Study On The Anti-Osteoporosis Activity And Mechanism Of Flavonoids From Cortex Mori

With an increase of the aging population in our society,the incidence of osteoporosis is rising year by year.Osteoporosis is a systemic bone disease associated with abnormal bone metabolism and reduced bone mineral density,ultimately leading to a high fracture risk.Osteoblasts and osteoclasts are involved as the basic regulatory units in the bone remodeling system.Bone homeostasis is dynamically regulated through the coordinated action of osteoclasts and osteoblasts.Increased osteoclast-induced bone resorption and inadequate osteoblast-mediated bone formation can break the dynamic balance and lead to the occurrence of osteoporosis.Therefore,the agents targeting osteoblast-osteoclast coupling that inhibit osteoclastic resorption and/or promote osteoblastic formation may become the promising candidates to prevent osteoporosis.Traditional Chinese medicine（TCM）plays an important role in the prevention and treatment of osteoporosis.TCM contains a large number of bioactive chemical ingredients,which is a treasure for people to explore active components,lead compounds and clinical drugs.Cortex Mori（Sangbaipi）is the dried root bark of Morus alba L.,which is rich in flavonoids.Our preliminary screening study found that Cortex Mori could inhibit the formation of osteoclasts,suggesting that it had the potential osteoprotective effect for osteoporosis.In this paper,the research on Cortex Mori was further carried out.The purpose of this study was to discover the bioactive ingredient with anti-osteoporosis effect from Cortex Mori and explore its potential mechanism by using cell models of osteoclasts and osteoblasts.The anti-osteoporosis activity in vivo of the compounds was further evaluated in the animal model.This study will provide technical support for the research and development of anti-osteoporosis drugs.The main content of this paper is as follows:1.Effect of Cortex Mori Extract on the Activity of Osteoblasts and OsteoclastsThe water extract（W）and ethanol extract（E）from Cortex Mori were prepared by reflux extraction,and the flavonoids in the ethanol extract were further enriched by macroporous resin and polyamide column chromatography to obtain purified extract R and PA.The content of flavonoids in Cortex Mori extract was determined by visible spectrophotometry,and the results were as follows:PA>R>E>W.MTT assay indicated that all of the extracts could promote the proliferation of MC3T3-E1Subclone 14 cell and the proliferation effect was enhanced with the increase of flavonoid content.The inducing conditions of osteoblasts were investigated and determined.The result of ALP activity indicated that extract E,R,PA exhibited significant stimulatory effect on MC3T3-E1 differentiation.Bone marrow cells were obtained and induced by 1α,25-（OH）₂VitD₃ to establish the cell model of osteoclasts.The four extracts within a certain range of doses were found to inhibit osteoclast formation,as demonstrated by a lower number of TRAP-positive multinuclear cells compared to the control group.2.Screening Study of Active Compound from Cortex Mori（Sangbaipi）Fifteen compounds derived from Cortex Mori were selected for the study of anti-osteoporosis activity.Among the compounds,there were eight flavonoids,and the rest seven belonged to distyrene and its glucosides,benzofurans,coumarins,alkaloids and pentacyclic triterpenoids,respectively.The results showed that the activity of flavonoids was better than that of non-flavonoids in general.Among eight flavonoids,seven compounds were found to stimulate osteoblast proliferation and inhibit osteoclast formation at the same time,including Sanggenon C,Sanggenon D,Mulberrin,Morusin,Kuwanon G,Kuwanon H and Morusignin L.Among seven non-flavonoids,resveratrol and oxyresveratrol were found to inhibit osteoclast formation,but the other five compounds could promote the formation of osteoclasts at different levels.3.Inhibitory Effect of SC,SD on Osteoclasts and its Molecular MechanismSanggenon C（SC）and Sanggenon D（SD）are stereoisomers of each other,and they are the main isopentenyl flavonoids in Cortex Mori.SC,SD were found to inhibit osteoclast formation and bone absorption,as demonstrated by a lower number of TRAP-positive multinuclear cells,and a fewer area of bone resorption pits compared to the control group.To further illustrate the effect of SC,SD on osteoclasts,we selected several genes that associated with bone development,including TRAF6,NFATc1,TRAP,CTSK and MMP9.The results of real-time PCR and western blotting indicated that TRAF6,NFATc1,TRAP,CTSK mRNA expression and TRAF6,CTSK protein expression were downregulated in SC,SD treated groups.4.Promoting Effect of SC,SD on Osteoblasts and its Molecular MechanismThe effect of SC,SD on cell proliferation was evaluated by MTT assay.The results indicated that SC,SD could promote the proliferation of MC3T3-E1 Subclone14 and the proliferation effect was enhanced with the increase concentration.Alkaline phosphatase（ALP）is an early phenotypic marker of osteoblastic differentiation.The formation of mineralized nodules is in the final stage of osteoblast differentiation.The promoting effect of SC,SD within a certain range of concentrations on the osteoblastic differentiation and mineralization was evaluated by using ALP assay and the mineralized nodules stained with Alizarin red.To elucidate the mechanism,the osteogenic related genes were examined,including Runx2,Collagen Ⅰ,Osteopontin,β-catenin,OPG and RANKL.The results of real-time PCR and western blotting indicated thatβ-catenin,Runx2,Collagen Ⅰ,Osteopontin mRNA expression and/or protein expression were upregulated in SC,SD treated groups.The expression of OPG/RANKL was also upregulated in SC treated groups.5.Effect of SC and SD on Prednisone-induced Osteoporosis in Zebrafish ModelThe anti-osteoporosis effect of SC,SD in vivo was evaluated in zebrafish model induced by prednisone.Compared to the model group,the integral optical density of vertebrate column increased significantly in SC（1μM、3μM、10μM）,SD（0.1μM、0.3μM、1μM）treated zebrafish.The improvement rate of vertebrae mineralization in10μM SC treated group was equivalent to that of the positive control group（etidronate disodium）.The results suggested that SC,SD could reverse the bone loss of zebrafish induced by prednisone and improve the bone mineral density（BMD）.In conclusion,through the study on Cortex Mori extract and the screening of active compounds from Cortex Mori,SC and SD were found to have a good activity as main isopentenyl flavonoids of Cortex Mori.The results indicated that SC,SD exhibited the anti-osteoporosis activity in both cell models in vitro and animal experiments in vivo.It was speculated that the anti-osteoporosis mechanism was as follows:（1）The inhibitory effect of SC,SD on osteoclasts was probably associated with the suppression of RANKL/RANK signaling pathway.SC,SD could inhibit the expression level of TRAF6 and NFATc1,subsequently the expression of osteoclast-specific genes such as TRAP,CTSK were downregulated.（2）The promoting effect of SC,SD on osteoblasts was probably related to the activation of Wnt/β-catenin signaling pathway.Subsequently this pathway led to the activation of Runx2,ultimately upregulated the expression of osteogenic related genes such as Collagen Ⅰ,Osteopontin.