Effect Of Bushen Jianpi Huoxue Recipe On Bone Formation In Rats Based On Sost/Wnt Signaling Pathway

ObjectiveThe role of Wnt signal pathway inhibitor Sost in bone formation in rats was studied by using adenovirus overexpression and silencing techniques.The effect of bushen jianpi huoxue recipe on Sost,Wnt2,OPG and BMP2 was explored.Methods1.Twenty-eight female Sprague-Dawley rats aged four months.2.The experiment group was divided into group A)blank control;group B)blank adenovirus;group C)overexpression of Sost adenovirus infection;group D)silencing Sost adenovirus infection,each group was divided into 7 rats.3.Administration method and dosage:the encapsulated rat adenovirus was injected intraperitoneally with the dose of virus titer 10¹⁰/200ul.A group of blank control group was not injected with the virus.Adenovirus group?group B?was intraperitoneally injected with non-loaded adenovirus?group B?,group with overexpression adenovirus with Sost?group C?was injected intraperitoneally with Sost adenovirus,and group with silent adenovirus with Sost?group D?was injected with adenovirus of silent Sost.4.One month later,rats in group C and group D were given intragastric administration of bushen jianpi huoxue recipe by stomach,rats in group A and group B were given the same amount of normal saline.After two months,the rats in each group were killed.The femur of rats assayed by Bone mineral density?BMD?.?1?The gene expression of Wnt2?OPG?Sost?BMP-2 was detected in the tissue of rats after adenovirus overexpression or silencing Sost assayed by real-time fluorescence quantitative PCR.?2?The expression of the pathway protein Wnt2?OPG?Sost?BMP-2 was detected by WB technique.Result:1.Adenovirus transfection into ratsAfter one week of adenovirus injection,fluorescent PCR was detected in serum of rats in each group.The results showed that the content of adenovirus in the remaining groups was between 26 and 30 except the blank control group.The recombinant adenovirus was successfully transfected into rats.2.Changes of BMD in femur of rats in each groupIn the blank control group and empty adenovirus group,changes of saline irrigation on rat bone mineral density had no significant effect?P>0.05?;the overexpression of Sost group,no case of gastric lavage and overexpression of Sost could reduce the bone mineral density?P<0.05?,and intragastric administration of bushen jianpi huoxue recipe.The bone density of rats was increased,the difference was statistically significant?P<0.05?;In the silent Sost group,there was no significant change in bone mineral density of the rats with silent Sost without gastric perfusion,and intragastric administration of bushen jianpi huoxue recipe,bone density increased,the difference has statistical significance?P<0.05?.3.The effect of bushen jianpi huoxue recipe on Wnt2,OPG,Sost and BMP-2mRNA in ratsIn blank control group and empty adenovirus group,normal saline did not affect the expression of Wnt2,OPG,Sost and BMP-2 mRNA in rat?P>0.05?.The overexpression of Sost could increase the expression of Sost mRNA?P<0.05?and decrease the expression of Wnt2 and BMP-2 mRNA?P<0.05;P<0.05?,but the expression of OPG mRNA did not change obviously.The expression of Wnt2 and BMP-2 mRNA was increased?P<0.05;P<0.05?and the expression of OPG and Sost mRNA was decreased?P<0.05;P<0.05?after oral administration of bushen jianpi huoxue recipe.In silent Sost group,silencing Sost could increase the expression of Wnt2 mRNA?P<0.05?and decrease the expression of Sost mRNA?P<0.05?,but the expression of OPG and BMP-2 mRNA did not change after gastric perfusion.bushen jianpi huoxue recipe could increase the expression of Wnt2and BMP-2 mRNA?P<0.05;P<0.05?,the expression of Sost and OPG mRNA was decreased?P<0.05;P<0.05?.4.The effect of bushen jianpi huoxue recipe on the protein of Wnt2,OPG,Sost and BMP-2 in ratsIn blank control group and empty adenovirus group,normal saline did not significantly affect the expression of Wnt2,OPG,Sost and BMP-2 protein in rats?P>0.05?.In the over-expressed Sost group,overexpression of Sost could increase the expression of Sost protein?P<0.05?and decrease the expression of BMP-2 protein?P<0.05?,but the expression of Wnt2 and OPG protein did not change significantly.The expression of BMP-2 protein was increased?P<0.05?and the expression of OPG and Sost protein was decreased?P<0.05;P<0.05?,but the expression of Wnt2 protein did not change significantly after oral administration of bushen jianpi huoxue recipe.In the silent Sost group,the silent Sost could increase the expression of Wnt2 and BMP-2 protein?P<0.05;P<0.05?,decrease the expression of Sost protein?P<0.05?,but the expression of OPG protein was not obvious.After gavage,bushen jianpi huoxue recipe could increase the expression of BMP-2 and Wnt2 protein?P<0.05?and decrease the expression of OPG and Sost protein?P<0.05;P<0.05?.Conclusion:1.Overexpression of Sost could decrease the bone mineral density of femur,increase the expression of Sost mRNA,decrease the expression of Wnt2 and BMP-2mRNA,increase the expression of Sost protein and decrease the expression of BMP-2 protein.2.Silencing Sost could increase the expression of Wnt2 mRNA and decrease the expression of Sost mRNA,increase the expression of Wnt2 and BMP-2 protein and decrease the expression of Sost protein.3.After the intervention of bushen jianpi huoxue recipe,the bone mineral density of femur in rats was increased,the expression of BMP-2 and Wnt2 mRNA was increased,the expression of OPG and Sost mRNA was decreased,the expression of Wnt2 and BMP-2 protein was increased and the expression of OPG and Sost protein was decreased.