| Atherosclerosis(AS)is the main pathological basis of cardiovascular disease.Rupture of vulnerable plaques and secondary thrombosis are major causes of death and disability after acute cardiovascular events.Chronic lipid-derived inflammation is an important feature of AS and one of the important pathological mechanisms of reduced plaque stability in advanced stages.NLRP3 inflammasome activation is an important core process in inducing inflammatory response and an important target for anti-AS inflammation research.Abnormal changes in DNA methylation affect the expression of AS-related genes,and many risk factors for AS are also related to abnormal DNA methylation.Therefore,the introduction of DNA methylation research into AS will help to elucidate the mechanism of abnormal expression of complex polygenes in AS from an epigenetic perspective.The reversibility of DNA methylation also has a guiding role in the development of drugs for the prevention and treatment of AS.The previous research of our research group found that the traditional Chinese medicine for nourishing qi and activating blood,and the traditional Chinese medicine for promoting blood circulation and detoxification can regulate the abnormal methylation of AS.According to the theory of traditional Chinese medicine,AS has the pathogenesis characteristics of deficiency,blood stasis and toxin.Combined with the clinical experience of my tutor,we found that the Ginseng-Polygonum cuspidatum compound can be used as the core component of the prescription for nourishing qi,promoting blood circulation and detoxifying,and has clinical efficacy against AS.But its anti-AS pharmacological effects and mechanisms are not clear.In this study,the possible mechanism of anti-AS of Ginseng-Polygonum cuspidatum compound was predicted by LC/MS technology and network pharmacology method.ApoE-/-mice were fed with high-fat diet to construct an AS mouse model.Based on genome-wide DNA methylation and TLR4/NF-κB/NEK7-NLRP3 pathway,the regulatory effect and mechanism of Ginseng-Polygonum cuspidatum compound on AS were investigated,aiming to systematically reveal the anti-AS effect characteristics and mechanism of Ginseng-Polygonum cuspidatum compound at the level of DNA methylation.This doctoral dissertation is divided into the following two parts.Part I Prediction of the anti-atherosclerosis mechanism of Ginseng-Polygonum cuspidatum compound based on LC/MS and network pharmacology.Objective:To preliminarily predict the action pathways and targets of the potential active components of Ginseng-Polygonum cuspidatum compound by means of LC/MS and network pharmacology,so as to provide research directions for subsequent experiments.Methods:Ginseng 500g and Polygonum cuspidatum 600g were extracted and freezedried to prepare Ginseng-Polygonum cuspidatum compound.The components of Ginseng-Polygonum cuspidatum compound were identified by means of ultra-high pressure liquid chromatography tandem quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS).In the Swiss Target Prediction database,set the parameter Probability>0 to predict the target of the compound components of GinsengPolygonum cuspidatum compound.The Gene Cards database was searched with the keyword "atherosclerosis" to establish the AS disease target database.The network analysis of the components-targets of Ginseng-Polygonum cuspidatum compound antiAS diseases was carried out by CytoScape software.Construct and analyze the interaction(PPI)between target proteins on the STRING database online platform.Finally,the KEGG pathway was enriched and the core targets were screened.Results:(1)19 compounds with well-defined structures were preliminarily identified by LC/MS method,including(+)-catechin,resveratrol,cis-resveratrol,polydipside,and emodin anthrone,emodin,luteolin,ω-hydroxyemodin,ginsenoside Rf,kaempferol,ginsenoside Rbl,ginsenoside Rb2,ginsenoside Ro,emodin methyl ether,cassia ketone,ginsenoside Re,20(S)-ginsenoside Rg2,apigenin,ginsenoside Rg5.A total of 318 targets were predicted and screened in the Swiss Target Prediction database.(2)A total of 4 076 AS disease targets were screened by the Gene Cards database,of which 225 drug targets intersected with the 318 predicted targets of Ginseng and Polygonum multiflorum.In the protein interaction network,there were 51 targets with a node degree value of more than 2 times of the median,which was the core target of AS treated with Ginseng-Polygonum cuspidatum compound.(3)After enriching the core target KEGG pathway,select the pathway with p<0.05.There are 9,8,and 7 core targets for the potential effects of Ginseng-Polygonum cuspidatum compound in inflammationrelated signaling pathways,such as Toll-like receptor signaling pathway,NOD-like receptor signaling pathway,and NF-κB signaling pathway.Among them,RELA is the common target of the three signaling pathways.Conclusion:Ginseng-Polygonum cuspidatum compound can act on a number of targets of multiple immune-inflammatory signaling pathways to exert anti-AS effect.Among them,the RELA-encoded protein NF-κBp65 is a key target of the three pathways,bridging the signal transduction of the upstream Toll-like receptor signaling pathway and the NOD-like receptor signaling pathway.The anti-AS effect of GinsengPolygonum cuspidatum compound mediated by TLR/NF-κB/NOD-like receptor signaling pathway will be further verified in animal experiments.Part Ⅱ Study on the mechanism of anti-atherosclerosis in miceObjective:To investigate the effect of Ginseng-Polygonum cuspidatum compound on atherosclerosis and genome-wide DNA methylation in mice from the whole animal level,and to reveal the possible epigenetic mechanism of its pharmacodynamic effect.Methods:(1)Construction of mouse AS disease model and group intervention:85 7-weekold male ApoE-/-mice were fed a high-fat diet for 9 weeks to establish an AS model.The plaque formation was observed by aortic gross oil red O staining as the model was successfully established.The AS ApoE-/-mice were randomly divided into model groups(MC),positive control group(simvastatin SS),low-dose group(GPL),mediumdose group(GPM),high-dose group(GPH).C57BL/6J mice with the same genetic background were used as blank control group(NC).After 12 weeks of gavage intervention,pharmacodynamic indexes were detected.(2)Assessing the pharmacodynamic effect of Ginseng-Polygonum cuspidatum compound on AS in mice:The common carotid artery pulse wave velocity(PWV),aortic root intima-media thickness(IMT),carotid artery and aortic diastolic and systolic vascular diameter changes(DC)were examined by small animal vascular ultrasound.The aorta was stained with Oil Red O to observe the plaque area,and the pathological tissue sections of the aortic sinus were stained with HE,Oil Red O,and Movat to observe and calculate the content of each structural component of the plaque;automatic biochemical analyzer was used to detect the fasting serum TC,LDL-C,HDL-C and TG contents,serum IL-1β,MCP-1,SOD1,Ox-LDL contents were detected by ELISA,and serum SOD activity was detected by WST-1 method.(3)Analysis of the regulatory effect of Ginseng-Polygonum cuspidatum compound on abnormal DNA methylation in AS mice:The aortic tissue of AS mice was selected to extract DNA.After passing the quality inspection,a sequencing DNA library was constructed.The DNA methylation of mouse aorta was detected by whole-genome methylation capture sequencing technology,and the degree of methylation difference>10%was set to screen for differentially methylated CpG sites.Differentially methylated CpG sites were annotated in different gene elements,and the regulatory effect of Ginseng-Polygonum cuspidatum compound on abnormal DNA methylation sites in AS mice was statistically analyzed.Pyrosequencing was used to confirm the regulatory effect of ginseng and knotweed compound on the methylation of candidate genes.(4)To analyze the effect of Ginseng-Polygonum cuspidatum compound on the expression of TLR/NF-κB/NOD-like receptor pathway-related targets:Mouse aorta samples were selected,and Western blot was used to detect the activation of NLRP3 inflammasome and the expression and phosphorylation levels of upstream TLR4/NF-κB signaling pathway-related target proteins.Pearson method was used to analyze the relationship between the expression of each target protein and serum IL-1βand the correlation between DNA methylation of candidate gene NEK7 and protein expression.Results:(1)Effects of Ginseng-Polygonum cuspidatum compound on the elasticity of arterial blood vessels in AS mice:Compared with the model group,the carotid artery PWV of the mice in the high-dose Ginseng-Polygonum cuspidatum compound group was significantly decreased(P<0.05).Aortic root IMT was significantly decreased in low,medium and high dose groups(P<0.01).The intravascular meridian difference of the carotid artery was significantly increased in the low and medium dose groups(p<0.05).(2)Effects of Ginseng-Polygonum cuspidatum compound on plaque stability in advanced stage of AS mice:Oil red O staining of gross aorta and HE staining of aortic sinus pathological sections showed the formation of aortic plaques in AS mice.Oil red O staining of pathological sections showed a large amount of lipid accumulation,and Mo vat staining showed unstable plaque formation with high plaque vulnerability index.Compared with the model group,the area of unstable plaques in the low,medium and high doses of Ginseng-Polygonum cuspidatum compound group was significantly smaller,and most of them were stable plaques with intact fibrous caps;the lipid core was smaller,containing a small amount of cholesterol crystals,foam cells and very few proteoglycans;the outer elastic lamina is curved,but there is also a slight rupture of elastic fibrolysis near the base of the plaque.Compared with the model group,there was no significant change in the plaque area in the low,medium and high doses of Ginseng-Polygonum cuspidatum compound(p>0.05).Compared with the model group,the plaque vulnerability index in the low,middle and high dose groups of Ginseng-Polygonum cuspidatum compound were significantly decreased to varying degrees(P<0.05,P<0.01,P<0.001).The content of foam cells in the plaques in the low and middle dose groups was significantly decreased(P<0.01).The contents of collagen and smooth muscle cells in the high-dose group were significantly increased(P<0.05).(3)Effects of Ginseng-Polygonum cuspidatum compound on four items of blood lipids,inflammation and antioxidant indexes in AS mice:Compared with the model group,the fasting serum TG content of the mice in the Ginseng-Polygonum cuspidatum compound low,middle and high group was significantly decreased(P<0.05、P<0.01).There was no statistical difference in serum TC and LDL-C in the low and medium dose groups,while the serum TC and LDL-C contents in the high dose group were significantly decreased(P<0.05),and there was no statistical difference in serum HDL-C among the groups(P>0.05).The serum IL-1β content in the low,middle and high group of Ginseng-Polygonum cuspidatum compound was significantly decreased(P<0.01 or P<0.001).The serum MCP-1 content in the middle-dose group was significantly decreased(P<0.01),and there was no statistical difference in MCP-1 between the other two groups.The content of serum Ox-LDL in the low and middle dose group of Ginseng-Polygonum cuspidatum compound was significantly decreased(P<0.05).The SOD activity in the middle-dose group was significantly increased,and the SOD1 content in the high-dose group was significantly increased(P<0.05).(4)The regulation of Ginseng-Polygonum cuspidatum compound on genome-wide DNA methylation in AS mice:The whole-genome DNA methylation capture and sequencing data were screened by the degree of methylation difference between the two groups>10%,and it was found that the proportion of abnormally hypermethylated CpG sites in the aortic DNA of AS mice was higher than that of abnormally hypomethylated CpG sites ratio(53.67%>46.33%).The proportion of abnormally hypermethylated sites on CpG islands in the non-intergenic region was 73.22%,and the proportion of abnormally hypermethylated sites on CpG islands in the promoter region was 74.06%.The proportions of abnormal DNA methylation CpG sites in AS regulated in the low,medium and high dose groups of Ginseng-Polygonum cuspidatum compound compound were 13.24%(10341/78122),12.62%(9857/78122)and 6.62%(5174/78122),respectively.Taking the abnormally methylated CpG sites in the model group as the control,the proportion of up-regulated abnormally hypomethylated CpG sites in each group was 46.63%(4822/10341),49.99%(4928/9857),and 51.33%(2656/5174),respectively.Compared with the methylation levels of abnormally hypomethylated CpG sites in the model group,the up-regulated levels of methylation in each group were 0.141±0.038,0.139±0.028,0.149±0.043,respectively,and there were statistical differences in the up-regulated levels(P<0.01).The proportion of down-regulated abnormal hypermethylation in each group was 52.89%(5469/10341),49.54%(4883/9857),46.04%(2382/5174),respectively.Compared with the methylation levels of abnormally hypermethylated CpG sites in the model group,the methylation down-regulation levels in each group were 0.124±0.021,0.128±0.021,and 0.13±0.025,respectively,and the down-regulation levels were statistically different(P<0.01).The chi-square test results of the ratio of up-regulated abnormally hypomethylated CpG sites and the ratio of down-regulated abnormally hypermethylated CpG sites in the low,middle and high dose group of GinsengPolygonum cuspidatum compound are as follows:Regarding the regulation of all abnormally methylated CpG sites,the middle and high dose groups showed that the ratio of up-regulated abnormally hypomethylated CpG sites was higher than that of down-regulated abnormally hypermethylated sites(P<0.01).In terms of the regulation of abnormally methylated CpG sites in all CpG islands,CpG islands in the promoter region,and CpG islands in the exon region,the ratio of up-regulated abnormally lowmethylated CpG sites in the low-,middle-and high-dose groups was significantly higher than that of down-regulated abnormally hypermethylated CpG sites(P<0.01).The KEGG pathway enrichment analysis results of genes annotated by differentially methylated CpG sites regulated by Ginseng-Polygonum cuspidatum compound are as follows:With P<0.05,the top 20 pathways in descending order of rich factor include estrogen signaling pathway,FcγR-mediated phagocytosis,leukocyte transendothelial migration signaling pathway,Hippo signaling pathway,phosphoinositide metabolism pathway,phosphatidylinositol alcohol signaling system pathway,etc.The proportion of regulated genes in the signaling pathway is up to 50%.The pathway with the largest absolute number of regulated genes is the PI3K-Akt signaling pathway.The methylation of 61 genes changed after the intervention of Ginseng-Polygonum cuspidatum compound,mainly encoding fibroblast growth factor receptor family,integrin family,interleukin family,etc.(5)The regulation of Ginseng-Polygonum cuspidatum compound on abnormal DNA methylation of candidate genes in AS mice:The results of pyrosequencing showed that the methylation levels of 3 sequencing sites of the TRIL gene were significantly up-regulated in the low and medium doses of Ginseng-Polygonum cuspidatum compound(P<0.01).The methylation levels of the five sequencing sites of FGFR3 gene in the low-dose group were signifi cantly up-regulated(P<0.05).The methylation level of NEK7 was significantly up-regulated in the high-dose group(P<0.05).Correlation analysis showed that the correlation coefficient of FGFR3 methylation levels detected by pyrosequencing and the results of genome-wide DNA methylation capture sequencing was R=0.3499005,p-value=4.766e-10.The correlation coefficient between TRIL methylation levels detected by pyrosequencing and the results of genome-wide DNA methylation capture sequencing was R=0.598239,p-value=1.138e-05.(6)Effects of Ginseng-Polygonum cuspidatum compound on the activation of NLRP3 inflammasome in AS mice:Compared with the model group,the protein levels of NLRP3,ASC/TMS1,and caspase-1 p20 in the aorta of the low,medium and high doses of Ginseng-Polygonum cuspidatum compound were significantly decreased(P<0.01).Correlation analysis also showed that serum IL-1β levels were significantly positively correlated with NLRP3,ASC/TMS1,and caspase-1 p20 protein levels(P<0.001).(7)Effects of Ginseng-Polygonum cuspidatum compound on NEK7-mediated NLRP3 inflammasome activation:Compared with the model group,the expression of NEK7 protein in the low-medium-high-dose group of Ginseng-Polygonum cuspidatum compound was significantly decreased(P<0.01).And correlation analysis showed that NEK7 DNA methylation was significantly negatively correlated with protein expression level(P<0.001).The expression level of NEK7 protein was positively correlated with the expression of NLRP3 inflammasome protein and serum IL-1β content(P<0.001).(8)Effects of Ginseng-Polygonum cuspidatum compound on TLR4/NF-κB pathway-mediated NLRP3 inflammasome activation:Compared with the model group,the protein expression levels of NF-κBp65,p-NF-κBp105/p50,p-IκBα,and pIKKα/β in the low,middle and high doses of Ginseng-Polygonum cuspidatum compound groups were significantly decreased(P<0.01).And the protein expression level was significantly positively correlated with the reduction of NLRP3 inflammasome activation(P<0.001).Conclusion:Ginseng-Polygonum cuspidatum compound has a good effect on improving the elasticity of blood vessels.It can significantly reduce plaque vulnerability by increasing the content of plaque smooth muscle and collagen.It delays the progression of plaque by regulating lipids,anti-inflammatory and increasing antioxidant activity.Ginseng-Polygonum cuspidatum compound has a bidirectional regulation of abnormal DNA methylation in AS mice,and the up-regulation of abnormal hypomethylation is significantly stronger than the down-regulation of abnormal hypermethylation.Ginseng Polygonatum compound up-regulates NEK7 DNA methylation,reduces the expression of NEK7 protein mediated by NF-κB pathway,and then inhibits NLRP3 inflammasome activation,which may be the epigenetics of Ginseng Polygonatum compound reducing serum IL-1β content and exerting anti-inflammatory effect mechanism. |
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