Studies On Effects And Mechanism Of Iridoid Glycoside And Anthraquinone Compounds From Morindae Officinalis Radix For Regulating Bone Metabolism

Osteoporosis(OP)is a metabolic bone disease characterized by reduced bone mass,destroyed bone microstructure as well as increased risks of bone fragility and fracture.Osteoporosis can be induced by multiple factors including estrogen deficiency,inflammation and oxidative stress in particular.Estrogen deficiency can induce bone loss and osteoporosis,Chronic inflammation can also perturb bone metabolism and accelerate bone loss with increased bone resorption and impaired bone formation.In many inflammatory diseases such as rheumatoid arthritis,inflammatory bowel disease and periodontitis,increased inflammatory cytokines can inhibit osteoblast activity and bone formation as well as increase the bone resorption of osteoclasts,resulting in bone loss induced by inflammation.Morindae officinalis Radix prepared from the dry radix of Morinda officinalis How.has the function of nourishing yin,tonifying kidney,strengthening bone and enhancing immunofunction in the treatment of impotence,menstrual disorders,osteoporosis,diabetes and inflammatory diseases such as rheumatoid arthritis and dermatitis.Monotropein as a major iridoid glycoside extracted from the root of Morind officinalis How.has been demonstrated to possess anti-inflammatory and anti-nociceptive activities in vivo.In our previous study,it's found that monotropein could inhibite the bone loss in OVX mice and promote the formation and differentiation of osteoblasts,demonstrating that monotropein has bone protective activity.Rubiadin and rubiadin-1-methyl ether(RBM)are the main anthraquinone compounds isolated from the root of Morinda officinalis How..Our previous study found that Rubiadin and RBM obtained from Morinda officinalis How.could decrease the TRAP and bone resorption of osteoclas.Nevertheless,the further effects of rubiadin and RBM on formation and differentiation of osteoclasts and the underlying mechanisms remain unclear.In the present study,we investigated the effects and the involved mechanism of monotropein on inflammatory bone loss as well as the effects of rubiadin and RBM on osteoclastogenesis and the underlying mechanisms.1 In vivo anti-inflammatory bone loss evaluation of monotropeinMonotropein was evaluated for the anti-inflammatory bone loss effects in mouse model induced by LPS.The index of serum and urine was determined by using assay kit.Themicroarchitecture and micro-structure parameters were measured by Micro-CT analysis.Compared with the control group,the levels of IL-6 and IL-1? were significantly increased,the trabecular bone gap was significantly increased,and the number of trabecular bone decreased significantly in the model group.However,Monotropein could significantly elevate the activity of ALP and PICP and decrease the levels of IL-6,IL-1?,TRACP and RANKL.What's more,mootropein could increase TB.N,BVF and BV/TV as well as decrease TB.SP.The results demonstrated that monotropein could increase bon formation and inhibit bone resorption.In addition,monotropein colud inhibit the expression of P-P65,TRAF6 and bone resorption related protions such as NFATC1,C-Fos,MMP9 and CtsK as well as promote the expression of I?B?.These results suggest that monotropein might be used as a therapeutic agent for the treatment of inflammatory bone loss.What's more,monotropein could promote the expression of BMP2,Wnt3 a,LRP5/6 and Osterix,increase the phosphorylation of GSK3? and the ratio of P-Smad1/5/8 and Smad1,and decrease the phosphorylation of ?-catenin,demonstrating that monotropein could promote the activiation of BMP and Wnt signaling pathway and increase bone formation.2 Investigation of effects and mechanism of monotropein on osteoblasts injuried by LPSMC3T3-E1 osteoblasts injured by LPS were adopted to investigate the antiinflammatory bone loss effect of monotropein.LPS significantly inhibited the proliferation,ALP activity and mineralization of MC3T3-E1 cells,promoted the expression of IL-6,NO and IL-1?,and increased the phosphorylation of IKK,I?B? and P65,along with the degradation of I?B? and nuclear translocation of P65 and P50.However,monotropein increased the proliferation and activity of alkaline phosphatase(ALP),bone matrix mineralization and the expression of bone matrix protein osteopontin(OPN)in osteoblastic MC3T3-E1 cells injured by LPS.In addition,monotropein significantly decreased the production of IL-6 and IL-1?,inhibited the nuclear translocation of P65 and NF-?B P50,and down-regulated the phosphorylation of NF-?B P65 and IKK,indicating that monotropein could attenuate inflammatory impairment to MC3T3-E1 cells by suppressing the activation of NF-?B pathway.All these results suggest that monotropein may prove to be a promising candidate for the prevention and treatment of inflammatory bone loss.3 Investigation of effects and mechanism of monotropein on osteoclasts induced by LPSThe LPS-induced osteoclasts were used to investigate the anti-inflammatory bone loss effect of monotroepin.Monotropein could effectively inhibit the formation and differentiation of osteoclasts as well as inhibit the expression of TRAP activity.Monotropein could significantly inhibit the expression of TRAF6 and P-P65,inhibit the degradation of I?B?,thus effectively downregulated the expression of osteoclastrelated proteins,including NFATC1,C-Fos,MMP9 and CtsK as shown by Western blot,indicating that monotropein could inhibit the bone resorption of osteoclasts.4 Investigation of effects and mechanism of anthraquinone compounds on osteoclasts induced by RANKLThe RANKL-induced osteoclasts were used to investigate the anti-osteoporosis effect of rubiadin and rubiadin-1-methyl ether in vitro.Rubiadin and rubiadin-1-methyl ether at the dose that did not affect the viability of cells significantly inhibited RANKL-induced osteoclastogenesis and actin ring formation of osteoclast,while rubiadin and rubiadin-1-methyl ether performed a stronger effect at the early stage.In addition,rubiadin and rubiadin-1-methyl ether downregulated the expression of osteoclastrelated proteins,including NFATC1,C-Fos,MMP9 and CtsK as shown by Western blot.Furthermore,rubiadin and rubiadin-1-methyl ether inhibited the phosphorylation of P65 and the degradation of I?Ba as well as decreased the nuclear translocation of P65.Collectively,the results suggest that rubiadin and rubiadin-1-methyl ether inhibit osteoclastic bone resorption may through blocking NF-?B pathway and may be promising agents for the prevention and treatment of bone diseases characterized by excessive bone resorption.