| Purpose:To confirm that WenShenGuShu formula may have the effects of anti-oxidative stress and treatment of osteoporosis.To clear the role of Fox0/Wnt pathway in osteoblast differentiation and bone formation.To confirm that WenShenGuShu formula have effect of anti-oxidative stress through regulation of Fox0/Wnt pathway to treat and prevent postmenopausal osteoporosis,establish its anti-oxidative stress mechanism,and find out the possible action targets.Objective to provide experimental basis for the study of the anti-oxidative stress effect of Bushen Wenyang therapy on the prevention and treatment of postmenopausal osteoporosis.Method:The model of postmenopausal osteoporosis was prepared by OVX,and one month after the operation,the rats were divided into groups randomly.WenShenGuShu formula groups and estradiol group were treated respectively,model group and sham operation group were given normal saline gavage.The course of treatment lasted for two months.After the last treatment,specimens were collected.And then we use Dual Energy X-ray absorptiometry evaluated the BMD;Micro-CT for bone histomorphometry determination;Serum bone metabolism indexes of PINP,BGP,TRACP5b,sRANKL,OPG were examined by ELISA;The content of MDA in serum and the activity of CAT and SOD in serum were detected by colorimetric method;The protein of p66Shc??-catenin?Wnt2?Fox03a?PPAR?2?Dlx5 in bone were measured by Western blot,The expression of osteogenic differentiation regulation factor gene Runx2,Osteorix,Dlx5 and the gene expression of bone formation related proteins OPG,Alpl,Coll1,OCN,sRANKL,FoxO/Wnt pathway key genes Gadd45a,Axin2,PPAR,y2were detected by RT-PCR.Results:1.bone mineral density,bone metabolism indexes and bone morphology:Compared with the sham operation group,proximal femur BMD and total BMD of model group rats were significantly decreased(P<0.05);Serum TRACP5b,BGP and PINP increased significantly(P<0.05),and the content of OPG and OPG/sRANKL were significantly lower(P<0.05);Bone microstructure was abnormal,BV/TV decreased by 53.8%(P<0.05),the bone mineral density of femoral metaphysis decreased by 27.6%(P<0.05),and Tb.N and Tb.Th were significantly decreased,while SMI and Tb.Sp were significantly increased(P<0.05).Compared with the model group,BMD of rats in estrogen and WenShenGuShu formula groups were significantly increased(P<0.05);WenShenGuShu formula group serum TRACP5b,PINP,BGP was significantly decreased(P<0.05),OPG content,OPG/sRANKL ratio was significantly increased(P<0.05),sRANKL content were lower,but have no statistical significance;The parameters of BV/TV,BMD and Tb.N,and Tb.Th were significantly increased in rats of medium dose WenShenGuShu formula and estrogen groups,and Tb.Sp,SMI decreased significantly(P<0.05),bone microstructure is obviously improved.2.Changes of anti-oxidative stress indicators:Compared with sham operation group,model group rats p66Shc protein expression was significantly higher,serum MDA activity was significantly increased(P<0.05),while the activity of CAT decreased(P<0.05),the activity of SOD decreased,but the difference was not statistically significant;Compared with model group,high and medium dose WenShenGuShu formula groups and estrogen group can significantly reduce the expression of p66sShc protein phosphorylation,The CAT activity of high and medium dose WenShenGuShu formula groups increased significantly(P<0.05),MDA activity significantly decreased in medium dose group rats(P<0.05).The activity of SOD was increased in each WenShenGuShu formula groups and estrogen group compared with model group,but there was no statistical significance.3.Effect on osteogenic differentiation in ovariectomized osteoporosis rat:compared with sham operation group,the PPAR y2 mRNA and protein expression in bone tissue of the model group were significantly increased(P<0.05),The mRNA expression of bone formation related regulatory factor and functional proteins Runx2,Osteorix,OPG,Coll1 and OCN were significantly decreased(P<0.05),Dlx5 mRNA and protein expression were also significantly decreased(P<0.05),osteoclast related sRANKLmRNA expression was significantly higher(P<0.05).Compared with the model group,estrogen and WenShenGuShu formula can significantly reduce the gene expression of PPARy2 and sRANKL(P<0.05),WenShenGuShu formula can also significantly reduce the expression of PPAR?2 protein(P<0.05),increased osteoblast related genes expression excepted Alpl gene(P<0.05),at the same time,improve the gene and protein expression of Dlx5(P<0.05).4 Effects on FoxO/Wnt signal pathway:compared with sham operation group,expression of Fox03a protein and Gadd45 gene in model group were significantly increased(P<0.05),and the expression of ?-catenin protein and Axin2 gene in Wnt signaling pathway decreased significantly(P<0.05),Wnt2 expression decreased but have not statistically significant.Compared with the model group,the expression of Fox03a protein and Gadd45 gene in estrogen and WenShen GuShu formula group were significantly decreased(P<0.05),but increased the Wnt2 and?-catenin protein and its target gene Axin2 expression in Wnt signaling pathway(P<0.05).The results showed that:WenShenGuShu formula have a positive regulation effect on Wnt signal pathway.Conclusion:The anti-oxidative stress effect of WenShenGuShu formula in prevention and treatment of postmenopausal osteoporosis is more explicit.The possible mechanism of the action is to inhibit the transcription of FoxO activated by oxidative stress through the anti-oxidative stress effect,and positive regulation of Wnt signaling pathway,so as to improve the ability of osteogenic differentiation and bone formation,and it can also reduce the high conversion level of bone metabolism and reduce bone resorption.This study can provide experimental basis for the research on the anti-oxidative stress effect of Bushen Wenyang therapy in prevention and treatment of postmenopausal osteoporosis.Provides new ideas for the further studies on age-related diseases.And further prove that the theory of Bushen anti-aging and "kidney dominate bone marrow". |
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