The Study On The Estrogenic Effect And Mechanism Of Incompatibility Of Veratrum Nigrum And Salvia Miltiorrhiza Bunge

ObjectiveTo evaluate the estrogen-like effect of Salvia miltiorrhiza bunge and its mechanism,and to evaluate the antagonistic effects on estrogen-like effect of Salvia miltiorrhiza bunge after treated with Veratrum nigrum,so as to provide a scientific basis for eighteen incompatible medicaments Veratrum nigrum and Salvia miltiorrhiza bunge according to theories of traditional Chinese medicine.MethodsEstrogen-like effects and the mechanism were studied using classical animal models,including immature mice model and ovariectomized(OVX)mice model.The estrogen-like effect was detected by observing estrous cycle,body weight gain,uterine weight,hormone levels in circulation,measuring the histologic structure changes and estrogen receptors(ER? and ER?)expression in reproductive target tissues.1.Immature mice modelTotally 100 21-day-old healthy Kunming female mice(12 ± 2)g were randomly divided into 10 groups:control group(Con),estradiol valerate(E2,0.1g/kg),E2 plus estrogen receptor antagonist(ICI,0.005g/kg),E2 plus Veratrum nigrum(VN,0.045g/kg),Salvia miltiorrhiza bunge(SM,1.6,3.2 g/kg),SM plus ICI,SM plus VN for 7 days respectively,with ten mice in each group.All mice were monitored by daily vaginal epithelium cells smear testing during the 7 days administration period.Blood was collected from the eye venous plexus and the serum was analyzed for estradiol(E2),luteinizing hormone(LH)and follicle-stimulating hormone(FSH)by enzymelinked immunosorbent assay(ELISA).The uterus and vagina were removed and weighed.The left horns of the uterus and the upper portion of vagina were stored at-80?.The right horns of the uterus and the under portion of vagina were fixed with 4%polyoxymethylene for 24 h.After that then,HE method was used to determine tissue pathological changes of uterus and vagina;the protein expression level of ER? and ER? in uterus and vagina was detected by the immunohistochemistry and Western blot technique;the mRNA expression level of ER? and ER? was detected by Realtime PCR.2.Ovariectomized mice modelTotally 110 four-week-old healthy Kunming female mice maintained normal 5-days estrous cycles as confirmed by daily vaginal epithelium cell smear testing until the ovariectomy was performed.In sham-operated negative controls,fat near the ovary was removed.The mice were randomly divided into eleven groups:sham operated(Sham),ovariectomized without treatment(OVX),estradiol valerate(E2,0.1g/kg),E2 plus estrogen receptor antagonist(ICI,0.005g/kg),E2 plus Veratrum nigrum(VN,0.045g/kg),Salvia miltiorrhiza bunge(SM,1.6,3.2 g/kg),SM plus ICI,SM plus VN for 4 weeks.Blood was collected from the eye venous plexus and the serum was analyzed for estradiol(E2),luteinizing hormone(LH)and follicle-stimulating hormone(FSH)by enzymelinked immunosorbent assay(ELISA).The uterus,vagina and adrenal gland were removed and weighed.The left horns of the uterus and the upper portion of vagina were stored at-80?.The right horns of the uterus and the under portion of vagina were fixed with 4%polyoxymethylene for 24 h.After that then,HE method was used to determine tissue pathological changes of uterus and vagina;the protein expression level of ER? and ER? in uterus and vagina was detected by the immunohistochemistry and Western blot technique;the mRNA expression level of ER? and ER? was detected by Realtime PCR.Results1.Estrogenic activity of Salvia miltiorrhiza bungeThe results showed treatment with SM at the dose of 1.6 and 3.2 g/kg resulted significant estrogenic activity on reproductive target tissues in immature mice and ovariectomized mice.Treatment with SM promoted sexual maturation in immature mice,and it reversed the atrophy caused by ovariectomy and restored morphology of reproductive target tissues;as indicated by prolonging estrous stage,increasing uterine weight,thickening the uterine endometrium and vaginal epithelial layers.In addition,SM could relieve the symptoms of body weight gain induced by estrogen decline in ovariectomized mice.2.The mechanism of estrogenic activity of Salvia miltiorrhiza bungeThe results show that these estrogenic effects may be mediated by stimulating the biosynthesis of estrogen in circulation and increasing the quantity of ERs in the target organs.Treatment with SM significantly raised the levels of circulating E2,and significantly down-regulate the levels of LH and FSH;SM induced significant up-regulations of ER? and ER ? on protein and mRNA levels in target tissues.3.The Antagonistic effects of Veratrum nigrum on the estrogenic activity of Salvia miltiorrhiza bungeVeratrum nigrum at the dose of 0.045 g/kg antagnized the estrogenic efficacy of SM on the growth and development of uterus and vagina in immature mice and reversing the atrophy of uterus and vagina in OVX mice as indicated by obvious degeneration of the cavities,endometrium and secretory glands in uterus,less cell layers being present in vagina.4.The mechanism of the Antagonistic effects of Veratrum nigrumVeratrum nigrum decreased the increase of the serum estradiol and the up-regulation of ER? and ER? expressions in the reproductive tissues in immature mice and OVX mice by treatment with SM.This activity is similar to estrogen receptor antagonist ICI 182,780.ConclusionThese findings provide that SM exerts estrogenic effect,and Veratrum nigrum can antagonize the estrogenic efficacy of SM by decreasing the increase of the serum estradiol and the up-regulation of ER? and ER?expressions by treatment with SM.Provide a scientific evidances for eighteen incompatible medicaments Veratrum nigrum and Salvia miltiorrhiza bunge according to theories of traditional Chinese medicine.