Effect Of Astragalin On Bone Loss After Ovariectomy In Mice And Its Mechanism

Background: Osteoporosis is a metabolic bone disease characterized by decreases bone density and strength due to excessive loss of bone protein and mineral content.Postmenopausal osteoporosis(PMOP)is the most common form of primary osteoporosis,and the incidence of PMOP has been in-creasing rapidly.The imbalance between osteogenesis by osteoblasts and osteoclastogenesis by osteoclasts contributes to the pathogenesis of PMOP.Estrogen is osteoprotective.On one hand,it targets osteoblasts and stimulates osteoblasts to secrete osteoprotegerin(OPG)which can inhibit osteoclastogenesis.On the other hand,estrogen directly inhibits preosteoclast differentiation and osteoclast formation and function and induces apoptosis of osteoclasts and preosteoclasts to reduce the number of osteoclasts.After menopause,withdrawal of estrogen leads to an increase of osteoclastogenesis and a decrease of osteogenesis.Thus,postmenopausal osteoporosis occurs.Osteoclasts overactivated by inflammation play a vital role in the imbalance.Thus,inhibiting osteoclastognenesis through suppressing inflammation is an important strategy for preventing and treating PMOP.Astragalin is the main component of Chinese herbal medicines,and it has been shown to have anti-inflammatory,anti-oxidative,immunomodulatory and other pharmacological effects.It can be used in a variety of inflammatory diseases such as rheumatoid arthritis and bronchial pneumonia.Since inflammation is closely related to the occurrence of PMOP,we believe that astragalin has the function of anti-osteoporosis and is expected to be a candidate for anti-osteoporosis.The aim of this study was to investigate the effects of astragalin on bone loss in ovariectomized mice and to explore its possible molecular mechanisms.Objective:1.To explore the inhibition effect of astragalin on bone loss in ovariectomized miceby intraperitoneal injection of astragalin.2.To investigate the effects of astragalin on osteoclast differentiation and function invitro;3.To explore the mechanism of astragalin affecting osteoclast differentiation andfunction.Methods:Part 1: The protective effect of intraperitoneal injection of astragalin on bone loss in ovariectomized mice.8 weeks old female C57BL/6 mice were randomly divided into three groups:(1)sham operation group(2)osteoporosis model group(3)osteoporosis model + astragalin treatment group.Except for sham operation group,the other two groups underwent ovariectomy(OVX).On the third day after surgery,the treatment group was intraperitoneally injected with Astragalin for 6 weeks.After 6 weeks,the mice were sacrificed,and the bilateral femurs were taken.The blood was taken from the heart and centrifuged to obtain serum.One side of the femur was decalcified and embedded,and paraffin sections were taken for HE and Trap staining;The other femur was fixed with paraformaldehyde for 3 days and then subjected to femoral Micro CT scanning.Serum Elisa assay was used to detect the expression levels of inflammatory cytokines and osteogenic and osteoclast-related markers in peripheral blood.Part 2: The effect of astragalin on the differentiation and function of osteoclasts in vitro.RANKL and M-CSF was used to induce osteoclast differentiation of primary bone marrow mononuclear cells and mononuclear macrophage cell line RAW264.7.The experiment was divided into five groups:(1)Blank group;(2)Cell + induction group;(3)Cell + induction + low concentration of astragalin group;(4)Cell + induction + medium concentration of astragalin group;(5)Cell + induction + high concentration of astragalin group.After induction,osteoclast differentiation was determined by tartrate-resistant acid phosphatase(TRAP)staining,and whether different concentrations of astragalin inhibited osteoclast actin ring formation by actin loop assay.The same number of mature osteoclasts were inoculated into the hydroxyapatite bone plate,and different concentrations of astragalin were added to observe whether the osteoclast function was inhibited by analysing the area of bone absorption pores.Part 3: In vitro experiments were conducted to investigate the molecular mechanism of astragalin in regulating osteoclast differentiation and function.The mature mononuclear macrophage cell line RAW264.7 was induced to differentiate into osteoclasts by RANKL+M-CSF.The experiment was divided into five groups:(1)Blank group;(2)Cell + induction group;(3)Cell + induction + astragalin group.Western blot was used to detect the expression of osteoclast differentiation-related proteins,including:TRAP,NFATC1,MMP-9,C-Src,and Catheosin K.The expression of NF-?B,MAPK andAKT pathway-associated marker proteins and phosphorylation levels in the downstream of RANK pathway were detected.Results:In vivo,we observed that bone loss was obvious in ovariectomized mice,which showed a significant decrease in bone mineral density,bone volume fraction,bone surface area and bone volume ratio,and the number of bone trabeculae in Micro CT.The number of trabecular bone decreased in HE staining of femur,and the number of osteoclasts on the surface of trabecular bone increased in TRAP staining.At the same time,the levels of IL-6,osteoclast markers TRAcp-5B and CTX-1 in serum were increased by serological detection,but there was no significant difference in the level of osteogenic related markers BGP between sham operation group and the OVX group.Injection of astragalin in vivo could reduce bone loss in ovariectomized mice,decrease the number of osteoclasts on the surface of trabecular bone stained with TRAP in the experimental group,and decrease the levels of inflammatory factor IL-6 and osteoclast related markers TRAcp-5B and CTX-1.In vitro,different concentrations of astragalin could inhibit the differentiation of bone marrow monocytes and monocyte macrophage cell line RAW264.7 into osteoclasts.The results showed that the number of osteoclast positive cells decreased and the formation of actin ring in osteoclasts was inhibited.At the same time,different concentrations of astragalin affected the function of osteoclasts,which showed that the absorption area of osteoclasts inoculated on hydroxyapatite bone plate was lower than that of simple induction group.Western-blot assay showed that the expression of osteoclast differentiation-related protein TRAP,NFATC1,MMP-9,C-Src,Catheosin K in RANKL and M-CSF induced group was significantly increased,but the expression was significantly inhibited after the intervention of astragalin.In RANKL and M-CSF induction group,the phosphorylation levels of NF-?B,MAPK and AKT pathway related marker proteins downstream of RANK pathway were significantly increased.After treatment with astragalin,the activation of these pathways was significantly inhibited and the phosphorylation level of related marker proteins was decreased.Conclusion:1.In vivo experiments,it was proved that the treatment with astragalin could reduce the bone loss after ovariectomy in mice,reduce the number of osteoclasts on the surface of bone trabecula,and reduce the levels of inflammatory factors and osteoclast markers in estrogen deficient mice in vivo.2.In vitro experiments showed that astragalin could inhibit the differentiation of osteoclasts induced by RANKL and the formation of actin protein ring of osteoclasts.And it can inhibit the bone resorption function of osteoclasts.3.In molecular mechanism,astragalin can inhibit the activation of NF-?B,MAPK and AKT pathway induced by RANKL,and inhibit the expression of markers related to osteoclasts,including TRAP,NFATc1,MMP-9,C-Src,Catheosin K.