| ObjectiveTo reveal the mechanism that how the method of nourishing the kidney-yin treat glucocorticoid induced osteoporosis in the aspect of the change of laws of the Wnt signal transduction pathway through in vivo and in vitro experiments,as well as to provide further experimental evidence for exploring the scientific connotation of the mechanism of"The kidney dominating bone ".Methods1 In vivo experimentFirst,fifty-five female Wistar rats were selected and divided into four groups randomly:twelve in the normal control group,fifteen in model group,thirteen in positive control group and fifteen in nourishing the kidney-yin group.Model group rats were injected with dexamethasone,(when using dexamethasone injection was diluted to 1mg/ml with normal saline,the delivery volume was 0.1ml/100g weight and the dose was 0.1mg/100g weight),while normal control rats were injected the corresponding dose of normal saline injection,2 times per week for 8 weeks.Positive control group and nourishing the kidney-yin group rats were treated with alfacalcidol tablets(the tablets will be crushed,prepared using distilled water as the suspension concentration 0.005μg/ml,the delivery volume was 1ml/100g weight and the dose was 0.005μg/100g weight) and Zuogui pills(drug concentration was 0.952g/ml,the delivery volume was 1ml/100g weight and the dose was 0.925g/100g weight) respectively,except for injecting dexamethasone in the above method.The drugs were given once a day and last six days.There has 1 day pause every six days.The whole period last 8 weeks.At the third day and the sixteenth day before the day all the rats were killed, hydrochloric acheomycin were respectively injected into the abdomen of the rats according to 30mg/kg to mark the bone with fluorescence.After Medication,The rats were anesthetized to get artery blood,then the blood was centrifuged to detect the level of BGP,E2,PTH and IGF- I in serum by enzyme-linked immunosorbent assay. After being killed the right tibia of the rats were retained for bone tissue section which weren't decalcified to evaluate the change of bone histomorphometry,including TBV%,TRS%,TFS%,MAR,OSW and mAR.At the same time the left tibia of the rats were used for paraffin tissue section after being decalcified to observe the protein and mRNA expression of Wntl,LRP-5 andβ-catenin by means of immunohistochemistry and in situ hybridization.2 In vitro experiment2.1 Preparation of serum containing Zuogui pillTwenty female Wistar rats were selected and allocated to two groups randomly: normal control group and Zuogui pill treated group,there were ten rats in every group. Zuogui pill treated group rats were injected with dexamethasone,twice per week and last for 8 weeks.After 8 weeks the rats were were administrated orally Zuogui pill twice one day,the rats in normal control group were fed with the same volume diluted water.The blood of all the rats was collected by abdominal aorta one hour later after those rats were administrated in the fifth time.The blood was placed quietly 1 hour, and then centrifuged at 2500r/min,25min.The serum was isolated and conserved at -20℃.We called the serum of rats in the Zuogui pill treated group the serum containing Zuogui pill.2.2 The detection of the effect on proliferation of osteoblast in different experimental conditions by MTT methodThere were six groups in this experiment:Osteoblasts +normal serum group,Osteoblasts +10-6 mol/L dexamethasone,Osteoblasts +10-7 mol/L dexamethasone,Osteoblasts +5%serum containing Zuogui pill,Osteoblasts +10%serum containing Zuogui pill,Osteoblasts +15%serum containing Zuogui pill,and eight wells in one group.Osteoblast suspension would be regulated to the concentration of 1×104/ml,and coveraged 200μl osteoblast suspension every well to 96-hole cell culture plat.After 12 hours treated the osteoblast with normal serum,10-6 mol/L dexamethasone,10-7 mol/L dexamethasone,5%serum containing Zuogui pill,10%serum containing Zuogui pill and 15%serum containing Zuogui pill respectively,all kinds of cells were incubated in 37℃,5%CO2 air atmosphere.After 96 hours,the optical density of the osteoblasts in each group was detected at the 570nm wavelength on microplate reader by MTT method.2.3 The detection of the protein and mRNA expression of Wnt3a,LRP-6 andβ-catenin of osteoblastsThere were three groups in this experiment:normal serum group,10-7 mol/L dexamethasone group and 15%serum containing Zuogui pill group.After incubating for 12 hours,treated the osteoblast with normal serum,10-7 mol/L dexamethasone,and 15%serum containing Zuogui pill respectively,After incubating for 96 hours,the western bloting and fluorescence quantitative PCR motheds were used to detect the protein and mRNA expression of Wnt3a,LRP-6 andβ-catenin.3 Statistical analysisResults are presented as Mean±SE.Differences between groups were analyzed using q test by one-way ANOVA.Results1 In vivo experiment1.1 Effects of nourishing kidney-Yin on the bone histomorphometry indicators in rats suffered from Glucocorticoid-induced osteoporosisAfter being injected dexamethasone for 8 weeks,TBV%of tibia of rats in model group significantly decreased,while TRS%significantly increased and TFS%,MAR, mAR and OSW significantly decreased compared with normal control group. Compared with model group,TBV%significantly increased,at the same time TFS%, MAR,mAR and OSW increased to a certain extent after being treated by prescriptions of Zuogui pill.1.2 Effects of nourishing kidney-Yin on protein and mRNA expression of Wntl, LRP-5 and B-cateninCompared with nomal control group,the protein and mRNA expression of Wntl, LRP-5 and B-catenin decreased obviously,after bing injected dexamethasone for 8 weeks. After being treated by prescriptions of Zuogui pill,the protein and mRNA expression of Wntl,LRP-5 and B-catenin significantly increased compared with model group.1.3 Effects of nourishing kidney-Yin on serum level of BGP,E2,PTH and IGF-â… After being injected dexamethasone for 8 weeks,the serum level of BGP,E2,and IGF- â… of rats in model group significantly decreased,while the serum level of PTH significantly increased.Compared with model group,the serum level of BGP,E2,and IGF-â… of rats treated with Zougui pills significantly increased,while their serum level of PTH significantly decreased.2 In vitro experiment2.1 Effect on proliferation of osteoblast in different experimental conditions by MTT methodCompared with nomal serum group,the optical density of the osteoblasts in Osteoblasts +10-6 mol/L dexamethasone,Osteoblasts +10-7 mol/L dexamethasone significantly decreased,and there was no significance change between them.Compared with the above groups,the optical density of the osteoblasts in Osteoblasts +5%serum containing Zuogui pill,Osteoblasts +10%serum containing Zuogui pill,Osteoblasts +15%serum containing Zuogui pill all increased obviously,and the 15%serum is better than 5%and 10%serum So the 10-7 mol/L dexamethasone and the 15%serum containing Zuogui pill were selected to be experimental conditions.2.2 Effects of nourishing kidney-Yin on protein expression of Wnt3α,LRP-6 andβ-cateninCompared with nomal serum group,the protein expression of Wnt3α,LRP-6 andβ-catenin decreased obviously,after bing treated by dexamethasone for 96 hours.After being incubated with serum containing Zuogui pill,the protein expression of Wnt3α, LRP-6 andβ-catenin significantly increased compared with 10-7 mol/L dexamethasone group.2.3 Effects of nourishing kidney-Yin on mRNA expression of Wnt3α,LRP-6 andβ-cateninBing treated by dexamethasone for 96 hours,the mRNA expression of Wnt3α, LRP-6 andβ-catenin decreased obviously compared with nomal serum group,After being incubated with serum containing Zuogui pill,the mRNA expression of Wnt3α, LRP-6 andβ-catenin significantly increased compared with 10-7 mol/L dexamethasone group. ConclusionThe method of nourishing the kidney-yin can prevent and treat glucocorticoid induced osteoporosis.The Mechanism of which is as the following three:First,to promote the proliferation of osteoblasts,so that increase bone formation. Second,increase the expression of the key signaling molecule:Wnt,LRP and theβ-caten in Wnt signal transduction pathway.The specific way is to enhance the Wnt signal,the combination of Wnt and its receptor:LRP is increased,so J3-catenin protein will not be degraded and can accumulated in the intracellular,then enter the nucleus,combine with the transcription factor TCF to activate the process of gene transcription.On the one hand,this facilitated the orientation,differentiation, development,proliferation of osteoblast and inhibition its apoptosis;on the other hand, through osteoblast-mediated effect to inhibite the activity of osteoclasts and eventually to increase bone formation and reduce bone resorption,which can finally restorate and control bone loss.Third,regulate the serum level of PTH,BGP,E2,IGF-1:improve the peripheral blood level of E2,IGF-1,BGP and reduce the level of PTH,So that the disordered state of these factors induced by glucocorticoid can be returned to normal,thereby preventing the loss of bone mass.
|
PDF Full Text Request