQLXSJ group(P<0.05),while the level of NOS3 increased significantly(P<0.01).Conclusions:1.YQLXSJ can inhibit VSMC proliferation,promote VEC repair,and reduce the level of inflammatory factor CRP in the abdominal aorta of rats after balloon injury.It has the effect of inhibiting neointimal hyperplasia and promoting reendothelialization.2.YQLXSJ can recall citrulline,spermidine,glutamine,glutathione,ornithine,PC(15:0/15:0),LysoPC(16:1/0:0),pyruvate and other metabolites.And YQLXSJ could comprehensively regulate the metabolic disorder after PCI by improving the bioavailability of arginine and NO,reducing inflammatory reaction and oxidative stress,and regulating the balance of energy metabolism.3.Quercetin,luteolin,festin,kaempferol,isorhamnetin and tanshinone Ⅱa are the key components of YQLXSJ.These components can stably combine with core target proteins NOS2,NOS3,ODC1,VEGFA and PLA2G2A to play a role in regulating amino acid metabolism,lipid metabolism,energy metabolism.YQLXSJ can promote vascular repair after PCI by interfereing with inflammation,oxidative stress,blood flow shear stress,angiogenesis,VSMC proliferation and other biological processes.