The Role Of Wnt/β-catenin Pathway And OPG/RANKL/RANK Axis In The Anti-osteoporosis Mechanism Of Metformin Combined With Total Flavonoids Of Rhizoma Drynariae

Osteoporosis（OP）is a metabolic bone disease characterized by bone loss and deterioration of bone tissue microstructure.Osteoporosis occurs more commonly in the elderly population,particularly in postmenopausal women.A decrease in estrogen levels in women triggers rapid bone loss resulting from an active bone turnover with an imbalance between bone formation and bone resorption.Currently,a wide range of drugs are available for the clinical prevention and management of osteoporosis;however,these drug therapies have numerous disadvantages.There is an urgent demand for alternative treatments with a good safety profile to increase bone mass and prevent fractures.Total flavonoids of Rhizoma Drynariae（TFRD）has been used as traditional Chinese patent medicine for treating osteoporosis.Moreover,TFRD is more effective in combination with other drugs to treat osteoporosis in clinic.Metformin（Met）,as a drug for the first-line treatment of type 2 diabetes,has a high safety and efficacy,and Met has played a huge potential in the treatment of diabetes osteoporosis.Therefore,we chose the combination of Met and TFRD to observe the anti-osteoporosis effects and mechanisms of Met combined with TFRD.Firstly,we analyzed the underlying mechanisms of Met and TFRD regulating osteocytes through transcriptomics and phosphorylated proteomics.Transcriptomic and phosphorylated proteomics results indicated that Met and TFRD affected calcium signaling pathway,phosphatidylinositol 3 kinase（PI3K）-protein kinase B（PKB/AKT）signaling pathway,Wnt signaling pathway,mitogen-activated protein kinase（MAPK）signaling pathway and osteoclast differentiation signaling pathway in osteocytes.The previous experimental researches and literature searches supported that Met and TFRD could act on Wnt/β-catenin signaling pathway to stimulate osteogenesis regulated through osteocytes;for osteoclastogenesis,Met and TFRD could act on osteoclast differentiation pathway to inhibit osteoclast function.Subsequently,we will use a series of in vivo and in vitro experiments to verify the anti-osteoporosis effects and mechanisms of Met and TFRD.In vitro,we selected the main types of bone tissue cells as the subject,namely osteoprogenitor cells（BMSCs）,osteoblast precursor cells（MC3T3-E1）,osteocytes（IDG-SW3）and osteoclasts（RAW264.7）.The optimal concentrations of Met and TFRD to stimulate osteocytes and inhibit osteoclasts were 0.1 m M and 1 mg/L,respectively,determined by MTT method.Subsequently,the effects of Met combined with TFRD on the differentiation of osteoblasts and osteoclasts were observed through scratch test,alkaline phosphatase（ALP）staining,mineralized nodule staining,immunofluorescence,tartrate resistant acid phosphatase（TRAP）staining,bone resorption lacuna and Western blot assay.In vitro experiments showed that Met combined with TFRD significantly promoted the migration of osteoprogenitor cells and stimulate the differentiation and maturation of osteoblast precursor cells.In addition,Met combined with TFRD significantly inhibited the expression of osteogenic inhibitor sclerostin（SOST）and dickkopf-1（DKK1）in osteocytes,and Met combined with TFRD also significantly downregulated receptor activator for nuclear factor-kappa B ligand（RANKL）/osteoprotegerin（OPG）ratio.In terms of osteoclast,Met combined with TFRD significantly inhibited osteoclast differentiation and bone resorption function.Our results showed that Met combined with TFRD stimulated osteoblast differentiation and inhibited osteoclast function,and more significant effect was showed in combination of Met and TFRD treatment compared to TFRD treatment alone.To further evaluate the anti-osteoporosis effects and mechanisms of Met combined with TFRD in vivo,we successfully replicated the osteoporotic model using ovariectomized rats.The osteoporotic rat were administrated by gavage 1 mg·kg^-1·day^-1 positive drug raloxifene（RLX）,100 mg·kg^-1·day^-1 Met,72 mg·kg^-1·day^-1TFRD and 100 mg·kg^-1·day^-1 Met+72 mg·kg^-1·day^-1 TFRD.After 10 weeks of treatment,rats were anesthetized and sacrificed,and their serum,long bones,and organs included the heart,liver,kidney,and brain were collected.In the experiment,serum biochemical indices of rats were detected.The anti-osteoporosis effects and mechanisms of Met combined with TFRD were observed through Micro CT,three-point bending test,HE staining,immunohistochemistry staining,and Western blot assay.The results of in vivo experiments showed that Met did not decrease blood glucose levels in osteoporotic rats,the combination of Met and TFRD treatment has no significant hepatorenal toxicity in osteoporotic rats.For bone metabolism,Met combined with TFRD significantly decreased the levels of bone resorption markers such as type-I collagen carboxy-terminal peptide（CTX-1）and TRAP in serum of osteoporotic rats,and significantly increased the level of bone formation marker,propeptide of type I procollagen（PINP）.In addition,Met combined with TFRD improved bone mineral density（BMD）and bone microstructure,and enhanced the mechanical properties of osteoporotic rats.On the one hand,Met combined with TFRD reduced SOST and DKK1 protein expression in bone tissue of osteoporotic rats.Moreover,the combined treatment stimulated Wnt10b andβ-catenin protein expression and inhibited the phosphorylation of glycogen synthesis kinase 3 beta（GSK3β）.Met combined with TFRD also upregulated the expression of ALP and Runx2 proteins.On the other hand,Met combined with TFRD reduced the RANKL/OPG ratio in bone tissue of osteoporotic rats.Besides,Met combined with TFRD inhibited receptor activator of nuclear factor-kappa B（RANK）and its downstream nuclear factor of activated T cells c1（NFATc1）and TRAP protein expression.These results indicated that Met combined with TFRD treatment improved bone quality and bone strength in osteoporotic rats,and the effect of the combined treatment was superior to TFRD treatment alone.It was verified that Met combined with TFRD could activate Wnt/β-catenin signaling pathway to stimulate bone formation,while decreasing RANKL/OPG ratio inhibited bone resorption.To sum up,this study elucidated the anti-osteoporosis effects and mechanisms of Met combined with TFRD.At the cellular level,Met combined with TFRD treatment was more effective than TFRD treatment alone in stimulating the differentiation of osteoprogenitor cells and osteoblast precursor cells,inhibiting osteoclastogenesis and bone resorption function.In vivo experiments have shown that Met combined with TFRD has a better effect on improving bone quality and biomechanical properties in osteoporotic rats than TFRD treatment alone.We demonstrated that osteogenic inhibitors SOST and DKK1 derived from osteocytes,as well as osteoclast transcription factors RANKL and OPG,play important roles in regulating osteogenic and osteoclastic signaling pathways.The results of in vitro and in vivo experiments confirmed that Met combined with TFRD had a positive therapeutic effect on osteoporosis.