Danshen enhanced the estrogenic effects of Qing E formula in ovariectomized rats

Abstract

Background: Menopause is characterized by a decrease in life quality due to the appearance of uncomfortable symptoms. Nowadays, Understanding menopause-associated pathophysiology and developing new strategies to improve the treatment of menopausal-associated symptoms is an important issue. Our study was to evaluate the synergistic effects of Danshen (salvia miltiorrhiza bunge) and the phytoestrogenic effects of 3 modified Qing E formulas, to explore a better formula for menopausal disorders.

Methods: 100 rats were randomized into 5 groups: Sham (Sham operation group), OVX (model group of ovariectomized rat), BDL (group with low concentration of Qing E Formula), BDH (group with high concentration of Qing E Formula) and BDD (group with high concentration of Qing E Formula Plus Danshen), receiving vehicle and extract of different modified Qing E formula respectively. The food intake, body weight, uterus weight, blood levels of triglycerides (TG), total cholesterol (TC) and cholesterol fractions were assessed. The mammary glands and uterus were morphologically analyzed. The bone density of tibias were measured by peripheral quantitative computed tomography (pQCT). Additionally, luciferase induction assays were performed in Hela cells with the mixtures derived from Qing E formula plus Danshen (BDD).

Results: Qing E formula plus Danshen significantly increased the uterus wet weight, enhanced the thickness of uterine wall, endometrial epithelium and glandular epithelium, improved trabecular bone and total density evidently, reduced the levels of low density lipoprotein cholesterol (LDL-C) and TG, possessed notable estrogen receptor beta (ERβ) and estrogen receptor alpha (ERα) agonist activity.

Conclusion: Qing E formula plus Danshen exerted more evident estrogen-like effects, thus it has a potential therapeutic use to treat menopausal disorders.

Keywords: Danshen; ERα; ERβ; Estrogenic effects; Menopause; Qing E formulaplus Danshen; Rats.

Fig. 1

Effects on the morphology of mammary. The longitudinal section of the mammary in each group. After the administration of 3 modified Qing E formulas, the mammary glands showed different degrees of slighter atrophy in comparison with the OVX group

Fig. 2

Effects on the morphology of uterine tissue. A1-A5 showed the transverse section of the uterus in each group. The endometrium thickness of all the ovariectomized rats was significantly decreased compared with the sham group, but mildly increased by all the formulas. Redline - the endometrium. (100×). B1-B5 showed transverse section of the uterus in each group. The endometrium epithelium thickness of all the ovariectomized rats was significantly decreased compared with the sham group, But significantly increased by Qing E formula plus Danshen. Arrow- epithelium. (400×). C1-C5 showed transverse section of the uterus in each group. The uterine glandular epithelium thickness of all the ovariectomized rats was significantly decreased compared with the sham group, But significantly increased by all the formulas. Arrow-uterine gland. (400×)

Fig. 3

Effects on the BMD. a showed that the density of trabecular bone and total bone density were significantly improved by Qing E formula Plus Danshen at epiphyseal line down to 3.0 mm; b showed that there was no apparent difference shown at the epiphyseal line down to 12.0 mm. ^★ P < 0.05 vs. Sham group; ^▲▲ P < 0.01 vs. OVX group; ^# P < 0.05 vs. BDH group

Fig. 4

Tomographic determination of BMD scanned by pQCT (3.0 mm & 12.0 mm down to the epiphyseal line of proximal tibia). The scale bar of BMD is from 0 to over 1000 shown at the left with color variation from black, gray, red, blue,…to white. In Fig. 4 - a, there was evidently higher intensity component with red and blue signal values in the cancellous bone area in group BDD comparing to OVX, which was mostly in gray; however, the BMD of cortical bone didn’t show any notable difference in fig. 4-b.

Fig. 5

Effects on the expression of estrogenic activities. Transcription of ERα and ERβ activated by the mixture of BDD in Hela cells. Hela cells were transiently cotransfected with pERE-luc, pRL-TK, and ERα or ERβ by lipofection. The cells received no treatment, treatment with 10 nM E₂, or treatment with varying mass concentrations of each of the mixture alone. Estrogen-stimulated luciferase activity of ERs was measured and the data was presented as the fold-increase over control + standard deviation, and fold-increase representative of three experiments done in triplicate. ** P < 0.01 vs. control; ^◆◆ P < 0.01 vs.E₂