| Objective1.Clinical study of syndrome is the spleen kidney yang deficiency syndrome in elderly female patients with primary osteoporosis by observing the pain(VAS score)and serum β-C-terminal telopeptide of type Ⅰ collagen(β-CTx),N-MID osteocalcin(N-MID)and procollagen I N-terminal peptide(T-PINP),the content changes of DKK-1 and Sost,to observe the clinical efficacy of Bushen Jianpi Huoxue Recipe in treatment of osteoporosis.2.The basic experiment using Bushen Jianpi Huoxue Recipe intervention by the expression of DKK-1 in osteoblasts transfected with Sost adenovirus,by observing cell activity,calcium ion concentration,the activity of ALP,BMP2,CTGF,FGF expression and the molecular mechanism of Bushen Jianpi Huoxue Recipe in the treatment of osteoporosis.Methods1.The clinical study①The clinical study of Guangzhou University of Chinese Medicine Affiliated Hospital orthopedics received primary osteoporosis in elderly female patients with 40 cases,the patients were divided into control group and treatment group,the control group was given oral Caltrate D and Calcitriol Soft Capsules;in the treatment group and control group in the same administration on the basis of oral Recipe of Bushen Jianpi Huoxue Recipe.①The object of study selected on the morning of the second day after administration of the morning before and after treatment,fasting venous blood,remove the serum,evaluate the markers N-MID,β-CTx and T-PINP,DKK-1 and beta Sost levels by ELISA;observed before and after treatment visual analog pain score(VAS score).2.Basic experimental researchThe cultured cells were divided into NC control group,Ad-DKK-1 group,Ad-Sost group,Ad-DKK-1+Ad-Sost group.NC control group with empty adenovirus transfection;group Ad-DKK-1 by packing overexpression of adenovirus transfected DKK-1;group Ad-Sost by packing overexpression of adenovirus transfected Sost;group Ad-DKK-1+Ad-Sost by overexpression of DKK-1 adenovirus packaging packaging and overexpression of Sost Cotransfection.With each group containing serum and blank serum intervention,the cell proliferation was detected by CKK-8,ALP was detected by ALP assay kit,flow-cytometry cell calcium concentration,the expression of BMP2,CTGF,Western blot detection of FGF.Results1.The clinical study① In the control group and the experimental group of the general data(including age,weight,height,BMI and bone density)with the mean and standard deviation to describe,first with the normality test data analysis shows that data with normal distribution,comparison between groups using t test to describe.Homogeneity of variance was used to test the homogeneity of variance in each group.There was no statistical difference between the experimental group and the control group(Levene).There was no statistically significant difference between the two groups,with a good comparability.②Bone metabolism index detection β-CTx,N-MID,T-PINP,DKK-1 and Sost in the experiment before the difference,no statistical difference between the two groups.The two groups after drug treatment,the expression of β-CTx was significantly decreased,but experimental group and treatment group after treatment,no significant difference(t=0.614,P=0.543),compared with the experimental group used traditional Chinese medicine Bushen Jianpi Huoxue Recipe,the control group decreased more significantly;N-MID expression was not changed,but increased;T-PINP expression was not obvious change,DKK-1 and Sost,but a decreasing tendency.③The VAS scores showed no significant difference in the degree of pain of the two groups of patients before treatment(t=0.831,P=0.411),after drug treatment,the pain symptoms of the two groups were improved,the pain of experimental group than the control group improved significantly(t=5.068,P=0.000).2.Experimental study①Changes of cell activity in each groupCompared with the blank serum group,four groups of serum containing drugs were respectively in the intervention group,and the activities of 24h,48h and 72h were enhanced in the group;②Changes of ALP activity in each groupFour compared with the blank serum group intervention,four groups of serum activity were improved(P=0.029,P=0.015,P=0.041,P=0.029),when the expression of DKK-1 and Sost were transfected into cells and co transfection,ALP activity trend is more obvious;③Changes of calcium ion in each groupAd-DKK-1 group and blank serum respectively intervention group Ad-Sost,group Ad-DKK-1+Ad-Sost three group,Ad-DKK-1 group,Ad-Sost group respectively,serum intervention,calciumion concentration of Ad-DKK-1+Ad-Sost group decreased(P=0.026,P=0.035,P=0.002),in the control group in the blank serum and serum containing drug intervention had no significant difference(P=0.108);④Changes of bone related proteins in each groupIn Western blot assay,BMP2 in serum of the four groups of relative expression compared with blank serum intervention group of the four relatively high expression level(P=0.000,P=0.000,P=0.000,P=0.001),the relative expression of CTGF was observed in Ad-DKK-1 group,serum Ad-Sost were intervention group,Ad-DKK-1+Ad-Sost group,relative the expression of Ad-DKK-1 of the three groups were compared with blank serum group,respectively Ad-Sost intervention group,expression of Ad-DKK-1+Ad-Sost group increased(P=0.000,P=0.000,P=0.000,NC)in the control group in the serum and blank serum no significant difference;in the observation of the relative expression of FGF in Ad-DKK-1 group,serum intervention respectively.The Ad-Sost group,Ad-DKK-1+Ad-Sost group,the relative expression of the three groups of Ad-DKK-1 group,the amount of serum were compared with blank intervention group Ad-Sost,Ad-DKK-1+Ad-Sost group expression increased(P=0.000,P=0.000,P=0.000),lower serum and blank serum intervention in the NC control group(P=0.047),DKK-1 and Sost over expression and co expression in cells containing serum and blank serum stem change prognosis comparison of protein is more obvious.Conclusion1.In the clinical study,experimental group and control group N-MID、T-PINP、DKK-1 and Sost before and after treatment showed no significant difference,but β-CTx in the control group and the experimental group had statistical significance before and after treatment,however the two groups after treatment was statistically significant.2.In the VAS score after treatment,there were statistically significant differences,especially in the experimental group to improve the effect of pain better.3.Bushen Jianpi Huoxue Recipe medicated serum can increase cell activity,reduce the concentration of calcium,inhibit cell apoptosis,enhance the activity of ALP,improve the expression of BMP2 and CTGF,the relative amount of FGF,is conducive to osteoblast mineralization,especially in the over expression of DKK-1 and overexpression of Sost after intervention.Bushen Jianpi Huoxue Recipe can regulate the metabolism of osteoblasts by regulating DKK-1 and Sost. |
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