| Purpose:This paper systematically discusses the correlation between kidney deficiency and blood stasis and postmenopausal osteoporosis,and clarifies the therapeutic effect and mechanism of tonifying kidney,invigorating qi and activating blood circulation and its prescription on postmenopausal osteoporosis.It is helpful to elucidate the new method of regulating bone formation and to develop effective prescriptions to improve bone metabolism,and to provide theoretical and experimental basis for clinical prevention and treatment of postmenopausal osteoporosis.Material and method:Part 1:theoretical studyThrough literature review and theoretical analysis,this paper discusses the close relationship between kidney's essence qi,blood,yin and yang and bone.,explains the basic pathogenesis of postmenopausal osteoporosis,and discusses the correlation between kidney essence and bone marrow mesenchymal stem cells from two aspects of origin and function.To further explore the important treatment of postmenopausal osteoporosis by tonifying the kidney,invigorating qi and activating blood circulation.Part 2:in vivo experiments90 female SD rats of SPF grade were randomly divided into 5 groups:normal group?n=18?,model blank group?n=18?,high dose group?n=18?,low dose group?n=18?and Fosamax group?n=18?.The animal model of postmenopausal osteoporosis was established by one-time removal of bilateral ovaries in rats,and no operation was performed in the normal group.The rats in the normal group and the model blank group were given 0.9%saline orally,and the other groups were given the corresponding drugs.The rats in the model blank group were given the medicine from the second day after the operation,once a day for12 weeks.After the last gavage,anaesthesia,open surgery,blood from the abdominal aorta,and 3000r/m after 15 minutes of centrifugation with in,the serum was taken out,the kidney and the femur of the left hind limb were taken out.The bone mineral density of the left hind limb of rats in each group was measured by dual energy X-ray,the activity of serum ALP,TRACP was measured by ELISA method,and the Wnt7b,Axin2,Runx2 mRNA and protein activity expression of bone and kidney tissue was detected by qRT-PCR and ELISA methods,Detection of Runx2,?-catenin protein expression in bone and kidney by Western method.Part 3:in vitro experimentsExperimental drugs:icariin and Rehmannioside D are the effective components of Epimedium brevicornu Maxim and Rehmannia glutinosa Libosch respectively,which are Chinese herbal medicines for tonifying kidney.Cornu cervi pantotrichum extract is a self-made freeze-dried powder of pilose antler in our laboratory,and Astragaloside IV is an effective ingredient of Astragali Radix,which is a Chinese herbal medicine for invigorating qi.Hydroxysafflor Yellow A is an active component of Carthamus tinctorius L,which is a Chinese herbal medicine for activating blood circulation.The rat bone marrow mesenchymal stem cells were divided into the following 9 groups:normal group,osteogenic induction group,tonifying kidney group,invigorating qi and activating blood circulation group,tonifying kidney and invigorating qi and activating blood circulation group,osteogenic induction add DKK-1 group,tonifying kidney add DKK-1group,invigorating qi and activating blood circulation add DKK-1 group,tonifying kidney and invigorating qi and activating blood circulation add DKK-1 group.The formation of mineralization nodules on the 15th day was observed by alizalin red staining.qRT-PCR method was used to detect the expression of Wnt7b,Axin2,Runx2 mRNA and ELISA method was used to detect the expression of Wnt7b,Axin2,Runx2 protein.Result:Part 1:theoretical studyThe fundamental disease of postmenopausal osteoporosis lies in the kidney,kidney deficiency and blood stasis is one of its common syndromes.From the point of view of"kidney deficiency and blood stasis",kidney essence deficiency will induce to qi,blood,yin and yang of kidney imbalance,blood stasis caused by deficiency,bone marrow is not born,bone marrow is reduced and bone is withered.Leading to postmenopausal osteoporosis.The method of tonifying kidney,invigorating qi and activating blood circulation can improve the pathogenesis of kidney deficiency and blood stasis by regulating qi and blood circulation through tonifying kidney and promoting blood circulation through regulating qi.It is an effective method to prevent and cure postmenopausal osteoporosis.Part 2:in vivo experiments1.The bone mineral density?BMD?of the left hind limb in the model blank group was significantly lower than that in the normal group?P<0.01?.Compared with the model blank group,the BMD of the left hind limb in each treatment group was significantly higher?P<0.01?.2.The results of bone metabolism showed that the levels of serum ALP and TRACP in the model blank group were significantly higher than those in the normal group?P<0.01?.Compared with the model blank group,the levels of serum ALP and TRACP in the treatment groups were significantly lower?P<0.01?,especially in the Fosamax group,and that in the high dose group of tonifying kidney,invigorating qi and activating blood circulation recipe was the second?P<0.01?.3.The results of qRT-PCR showed that the relative expression of mRNA in Wnt7b,Axin2,Runx2 of bone and kidney tissue in the model blank group was significantly lower than that of normal group?P<0.05?.Compared with the model blank group,the relative expression of mRNA in Wnt7b,Axin2,Runx2 of high dose group of tonifying kidney,invigorating qi and activating blood circulation recipe and Fosamax group was significantly up-regulated?P<0.05?.Compared with the low dose group,the relative expression of Wnt7b,Axin2 mRNA in bone and kidney tissue was significantly up-regulated in the high dose group of tonifying kidney,invigorating qi and activating blood circulation recipe?P<0.05?.The relative expression of mRNA in Runx2 of renal tissue was significantly up-regulated?P<0.05?,but there was no significant difference in mRNA expression of Runx2 in bone tissue?P>0.05?.4.The results of ELISA showed that compared with normal group,the expression of Wnt7b,Axin2,Runx2 protein in bone and kidney tissue was significantly decreased in model blank group?P<0.01?.Compared with the model blank group,the expression of Wnt7b,Axin2,Runx2 protein in the high dose group of tonifying kidney,invigorating qi and activating blood circulation recipe and Fosamax group were up-regulated obviously?P<0.01?.5.The results of Western showed that the expression of Runx2,?-catenin protein in the model blank group of bone and kidney tissue was significantly lower than that of normal group?P<0.05?.Compared with model blank group,the expression of Runx2,?-catenin protein in bone and kidney tissue of Fosamax group and high dose group of tonifying kidney,invigorating qi and activating blood circulation recipe was significantly up-regulated?P<0.05?.The expression of Runx2 and?-catenin protein in the high dose group of tonifying kidney,invigorating qi and activating blood circulation recipe was significantly higher than that in the low dose group of tonifying kidney,invigorating qi and activating blood circulation recipe?P<0.05?.Part 3:in vitro experiments1.The results of alizalin red staining on the 15th day showed that the number of mineralized nodules in osteogenic induction group was the largest and the volume was larger than that in other groups.The mineralization nodules in tonifying kidney group,invigorating qi and activating blood circulation group and tonifying kidney,invigorating qi and activating blood circulation group were more than those in normal group,and the volume was significantly larger,especially in tonifying kidney,invigorating qi and activating blood circulation group.After adding DKK-1 to osteogenic induction group,tonifying kidney group,invigorating qi and activating blood circulation group and tonifying kidney,invigorating qi and activating blood circulation group,the number of mineralization nodules decreased significantly,the volume was small,and the mineralization level decreased.2.The results of qRT-PCR showed that the Wnt7b,Axin2 and Runx2 mRNA relative expression of osteoblasts in osteogenic induction group,tonifying kidney,invigorating qi and activating blood circulation group and tonifying kidney,invigorating qi and activating blood circulation add DKK-1 group was up-regulated?P<0.05?.Compared with osteogenic induction group,the Wnt7b mRNA relative expression of osteoblasts in osteogenic induction add DKK-1 group and invigorating qi and activating blood circulation add DKK-1 group was down-regulated?P<0.05?,the Axin2 mRNA ralative expression of tonifying kidney group,invigorating qi and activating blood circulation group,osteogenic induction add DKK-1 group,tonifying kidney add DKK-1 group and invigorating qi and activating blood circulation add DKK-1 group was down-regulated?P<0.05?.The relative expression of Runx2 mRNA in invigorating qi and activating blood circulation group,osteogenic induction add DKK-1 group and invigorating qi and activating blood circulation add DKK-1 group was down-regulated?P<0.05?.After adding DKK-1,the relative expressions of Wnt7b,Axin2 and Runx2 mRNA in tonifying kidney group,invigorating qi and activating blood circulation group and intonifying kidney,invigorating qi and activating blood circulation group were down-regulated?P<0.05?.3.The results of ELISA showed that the expression of Wnt7b,Axin2 and Runx2 protein in osteoblasts was up-regulated in osteogenic induction group,tonifying kidney group and tonifying kidney,invigorating qi and activating blood circulation group?P<0.05?.Compared with osteogenic induction group,the expression of Wnt7b,Axin2 and Runx2 protein in osteoblasts was down-regulated in invigorating qi and activating blood circulation group,tonifying kidney,invigorating qi and activating blood circulation group,osteogenic induction add DKK-1 group,tonifying kidney add DKK-1 group,invigorating qi and activating blood circulation add DKK-1 group,tonifying kidney,invigorating qi and activating blood circulation add DKK-1 group?P<0.05?.After adding DKK-1,in tonifying kidney group,invigorating qi and activating blood circulation group and tonifying kidney,invigorating qi and activating blood circulation group,the protein expressions of Wnt7b,Axin2 and Runx2were down-regulated?P<0.05?.Conclusion:1.Kidney deficiency is the fundamental pathogenesis of postmenopausal osteoporosis,and blood stasis is the key pathogenesis of postmenopausal osteoporosis.The method of tonifying kidney,invigorating qi and activating blood circulation treatment and its compound tonify the kidney and generate essence through invigorating qi,and then promote the operation of qi and blood,regulate the balance of yin and yang,thereby improving the changes of postmenopausal osteoporosis kidney deficiency and blood stasis.The theoretical basis is sufficient.2.Tonifying kidney,invigorating qi and activating blood recipe can increase the bone mineral density of femur and significantly decrease the expression of ALP and TRACP in serum of postmenopausal osteoporosis rats,which indicates that Tonifying kidney,invigorating qi and activating blood recipe could improve bone mineral density and bone conversion rate in postmenopausal osteoporosis rats,thus effectively preventing and treating postmenopausal osteoporosis.3.The mechanism of tonifying kidney,invigorating qi and activating blood recipe is to up-regulate the expression of Wnt7b,Axin2,Runx2 mRNA and protein in bone and kidney tissue of postmenopausal osteoporosis rats,and the expression of?-catenin protein in bone and kidney tissue is dose-dependent.Wnt7b/?-catenin signaling pathway related factors are up-regulated to promote osteogenic differentiation,which plays a preventive and therapeutic role in postmenopausal osteoporosis.4.The functional combination of effective components of tonifying kidney,invigorating qi and activating blood circulation can enhance the formation of mineralization nodules,promote osteogenic differentiation of BMSCs and maintain normal bone mass.5.The functional combination of effective components of tonifying kidney,invigorating qi and activating blood circulation can up-regulate the expression of Wnt7b,Axin2,Runx2mRNA and protein in Wnt7b/?-catenin signaling pathway to promote osteogenic differentiation of BMSCs. |
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