| Objective:Applied high-throughput molecular biology technology, from a macroscopic point of view of whole genome, explored molecular mechanism of nourishing Yin benefiting Qi and transforming stasis method blocking the "metabolic memory" of spontaneously diabetic GK rats, to expand the prevention and treatment of diabetic vascular disease, and reveal the theoretical advantages of the holistic concept of traditional Chinese medicine.Methods:50 GK rats were randomly divided into 5 groups:model group(M), glucophage group (G), Shen-Qi Compound high dose group (SH), Shen-Qi Compound compound middle dose group (SM), Shen-Qi Compound low dose group (SL). The diabetic metabolic memory model was established for 12 consecutive weeks with high fat diet. Another 10 normal Wistar rats were set as the blank group (C), which were fed with normal diet, parallel control.12 weeks after modeling, SH, SM and SL group were treated with high, middle and low dose Shen-Qi Compound extract gavage, group G treated with metformin suspension gavage, groups C and M were treated with saline. Animal body mass, water intake, food intake and fasting blood glucose (FPG) were monitored every week during the drug intervention. After 16 weeks of intervention, all groups were executed. Masson and HE staining were used to observe the pathological changes of aorta in each group. TUNEL staining were used to observe the apoptosis of endothelial cells in blood vessels; Carried out the detections of aortic tissue homogenate SOD activity, MDA content, GSH-Px activity; serum 8-iso-PGF2a, ox-LDL concentration, the aortic tissue Affymetrix IVT expression profile chip and DNA methylation immune coprecipitation sequencing, the differentially expressed genes were enriched with GO and Passway, the biological significance of the detection results was further analyzed.Results:After 16 weeks of treatment, group M compared with group C, body weight decreased, water intake increased, food-intake had no significant difference, FPG increased significantly. Water-intake, food-intake, FPG declined in group SH, SM and SL, compared with group M. The water-intake were less than that of group G, and food intake, FPG were more than that of group G. In group SH, SM and SL body weight increased compared with group M. Collagen fiber of aorta aortic pathological changing degree:M>SL>G>SM>SH>C. The thickness of aortic membrane in G, SH, SM and SL was significantly higher than that of group M, collagen fiber and lumen area ratio were significantly less than group M, group SH had the lowest proportion. Vascular endothelial cell apoptosis degree:M>SL>SM>G>SH>C. In group G, SH, SM and SL, the aortic film thickness was significantly higher than that of group M, collagen fibers and lumen area ratio were significantly lower than that in group M, the lowest proportion of collagen fibers in group SH. Vascular endothelial cell apoptosis degree:M> SL>SM>G>SH>C; Group G, SM and SL, the aortic homogenate MDA content was significantly lower than that of group M, group SM was the lowest. In SOD, aortic GSH-Px activity and serum 8-iso-PGF2a concentration was no significant difference between groups.Through whole genome expression profiles and the DNA methylation detection, group M and group C compared gene expression and DNA methylation level are widely different, main trend is widespread DNA methylation level increased and mRNA expression decreased. The degree of methylation of G, SH, SM, SL were significantly lower than that in group M. In group G, SH, SM, mRNA expression significantly higher than that in M group, the SH group was significantly higher than that of G group. The Passway screened out from KEGG analysis of differentially expressed genes between group SH and M and the Passway from group M and C were highly coincident. The number of Passway enriched in the two groups was more than that of SM and M group, the number of Passway enriched in group G and M was the least. DNA methylation data GO among groups and KEGG enrichment involved in gene function and Passway and mRNA levels control is basically the same, mainly related to hormone secretion, cardiovascular disease, immune regulation, cell migration, adhesion, proliferation and differentiation. Compared to the level in group M, in group SH and G the expressing levels of aortic tissue Map2k1 gene decreased significantly. Racl gene expression in SH group increased significantly, and the upstream regulatory gene Vav family expression also increased, and it was not found the difference between group G and M in Racl expression, Vav family expression in group G decreased compared with group M.Conclusions:Nourishing Yin benefiting Qi and transforming stasis method can effectively inhibit the diabetic "metabolic memory",thus delaying the occurrence and development of metabolic memory in a rat model of atherosclerosis, and the role of play depends on sufficient drug concentration. Nourishing Yin benefiting Qi and transforming stasis method may improve the "metabolic memory" by correcting the DNA hyper methylation status of animals, and extensively and accurately restore the expression level of mRNA, and thus play a role in blocking the "metabolic memory". By promoting the expression of Racl and inhibiting the expression of Map2k1, through the regulation of cell proliferation and adhesion function, nourishing Yin benefiting Qi and transforming stasis method may regulate the growth of vascular endothelial cells in a benign way, and are involved in blocking diabetes "metabolic memory". Nourishing Yin benefiting Qi and transforming stasis method showed better in blocking metabolic memory effect of diabetes, which may due to the advantage TCM theory of strengthening healthy Qi to eliminate pathogens. |
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