Effect Of Bushen Jianpi Huoxue Recipe On Bone Formation In Rats Based On DKK1/Wnt Signal Pathway

Objective:Intervention of Silencing DKK1 Gene Rats by Bushen Jianpi Huoxue Recipe to investigate the effects of Bushen Jianpi Huoxue Recipe on Wnt signaling pathway related factors LRP6,?-catenin,APC,RUNX2 and OPG in rats.Methods:1.Experimental animals:18 female SD rats of 3 months old.2.Experimental group:Rats were randomly divided into A.blank control group(NC),B.empty adenovirus group(Ad-EV),C.silent DKK1 adenovirus infection group(Ad-shDKK1),6 rats in each group.Marked as 1 to 6.3.Adenovirus transfection and dose:The rat adenovirus containing the silenced DKK1 gene was injected into the tail vein,and each rat was injected with 200 ul,and the virus titer was(1010).Group A(blank control group)was not injected with virus,and an equal volume of normal saline was injected;group B(empty adenovirus group)was injected with empty adenovirus;group C(silent DKK1 group)was injected with silencing DKK1 adenovirus.4.One month after transfection of adenovirus,each group received Bushen Jianpi Huoxue Recipe for 1 to 3,and the same volume of normal saline was administered from 4 to 6 for 2 months.Two months later,rats in each group were sacrificed and the femur bone density(BMD)was measured.Real-time quantitative PCR was used to detect the expression of DKK1,LRP6,?-catenin,APC,RUNX2 and OPG genes in bone tissue after silencing DKKI adenovirus-transfected rats.The expression of DKK1,?-catenin,RUNX2 and OPG protein was detected by Western Blot.Results:1.One week after transfection of adenovirus,qualitative detection of serum fluorescent PCR was performed in each group.The results showed that except group A(blank control group),adenovirus Transfection was successful in group B(empty adenovirus group)and group C(silent DKK1 group).2.The femoral bone density of group C(silent DKKI group)was higher than that of group A(blank control group)and group B(empty adenovirus group)(P<0.05);There was no significant difference in bone mineral density between group A(blank control group)and group B(empty adenovirus group)(P>0.05).In each group,the femur bone density of rats with Bushen Jianpi Huoxue Recipe was higher than that of normal saline rats(P<0.05).3.Compared with group A(blank control group)and group B(empty adenovirus group),LRP6,?-catenin,RUNX2,OPG mRNA expression increased in group C(silent DKK1 group),?-catenin,RUNX2,OPG protein expression increased and the APC mRNA expression decreased(P<0.05).4.In each group,the expression of LRP6,?-catenin,RUNX2 and OPG mRNA increased,the expression of ?-catenin,RUNX2 and OPG protein increased and the expression of APC mRNA was decreased in Bushen Jianpi Huoxue Recipe rats compared with normal saline rats.(P<0.05).Conclusion:1.Silencing the DKK1 gene can increase bone density in rats.2.Silencing DKK1 gene can increase the expression of LRP6,?-catenin,RUNX2 and OPG mRNA,increase the expression of ?-catenin,RUNX2 and OPG protein,and decrease the expression of APC mRNA.3.After intervention with Bushen Jianpi Huoxue Recipe,the expression levels of LRP6,?-catenin,RUNX2 and OPG mRNA were increased,the expression of ?-catenin,RUNX2 and OPG protein was increased,the expression of DKK1 and APC mRNA was decreased,and the expression of DKK1 protein was decreased.