OBJECTIVE To investigate the effect of Yangyin Huoxue Prescription and its functional units on the process of Atherosclerosis (AS) from the perspective of DNA methylation.
METHODS ApoE-/- mice were randomly divided into the control group, model group, simvastatin group, Yangyin Huoxue Prescription group, Yangyin group, Qingre group and Huoxue group. The mice in the control group were fed with normal diet and the mice in the other groups were fed with high-fat diet. At the 10th, 12th, 14th, 16th and 18th week, the control group was injected with an equal volume of sterile PBS, and the other groups were injected with 10 μg LPS intraperitoneally. Intragastric administration was carried out simultaneously with high-fat diet feeding and ended at the 20th week when evaluations were carried out. The area of aortic plaque in mice was observed by oil red O staining; the levels of TC, TG, LDL-C, HDL-C and non-HDL-C in mice were detected by kits; HE staining was used to observe the stenosis and lumen area of coronary artery in mice; the mRNA expression of DNA methylation enzymes Tet1, Tet2, Tet3, Dnmt1, Dnmt3a and Dnmt3b in spleen/aorta of mice was detected by qPCR; western blot was used to detect the expression of Dnmt1, Dnmt3a and Dnmt3b proteins in the spleen/aorta of mice; ELISA was used to detect the changes of cytokines IL-6, IL-2 and TGF-β1.
RESULTS Compared with the model group, Yangyin Huoxue Prescription could significantly reduce the plaque area and inhibit coronary stenosis (P < 0.01), reduce the levels of TG and LDL-C (P < 0.01), increase the level of HDL-C (P < 0.05), reduce the expression of Dnmt1 and Dnmt3a proteins in spleen (P < 0.05), reduce the expression of Dnmt1 mRNA and Dnmt3b mRNA in aorta (P < 0.001, P < 0.05) and the expression of Dnmt1 and Dnmt3b proteins in aorta (P < 0.05). The level of serum IL-6 was down-regulated (P < 0.05) and TGF-β1 was up-regulated (P < 0.05). Compared with the model group, each efficacy unit group reduced plaque area and regulated blood lipid levels; the Yangyin group decreased the expression of Dnmt3b mRNA in spleen (P < 0.05), reduced the expression of Dnmt3b mRNA (P < 0.001) and Dnmt3a protein in aorta (P < 0.05), and increased TGF-β1 (P < 0.05). The Qingre group decreased the expression of Dnmt1 protein in spleen (P < 0.05), Dnmt1 and Dnmt3b mRNA in aorta (P < 0.05) and the expression of Dnmt1, Dnmt3a and Dnmt3b proteins (P < 0.05), reduced the level of IL-6 (P < 0.05) and increased the level of TGF-β1 (P < 0.05). In the Huoxue group, the expression of Dnmt3a and Dnmt3b protein in aorta was decreased (P < 0.05), and the level of IL-6 was decreased (P < 0.05).
CONCLUSION Yangyin Huoxue Prescription can effectively reduce the formation of AS plaques in ApoE-/-mice induced by high fat combined with LPS injection by regulating blood lipids, reducing the expression of DNA methylase Dnmts and improving the level of inflammation.
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