Gene expression profiling on the molecular action of danshen-gegen formula in a randomized placebo-controlled trial of postmenopausal women with hypercholesterolemia

Abstract

The Danshen-Gegen formula (DG) is a traditional Chinese herbal formula which has long been used to treat cardiovascular disease. DG was found to be a cardiovascular tonic in our recent research. However, a comprehensive investigation of the molecular mechanism of DG in cardiovascular disease has not been performed. The aim of this study was to clarify the transcriptional profiling of genes modulated by DG on postmenopausal women by using DNAmicroarray technology. We obtained 29 whole blood samples both from DG-treated and placebo-treated subjects. Blood lipid profile and intima-media thickness (IMT) were measured. Affymetrix GeneChip was used to identify differentially expressed genes (DEGs), followed by validation by the real-time PCR method. The results showed that DG-treated group has a significant improvement in IMT and lipid profile as compared to placebo-treated group. For the genomic study, the DG-treated group has a higher number of DEGs identified as compared to the placebo-treated group. Two important biological processes of "regulation of systemic arterial blood pressure by hormone" and "regulation of smooth muscle proliferation" have been identified by GePS in the DG-treated group. No significant biological process and cellular components were identified in the placebo-treated group. This genomic study on the molecular action of DG in postmenopausal women gathered sufficient molecular targets and pathways to reveal that DG could improve neointima thickening and hypertension.

Figure 1

(a) Percentage changes from baseline in IMT at 6-month and 12-month DG or placebo treatment, *P < 0.05; (b) IMT of subjects at baseline, 6-month, and 12-month DG or placebo treatment (in mm).

Figure 2

(a) TC, LDL, HDL, and TG in mmol/L at 12 months compared with baseline, **P < 0.01; (b) percentage change in blood lipid profile (TC, LDL, HDL, and TG) of subjects after 12-month DG or placebo treatment.

Figure 3

Venn diagram of differential expressed genes (DEGs) in the DG-treated group and the placebo-treated group. Nonoverlapped area is the number of DEGs found only in corresponding group. Overlapped area is the number of common DEGs shared between the DG-treated group (n = 3) and the placebo group (n = 3).

Figure 4

PCA analysis of Pre and Post samples of the DG-treated group and the placebo-treated group. Each Pre and Post sample of each group was repeated in three individual hybridizations. The cluster of three red balls shows the intraexperimental variation of each sample.

Figure 5

Cluster analyses of Pre and Post samples in three individual subjects of the DG-treated group and the placebo-treated group. The overlapped area of the clusters (red and blue) indicates the greater similarity of the gene expression profile.

Figure 6

A pathway of “regulation of systemic blood pressure by hormone” was generated by the GePS program from Genomatix that connects the DEGs found after DG treatment for 12 months.

Figure 7

A pathway of “regulation of smooth muscle cell proliferation” was generated by the GePS program from Genomatix that connects the DEGs found after DG treatment for 12 months.

Figure 8

Gene expression level of the 11 DEGs after DG and placebo treatment. The RNA from the remaining subjects in the treatment group (n = 18) and the placebo group (n = 11) was used. The y-axis represents fold change (posttreatment versus pretreatment). The bar on the column stands for the mean ± S.E.M., *P < 0.05 as compared to the placebo group.

Figure 9

qPCR analyses of the nine co-citation genes after DG and placebo treatment. The RNA from the remaining subjects in the treatment group (n = 18) and the placebo group (n = 11) was used. The y-axis represents fold change (posttreatment versus pretreatment). The bar on the column stands for the mean ± S.E.M., *P < 0.05 as compared to the placebo group.

Figure 10

Integration of DEGs in two biological processes (blood pressure and smooth muscle proliferation) by using GePS. Two core DEGs (NOS and EDN1) are identified by highlighting in orange color.