Effects Of Neohesperidin On Rankl-Induced Osteoclast Differentiation And Osteoporosis In Ovariectomizied Mice

Osteoporosis is a systematic skeletal disease. It is characterized by the destruction of the micro structure of bone tissue, decline in bone mass and bone strength and increase in bone fragility. Osteoporotic fracture occurred easily in osteoporosis patients even with a slight trauma or during daily activity. It is prone to induce serious consequences and make burden to family and society.Bone remodeling is the most important process of osteogenesis and bone metabolism. It is modulated by the balance between osteoblast bone formation and osteoclast bone absorption. Long-term and excessive absorption break this balance and cause diseases due to increasing bone absorption like osteoporosis. Excessive bone absorption of osteoclast plays an important role in osteoporosis. Therefore, inhibition of osteoclast differentiation or activity can be an effective way to therapy osteoporosis.Osteoclasts originate from bone marrow macrophages. The RANKL/RANK signaling pathway is closely related to the osctoclast differentiation and maturation. NF-κB is an important regulator of cell’s proliferation and differentiation which belongs to dimeric transcription factors and is held in an inactive state binding with IκB-α NFATcl is the main transcription factor of osteoclast differentiation and mediates the cell fusion and osteoclast differentiation by gene expression procedure. NF-κB and NFAT interact with each other and the expression of these two factors can reflect the activation of RANKL/RANK signaling pathway.TRACPSb is a marker of osteoclast and bone absorption which originated from osteoclast. It transports the products of bone matrix degradation from the resorption lacuna to a functional secretory domain to destroy the bone organic matrix components. Cathepsin K is the main Collagenolytic protease in physiological and pathological bone degradation with the cleavage action on type Ⅰ and type Ⅱ collagen. It is expressed by osteoclasts and plays an special role in bone absorption.Intracellular calcium oscillations are widespread pattern of the signal transduction in excitable and non-excitable cells. RANKL can induce calcium oscillations to activate calcineurin-mediated NFATcl and trigger a sustained NFATcl-dependened osteoclast differentiation procedure to promote osteoclast differentiation.Currently it is considered the ovariectomzied mice model is similar to human conditions which can be used to study and intervene the reasons of bone absorption and suitable for the evaluation of potential therapeutic drugs to prevent osteoporosis. After ovariectomzied, the mice’s bone mineral density reduced and the bone micro structures are destructed and these ovariectomzied mice are suitable for study as a model of postmenopausal osteoporosis.Neohesperidin is Citrus extract which belongs to the flavonoids. It is a latural antioxidant with the functions of anti-inflammation, anti-tumor and protecting cardiovascular system. But there is no related research on osteoporosis inhibition.In this study, we performed a series of in vitro assays to analyze the neohesperidin inhibition effects on RANKL-induced osteoclast differentiation. we observed the number of osteoclast differentiation by dose-dependent assays; analyzed the cell apoptosis by flow cytometry; measured the expressing of TRACPand Cathepsin K on osteoclasts by qPCR; detected the osteoclast absorption by hydroxyapatiteanalyzed NF-κB, NFAT and IκB-α in RANKL/RANK signaling pathway by luciferase and western blot assays; analyzed the osteoclast differentiation by calcium oscillation assay.In vivo assay, we observed the neohesperidin effects on bone conditions in OVX mice. The results are analyzed using the following parameters respectively: bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular spacing (Tb.Sp).The study includes two parts. Part I The Effects of Neohesperidin on RANKL-induced Osteoclast DifferentiationObjective:To study the effects of neohesperidin on RANKL-induced osteoclast differentiation and functions.Methods:Bone marrow macrophages were induced by RANKL with different concentrations of neohesperidin and stained by TRAP, calculated the number of TRAP positive osteoclast in dose-dependent manners under the optical microscope; RAW264.7 cells were induced by RANKL with different concentrations of neohesperidin, analyzed apoptosis by flow cytometry to calculated the rate of healthy cells; Bone marrow macrophages were induced by RANKL with 10μM neohesperidin, measured the osteoclast expression of TRACPand Cathepsin K by qPCR; Bone marrow macrophages were cultured in the hydroxyapatite-coated plate by RANKL and 10μM neohesperidin,observed the absorption area of hydroxyapatite-coated and analyzed by ImageJ;P3K Luc cells transfected with p-NF-κB-TA-Luc and Raw264.7 cells transfected with p-NFAT-TALuc were induced by RANKL with different concentrations of neohesperidin, measured the expression of NF-κB and NFAT with BMG Polar Star Optima by luciferase; Bone marrow macrophages were induced by RANKL with 10μM neohesperidin, measured the short-term expression of IκB-a and the long-term expression of NFAT by western blot and analyzed by Image J; Bone marrow macrophages were induced by RANKL with 10μM neohesperidin, observed the average peak height of oscillation cells by inverted fluorescence microscope and analyzed Nikon Basic Research.Results:Neohesperidin inhibited osteoclast differentiation in dose-dependent manner; the rate of healthy cells were kept in the high level of 90% as well as necrosis and apoptosis were kept in the low level in different concentrations of neohesperidin; neohesperidin inhibited the expression of TRACP and Cathepsin K; neohesperidin inhibited the expression of NF-κB and NFAT in dose-dependent manner; neohesperidin inhibited the degradation of IκB-α; neohesperidin inhibited osteoclast calcium oscillation.Conclusions:Neohesperidin inhibits osteoclast differentiation and functions in many aspects. It is a safe and effective inhibitor of osteoclasts.Part II The Effects of Neohesperidin on Osteoporosis in Ovariectomized MiceObjective:To study the effects of neohesperidin on osteoporosis in ovariectomzied mice。Methods:30 female C57BL6J mice aged 6 weeks were randomly divided into sham group (SHAM), model group (OVX), estrogen group (E2), neohesperidin low dosage group (NE1) and neohesperidin high dosage group (NE2).6 mice in each group. Weighed each mouse, intraperitoneally injected 10% chloral hydrate Opened the both sides of abdominal skin and muscle in sham group, closed abdominal cavity without removal of the ovaries. In the rest of the four groups, opened the abdominal cavity, ligated and resected the both sides of ovaries completely and closed abdominal cavity. Given gentamicin in each mouse after operation and fed water and food.1 week later, Intraperitoneally injected drugs based on the weight of each mouse every other day. Sham group and OVX group received saline, E2 group received 0.1mg/kg of estrogen, NE1 group received 3mg/kg of neohesperidin and NE2 group received 6mg/kg of neohesperidin. After 7 weeks, each mouse underwent ether anesthesia, took the one side of tibial and immersed in 4%FPA solution. Washed the tibials twice with 1XPBS solution after 20-24 hours. One side of tibial immersed and preserved with 1XPBS solution in 1.5ml tube. Comparatively observed bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular spacing (Tb.Sp) in each group by Micro-CT. Results:Neohesperidin increased BV/TV of OVX mice and BV/TV basically recovered to normal levels which was same as estrogen in a dose-dependent manner; no significant changed in Tb.Th in each group; high dose neohesperidin reduced Tb.Sp of OVX mice and Tb.Sp basically recovered to normal levels which was same as estrogen; neohesperidin increased Tb.N of OVX mice and Tb.N basically recovered to normal levels in a dose-dependent manner and the effect of high dose of neohesperidin was same as estrogen. Conclusions:Neohesperidin inhibits the development of osteoporosis in OVX mice and might be a potential drug for the treatment of osteoporosis.