The Experiment Of GENGNIANGANLUYIN Plusing Low-dose Estrogen Treats Postmenopausal Osteoporosis

Postmenopausal osteoporosis(PMO)is a frequently encountered disease of which incidence may get to 25%in post-menopause female,and its treatment of choice is hormone replacement therapy(HRT)because of its close correlation with estrogen.Lots facts certificated that HRT can relieve PMO effectively.However,long-term use of HRT may contribute to some of the risks such as endometrial carcinoma,breast carcinoma,irregular vagina bleeding,and weight gain and so on,which limits its application.Other osteoporotic pharmacological interventions including selective estrogen receptor modulators,phytoestrogen,calcium and vitamin D,diphosphonate, parathyrin,calcitionin can not reveal satisfactory effects due to their drug action and adverse reaction.Chinese drugs pharmaceutics,with mild drug property and little side effects,is suitable for long-term use.Researchers have exploring the application of traditional Chinese medicine in PMO using modern medicine approaches since recent years,yet,the mechanisms involved are still not clear.Some results indicated that their anti-PMO correlate with phytoestrogen.Current researches about Chinese drugs pharmaceutics are mainly limited in bone histomorphology and metrology and so on,yet,are short of gene and molecular biology level mechanism of bone metabolism adjustment. In this study,the complex prescription,GENGNIANGANLUYIN (consisted mainly of several phyto-estrogenlike traditional Chinese medicine), was investigated to observe its effects on bone metabolism related gene of osteoblasts from estrogen receptor(ER)hypotype basing on the Chinese medical science theory and modern pharmacology.The purpose is to research the effects mechanism of menopause-manna-drink on bone metabolism from molecular level.Moreover,in order to approach a optimum treatment, GENGNIANGANLUYIN combining low dose-estrogen was used to treat PMO in vivo and vitro study.1.The effects of GENGNIANGANLUYIN on osteoblastObjective:To observe the effects of GENGNIANGANLUYIN on bone metabolism related gene of osteoblasts from estrogen receptor(ER)hypotype aspect and approach the effects mechanism from molecular level.Method:ovariectomized rat serum containing GENGNIANGANLUYIN was prepared and added to osteoblast that had treat by ERαsiRNA, ERβsiRNA,random RNA and liposome respectively;ovariectomized rat serum without GENGNIANGANLUYIN was added to osteoblast as control. RT-PCR detected ERα,ERβ,transforming growth factor-β(TGF-β),bone morphogenetic protein-2(BMP-2),osteoprotegerin(OPG)mRNA expression; Westen-blot analyzed ERα,ERβprotein expression;Elisa evaluated TGF-β, BMP-2,ALP,Col-â… ,OPG protein contents in osteoblast supernatant.Results:ERαsiRNA and ERβsiRNA could silence ERαand ERβexpression and random RNA and liposome had no effects on ERαand ERβexpression.Serum containing GENGNIANGANLUYIN had no effects on ERαand ERβexpression,yet,it up-regulated TGF-β,BMP-2 mRNA expression and TGF-β,BMP-2,ALP,Col-â… ,OPG protein contents in osteoblast supernatant that can be silenced by ERβsiRNA but not ERαsiRNA. Random RNA and liposome had no obviously up-regulation on these factors. Serum containing GENGNIANGANLUYIN had no obviously up-regulation on OPG;ERαand ERβsilencing had no effects on OPG too.Conclusion:GENGNIANGANLUYIN can not up-regulate ERαand ERβexpression;it can promote TGF-β,BMP-2,ALP,Col-â… expression through ERβ;it had no obviously up-regulation on OPG.2.The effects of GENGNIANGANLUYIN plusing low dose estrogen on osteoblastsObjective:To observe the effects of GENGNIANGANLUYIN combining low dose estrogen on ER and bone metabolism related gene of osteoblasts and approach the possibility of integrated traditional and western medicine treating PMO from molecular level.Method:osteoblast was treated by Serum containing GENGNIANGANLUYIN combining 1/16,1/8,1/4,1/2 optimal dose estrogen,the cell treated by ovariectomized rat serum without GENGNIANGANLUYIN,Serum containing GENGNIANGANLUYIN,and optimal dose estrogen respectively as control.MTT observed osteoblast proliferation;alkaline phosphatase(ALP)kit measured ALP activity; RT-PCR detected ERα,ERβ,TGF-β,BMP-2,OPG mRNA expression;Elisa evaluated TGF-β,BMP-2,ALP,Col-â… ,OPG protein contents in osteoblast supernatant.Results:Serum containing GENGNIANGANLUYIN can promote not only osteoblast proliferation and ALP activity,but also TGF-β,BMP-2,ALP, Col-â… expression.Furthermore,combined low dose estrogen,it can get more obviously effects,which combined 1/8-1/4 normal dose estrogen can reach the effectiveness of normal dose estrogen.Serum containing GENGNIANGANLUYIN had no effects on ERαand ERβexpression.Yet, combined low dose estrogen,it up-regulated ERα,ERβand OPG expression obviously,which combined 1/8-1/4 normal dose estrogen can reach the effectiveness of normal dose estrogen.Conclusion:GENGNIANGANLUYIN combining low dose estrogen can promote osteoblast proliferation and alkaline phosphatase activities, up-regulate ER and bone metabolism related gene expression.3.The prevention of GENGNIANGANLUYIN plusing low dose estrogen on osteoporosis of ovariectomized ratsObjective:To observe the effects of GENGNIANGANLUYIN combining low dose estrogen on osteoporosis rats and approach the possibility of integrated traditional and western medicine treating PMO.Method:ovariectomized rats was treated by GENGNIANGANLUYIN, GENGNIANGANLUYIN combining 1/16,1/8,1/4,1/2 optimal dose estrogen,and optimal dose estrogen respectively for 6 month, ovariectomized rats without treatment and sham operated rats being control. Radioimmunity detected serum E₂,FSH and LH contents,bone density apparatus analysis femur density.Observe endometrium and mammary gland histology and analysis femur using morphometry.Result:GENGNIANGANLUYIN combining low dose estrogen can increase PMO rat serum hormone,elevate femoral bone mass,improve femoral bone trabecula morphous,increase substantia corticalis ossium, decrease bone trabecula spacing.GENGNIANGANLUYIN combining 1/8-1/4 normal dose estrogen can reach the effectiveness of normal dose estrogen, furthermore,have no obvious stimulation on endometria and mammary gland.Conclusion:GENGNIANGANLUYIN combining low dose estrogen can ameliorate PMO rat serum hormone,elevate femoral bone mass,improve femoral bone trabecula morphous.GENGNIANGANLUYIN combining 1/8-1/4 normal dose estrogen can reach the effectiveness of normal dose estrogen, furthermore,have no obvious stimulation on endometria and mammary gland.In short,this study approached not only the mechanism of GENGNIANGANLUYIN on bone metabolism,but also the possibility of GENGNIANGANLUYIN combining low dose-estrogen to treat PMO.The objective is to provide a new idea and theory foundation to treat PMO integrating traditional and western medicine.