Study On The Anti-osteoporosis Of Tiliroside

Background: Osteoporosis is an osteolytic bone disease characterized by enhanced bone resorption function of osteoclasts.There are many kinds of cells that can promote bone formation,but osteoclasts are unique cells that can perform bone resorption.Currently,there are a variety of drugs used to prevent and treat osteoporosis.However,due to the strong side effects or general efficacy of these drugs,there is an urgent need to develop new potential anti-osteoporosis drugs.Chinese herbal medicine monomers are natural active ingredients,and most of the flavonoids have anti-inflammatory effects,and also have the characteristics of wide sources,low prices,and toxic and side effects.They have gradually become a hot spot for the study of osteoporosis prevention and treatment drugs.Tiliroside(Tle),extracted from Oriental Paperbush flower,is a kind of flavonoid natural compound with a wide range of pharmacological functions,including anti‐inflammatory,antithrombotic,antiviral,antioxidation and anticarcinogenic activities.A recent study demonstrated neuroprotective effects of the compound.However,there are very few studies on the potential application of Tle in osteoporosis.In this study,we investigated the role of Tle in osteoclastogenesis and bone loss.Experimental methods:(1)In vitro experiment: 1)Extract the required bone marrow-derived macrophages(BMMs)from C57/BL6 mice at 6-8 weeks for 3days of in vitro cell culture(M-CSF stimulation)and passaging,which can be used for subsequent cell experiments.Under the stimulation of M-CSF,after 48 hours of incubation,CCK-8 kit was used to detect whether porcine can affect the proliferation of BMMs.Stimulate the differentiation of osteoclasts under the stimulation of RANKL,and intervene in the process of adding Tiliroside to test whether Tiliroside can affect the differentiation of osteoclasts.2)To detect the effect of Tiliroside on the bone resorption function of osteoclasts.After RANKL stimulation for 3-4 days,BMMs were induced to differentiate into small osteoclasts,which were then passaged to hydroxyapatite,and cultured until large osteoclasts appeared.Observe the area of ??bone resorption.The expression level of F-actin belt of osteoclast bone resorption function protein was further tested to investigate the mechanism of the influence of Tiliroside on bone resorption function.3)The stimulation of RANKL induces osteoclast differentiation for 5 days,during which gradient concentration of Tiliroside is added to treat samples.Then use RT-PCR instrument to detect the expression level of osteoclast differentiation-related genes.4)Use WB experiments to investigate the possible molecular mechanism of the influence of Tiliroside on osteoclast differentiation and bone resorption function,in order to clarify the signal pathway of Tiliroside.(2)In vivo experiments: 32 eight-week-old male C57/BL6 mice were randomly divided into four equal groups: Sham group,OVX group,OVX with Tle at low dose(5 mg/kg body weight)and high dose(10 mg/kg body weight).After a week’s rest,the mice were treated with the corresponding concentration of Tiliroside for 6 weeks,then the material(tibia)was scanned,the tibia was scanned with micro-CT for three-dimensional analysis and bone parameter analysis,and the tissue sections were analyzed for histomorphology and pathology.Result:(1)In vitro experiment: 1)Agaridin significantly inhibits the osteoclast differentiation induced by RANKL;2)Agaridin significantly inhibits the bone resorption function of osteoclasts;3)Agaridin significantly inhibits the expression of specific genes related to osteoclast differentiation;4)Ginsenoside affects osteoclast differentiation and function by inhibiting NFATc1 and MAPK signaling pathways.(2)In vivo experiment: Tiliroside significantly rescued bone loss in OVX-induced osteoporosis model mice.Conclusion: This study shows that porcine glycoside inhibits RANKL-induced osteoclast formation by inhibiting MAPK and NFATc1 signaling pathways,and can inhibit osteoclast bone resorption function,and is expected to become a potential new drug for preventing osteoporosis.