Based On The "kidney-marrow-bone" Theory And The BMSCs Wnt/β-catenin Signaling Pathway,exploring The Osteoprotective Effect Of Rhizoma Drynariae On Ovariectomized Rats

Objective:To investigate the osteoprotective effect of Rhizoma Drynariae on ovariectomized rats based on the theory of " kidney-marrow-bone" and the Wnt/β-catenin signaling pathway of BMSCs,including its promotion of bone formation and inhibition of bone resorption.Methdos:This experiment was divided into three parts.Experiment 1: Using Micro-CT,bone tissue section,and determination of TRAP,MMP-9,CTX-1 to observe the effect of Rhizoma Drynariae on ovariectomized rats bone,including microstructure,intra-bone adipose tissue and serum bone resorption indicators.Experiment 2: Western-Blot and Real-time PCR techniques were used to observe the effects of Rhizoma Drynariae on Wnt/β-catenin signaling pathway-related factors,such as Wnt3 a,Wnt1,β-catenin,Cyclin D,C-myc,lef1,PPAR-γ and C/EBP-α.Using ALP staining and quantitative analysis,alizarin red calcium nodule staining and quantitative analysis,oil red staining and quantitative analysis to observe the effect of Rhizoma Drynariae on osteogenic differentiation and adipogenic differentiation of BMSCs.Experiment 3: Establish a coculture system of BMSCs and OC.After Hematoxylin-Eosin(HE)staining and Tartrate Resistant Acid Phosphatase(TRAP)staining of osteoclasts,the differentiation and maturation of osteoclasts were observed under microscope,meanwhile osteoclasts were counted.In order to observe the effect of BMSCs on the proliferation and activity of osteoclasts,the markers of Wnt10 b,β-catenin,OPG,RANKL and TRAP in BMSCs were detected by Western-Blot and PCR,and OPG,RANKL content in the coculture were detected by ELISA.Results:Experiment 1: Rhizoma Drynariae can improve bone mineral density and bone microstructure,reduce adipose tissue in the marrow cavity,and serum bone resorption index(TRAP,MMP-9,CTX-1).Experiment 2: Rhizoma Drynariae can activate the Wnt/β-catenin signaling pathway,increase the expression of Wnt1,Wnt3 a,β-catenin,C-myc and Cyclin D genes in the signaling pathway,and increase the protein content of Wnt3 a,Wnt1,β-catenin and lef1.At the same time,it can also reduce the gene expression and protein content of PPAR-γ and reduce the content of C/EBP-α protein.Under osteogenic induction,Rhizoma Drynariae can increase the quantitative results of ALP staining and the quantitative results of bilirubin staining in BMSCs.Under the induction of adipogenesis,Rhizoma Drynariae can reduce the quantitative results of oil red staining.Experiment 3: According to the morphology of osteoclasts and the number of osteoclasts: Rhizoma Drynariae can inhibit the number and differentiation of osteoclasts in the co-culture system,promote the content of Wnt10 b,β-catenin,OPG protein and gene expression in BMSCs,inhibit the content of RANKL protein and gene expression in BMSCs,and inhibit TRAP gene expression in osteoclasts.The results of ELISA showed that the content of OPG in the culture medium was increased and the RANKL content was inhibited after the gavaging.Conclusion:1.Rhizoma Drynariae has obvious osteoprotective effect.Based on the study of BMSCs Wnt/β-catenin pathway,it can activate the Wnt/β-catenin signaling pathway,regulate the relationship between β-catenin and PPAR-γ,regulate the differentiation of BMSCs,promote osteogenic differentiation and inhibit adipogenic differentiation,promote bone formation.At the same time,Rhizoma Drynariae may act on BMSCs through Wnt/β-catenin signaling pathway,regulate OPG and RANKL secretion,inhibit OPG/RANKL/RANK pathway in osteoclasts,inhibit osteoclast differentiation and maturation,and inhibit bone resorption.In short,Rhizoma Drynariae has osteoprotective effect by promoting bone formation and inhibiting bone resorption.2.According to the traditional Chinese medicine "kidney-marrowbone" theoretical system,we consider that BMSCs can be associated with the "marrow" of traditional Chinese medicine,at least can be identified as the cytological basis of the "marrow" of traditional Chinese medicine.At the same time,we further propose that under the traditional Chinese medicine "kidney-marrow-bone" theory system,it may be considered to associate "kidney" or " kidney essence" with various signal pathways in modern medicine that regulate the proliferation and differentiation of BMSCs,such as Wnt/β-catenin signaling pathway,BMP signaling pathway.At least it can be regarded as a specific action pathway or mechanism of "kidney essence" nourishing the "marrow",further enriching and developing the traditional Chinese medicine " kidney dominates the bone and derives the marrow"," replenishing the kidney,filling the bone marrow,and strengthening the bones" theory.