| Atherosclerosis(AS)is one of the underlying causes of coronary heart disease.Over 75%of acute coronary syndrome(ACS)events are caused by vulnerable plaques.The thinning of the fibrous cap and the enlargement of necrotic core can be aggravated by inflammatory,cholesterol deposition and apoptosis.Emerging studies have shown that abnormal methylation of DNA plays a very important part in the endothelial injury,foam cell formation,vascular smooth muscle cell proliferation and apoptosis.DNA methylation induces changes in gene expression without changing the DNA sequence,and gives personalized characteristics to species.At the same time,hypomethylation and hypermethylation characteristics coincide with the theory of Yin-Yang in traditional Chinese medicine(TCM).Therefore,it is expected to further elaborate the mechanism of TCM through the study of DNA methylation affecting the occurrence and development of AS and related mechanisms.Promoting blood circulation and detoxification is one of the basic treatment methods for preventing and treating atherosclerosis.The treatment of activating blood and detoxifying can stabilize vulnerable plaques through anti-inflammatory and lipidlowering.Ligusticum chuanxiong Hort.and Gardenia jasminoides Ellis(ZZCX)herb pair have the effect of promoting blood circulation and detoxifying,which is the core drug pair for differentiation and treatment of blood stasis and toxin of coronary heart disease(CHD).What is the effect of ZZCX on stabilizing ApoE-/-mice vulnerable plaque?Can ZZCX affect DNA methylation through apoptosis via MAPK signaling pathway?All these questions remain to be answered.Based on this,this research carried out pharmacodynamic material basis,bioinformatics,molecular mechanisms.To investigate the effect of blood components of ZZCX on AS vulnerable plaque and its relationship with DNA methylation and apoptosis.1 Clinical researchBased on the theory of "blood stasis and toxin",to explore the drug use characteristics of Professor Xu Fengqin in treating patients with coronary heart diseaseObjective:To explore the clinical medication rules of Professor Xu Fengqin in the differentiation and treatment of "blood stasis and toxin syndrome" of CHD,and to provide basis for subsequent experimental research.Methods:Medical cases of CHD with "blood stasis and toxin syndrome" treated by HIS outpatient system tutor in Xiyuan Hospital,China Academy of Chinese Medical Sciences were retrospectively analyzed.With the help of Ancient and Modern Medical Records Cloud platform V2.3.5,association rules and complex network analysis were adopted to explore the core drug pairs and prescriptions for CHD based on the theory of "blood stasis and toxin".Results:A total of 211 medical cases involving 195 kinds of TCM were included in this study.Professor Xu Fengqin distinguished the treatment of CHD "blood stasis and toxin syndrome" four properties with warm,flat and cold;The five taste are equal in both pungent,bitter and sweetness;The main effect is eliminat dampness resolv phlegm,wind-expelling and pain-alleviating,activate blood and remove stasis.In terms of medication rule,Ligusticum chuanxiong Hort.,Gardenia jasminoides Ellis was the most frequently used single herb for promoting blood circulation and detoxification.The high frequency drugs are Paeonia lactiflora Pall.-Ligusticum chuanxiong Hort.,Paeonia lactiflora Pall.-Salvia miltiorrhiza Bge.,Astragalus membranaceus-Salvia miltiorrhiza Bge.,Gardenia jasminoides Ellis-Ligusticum chuanxiong Hort.,Astragalus membranaceus-Codonopsis pilosula,etc.The core prescriptions of complex network analysis included 9 drugs,including Ligusticum chuanxiong Hort.,Gardenia jasminoides Ellis,Pinellia ternata,Salvia miltiorrhiza Bge.,Paeonia lactiflora Pall.,Astragalus membranaceus,Codonopsis pilosula,Citrus reticulata Blanco and Curcuma Longa L.Conclusion:ZZCX is a common drug pair for promoting blood circulation and detoxification,throughout the treatment of AS stasis and toxin syndrome.Professor Xu Fengqin not only promotes blood circulation and detoxification,but also attaches importance to the application of regulating qi-flowing for eliminating phlegm.The prescription is based on the principle of "promoting blood circulation and detoxification,regulating qi-flowing for eliminating phlegm".2 Experimental ResearchPart 1.Analysis of active components and blood components of ZZCX by UPLCQ-TOF-MSE techniqueObjective:To determine the chemical constituents of ZZCX in vitro and in vivo.Methods:Firstly,the database of chemical constituents of ZZCX was established through literature retrieval.The information of ZZCX and negative control samples was analyzed by using the UPLC-Q-TOF-MSE technology.The components were compared and inferred by using Progenesis QI software,and some standard samples were compared and verified to identify chemical constituents.Secondly,according to the pharmacological strategy of biological prescription analysis,the same technique was used to conduct qualitative analysis on the active components of ZZCX that could be absorbed into blood by C57BL/6J mice.Results:In this research,total of 20 chemical constituents including 3-nbutylphthalide,4-Hydroxy-3-butylphthalide,Caffeic acid,Crocetin,Crocin Ⅰ,CrocinⅡ,Crocin Ⅲ,Ferulic acid,Gardenoside,Genipin 1-gentiobioside,Geniposide,Geniposidic acid,Levistilide A,Ligustilide,N-butylidenephthalide,Vanillic acid,Palmitic acid,and Quinic acid were analysed and charactered by the UPLC-Q-TOFMSE technology and Progenesis QI software.In addition to crocin Ⅰ and Ⅱ,18 prototype compositions,including iridoid,diterpenes,organic acids,phthalides and lactones,were absorbed into blood.Conclusion:In this research,20 components in vitro and 18 prototype components in vivo were analyzed and characterized by UPLC-Q-TOF-MSE technique.The method was stable and reliable,which provided basis for elucidating the pharmacodynamic substance basis,clinical application and follow-up anti-AS mechanism research.Part 2:The pharmacodynamic study of ZZCX vulnerable atherosclerotic plaque Objective:To observe the effect of ZZCX on vulnerable atherosclerotic plaque in ApoE-/-mice,and to clarify its characteristics.Methods:ApoE-/-mice were fed with high fat for 9 weeks,and were divided into model group,simvastatin group(SIM group),DNA methyltransferase inhibitor group(DAC group),ZZCX high dose group(ZZCX-H group),ZZCX medium dose group(ZZCX-M group),ZZCX low dose group(ZZCX-L group)according to low density lipoprotein cholesterol(LDL-C)level by block random number table method,and C57BL/6J mice as control group;the corresponding drugs were given for 12 weeks.The common carotid artery pulse wave velocity(PWV),intima-media thickness(IMT)were measured by ultrasound in small animals;Plaque area and distribution were assessed by oil red O staining;HE staining was used to evaluate the fibrous cap and necrotic core area of plaque in the aortic sinus,and Movat five-color staining was used to evaluate the content of foam cells and collagen fibers.The levels of serum total cholesterol(TC),LDL-C,triglyceride(TG)and high density lipoprotein cholesterol(HDL-C)levels of ApoE-/-mice were detected by automatic biochemical analyzer(AU 480);The levels of tumor necrosis factor α(TNF-α),interleukin-6(IL6),Intercellular Cell adhesion molecule-1(ICAM-1)were detected by enzyme linked immunosorbent assay(ELISA).The above detection methods were used to comprehensively evaluate the intervention effect of ZZCX on ApoE-/-mice atherosclerosis.Results:Compared with the model group,the level of PWV in SIM group,ZZCXH group,ZZCX-M group,and ZZCX-L group were decreased(P<0.05);Compared with the model group,the level of IMT in control group,SIM group,ZZCX-H group,ZZCX-M group,and ZZCX-L group were significantly decreased(P<0.01);Aortic gross oil-red O staining showed that compared with the model group,the level of gross oil-red O lipid in each intervention groups were decreased(P<0.01,P<0.05);Quantitative analysis of the ratio of necrotic core area in aortic sinus to plaque area by HE staining showed that,compared with the model group,the necrotic core level of SIM group,ZZCX-M group,and ZZCX-L group were decreased(P<0.01,P<0.05);Movat staining in the sinus of aorta showed that compared with the model group,the proportion of foam cells in the intervention groups were decreased(P<0.01);Compared with model group,the level of collagen fiber in SIM group,ZZCXH group,ZZCX-M group,and ZZCX-L group were increased(P<0.01,P<0.05);Compared with the model group,the levels of LDL-C and TC in control group,SIM group,and ZZCX-H group were decreased(P<0.01,P<0.05),the level of TG in control group and each intervention groups decreased(P<0.01),and the level of HDL-C in DAC group and ZZCX-M group were increased(P<0.05);Compared with the model group,the levels of IL-6 and ICAM-1 in control group,SIM group,DAC group,ZZCX-H group,and ZZCX-M group were decreased(P<0.01,P<0.05),the level of TNF-α in control group,SIM group,ZZCX-H group,and ZZCXM group were decreased(P<0.01,P<0.05).Conclusion:In this research,pharmacodynamic evaluation was conducted on the treatment of atherosclerosis with ZZCX.ZZCX could slow down the PWV of the common carotid artery and the IMT of the aortic arch in ApoE-/-mice,reduce the gross lipid content of the aorta,the area of necrotic core and the infiltration level of foam cells,and increase the content of collagen fiber,lowering the levels of blood lipids and inflammatory factors.can stabilize AS vulnerable plaques through multiple ways.ZZCX plays a role in stabilizing vulnerable atherosclerotic plaque through multiple channels.Part 3:Genome-wide methylation sequencing analysis of ZZCX against atherosclerosisObjective:The characteristics,differential methylation sites and pathways of ZZCX regulating DNA methylation by SureSelectXT methylation targeting sequence capture platform.Methods:Based on research in mice aortic arch as the research object,using the Agilent SureSelectXT Methyl-Seq Application,observe the control group,model group,DAC group,ZZCX-H group,ZZCX-M group and ZZCX-L group differences in global methylation site characteristics,screening of difference in methylation sites and areas methylation,GO and KEGG functional enrichment analysis were performed.CB-dock and Discovery Studio were used to conduct molecular docking prediction between the blood components of ZZCX detected in part 1 and the selected differential methylation genes and core proteins of signal pathway.Results:According to the P value<0.05,|diff|>0.1 threshold,the model group than in control group identified 80736 different methylation sites,including hypermethylation sites 40957(50.73%),hypomethylation site 39779(49.27%);Compared with the model group,the positive drug DAC group showed a significant trend of demethylation,and the hypomethylated sites accounted for 62.53%of the total differential methylation sites.Compared with the model group,the hypermethylation sites and hypomethylation sites in the ZZCX-H group,ZZCX-M group and ZZCX-L group accounted for about 50%of the total differential methylation sites,respectively.ZZCX showed bidirectional methylation regulation characteristics.Functional enrichment analysis of differential target genes showed that ZZCX could regulate the positive transcriptional regulation of RNA polymeraseⅡ promoter,the positive regulation of GTase activity,the positive regulation of transcriptional DNA templarization,intracellular signal transduction and other biological processes.Regulation of MAPK,PI3K-Akt,Hippo,Notch,TNF,HIF-1,Apoptosis and other signaling pathways play a role in regulating AS DNA methylation.Among them,all the three dose groups of ZZCX could regulate MAPK signaling pathway,which is the key pathway of the regulation of ZZCX.The core genes such as Fibroblast growth factors 3(FGFR3),RAC-alpha serine/threonineprotein kinase 1(AKT1)and Ras-related protein Rap-1A(RAP1A),were screened by protein-protein interaction network on MAPK signaling pathway.Molecular docking results showed that at least 13 blood components of ZZCX were closely connected with differential methylation genes FGFR3,AKT1 and RAP1 A,with Vina score<-7.0kcal/mol.At least 7 blood components and core proteins of MAPK signaling pathway ERK1,ERK2,JNK1,JNK2 and P38MAPK docking Vina score<7.0kcal/mol,molecular docking interactions were mainly hydrogen bond,van der Waals and Pi-Alkyl.Conclusion:ZZCX showed bidirectional regulation of DNA differential methylation genes,and treated atherosclerosis through multi-target and multi-approach.Differential methylation genes involve MAPK,PI3K-AKT,Apoptosis,Hippo and other signaling pathways.Molecular docking showed that the blood components of ZZCX were closely bound with differential methylation genes and core proteins of MAPK signaling pathway,which could be further verified.Part 4:To explore the anti-atherosclerosis mechanism of ZZCX based on FGFR3 methylation regulation of MAPK/ERK signaling pathwayObjective:To explore the mechanism of FGFR3 promoter methylation regulated by ZZCX and its effect on downstream MAPK/ERK signaling pathway,thus inhibiting apoptosis.Methods:Based on the prediction results of part 3,methylation levels of FGFR3 gene promoter was verified by Pyrosequencing technology.Using Western blot detected the expression levels of P-FGFR3/FGFR3,Bcl-2,Bax,Cleaved Caspase-3 and core proteins of MAPK signaling pathway P-ERK1/2/ERK1/2 and PMEK1/2/MEK1/2.Real-time quantitative PCR was used to detect the expression level of FGFR3.Immunofluorescence technique was used for TUNEL staining of apoptosis.The purpose of this study was to explore the downstream biological effects of ZZCX regulating DNA methylation.Results:Pyrosequencing showed that the methylation level of FGFR3 Chr5.33723633 promoter was increased in control group,ZZCX-M group and ZZCX-L group compared with model group(P<0.01,P<0.05),the methylation level of FGFR3 Chr5.33723644 promoter in control group,ZZCX-H group and ZZCX-L group increased(P<0.01,P<0.05),the methylation level of FGFR3 promoter regions Chr5.33723648 and Chr5.33723657 in control group and ZZCX-L group were increased(P<0.01,P<0.05).Compared with model group,the methylation level of AKT1 Chr12.112659315 promoter in control group and ZZCX-L group decreased(P<0.05).WB and qPCR experiments showed that compared with model group,PFGFR3/FGFR3 protein relative expression and FGFR3 mRNA were decreased in control group,ZZCX-H group,ZZCX-M group and ZZCX-L group(P<0.01).Compared with model group,the relative protein expression levels of PERK1/2/ERK1/2 and P-MEK1/2/MEK1/2 in control group,ZZCX-H group,ZZCXM group and ZZCX-L group were decreased(P<0.01,P<0.05).Compared with the model group,the relative expression levels of Bax protein were decreased in the control group,and ZZCX-H group(P<0.01,P<0.05),the relative expression levels of Cleaved Caspase-3 protein were decreased in the control group,ZZCX-H group,ZZCX-M group and ZZCX-L group(P<0.01,P<0.05),the relative expression of Bcl2 protein was increased(P<0.01,P<0.05),and the relative expression of Bax/Bcl-2 protein was decreased(P<0.01);TUNEL results showed that compared with the model group,the positive rate of TUNEL was lower in control group,ZZCX-H group,ZZCX-M group and ZZCX-L group(P<0.01).Conclusion:ZZCX can increase the methylation level of FGFR3 gene promoter region in aorta tissue of mice,and down-regulate the mRNA and protein phosphorylation levels of FGFR3.The mechanism of ZZCX stabilization of ASvulnerable plaques may be related to the increase of FGFR3 gene promoter methylation level,down-regulation of gene expression,regulation of MAPK/ERK signaling pathway and ultimately increase Bcl-2 expression,decrease Bax,Cleaved caspase-3 expression and apoptosis index to inhibit apoptosis. |
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