Role Of Osteocytes In The Antagonism Of Total Flavonoids And Active Monomers Of Rhizoma Drynariae Against Retinoic Acid Induced Bone Loss In Rats

Osteoporosis（OP）is a common chronic bone metabolic disease characterized by damage to bone microstructure,decreased bone mass and bone mineral density（BMD）,along with an increased risk of fracture.Drugs used in the treatment of osteoporosis include drugs that inhibit bone resorption,drugs that promote bone formation and other supplements,but these drugs have shortcomings such as long medication cycle and serious side effects.Therefore,traditional Chinese medicine for the treatment of osteoporosis has become an emerging therapy.Total flavonoids of Rhizoma Drynariae（TFRD）is a commonly used orthopedic medicine in clinical practice,known as phytoestrogens.Previous studies have shown that TFRD and its monomers Naringin（Nar）,Neoeocitirin（Neo）,and Naringenin（NG）play important roles in bone metabolism.However,previous studies paid more attention to the role of TFRD and its monomers in osteoblasts and osteoclasts,with limited studies on osteocytes.Therefore,we investigated the effects of TFRD and its monomers on osteocyte vitality and expression of bone metabolism related factors.At the same time,we compared and analyzed the anti-osteoporotic effects of TFRD and its monomers,and analyzed the effects and related pathways of TFRD and its active monomers on the regulation of osteoclast differentiation by bone cells.Firstly,we identified the content of active monomers Nar and Neo in the total flavonoids of Rhizoma Drynariae.Considering that NG is a glycoside of Nar,high-performance liquid chromatography（HPLC）was used in this study to detect the content of the two most abundant monomer components,Nar and Neo,in TFRD.The linear relationship of this method is good,and the precision,repetition rate,and recovery rate of this method all meet the requirements.We detected 272.624 mg of Nar and 217.434mg of Neo in TFRD,both accounting for over 20%.Given that osteocytes are the main source of receptor activator for nuclear factor-κB ligand（RANKL）that regulates osteoclast differentiation,there are few studies on the effects of active monomers targeting TFRD on osteocytes.This study systematically analyzed the effects of different concentrations of Neo,NG,and Nar on osteocyte activity,and identified the optimal concentrations of the three monomers for stimulating osteocytes.The results showed that 5μM Neo,10μM NG,and 50μM Nar significantly stimulated osteocyte activity.On this basis,the effects of the three active monomers on the activity of major types of cells in bone tissue,such as osteocytes,osteoblasts,and osteoclasts,were identified over different experimental periods.The results showed that Nar inhibited osteoclast activity to varying degrees and stimulated osteocyte activity,but its stimulating effect on osteoblasts was not significant.Long-term exposure to Neo significantly stimulated osteoblasts and osteocytes,while inhibiting osteoclasts,showing the effect characteristics of Nar and Neo on osteogenesis and osteoclastogenesis.We detected the gene expression changes of osteoblast inhibitor sclerostin（SOST）,Dickkopf-related protein 1（DKK1）,and RANKL in osteocytes after treatment with Neo,NG,and Nar by PCR.The results showed that Neo and Nar significantly inhibited the expression of SOST and RANKL genes secreted by osteocytes,while NG significantly inhibited the expression of DKK1 gene.These results suggest that these three monomers also have a certain effect on regulating osteogenesis and osteoclastogenesis.Next,we successfully reproduced a rat model of osteoporosis with significant bone loss by administering retinoic acid（RA）at a dose of 90 mg?kg^-1?day^-1 for 14consecutive days through gavage.After subcutaneous injection of 0.1 mg?kg^-1?day^-1alendronate sodium（Al）,gavage with 58 mg?kg^-1?day^-1 TFRD,50 mg?kg^-1?day^-1NG,and 50 mg?kg^-1?day^-1 Nar for 30 consecutive days,the rats were sacrificed and their serum,long bones,and spines were collected.The biochemical parameters of serum were detected during the experiment,and the effects of TFRD and its monomers on antagonizing bone loss and treating osteoporosis were detected through H&E staining,Micro-CT,tartrate-resistant acid phosphatase（TRAP）staining,immunohistochemical staining,RT-PCR,and Western blot experiments.On the basis of confirming that TFRD,NG,and Nar had no significant effects on the liver and kidney functions of rats,it was demonstrated that TFRD,NG,and Nar could increase bone mineral density in different parts of osteoporotic rats to a certain extent,improve the bone microstructure of tibial metaphysis,and reduce the expression of SOST protein in bone tissue.In addition,TFRD,NG,and Nar treatment also decreased the number of osteoclasts expressing TRAP in bone tissue.These results reflect the inhibitory effect of TFRD,NG,and Nar on bone resorption.On this basis,this study analyzed the expression changes of osteogenic and osteoclastic related factors in bone tissue from different experimental groups.It was found that TFRD,NG,and Nar significantly inhibited the expression of SOST gene in bone tissue,while stimulating the expression of osteogenic regulatory factors Wnt10b andβ-catenin genes to varying degrees,suggesting that it may stimulate osteogenesis through the Wnt/β-catenin signaling pathway.In terms of osteoclasts,TFRD and Nar both significantly inhibited the expression of TRAP gene in bone tissue.We detected changes in these bone formation and bone resorption related factors at the protein level,showing that TFRD,NG,and Nar decreased SOST,TRAP,RANKL,and nuclear factorκB（NF-κB）protein levels in bone tissue.Nar can significantly increase the expression levels of osteogenic-related ALP and PTH1R proteins.From the gene and protein levels,it was verified that TFRD,NG,and Nar can not only reduce bone resorption,but also stimulate bone formation by inhibiting SOST secretion by bone cells.These results demonstrate the antagonistic effects of TFRD,NG,and Nar on osteoporosis and their characteristics of bone formation and resorption,with Nar having a better effect.In this study,we observed that TFRD monomers NG and Nar inhibit the expression of osteoclast differentiation regulatory proteins in bone tissues to varying degrees,but the direct inhibitory effect of TFRD on them is not obvious.Therefore,this study further analyzed the effects of TFRD,NG,and Nar on osteoclast regulation in bone cells.We used Transwell chambers to co-cultivate bone cells and osteoclasts,and denosumab（DMAb）was used as a positive control drug.The differentiation of osteoclasts in the upper layer was observed by TRAP staining,F-actin staining,and molecular biology techniques,as well as the expression changes of osteoclast differentiation markers and key regulatory factors.The results showed that TFRD,NG,Nar,and positive drugs can inhibit the expression of NFATc1,among which Nar and positive drugs have a more obvious inhibitory effect.The expression of RANK upstream of NFATc1 was significantly decreased in NG,Nar,and positive drug groups.Correspondingly,Nar and positive drugs have a stronger inhibitory effect on CTSK and TRAP than TFRD and NG,which can significantly inhibit osteoclast differentiation.Combining the above results,it can be seen that the inhibitory effect of TFRD,NG,and Nar on osteoclast regulation in bone cells can be achieved by reducing the RANK/NFATc1 pathway.Among them,Nar has a significantly better inhibitory effect on osteoclast differentiation than TFRD and NG,and the inhibitory effect is closer to that of positive drugs.In summary,this study clarified that TFRD and its monomers NG and Nar have good anti-osteoporotic effects,and Nar is a more promising and safer bone protective agent.It was also found that Nar can not only directly inhibit bone resorption,but also exert its inhibitory effect on osteoclast function by affecting the osteocyte-mediated osteoclast differentiation pathway.