The Study On The Estrogenic Effect And Mechanism Of Incompatibility Of Veratrum Nigrum And Panax Ginseng

ObjectiveWith the progress of human society and the growing awareness of drug safety,the essence of incompatibility already attracted broad attention in the world in recent years.Incompatibility of traditional Chinese medicine,is ancient and has been unable to clear proposition,Eighteen against incompatibility is an important part of traditional Chinese medicine,the staff who engaged in medicine no wonder.A representative of drug pairs,Panax ginseng and Veratrum nigrum,is made up of emetic medicine and tonic medicine.The perspective of traditional Chinese medicine considers Veratrum nigrum as an emetic,which contains various steroid ingredients,can strongly irritate the gastric mucosa.Panax ginseng as a tonic medicine,is unsuitable used with the emetic.The aim of our study is to evaluate the estrogen-like effect of Panax ginseng and its mechanism,and to evaluate the antagonistic effects on estrogen-like effect of Panax ginseng after treated with Veratrum nigrum.MethodsEstrogen-like effects and the mechanism were studied using classical animal models in vivo,including immature mice model and ovariectomized(OVX)mice model.To evaluate the estrogenic efficacy 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.In vitro,the proliferative effect and estrogen receptors expression of MCF-7 cells were measured.1.Immature mice model21-day-old female immature mice(12 ± 2)g were randomly divided into eleven groups by random number table method:control group(Con),treatment with 0.154 mg/kg estradiol valerate(EV)and treatment with GS intragastrically at a daily dose of 0.5,1.0,1.5,3.0,6.0,12.0,18.0,24.0 or 30.0 g/kg(n=10 in per group)for 7 days.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 a and ER? in uterus and vagina was detected by the immunohistochemistry and Western blot technique.2.Ovariectomized mice model.Four-week-old female Kunming(KM)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 assigned to several groups:ovariectomized without treatment(OVX),sham operated(Sham),treatment with 0.154 mg/kg estradiol valerate(EV),treatment with 0.045 g/kg Veratrum nigrum(VT),treatment with estradiol valerate and Veratrum nigrum(EV + VT),treatment with estradiol valerate and ICI 182,780(EV + ICI),treatment with Panax ginseng(GS)at a daily dose of 12.0 g/kg,18.0 g/kg or 24.0 g/kg per day,treatment with Panax ginseng and Veratrum nigrum(GS + VT),treatment with Panax ginseng and ICI 182,780(GS + ICI)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 a and ER ? was detected by Realtime PCR.3.MCF-7 cell modelIn vitro,the proliferative effect of MCF-7 cells,the positive cell of estragon receptor,was determined by MTT method.Three days before testing,the cells were cultured in DMEM without phenol red for purpose of depleting the intracellular estragon.The cells in logarithmic phase were digested by trypsin enzyme(0.05%),and cultured in DMEM without phenol red.Then,they were inoculated to the 96-well plate at 200?L per well.After 24 hours,water extract of Panax ginseng(GS)and Veratrum nigrum(VT)were respectively added into 0.0001 mg/mL,0.001 mg/mL,0.41 mg/mL,0.1 mg/mL.The 17?-estradiol was also added into the plate at the dose of 10-9 M as well as the ICI 182,780 at the dose of 0.1 ?M.48h after culturing,5%MTT was add into every well at lO?L.After 4h incubation,nutrient solution was absorbed and the DMSO was added per well at 150 ?L?At 490nm,MTT was used to measure the absorption value of the wells,and calculated the average absorbance value and proliferation rate.MCF-7 cell were cultured as described above and seeded in 10cm dishes,then cells were treated with water extract of Panax ginseng(GS)and Veratrum nigrum(VT).After cultured for 48h,cells were collected to extract cellular proteins.The protein expression level of ER? and ER? in MCF-7 cell was detected by Western blot technique.Results1.Estrogenic activity of Panax ginsengIn vivo,we comprehensively characterize the estrogenic effects of Panax ginseng on reproductive target tissues in immature mice and ovariectomized mice.The results showed treatment with Panax ginseng at the dose of 12.0,18.0 and 24.0 g/kg resulted significant estrogenic activity.Treatment with Panax ginseng 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,Panax ginseng could relieve the symptoms of body weight gain induced by estrogen decline.In vitro,Panax ginseng promoted the proliferation of MCF-7 cells,and this activity can be suppressed by estrogen receptor antagonist ICI 182,780.2.The mechanism of estrogenic activity of Panax ginsengIn vivo,the 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 Panax ginseng significantly raised the levels of circulating E2,and significantly down-regulate the levels of LH and FSH.There was a trend of this decreasing with an increasing dose of ginseng;Panax ginseng induced significant up-regulations of ER? and ER? on protein and mRNA levels in target tissues.It is worth mentioning that ER? upregulation induced by ginseng extract was stronger than that of ER?,suggesting that Panax ginseng may bind to ER?with higher selectivity than ER? in reproductive target tissues.In vitro,Panax ginseng induced significant up-regulations of ER? and ER? on protein levels in MCF-7 cell.This activity could be suppressed by estrogen receptor antagonist ICI 182,780,further suggested that it might produce estrogen-like effect through the pathway of estrogen receptor.3.The Antagonistic effects of Veratrum nigrum on the estrogenic activity of Panax ginsengAs was expected,Veratrum nigrum at the dose of 0.045 g/kg antagnized the estrogenic efficacy of Panax ginseng on 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.In vitro,Veratrum nigrum(0.0001 mg/mL?0.01 mg/mL)antagnized the estrogenic efficacy of Panax ginseng(0.001 mg/mL?0.1 mg/mL)on promoting the proliferation of MCF-7 cells,and this effection is similar to estrogen receptor antagonist ICI 182,780.Moreover,the antagonistic effect of co-fried water extract was better than combination with single-fried water extract.The antagonistic effect was most significant when the compatibility proportion between Panax ginseng and Veratrum nigrum was 10:1.4.The mechanism of the Antagonistic effects of Veratrum nigrumIn vivo,Veratrum nigrum decreased the increase of the serum estradiol and the up-regulation of ER? and ER? expressions in the reproductive tissues in OVX mice by treatment with Panax ginseng.In vitro,Veratrum nigrum decreased the up-regulation of ER? and ER? expressions in the MCF-7 cell by treatment with Panax ginseng.This activity is similar to estrogen receptor antagonist ICI 182,780.ConclusionThese findings provide systematic evidences that Panax ginseng exert estrogenic effects.And Veratrum nigrum antagnized the estrogenic efficacy of Panax ginseng by decreasing the increase of the serum estradiol and the up-regulation of ER? and ER? expressions by treatment with Panax ginseng.Provide a scientific evidances for eighteen incompatible medicaments according to theories of traditional Chinese medicine.