Effect of the Interaction of Veratrum Nigrum with Panax Ginseng on Estrogenic Activity In Vivo and In Vitro

Panax ginseng (GS) and Veratrum nigrum (VN) are representative of incompatible pairs in “eighteen antagonistic medicaments” that have been recorded in the Chinese medicinal literature for over 2,000 years. However, evidence linking interference effects with combination use is scare. Based on the estrogen-like effect of GS described in our previous studies, we undertake a characterization of the interaction on estrogenic activity of GS and VN using in vivo models of immature and ovariectomized (OVX) mice and in vitro studies with MCF-7 cells for further mechanism. VN decreased the estrogenic efficacy of GS on promoting the development of the uterus and vagina in immature mice, and reversing the atrophy of reproductive tissues in OVX mice. VN interfered with the estrogenic efficacy of GS by decreasing the increase of the serum estradiol and the up-regulation of ERα and ERβ expressions by treatment with GS. And VN antagonized the estrogenic efficacy of GS on promoting the viability of MCF-7 cells and up-regulation of protein and gene expressions of ERs. In conclusion, this study provided evidence that GS and VN decreased effects on estrogenic activity, which might be related to regulation of estrogen secretion and ERs.


2
Uterine weight was significantly decreased in immature mice treated with ICI, 182780 (P < 0.001). Treatment of immature mice with VN had modest inhibited effects on uterine weight, the medium dose 0.045 g/kg treatment resulted significant differences compared with untreated controls (P <0.05). Additional, treatment of immature mice with VN at 0.045 g/kg or with ICI, significantly decreased circulating E 2 level compared to those of untreated immature mice (P < 0.010 or 0.001) (Fig. S1), suggesting that VN at 0.045 g/kg has significant anti-estrogenic activity. These data prompted further studies to elucidate the activity and mechanism of VN at tat 0.045 g/kg Fig.S1 The effects of Veratrum nigrum (VN) on uterine weight and serum E 2. The uterine weights and serum E 2 of immature mice were measured at the end of the 7-day treatment period.. Data are the mean and standard deviation (SD) of samples from 10 mice. P values are for the one-way analysis of variance (ANOVA) comparing the treatment group with untreated mice. (***) P <0.001, (**) P < 0.01, and (*) P < 0.05 compared with the Con.

Effect of VN on histopathology of uterus and vagina
Histological analysis of uterine sections revealed treatment of immature mice with ICI or VN 0.045 g/kg substantially changed uterine morphology (Fig.S2), as indicated by thin of the uterine endometrium, decreased number of glands, and narrowed glandular cavities compared with untreated controls. Fig.S2 shows microscopic preparations of representative vagina from one animal per treatment 3 group, compared with untreated immature mice, treatment with ICI or VN 0.045 g/kg decreased epithelial thickness and also the number of cell layers. Taken together, these studies provide evidences that VN has anti-estrogenic activity, comparable to that of classical estrogen receptor antagonist ICI, 182780.

Effect of VN on ERs expression by immunohistochemistry in uterus and vagina
The expressions of ER and ER in the uterus and vagina from each group and quantitative analysis are shown in Fig

VN inhibited MCF-7 cell viability
To investigate the molecular basis of VN activity in more detail, we used MCF-7 human breast cancer cells as a model because it is estrogen receptor positive cell.

Conclusion:
Our findings suggest that VN had anti-estrogenic effect on reproductive tissues in immature mice by inhibited biosynthesis of estrogen in circulation, down-regulating ERs and maybe through ER-ERE-dependent pathway.

Transfection and reporter assay of estrogen receptor-subtype selectivity
HEK 293 cells were stably transfected with human estrogen receptor α /β (hER α /β ) and the estrogen response element (ERE) plasmid (kindly provided by Professor Yung-Chi Cheng, Yale University), and the luciferase reporter assay system from Promega (WI, USA) was used to evaluate the formation of functional ER α /β -ERE 8 complexes. The cells were maintained and primed to minimize the effects of endogenous estrogens as described above and then seeded (1 × 10 5 cells/100 μ L/well) in 96-well plates. The test samples with or without 17β -estradiol were added to three replicate wells, as described for the MTT assay of MCF-7 cell proliferation, and was incubated for 24 h. Finally, the growth medium was carefully removed and 50 μ L of lysis buffer per well was added, and the plate was rocked for 15 min. Twenty microliters of the detached cell solution was then transferred to a white micro well plate. Luciferase assay reagent (50 μ L) was added to each well, and luciferase activity was measured immediately. Activity of the luciferase reporter gene was expressed relative to the DMSO control. Results reported are the mean ± standard deviation of three replicate determinations from a representative assay.