| Background:The prevalence of atherosclerotic cardiovascular disease(ASCVD)is increasing rapidly over the past few decades and is now considered as an epidemic worldwide.Western medicine is currently the mainstream therapy for ASCVD,however,there was still a high incidence of cardiovascular events after receiving optimized drug therapy.Therefore,it is imperative to find a new drug to treat ASCVD.In recent years,Traditional Chinese medicine(TCM)has made many progressions in preventing and curing cardiovascular diseases.In the field of basic research,TCM has made great progress in improving vascular endothelial function,plaque stability,platelet activation,ischemia-reperfusion,ischemic preconditioning,left ventricular remodeling,vascular remodeling and microcirculation function by using of promoting blood circulation,activating meridians and benefiting Qi.Tongxinluo capsule(TXL)has been used for the treatment of ASCVD.It has been thought to have clinical benefits for patients with ASCVD,including reduction the incidence of acute myocardial infarction(AMI)Previous studies have shown that addition of TXL to conventional Western medicine may appears to reduce the risk of target lesion revascularization and angina attacks Meanwhile,basic research indicated that TXL could stabilize atherosclerotic(AS)lesions,protecting the myocardium from ischemia reperfusion injury,reducing the size of myocardial necrosis and no reflow,and improving cardiac performance.In our previous studies.Most of the current research on TXL focuses on its role in reducing blood lipids and inhibiting inflammation.However,due to the complicated composition,there should be numerous mechanisms involved in the anti-AS effect of TXL.Genechip is a potential tool to study the large-scale expression,function of the genes within an organism in health and disease.In this study,we used mouse genechip to detect the various mRNA levels of genes in an atherosclerotic animal model with TXL treatment We aim to investigate whether TXL can modulate the mRNA of atherosclerotic vascular as a whole and to synthetically analyze the underlying mechanismObjectives1.To clarify the influence of TXL on AS plaque formation in apoE-/-mice;2.To explore the possible mechanism of TXL exerting anti-AS effect.Methods1.TXL solution preparationIn vivo experiment,TXL ultrafine powder was prepared and mixed into normal saline suspension of different concentrations.Mice were given 0.1 ml of suspension daily by gavage.The high-fat feeding(HFD)group was given normal saline of the same volume.In vitro experiment,TXL medium solution was used to prepare TXL storage solution2.Experiment groupingA total of 100 apoE-/-mice(12 weeks old,male)were randomly assigned to the following groups(n=20 per group):Control group,(High fat diet)HFD group,low-dose TXL(TXL-L)group,medium-dose TXL(TXL-M)group,high-dose TXL(TXL-H)group.The TXL-L,TXL-M and TXL-H groups were given TXL superfine powder orally at 0.38,0.75,1.5 g/kg/d,respectively for 16 weeks and the HFD group were given an equal volume of saline.HFD and TXL groups were fed on an atherogenic diet and the Control group were fed a normal diet for the entire protocol3.Serum Biological MeasurementsThe level of serum lipids and glucose were measured by enzymatic methods.4.Histopathological analysisThe en face aortic were stained with Oil-red O and hematoxylin-eosin staining to assess burden and distribution of AS.Tissue samples from the aortic roots were cut into serial sections 5 ?m thick,stained with sirius red and Oil-red-O and reacted with the primary antibodies against macrophages/monocytes(MOMA-2),smooth muscle cells(SMCs),tumor necrosis factor-a(TNF-?),interleukin 6(IL-6),matrix metalloproteinase 2(MMP-2).Immunohistochemical staining involved standard techniques.In brief,The activity of endogenous peroxidase was inhibited by 3%H202 incubation.Then blocked with 5%goat serum and incubated 1 hours at room temperature with primary antibodies.After washing 3 times with PBS,the sections were incubated with a secondary antibody at room temperature.Immunohistochemical staining was visualized by use of a diaminobenzidine kit according to the manufacturer's instructions.Nucleus counterstaining involved hematoxylin stain.Immunohistochemical staining and histological was analyzed by using Image-Pro Plus 6.0(IPP 6.0,Media Cybernetics,MD,USA).All measurements were performed under the same conditions.5.Microarray AnalysisAgilent Amp Labeling Protocol(Version 5.7,Agilent Technologies)was used to amplify and transcribe total RNA from aortic tissues from the control group,NS group and TXL-H group to form fluorescent CRN AS.The labeled RNA was hybridized to a mouse genome-wide Oligo chip(4x44K,Agilent Technologies).After cleaning the slides,the array was scanned with Agilent scanner G2505B.6.Bioinformatics AnalysisAnnotation of differentially expressed proteins including the CC,MF,and BP was obtained from the GO database and PANTHER analysis.Biosignaling pathway analysis was performed using KEGG database.The identified protein reciprocity networks were analyzed using a search tool that retrieves the interacting gene/protein(STRING)software.7.Cell culturePrimary macrophages of mice were cultured in vitro,TXL solution of different concentrations was added pretreatment cell for 24 hours,then ox-LDL(40 g/mL)was added to stimulate the cells for 4 hours.The mRNA of macrophages was extracted for subsequent analysis8.Statistical analysisContinuous data were represented as mean±SEM.Categorical data were expressed as number(%).Two-sample t test or one-way ANOVA was used for normally distributed continuous variables,Wilcoxon rank-sum test was used for non-normally distributed continuous variables.Bonferroni-correction was used to correct p values for multiple comparisons.SPSS 16.0 was used for all analyses(SPSS Inc.,Chicago,IL,USA).p<0.05 was considered statistically significant.Result1.Lipid and glucose levelThe serum TC,TG,LDL-C and glucose concentration were significantly increased in HFD group(compared to Control group).However,there was no difference in these parameters between the HFD and TXL groups.2.HistopathologyThe cross-sectional plaque area of the aortic sinus and the relative en face lesion area of the entire aorta were significantly increased in HFD group and were reduced in all of the TXL treatment groups.As well,TXL dose-dependently increased the intraplaque content of smooth muscle cells and collagen but reduced that of lipids and macrophages relative to the HFD group.Consequently,the plaque vulnerability index was dramatically decreased after TXL treatment.Furthermore,TXL treatment dose-dependently decreased the expression of inflammatory cytokines such as TNF-a,IL-6 and MMP-2as compared with the control group.3.Gene Expression ProfilesThere were 3284 differentially expressed genes between the HFD the Control group,of which 2215 genes were up-regulated(>2-fold)and 1069 genes were down-regulated(<0.5-fold)in the HFD group.Meanwhile,632 differentially expressed genes were identified between TXL group and HFD group,there were 415 up-regulated(>2-fold)and 217 down-regulated(<0.5-fold)in TXL group.In pairwise comparisons,we identified 114 genes were significantly changed by atherosclerosis and reversed by TXL,of which 48 genes were upregulated by atherosclerosis and downregulated by TXL and 56 genes were downregulated significantly in atherosclerosis and TXL reversed the changes.4.Bioinformatics analysis of differentially expressed genesThe results showed that the majority of the genes have an intracellular distribution,involving intracellular organelle,nucleus,mitochondrion.According to the analysis of MF,the differentially expressed proteins were categorized into different groups.The top category detected was related to binding,including protein binding,ion binding,organic cyclic compound binding,small molecule binding and receptor binding.The other genes were categorized to activity,catalytic activity and hydrolase activity.In addition,according to the analysis of BP,the majority of the genes were categorized into a metabolic processing,including lipid metabolic,fatty acid metabolic and glucose metabolic.The other proteins were categorized to response to stimulus,cell differentiation,immune system process,inflammatory response.Kyoto Encyclopedia of Genes and Genomes enrichment analysis was then implemented to test proteomics pathway enrichment.KEGG enrichment indicated that the 114 differentially expressed genes were significantly associated with metabolic pathways and PPAR signaling pathway,peroxisome,natural killer cell mediated cytotoxicity,and fatty acid degradation.Conclusion1.TXL can significantly slow down the progression of aortic plaque in mice,inhibit the expression of inflammatory factors in plaques,and increase the stability of plaques;2.TXL intervention can play an anti-AS role through multiple targets.In addition to proven mechanisms such AS lipid lowering,inflammation inhibition and oxidative stress response,TXL plays a cardiovascular protective role by regulating neuroendocrine,immune response and metabolism of the body.BackgroundThe prevalence of coronary heart disease is increasing rapidly over the past few decades and is now considered as an epidemic worldwide.Percutaneous coronary intervention(PCI),as an important treatment of coronary heart disease(CHD),can remarkably relieve the stenosis of coronary artery criminals in a short time,greatly alleviate the symptoms of patients and improve their quality of life.Since the percutaneous coronary angioplasty was first performed in 1977,PCI has rapidly evolved as the first-line treatment to restore myocardial perfusion and reduce short-term mortality and improving the prognosis of patients with CHD.Although the benefit of percutaneous coronary interventions(PCIs)for patients presenting with acute coronary syndromes(ACS)has been established in numerous studies,however,whether patients with stable ischemic heart disease(SIHD)should be treated with PCI is highly controversial.Recently reported that ISCHEMIA trial identified no significant difference in the primary outcome endpoints in patients with moderate or severe SIHD randomized to either optimal medical treatment(OMT)or OMT plus PCI.Among factors that may offset the benefit of PCI in patients with SIHD,a rapid progression of non-target lesions(NTLs)after PCI has gained increased attention.In the PROSPECT trial,cardiovascular events were related to target lesions in 12.9%and to NTLs in 11.6%of patients undergoing PCI during follow-up.Tsiamis et al.reported that in patients undergoing PCI,half patients developed new NTLs during follow-up.These studies suggested that the non-target lesions(NTLs)progression seems to accelerated after percutaneous coronary intervention.However,it remains unknown whether stenting per se may promote the progression of NTLs.In this study,we used a atherosclerotic model of New Zealand rabbits to investigate the relationship between stent implantation and non-target lesion progression,and to explore its possible pathogenesisObjectives1.To observe the relationship between stent inplantation and the progression of NTLs;2.To explore the potential mechanisms of stent implantation on the progression of NTLs.Methods1.Animal modelA total of 30 purebred male New Zealand rabbits.were randomly divided into three groups:a stenting group that received angiography,intravascular ultrasonography(IVUS)and bare metal stent implantation,a sham group that received angiography and IVUS without stent implantation,and a control group that received none of the three invasive procedures.2.Angiography and stent implantation in rabbitsIn the sham and stenting groups,aortic angiography was performed via the left femoral artery access to identify the most stenotic lumen in the abdominal aorta as the site of the target lesion,and the abdominal aortic segment of 50mm in length at a distance>10mm from the proximal end of the most stenotic segment or implanted stent in the sham or stenting group,respectively,was defined the site of NTLs.A standard bare metal stent was installed in the target lesion in the stenting group.Intravascular ultrasonography(IVUS)in rabbitsThe IVUS images of the target lesion and NTLs were analyzed and the maximal and mean values of plaque area and plaque burden in the target lesion and NTLs were calculated.Histological and immunohistochemical staining in rabbits The abdominal aortic segment of 50mm in length at a distance>10mm from the proximal end of the most stenotic segment or implanted stent in the sham or stentinggroup,respectively,was defined as the site of NTLs.The abdominal aortic segment of 50mm in length at a distance>20mm from the common iliac artery bifurcation,which corresponded to the most sites of NTLs in the sham and stenting groups,was used for histopathological studies in the control group.The maximal and mean values of plaque area and plaque burden in the NTLs were calculated in each group of rabbitsEnzyme-linked immunosorbent assay(ELISA)in rabbits Serum levels of lipid profile were measured by an enzymatic method.The concentration of inflammatory cytokines was determined by using ELISA kits.Proteomic analysis in rabbitsSerum total proteins were extracted from 3 blood samples in each of the stenting and sham groups for proteomic analysis 3 months after angiography.Gene Ontology(GO)and KEGG(Kyoto Encyclopedia of Genes and Genomes)were used to analyze the protein family and pathway.Clinical study populationA total of 147 consecutive patients with unstable angina pectoris who underwent coronary angiography(CAG)were enrolled after satisfying inclusion and exclusion criteria.All patients were divided into two groups:CAG group and PCI group.Informed consent was received from all enrolled patients.Blood sample collectionBlood samples in fasting state were collected from all patients before and 1 day and 1 month after CAG in heparin-containing vacutainer tubes for subsequent experiments.Cytokine detectionSerum levels of TC,TG,LDL-C and HDL-C were measured by an enzymatic method on fully automatic clinical biochemical analyzer(Theromo Scientific,USA).Bio-plex ProYM human cytokine 27-plex immunoassay Bio-Plex assay(Bio-Rad,CA,USA,Cat No:M500KCAFoY)was utilized to measure the concentrations of 19 cytokines.The cytokines included interleukin-1?(IL-1?),IL-1RA,IL-5,IL-6,IL-8,IL-10,IL-12p70,IL-13,IL-15,TNF-?,interferon-?(IFN-?),macrophage inflammatory protein 1?(MIP-1?),macrophage inflammatory protein-1?(MIP-1?),C-reactive protein(CRP),serum amyloid A(SAA1),monocyte chemotactic protein-1(MCP-1),IFN-?-inducible protein 10(IP-10),basic fibroblast growth factor(Basic-FGF)and platelet derived growth factor(PDGF-BB).Co-culture of patient's serum and cell linesTHP-1 cells were cultured in RPMI 1640(HyClone,Logan,UT,USA)containing 10%fetal bovine serum(FBS)(Wisent,Quebec,Canada).HUVECs were cultured in endothelial cell medium(ECM)containing 5%FBS and 1%endothelial cell growth supplement(Sciencell,Califonia,USA).Monocyte adhesion testTo examine the pro-inflammatory effects of the serum from patients undergoing CAG or PCI,we performed monocyte adhesion test and quantitated the expression of inflammatory cytokines by western blot.For monocyte adhesion test,HUVECs showing 80%fusion growth were pretreated for 12hrs and 24hrs,respectively.Adherent cells were counted in five random fields per well.The total protein of HUVECs was collected after pretreatment for 24hrs with serum from patients 1 day after CAG,and the expression of inflammation-related cytokines were quantitated by western blot.Western blot analysisTo examine the effect of cytokines related to monocyte adhesion and acute phase reaction,the protein expression of the following cytokines in HUVECs were determined by western blot:intercellular adhesion molecule-1(ICAM-1),VCAM-1,TNF-?,IL-6,SAA1,CRP;phospho-p65/p65,phospho-signal transducer and activator of transcription 3(p-STAT3)/STAT3,All experiments were repeated for at least 5 times and the mean values calculated for statistical analysis.3.Statistical analysisContinuous data were represented as mean±SEM.Categorical data were expressed as number(%).Two-sample t test was used for.normally distributed continuous variables and Wilcoxon rank-sum test for.non-normally distributed continuous variables.The chi-square test or Fisher exact test was used for comparing categorical data.All analyses were performed with SPSS 16.0(SPSS Inc.,Chicago,IL,USA).Results1.Plaque burden and area of NTLs were rapidly increased after stent implantation in rabbitsIVUS images showed that the plaque burden and area of NTLs were increased significantly 3 months after angiography compared with that at baseline in both sham and stenting groups.From week 10 to week 22,the plaque volume increased from 96.03 mm3 to 140.44 mm3(by 46.25%)and the plaque burden from 18.53%to 26.47%(by 42.85%)in the sham group.By comparison,the plaque volume increased from 105.52 mm3 to 182.96 mm3(by 73.39%)and the plaque burden from 19.04%to 35.14%(by 84.56%)in the stenting group.Thus,the net increase in plaque volume and burden of NTLs from week 10 to week 22 was more significant in the stenting than the sham groups,with a relative increase in plaque volume by 30.28%and in plaque burden by 32.75%in the former versus the latter groupPathological studies showed that plaque burden in the stenting group was substantially larger than that in the sham or the control group in all 5 sub-segments The mean or maximal plaque burden and area of the NTLs were all significantly larger in the stenting group than those in the sham or control group.The relative increase in mean plaque area and burden was 55.9%and 27.3%,respectively,in the stenting group versus the sham group.2.Stent implantation increased plaque vulnerability of NTLsImmunohistochemical staining of the NTLs in 3 groups of rabbits exhibited no significant difference in the relative contents of lipids,macrophages,SMCs and collagen of NTLs between sham and control groups,whereas the relative contents of lipids and macrophages were higher and those of SMCs and collagen lower in the stenting group than the corresponding values in the sham or control group.Consequently,the vulnerability index in NTLs was markedly increased in the stenting group than that in the sham or control group3.Stent implantation induced vascular inflammation in rabbitsThe protein expression of TNF-?,IL-6,MCP-1 and VCAM-1 in the NTLs showed no difference between sham and control groups,but was remarkably higher in the stenting group than the sham or control group.4.Stent implantation induced persistent systemic inflammation in rabbitsThe serum levels of TNF-?,IL-6,MCP-1 and VCAM-1 showed a stepwise and remarkable uprising from baseline to 1 day to 3 months in the stenting group.As a result,the serum levels of TNF-?,IL-6,MCP-1 and VCAM-1 were much higher in the stenting group than the sham and control groups 3 months after angiography.These results suggested that stent implantation triggered a persistent systemic inflammation in these rabbits.5.Proteomics analysisTo explore the mechanism of the persistent vascular and systemic inflammation in the stenting group,we used proteomics analysis of the serum from the stenting and sham groups of rabbits 3 months after angiography(n=3 in each group).Compared with the sham group,the expression of multiple acute phase proteins(APPs)in stent group was significantly up-regulated,including serum amyloid A protein 1(SAA1),SAA4,SAA,CRP,lipopolysaccharide-binding protein(LBP),?1-acidglycoprotein(AAG).GO analysis showed that most DEPs involved extracellular distribution and KEGG analysis showed that these DEPs were mainly involved in immunity and inflammation-related pathways,including complement,staphylococcus aureus infection,NF-?B signaling,peroxisome proliferator-activated receptor(PPAR)and Jak-STAT signaling.General condition of patientsThere was no noticeable difference in baseline characteristics between patients undergoing CAG and PCI.In the CAG group of patients,serum levels of TC,TG,LDL-C and HDL-C did not show significant changes before and 1 month after CAG.However,in the PCI group of patients,serum levels of TC and LDL-C declined 1 month after PCI relative to baseline levels,probably due to an intensive statin treatment after PCI in this group.The expression of CRP and SAA1 increased after PCIIn CAG group,serum levels of SAA1 and CRP increased rapidly 1 day after CAG but returned to baseline level 1 month after CAG.In PCI group,however,although SAA1 and CRP exhibited a surge 1 day after PCI,CRP remained higher than baseline 1 month after PCI.The proportion of patients at high risk of CRP increased after PCIWe further divided CRP values into 3 categories:high(CRP>3mg/l),moderate(CRP1-3mg/l)and low(CRP<1mg/l)levels,and found that the percentage of patients with a high level CRP was 18.87%and 18.09%before intervention,20.76%and 41.49%I day after intervention and 13.21%and 28.72%1 month after intervention in the CAG and PCI groups,respectively.The expression level of inflammatory related factors was increased after PCIThe serum levels of other 17 inflammation-related cytokines showed no substantial changes over time in the CAG group,but a huge increase 1 day and/or 1 month after PCI except for MCP-1,MIP-1? and MIP-1?.In particular,the serum levels of IL-6,IL-8,IL-10,IL-13,TNF-? and IP-10 1 month after PCI remained as high as,or even higher than,the corresponding values 1 day after PCI.These results were consistent with our animal findings and clearly indicated that patients after PCI exhibited persistent systemic inflammation.Serum of patients after PCI promotes adhesion to endothelium-mononuclear cells HUVECs were divided into two groups:group A and group B,in which HUVECs were stimulated with serum of patients in the CAG group and PCI group,respectively,for 24 hrs.Monocyte adhesion assay demonstrated that the adhesion ability of THP-1 cells was dramatically enhanced after THP-1 cells were co-cultured with HUVECs in group B for 24 hrs in comparison with THP-1 cells co-cultured with HUVECs in group A,and the protein expression of ICAM-1,VCAM-1,TNF-?,IL-6,SAA1 and CRP was significantly increased in group B.The acute phase reaction was activated after PCIThe protein expression of TNF-?,IL-6,SAA1 and CRP was significantly increased in HUVECs in group B.These results suggested that the circulating APPs in patients receiving PCI may stimulate HUVECs to produce inflammatory cytokine,leading to an enhanced vascular inflammation.Meanwhile our results showed that the phosphorylated protein levels of both NF-?B and STAT3 were significantly higher in HUVECs of group B than group A.The effect of treatment with NF-?B or STAT3 inhibitor on the expression of APPs and inflammatory cytokines.Treatment of both groups of HUVECs with NF-?B inhibitor BAY11-7082 or STAT3 inhibitor WP1066 exerted no effect on the protein expression levels of TNF-?,IL-6,SAA1 and CRP in HUVECs of group A but substantially lowered these protein expression levels of group B.Conclusion1.Plaque burden and volume in the NTLs were significantly increased in a rabbit model of atherosclerosis 3 months after stent implantation;2.Stent implantation induced persistent vascular and systemic inflammation3.Activation of ARP were responsible for stenting-triggered inflammation in these animals;... |
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