The Methylation Nature Of Heat-toxic Blood Stasis Syndrome In Coronary Heart Disease And The Molecular Mechanism Of Gardenia And Chuanxiong Against A

ObjectivesHeat toxicity and blood stasis syndrome(HTBSS)is a common syndrome associated with coronary heart disease(CHD).One of the commonly used treatment methods for CHD is clearing heat and detoxifying,as well as promoting blood circulation and removing blood stasis.However,the biological mechanisms underlying heat toxicity and blood stasis syndrome in coronary heart disease are still not fully understood,and further research is needed to elucidate the biological processes involved in clearing heat and toxins,promoting blood circulation,and reducing blood stasis.In this study,the Infinium Methylation EPIC BeadChip technique was utilized to identify specific methylated genes associated with HTBSS CHD through whole-genome DNA methylation analysis.Bioinformatics analysis was then performed to gain insight into the biological nature of HTBSS CHD.In addition to observing the lipid-lowering and anti-inflammatory effects of Zhizichuanxiong freeze-dried powder(ZCFDP),an in vitro experiment was conducted to investigate its anti-atherosclerosis(AS)effect.The experiment aimed to clarify the biological connotation of clearing heat,detoxification,and activating blood circulation while removing blood stasis.To achieve this,the molecular mechanism of anti-AS in vitro of ZCFDP was investigated using a combination of methylation capture and transcriptome sequencing technology.Methods(1)In this study,we used the Infinium Methylation EPIC BeadChip technique to detect DNA Methylation in peripheral blood leukocytes of 10 patients with HTBSS CHD,10 patients with non-heat toxicity blood and stasis syndrome(NHTBSS)CHD,and 19 healthy volunteers(HV).The IMA package of R software was utilized to calculate the values of methylation sites of samples in each group.We then compared the differentially methylation sites(DMSs)and differentially methylation genes(DMGs)among the groups.The study analyzed two sets of DMGs concerning CHD.Set A included DMGs from the HTBSS CHD group compared to the HV group,while set B included DMGs from the NHTBSS CHD group compared to the HV group.The genes that were common to both sets A and B were grouped in set C,which represents DMGs specifically associated with CHD.The genes in set A,excluding those in set C,were grouped as set D,which represents DMGs associated with the coronary heart disease syndrome of heat toxicity and blood stasis.Further analysis of these genes(set D)using KEGG and GO enrichment analysis,as well as protein-protein network interaction analysis,was conducted to gain a better understanding of their role in the CHD.(2)The foam cells model was established after RAW264.7 cells were treated with oxygenized low-density lipoprotein(ox-LDL)for 48 hours.The lipid phagocytosis of foam cells was observed by oil-red O staining.The proliferation of Raw 264.7 cells treated with ZCFDP at different concentrations was detected by the CCK-8 method to determine the concentration of ZCFDP treated in subsequent in vitro experiments.The cells were divided into five groups:control group(no treatment),model group(80μg/mL ox-LDL),ZCFDP low-dose group(80 μg/mL ox-LDL and 0.1 g/L ZCFDP),ZCFDP medium-dose group(80μg/mL ox-LDL and 0.3 g/L ZCFDP)and ZCFDP high-dose group(80 μg/mL ox-LDL and 0.9g/L ZCFDP).The contents of total cholesterol(TC)and free cholesterol(FC)were determined by Gas chromatography-mass spectrometry(GC-MS).Enzyme-linked immunosorbent assay(ELISA)was used to check the expressions of inflammatory factors including interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),and intercellular cell adhesion molecule-1(ICAM-1).(3)The cells were divided into three groups:control group(no treatment),model group(80μg/mL ox-LDL),and ZCFDP high-dose group(80μg/mL ox-LDL and 0.9g/L ZCFDP).The DNA and RNA of each group of cells were extracted,and the DNA methylation and gene expression levels of each group of cells were detected by methylation capture sequencing(MC-seq)and RNA sequencing(RNA-Seq)methods.DMSs and differentially expressed genes(DEGs)were identified using the open-source R package dispersion shrinkage for sequencing data(DSS).Kyoto Encyclopedia of Genes and Genomes(KEGG)and Gene Ontology(GO)enrichment analysis were used to explore the bioinformatics implication.The targeted signaling pathway was verified using western blotting(WB).Results(1)In comparison to the HV group,the HTBSS CHD group had 1,398DMSs(852 DMGs).Among these,there were 154 hypermethylated genes and 698 hypomethylated genes.In comparison to the HV group,the HTBSS CHD group exhibited differential hypermethylation in the top 20 genes such as MAP2K5,ATP6V1D,CDC42BPA,MEIOB,and FRMPD4.Conversely,the top 20 differentially hypomethylated genes in the HTBSS CHD group compared to the HV group were IGSF21,Clorf57,CCDC85C,FLG2,and SLC4A8.Compared with the Health group,there were 979 DMSs(552 DMGs)in the NHTBSS CHD group,including 96 hypermethylated genes and 456 hypomethylated genes.Compared with the HV group,the top 20 differentially hypermethylated genes in the CHD group included SCO1,RBM33,FBXW7,ADIPOR2,and OR5H15.Compared with the HV group,the top 20 differentially hypomethylated genes in the NHTBSS CHD group included RBM33,SFI1,NFATC1,IGSF21,and TRPS1.The DNA methylation sites of both HTBSS CHD patients and NHTBSS CHD patients showed an overall tendency towards abnormally hypomethylation sites,accompanied by abnormal hypermethylation imbalance.678 DMGs representing HTBSS CHD,of which the hypermethylated genes included MAP2K5,ATP6V1D,CDC42BPA,MEIOB,FRMPD4,DLG1,GZF1,and COTL1,and the hypomethylated genes included Clorf57,CCDC85C,FLG2,MICA,GALNT9,RNF17,ATXN7L1,and ARHGAP42.The KEGG enrichment analysis on HTBSS CHD DMGs mainly included:Metabolic pathways,Phosphatidylinositol signaling system,AMPK signaling pathway,Rapl signaling pathway,PI3K-Akt signaling pathway,and Cell adhesion molecules.The results of GO enrichment analysis regarding HTBSS CHD DMGs in terms of biological processes were mainly related to the regulation of gene expression,inositol phosphate dephosphorylation,NF-κB transcription factor activity,and inflammatory response.A total of 97 HTBSS CHD core differentially methylated genes(cDMGs)were screened by PPI analysis.The KEGG pathway enrichment analysis of these cDMGs revealed significant enrichment in pathways such as the AMPK signaling pathway,Adipocytokine signaling pathway,PI3K-Akt signaling pathway,MAPK signaling pathway,and cell adhesion molecules.136 DMGs with RDXY CHDS located in the Promoter-TSS region were screened using the same inter-group comparison method.The results of the KEGG enrichment analysis of 136 HTBSS CHD DMGs located in the promoter-TSS region included:Metabolic pathways,Toll-like receptor signaling pathway,NF-κB signaling pathway,PI3K-Akt signaling pathway,Hippo signaling pathway,Jak-STAT signaling pathway.(2)Oil red O staining showed a large number of orange-red lipid granules in Raw264.7 cells treated with 80μg/mL ox-LDL for 48 hours.The CCK-8 assay showed that ZCFDP had a cytotoxic effect on Raw264.7 cells.In the experiment,the concentration of ZCFDP below 0.9 g/L was selected as the treatment concentration.Compared with the control group,the expression levels of TC and FC in the model group were significantly increased(P<0.01).Compared with the model group,the expressions of TC and FC in the ZCFDP-H,ZCFDP-M,and ZCFDP-L groups were significantly decreased(P<0.01).Compared with the control group,the contents of IL-6,ICAM-1,and TNF-α in the cell supernatant of the model group were significantly increased(P<0.01).Compared with the model group,the contents of IL-6 and ICAM-1 in the ZCFDP-H group,ZCFDP-M group,and ZCFDP-L group were significantly decreased(P<0.01),and the expression of TNF-α in ZCFDP-H group and ZCFDP-M group was significantly decreased(P<0.01).(3)The MC-seq analysis identified 38,368 DMSs between the model and control groups.Of these,23,600 sites were found to be hypomethylated in genes,while 14,768 sites were hypermethylated.The differentially methylated genes in the model group were found to be relatively hypomethylated compared to the control group.Compared to the model group,the ZCFDP-H group exhibited a total of 35,678 DMSs,with 18,889 sites showing hypermethylation and 16,789 sites showing hypomethylation.In the comparison between the model group and the control group,a total of 4,355 DMGs were identified in the promoter-TSS region,with 2,524 genes showing hypomethylation and 1,831 genes showing hypermethylation.In the comparison between the ZCFDP-H group to the model group,we identified 4,205 DMGs located in the promotor-TSS region.Of these,2,165 were hypermethylated genes and 2,040 were hypomethylated genes.Treatment with ZCFDP-H resulted in the reversal of 994 ox-LDL-induced abnormal DNA methylation genes.Specifically,435 hypermethylated genes showed decreased methylation levels after ZCFDP-H treatment,while 559 hypomethylated genes showed increased methylation levels.ZCFDP-H treatment was capable of reversing changes in DNA methylation levels in ox-LDL-induced foam cells in both whole-genome analysis and promoter-TSS region.RNA-seq analysis showed that the model group had a total of 896 DEGs,with 341 up-regulated genes and 555 down-regulated genes compared to the control group.In comparison to the model group,the ZCFDP-H group exhibited a total of 806 DEGs,with 570 genes being up-regulated and 236 genes being down-regulated.The RNA-seq results revealed that ZCFDP-H treatment reversed a total of 133 DEGs in the pairwise comparisons between the ZCFDP-H group and the model group and between the model group and the control group.Among these DEGs,33 that were up-regulated by ox-LDL were down-regulated by ZCFDP-H treatment,while 100 DEGs that were down-regulated by ox-LDL were up-regulated by ZCFDP-H treatment.In the comparison between the ZCFDP-H group and the model group,a total of 96 genes(DEG/DMSs)were identified in the promotor-TSS region.These genes had methylation levels that were negatively correlated with gene differences,as revealed by the correlation analysis of MC-seq and RNA-seq data.This study analyzed 96 genes(DEG/DMSs),out of which 69 genes showed high expression and hypomethylation,such as Lrp2,Wnt2b,Lnx9,Chst7,and Opr11.On the other hand,27 genes exhibited low expression and hypermethylation,including Ctgf,Sema7a,Atplb2,Fnl,and Col1a2.Correlation analysis revealed a significant negative correlation between the degree of expression differences and the methylation levels of Atg13,ORP5,Zfp36,and Slc27a1.KEGG enrichment analysis revealed that 96 genes(DEG/DMSs)were significantly associated with several signaling pathways including PI3K-Akt,Focal adhesion,Hippo,ECM-receptor interaction,and Rap1 signaling pathways.Furthermore,GO enrichment analysis indicated that the DEG/DMSs genes were functionally linked to cardiovascular diseases.Western blot results showed that compared with the control group,the relative expression levels of p-PI3K/PI3K,p-Akt/Akt,p-p65/p65,and p-IκBα/IκBα were significantly increased(P<0.001)in the model group.Compared with the model group,the relative protein expression levels of p-PI3K/PI3K,p-Akt/Akt,and p-IκBα/IκBα were decreased(P<0.05 or P<0.01)in ZCFDP-H and ZCFDP-M groups.Compared with the model group,the p-p65/p65 relative protein expression levels were significantly decreased(P<0.001)in ZCFDP-H,ZCFDP-M,and ZCFDP-L groups.Conclusions(1)In this study,the Infinium Methylation EPIC BeadChip 850K chip technique was used to select 678 HTBSS CHD DMGs,97 HTBSS CHD cDMGs,and 136 HTBSS CHD DMGs(promoter-TSS).The results showed that differential methylation genes associated with heat toxicity and blood stasis syndrome in coronary heart disease were linked to various signaling pathways involved in inflammatory and immune responses.These pathways included the PI3K-Akt signaling pathway,the AMPK signaling pathway,and cell adhesion molecules.The above contents preliminarily revealed the biological nature of heat toxicity and blood stasis syndrome of coronary heart disease.(2)In vitro studies have shown that ZCFDP has a strong anti-atherosclerosis effect.ZCFDP reduces the contents of cholesterol and free cholesterol in foam cells and also decreases the expressions of inflammatory cytokines such as IL-6,ICAM-1,and TNF-α.(3)This study found that ZCFDP can regulate abnormal DNA methylation,leading to changes in gene expression levels.Additionally,ZCFDP plays an anti-AS role by inhibiting the levels of proteins related to the PI3K-Akt signaling pathway and the NF-κB signaling pathway.The genes FN1,Colla2,FLT1,and SPP1 identified in this study may serve as potential therapeutic targets for ZCFDP against AS.These results provide a foundation for future research.