The Study Of Estrogen-like Effects Of QiBiaoMeiRan Formula And Mechanism Action

Objective:To evaluate the potential estrogen-like effects of QiBiaoMeiRan(QBMR)formula,and investigate the mechanism of its estrogen receptors(ERs).Method:Estrogen-like effects and the receptor mechanism of QBMR formula were studied through the in-vitro assays binding with the experiments in immature mice and ovariectomized(OVX)rats.Immature mice model:In the study,50 healthy female mice at 21 days of age were fed adaptively two days.And then they were randomly divided into three groups,including the medicated groups respectively treated with QBMR at doses of 0.113,0.056,and 0.028 g/ml per day for 6 weeks,the normal control treated with isovolumetric distilled water,and positive control group(0.14mg/ml EV).After that then,HE method was used to determine tissue pathological changes of the immature rats;the protein expression level of ERaand ERβin mammary gland,uterus and vagina was detected by the immunohistochemistry and Western blot technique.OVX rats model:60 healthy mature female rats were randomly divided into OVX group,Sham group,Estradiol Valerate(EV)group,the QBMR groups at doses of 0.35g/kg,0.18g/kg and 0.088g/kg.In addition to the Sham group removed the fat nearly the ovary,the others were treated by bilateral oophorectomy after anesthetized by 10%chloral hydrate(0.3ml/100g).After the wound healing fully,dioestrum lasted for five days suggesting that the castration was success through observing the vaginal epithelial cells stained by methylene blue.For 8 weeks,the OVX group and Sham group was administrated with the isovolumetric distilled water;the QBMR groups was treated at corresponding dose;the positive group was treated at dose of O.llmg/kg.After that then,HE method was used to determine the tissue pathological changes of OVX rats;the protein expression level of ERaand ERβ in mammary gland,uterus and vagina was detected by the immunohistochemistry and Western blot technique.As the preceding method to administrate drugs,the weight and rectal temperature were measured per day in the OVX rats,for 8 weeks.The radio immunoassay was used to determine the contents of bone gla-containing protein(BGP),calcium(Ca),phosphorus(P).Alkaline phosphatase(ALP).And the content of procollagen I in the OVX rats was analyzed by enzyme linked immunosorbent assays.HE and TRAP method was used to determine the bone pathological changes.The dual energy x-ray absorptiometry was used to determine the bone mineral density.The Micro-CT was used to detect the bone histomorphometry,and analyze bone volume fraction(BV/TV),bone trabecular thickness(Tb/Th),bone trabecular number(Tb.N),trabecular bone isolation(Tb.Sp)and bone mineral density(BMD).In vitro,the proliferative effect of MCF-7 cells,the positive cell of estragon receptor,resulting from treating with the QBMR formula was determined by MMT 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 tiypsin enzyme(0.25%),and cultured in in DMEM without phenol red.Then,they were inoculated to the 96-well plate at 200μl per well.After 24 hours,water extract,95%ethanol extract,70%ethanol extract and ethyl acetate extract of QBMR were respectively added into 0.0001mg/ml,0.001 mg/ml,0.01 mg/ml,0.1 mg/ml.The 17(3-estradiol was also added into the plate at the dose of 10-8M,10-9 M as well as the ICI 182,780 at the dose of 0.1 μM.48h after culturing,5%MTT was add into every well at 10μL.After 4h incubation,nutrient solution was absorbed and the DMSO was added per well at 150 μl At 490nm,MTT was used to measure the absorption value of the wells,and calculated the average absorbance value and proliferation rate.Results:1.Effects on immature miceFor immature mice treated by QBMR,the cornification of vaginal cells was presented and estrum arrived early.The uterine organ coefficient increased,E2 level in plasma was elevated and contents of LH become lower as well as FSH.Histopathological observation result showed that the QBMR could promote uterine development of the immature mice,largen the lumen,thicken endometrium,loosen lamina propria,increase the glands number obviously,secretory phase arrive early.In the immature mice,QBMR could also cause the vaginal epithelium to present proliferation and multilayer squamous cell,and the surface height and Cornification index appeared to increase.Immunohistochemistry and western blot results indicated that QBMR could obviously elevated the expression level of ERα and ERβ in uterus and vagina,compared with normal control group,and enhance the ERs expression in uterine cavity epithelium,glandular epithelium and endometrial stroma,vaginal squamous epithelium,cuticulated epithelium,mammary gland epithelial.2.Effects on OVX ratsThe uterus in OVX rats became smaller and the uterus cavity narrowed as well.Endometrium became thinner and the number of glands decreased significantly.But 8 weeks after treatment of QBMR,these symptoms improved obviously.As well as the vagina,compared with OVX rats,in rats of administrated with QBMR,the vaginal epithelium presented proliferation and multilayer squamous cell,and the surface height and cornification index appeared to increase significantly.8 weeks after the treatment with QBMR,compared with mammary gland atrophy in OVX rats,the duct cavity of mammary glands was expanded,the epithelium thickness also increased significantly and the secreta was found around them.In OVX rats,the uterus and adrenal gland index,and serum E2 level sharply decreased as well as the level of LH and FSH.By contrast.8 weeks after continuously administrating QBMR.the uterus and adrenal gland index increased significantly as well as the E2 levels,the LH and FSH levels still decreased.Immunohistochemistry results indicated that the expression of ERaand ERβin target organs of ovariectomized rats weakened,but the expression levels were enhanced apparently after QBMR interfering.Furthermore,the western blot and real-time results also indicated the expression of ERaand ER(3 was elevated significantly.In Reproductive target organs,the expression of ERa protein and mRNA were all stronger then ERP,suggesting that proliferation and differentiation of target organ cells might be mediated by ERa,and the estrogen-like effect could be achieved by up-regulate ERβ/ERα.3.In vitro testThe classical E-screen method was used to screen the activity of water extract,95%ethanol extract,70%ethanol extract and ethyl acetate extract of QBMR.And the activity of 70%ethanol extract was confirmed as the strongest by MTT experiment.70%ethanol extract could be contribute to MCF-7 cells prominently proliferate,and be suppressed by estrogen receptor antagonist,further suggested that QBMR might produce phytoestrogen-like effect through the pathway of estrogen receptor.4.Effects on perimenopausal syndrome of OVX ratsBefore administration,the average weight of all groups is similar.However,8 weeks after administration,the weight of rats in sham group significantly increased.3 weeks after administrating QBMR,the rats’ weight was suppressed,and there was negative correlation between dose and weight.The rectal temperature was also reduced suggesting that the QBMR could relief the menopausal hot flashes.Biochemical markers of bone metabolism showed that QBMR groups appeared to up-regulate the concentration of Ca+ and P+,mediate bone metabolism,increase the ALP and BGP levels in serum,and decrease CTX-I value.The QBMR could prompt bone formation and suppress bone resorption.The dual energy x-ray absorptiometry results suggested that QBMR might elevate the BMD value of lumbar.In Micro-CT test,the QBMR middle and low dose group showed that trabecular bone become denser,compared with the OVX group,and the continuity,porosity and microstructure is improved significantly.The values of BV/TV,Tb.Th,Tb.N,Tb.Sp,BMD of thighbone was elevated obviously.The statistical result of the number of osteoclast with TRAP staining indicated that the osteoclast of the QBMR middle and low dose group decreased sharply compared with OVX group.Compared with Sham group,histopathology found that bone tissue significantly degraded,in OVX rats,trabecular bone and bone cortical thinned,structural form was poor,the distortion and rupture appeared,fat vacuoles in the bone marrow cavity increased,suggesting that it was typical osteoporosis changes.After treatment with QBMR,trabecular bone did not thin,its arrangement was neat and interconnected into a network,the number of trabecular bone increased and trabecular space became big in local region.Conclusion:QBMR formula could promote the target organs of immature mice mature,antagonize the estrogen level reduction and the atrophy of uterus,vagina and mammal glands after ovariectomy,obviously up-regulate the expression of ERs protein and genes of the uterus and vagina of ovariectomized mice and the reproductive target organs of ovariectomized rats.And there was the correlation with dose.Expression of ERs of MCF-7 cells,the estrogen receptor-positive,was up-regulated,the effect antagonizing specifically ICI182780,the estrogen receptor antagonist,suggested that QBMR estrogen-like effect were mediated by receptor pathway.The ovariectomized rats’ weight significantly increased as well as rectal temperature rise.While OVX rats were treated with QBMR,their weight and rectal temperature dropped significantly suggested that QBMR could alleviate perimenopausal symptoms of hot flashes and obesity.QBMR formula also could improve bone microstructure of OVX rats,increase the trabecular bone density and thickness and the lumbar vertebrae bone density,reduce bone mineralization degree and the number of osteoclasts,and regulate the balance of bone resorption and bone formation.