Bushen Jianpi Huoxue Recipe Affect On Osteoblast Of Wnt Pathway Inhibitory Factors Transfection

Objective:1.Observe Bushen Jianpi Huoxue Recipe to have an effect on expressions of ALP,OPG,OPN,BMP-2 in osteoblast;2.Explore the mechanism of Bushen Jianpi Huoxue Recipe to differentiate in osteoblast;Methods:1.Traditional Chinese medicine intervened primary osteoblast extracted through trypsin digestion?1?Blank serum,Bushen Jianpi Huoxue Recipe serum?high,medium and low?intervened primary osteoblast respectively,the activity of osteoblast was detected by CCK-8;?2?Blank serum,Bushen Jianpi Huoxue Recipe serum?medium and low?and alendronate sodium vitamin D₃?Positive medicine?intervened primary osteoblast respectively,the expression of ALP in osteoblast was detected by BCIP-NBT;?3?Blank serum,Bushen Jianpi Huoxue Recipe medium serum and Positive medicine serum intervened osteoblast respectively,the mRNA expressions of OPG,OPN,BMP-2 were detected by RT-qPCR;the protein expressions of OPG and BMP-2 were detected by WB.Silenting adenovirus of DKK1 and Sost transfected MG-63 cell,and Chinese medicine intervened MG-63 cell adenovirus transfected vector adenovirus?silencing DKK1 adenovirus?sh-DKK1??silencing Sost adenovirus?sh-Sost??silencing DKK1+Sost adenovirus?sh-DKK1+Sost?transfected MG-63 cell respectively:blank serum,Bushen Jianpi Huoxue Recipe serum intervented MG-63 cell respectively,ALP activity was detected by ALP detection kit,Calcium ion concentration was detected by Calcium OtangeTM Indicators probe,the protein expressions of BMP-2,CTGF?FGF2?Runx2,TNF-?were detected by WB.Result:1.Bushen Jianpi Huoxue Recipe intervented primary osteoblast?1?cell activity was on the rise with high,medium and low doses of serum at 12h,24h,48h,cell activity had downtrend at 72h,compared with blank serum,cell activity in medium serum group was the strongest at 48h;?2?ALP staining positive rates of medicated serum group were higher than blank serum group;while compared with low serum and positive serum,ALP staining positive rates of medicated serum group was higher;?3?Compared with blank serum group,the mRNA expressions of OPG,OPN,and BMP-2 were increased in medium serum group;Compared with blank serum group,the mRNA expression of BMP-2 was decreased in positive serum;?4?Compared with blank serum group,the protein expressions of OPG and BMP-2were increased in medium serum group,and the protein expression of OPG was decreased in positive serum.2.Silenting adenovirus of DKK1 and Sost intervened MG-63 cell,and Bushen Jianpi Huoxue Recipe intervened it.?1?Compared with blank serum,respectively,the ALP activity of NC control group,sh-DKK1 group,sh-Sost group,sh-DKK1+sh-Sost group were increased with the medicated serum interventing MG-63 cell;?2?Compared with blank serum respectively,the calcium concentration in NC control group,sh-DKK1 group,sh-Sost group,sh-DKK1+sh-Sost group were decreased because of the medicated serum interventing in MG-63 cell;?3?Compared with blank serum respectively,the protein expressions of BMP-2,CTGF,FGF2,Runx2 were increased in NC control group,sh-DKK1 group,sh-DKK1+sh-Sost group,the protein expression of TNF-?was decreased,which was interfered by medicated serum;compared with the blank serum,the protein expressions of CTGF,FGF2,Runx2 in sh-Sost group that under the medicated serum intervention were increased,the protein expression of TNF-?was decreased.Conclusion:1.In the primary osteoblast,the ALP expression,the mRNA expressions of OPG,OPN,BMP-2 and the protein expressions of OPG and BMP-2 were increased with the Bushen Jianpi Huoxue Recipe,which can promote osteoblast differentiation.2.Because of the Bushen Jianpi Huoxue Recipe,the ALP activity and the protein expressions of BMP-2,CTGF,FGF2,Runx2 were increased,while the calcium ion concentration and the protein expression of TNF-?were decreased in MG-63 cell whose DKK1 and Sost were silenting.