The Efficacy Study Of Herbal Pair Radix Achyranthis Bidentatae And Eucommia Ulmoides Oliv. For Preventing Glucocorticoid Induced Osteoporosis By Uniform Design

Objectives:To expolore the mechanism of glucocorticoid induced osteoporosis（GIOP）by studying the effects of high dose of dexamethasone on Zebrafish’s bone mineralization and osteoblast differentiation,on MLO-Y4 osteocyte-like cells proliferation and apoptosis;To evaluate the best ratio of herbal pair,Radix Achyranthis Bidentatae（RAB）and Eucommia ulmoides Oliv.（EU）via observing the interventions in excess dexamethasone by uniform design;To provide experimental evidence for the prevention and treatment of GIOP with herbal pair RAB and EU.Methods:1.The water extracts（lyophilized powder）of RAB and EU were evaluated by high performance liquid chromatography（HPLC）.2.In vivo study:Zebrafish osteoporosis model was induced by 10^-5 mol/L dexamethasone（Dex）in vivo.Toxicity experiment was carried out by preventing in different concentrations of water extracts from herbal pair RAB and EU which is prepared with different mass proportions（1:3,2:2,3:1,respectively）.The concentration of less than median lethal dose(LD₅₀)was selected for subsequent experiments.According to mathematical model"uniform design",varying doses of RAB and EU have been combined to form 7 types of medications for the interventions of GIOP.After 96 hours of intervention,the efficacy was determined by calculating zebrafish vertebral area and sum brightness via alizarin red staining,detecting alkaline phosphatase（ALP）activity.Then the significant proportions and doses were selected for verification study.3.In vitro study:MLO-Y4 cells were divided into 6 groups:control group,model group(10^-5mol/L Dex),alendronate sodium trihydrate（ALN）group(10^-6mol/L),RAB group（10μg/m L）,EU group（10μg/m L）and herbal pair RAB and EU group（10μg/m L,ratio 1:1）.After 48 hours of drugs interventions,the cells proliferation and apoptosis were detected by CCK-8 and Annexin V-PI methods.And the protective effect of herbal pair RAB and EU on model cells were observed.Results:1.Quality evaluation of RAB and EU:The water extracts of RAB and EU both were accorded with Chinese pharmacopoeia standards and suitable for subsequent experiments.2.Toxicity experiments:LD₅₀ of herbal pair RAB and EU at ratio of 2:2 was 300μg/m L.While under the same concentrations,the mortality rate is higher when the proportion of RAB is more than EU relatively.The subsequent experiments results with less than LD₅₀concentration showed that different proportions of RAB and EU had the effects of preventing GIOP.The concentration below 200μg/m L was the safe range concentration.When the ratio was 2:2,the intervention effect of RAB and EU was stable.3.The Uniform design experiment:The herbal pair RAB and EU had significant intervention effect on zebrafish GIOP model.Compared with the model group,when combination of 50μg/m L RAB and 50μg/m L EU were at ratio of 1:1,the interventions of bone mineralization area,sum brightness and ALP activity of zebrafish GIOP model was the most significant（P<0.05）,which was better than that of single herb or ALN.4.Cell proliferation and apoptosis experiment:Compared with model group,MLO-Y4cells in ALN group,RAB group,EU group and herbal pair RAB and EU group were all proliferated and the total necrosis rate（PI⁺）was decreased.The cell proliferation of herbal pair RAB and EU group was significantly higher than model group（P<0.05）,and the total necrosis rate was significantly lower than model group（P<0.01）.The effects of herbal pair RAB and EU were better than single herb or ALN.Conclusion:1.From the in vivo study of zebrafish GIOP model,it was concluded that the herbal pair RAB and EU had an intervention effect on zebrafish GIOP model.The optimum concentration is 50μg/m L RAB and 50μg/m L EU.The best concentration ratio is 1:1,which is consistent with the compatibility ratio of RAB and EU in the classical herbal formula Duhuo Jisheng decoction.2.From the in vitro study of GIOP pathological model of MLO-Y4 cells,it was concluded that the herbal pair RAB and EU had a protective effect on GIOP pathological model of MLO-Y4 cells.