Study On The Effect And Mechanism Of Sesamin On Osteoclastogenesis And Inflammatory Osteolysis

Background and ObjectiveUnder normal physiological conditions,bone formation mediated by osteoblasts and bone resorption mediated by osteoclasts are in dynamic balance to maintain bone homeostasis.Osteoclasts are the only known multinucleated giant cells with bone resorption function in the skeletal system.The normal function and number of osteoclasts are essential for the maintenance of bone homeostasis.However,inflammation often causes abnormalities in the number and function of osteoclasts,resulting in excessive bone resorption,leading to the occurrence of many inflammatory osteolysis diseases,including rheumatoid arthritis,infectious osteolysis,aseptic loosening of joint prostheses,and periodontitis.At present,bisphosphonates are the first choice for the treatment of inflammatory osteolysis,but the clinical efficacy is poor,and long-term use will cause mandibular osteonecrosis,non-specific fracture of femur and other side effects.Other anti-bone resorption drugs such as denomumab have been found in clinical trials to be associated with the risk of malignancy,and these factors greatly limit their use.Therefore,it is of great significance to study the pathogenesis of inflammatory osteolysis and explore new prevention and treatment methods.Plant monomers,which exist widely in nature,have a variety of pharmacological and biological properties,and have relatively few safety concerns,making these agents perfect candidates for drug design,and have received increasing attention from researchers in recent years.We have previously found that praeruptorin C has a strong inhibitory effect on osteoclasts in vitro and can effectively relieve bone loss in mice after ovariectomy.However,it contains many other active ingredients,and the effects of the active ingredients will affect each other,and the cost of extraction,isolation and purification is high,so it is not suitable for the treatment of inflammatory osteolysis.Sesamin is a lignan isolated from sesame,which belongs to the same phenylpropanoid group with praeruptorin C.These compounds contain one or several natural components of C6 or C3 in its molecular structure,and has been reported to have antioxidant,anti-inflammatory,neuroprotective,anticancer and immunomodulatory effects.Sesamin may also inhibit osteoclasts by acting on the RANKL/JNK/AP-1 signaling axis due to the similar structure between sesamin and praeruptorin C.These effects of sesamin suggest that it may have the potential to prevent inflammatory osteolysis as a natural product.Therefore,this study explored the effects of sesamin on osteoclast formation,differentiation and bone resorption capacity through cell experiments,and at the same time,explored its possible molecular level mechanism.Combined with the cranial osteolysis model in mice,the effect and mechanism of sesamin in prevention and treatment of inflammatory osteolysis disease were further investigated.Methods:(1)Cell experiment: Bone marrow macrophages(BMMs)were extracted from the bone marrow of healthy female C57/BL6 mice,and these cells were used as study cells.Then,different concentrations of sesamin were added for intervention,and the effects of sesamin on BMMs proliferation were explored by CCK-8 experiment,and the safe concentration was selected for follow-up experiments.Then,30ng/m L MCSF and 100ng/m L RANKL were used to induce the directed differentiation of mouse primary macrophages into osteoclasts.At the same time,different concentrations of sesamin were added for stimulation.The formation of osteoclasts was observed by TRAP staining to explore the effect of sesamin on osteoclast differentiation.We also used Rhodamine-labeled ghost pen cyclic peptide to stain the F-actin ring of osteoclasts;we inoculated osteoclasts on bovine bone slices to observe the formation of bone resorption lacunae under scanning electron microscopy;we further verified the effect of different concentrations of sesamin on bone resorption function of osteoclasts.Real-time PCR was used to detect m RNA expression levels of osteoclast specific genes under the intervention of sesamin,and further to investigate the inhibitory effect of sesamin on RANKL-induced osteoclast differentiation in vitro.Finally,the specific mechanism of sesamin inhibiting RANKL-mediated osteoclast differentiation and bone resorbtion in vitro was further explored by using Western blot assay and computer molecular interconnecting technology,and the effects and regulation of sesamin on NF-κB signaling pathway and MAPK signaling pathway in osteoclasts were mainly explored.(2)Animal experiments: 18 C57/BL6 mices were divided into three groups:Sham group(injected with PBS),5mg/Kg LPS group,and 5mg/Kg LPS+20mg/Kg sesamin group.LPS was injected into the top of the skull to induce cranial bone loss in mice,simulating the inflammatory osteolysis model.The inhibitory effect of sesamin on LPS induced osteolysis was investigated by Micro-CT,tissue staining and blood analysis,and the possible mechanism was discussed.Results:(1)In vitro experiment: after treatment with sesamin for 48 h,no significant cytotoxic effect was observed on BMMs cells at concentrations below or equal to 50μM,while a concentration of 100 μM significantly inhibited the survival of BMMs.Therefore,sesamin concentration below 50μM was selected as the safe concentration for follow-up experiments.The BMMs cells were stimulated with sesamin in differentiation.In the control group,after TRAP staining,the primary macrophages differentiated into TRAP positive osteoclasts,which were dyed wine red.The osteoclasts showed typical multi-nucleated giant cells with complete morphology.In the experimental group,the differentiation of osteoclasts was inhibited after the addition of sesamin,and the number of TRAP positive osteoclasts was significantly reduced,and the reduction was concentration-dependent,that is,the higher in the concentration,the less in the number of osteoclasts.At the same time,the volume of TRAP positive osteoclasts decreased significantly,and the decrease became more obvious with the increase of sesamin concentration.The inhibitory effect was enhanced with the increase of drug concentration in concentrationdependent manner.Actin rings were stained with rhodamine labeled ghost pen cyclic peptide.The results showed that the actin rings of the osteoclasts in the control group were large in volume,complete in shape and more in number.In the experimental group,after different concentration of sesamin treatment,the volume and number of osteoclast actin rings were significantly reduced,and the shape of osteoclast actin rings became irregular and incomplete.The results showed that sesamin could significantly inhibit the actin ring formation of osteoclasts in a concentrationdependent manner,and the inhibitory effect was proportional to the concentration.The formation of bone resorption lacunae was observed under scanning electron microscopy.A large number of bone resorption lacunae were observed in the control group,while the number of bone resorption lacunae was significantly reduced in the intervention group with sesamin added,and the degree of reduction increased with the increase of sesamin concentration.Real-time PCR showed that sesamin could inhibit the expression of specific genes of osteoclasts in a dose-dependent manner,that is,the greater in the concentration of sesamin,the stronger in the inhibition effect.The final Western blot assay showed that sesamin had no effect on RANKL-induced P38 and JNK pathways,but inhibited osteoclast differentiation and bone resorptive function by inhibiting ERK and NFκB pathways.The same results were obtained for molecular docking.(2)Animal experiments: microscopic CT scan and 3D reconstruction of cranial bones showed that a large amount of bone erosion was observed in the LPS group,and sesamin treatment effectively reduced the severity of osteolysis.The BV/TV ratio in the sesamin treatment group was lower than that in the sham operation group,but significantly increased than that in the control group.The percentage of porosity in the sesamin treatment group was higher than that in the sham operation group,but significantly reduced than that in the control group.HE and TRAP showed an increase in the number of trap-positive osteoclasts and a significant increase in the surface of osteoclasts versus the surface of bone(Oc S/BS)in the LPS group,leading to significant bone erosion and trabecular microstructure damage.Compared with the LPS group,the sesamin treatment group significantly protected the skull from bone erosion by reducing the number of osteoclasts and Oc S/BS.Quantitative analysis of sesamin on reducing bone erosion area and osteoclast accumulation showed similar results as micro CT results.The serum levels of these three proinflammatory cytokines were determined by ELISA.The results showed that sesamin could significantly reduce the production of IL-1β,TNF-α and IL-6 proinflammatory cytokines in serum of mice.Conclusions:1.Sesamin could inhibit RANKL-induced osteoclast differentiation and bone resorption.2.Sesamin inhibits RANKL-induced osteoclast differentiation and bone resorption mediated by NF-κB and ERK signaling pathways.3.Sesamin could inhibit LPS-induced inflammatory osteolysis of mouse skull,and the possible mechanism is that sesamin inhibits LPS-induced osteoclast activation and inflammatory cytokine production.