| Objective: Osteoclasts derived from the mononuclear-macrophage system of human blood are the only cells in the body that have bone resorption capacity found in the current research.By secreting many types of acids and collagenases,osteoclasts can achieve Hydrolysis at the molecular level,digesting related mineral complexes,is self-evident for the maintenance,repair,and remodeling of bones.Osteoporosis is mainly due to the excessive proliferation and functional activation of osteoclasts,the proliferation of osteoblasts is inhibited and the degree of differentiation is reduced,resulting in the bone resorption mediated by the excessive activation of osteoclasts is much higher than that of osteoblasts.The mediated bone formation breaks the dynamic balance of osteogenesis and osteoclasts,so that the body’s bone tissue metabolism is in a high-conversion state,and ultimately leads to the loss of body bone.According to the findings of the basic research of traditional Chinese medicine,the main way of treating osteoporosis with traditional Chinese medicine monomer is to regulate and inhibit the maturation and differentiation of osteoclasts,promote the proliferation and functional activation of osteoblasts,affect the effects of related cytokines,and regulate the micro-Elements balance,estrogen balance,repair bone structure and other aspects.Arctigenin(Arctigenin,AGN)is a natural drug monomer subordinate to phytoestrogens and lignins.It has many functions such as eliminating inflammation,inhibiting tumor differentiation and metastasis,and resisting tissue oxidation.The latest research shows that arctigenin can inhibit the differentiation and maturation of osteoclasts in mouse bone marrow macrophages(BMMs)culture.However,the specific aspects of osteoclast differentiation affected by arctigenin and its potential molecular regulation mechanism still need to be further analyzed and explored.Therefore,the main purpose of this experiment is to study the influence of arctigenin on the differentiation of osteoclasts,and to analyze its potential molecular regulation mechanism in depth,and to carry out related drug toxicity,bone resorption and actin ring at the same time.And other experiments,in order to develop and find new drug targets as the basis for the treatment of related bone diseases caused by the pathological overexpression of osteoclast activity,and the potential for diagnosis and treatment of anti-osteoporosis and reducing the occurrence of fragility fractures.Broaden the guidance and provide more precise and reliable choices.Methods: 1.Establish a method for inducing differentiation of bone marrow-derived macrophages: Obtain bone marrow-derived macrophages from rat femurs and tibias,culture them in a medium containing recombinant RANKL and M-CSF to induce them to differentiate into osteoclasts;2.Tartrate-resistant acid phosphatase(TRAP)kit was used to detect the number of successfully differentiated osteoclasts,and the cytotoxicity of arctigenin at different doses to bone marrow-derived macrophages and osteoclasts was determined by the cell viability(XTT)method;3.Detect the effects of different doses of arctigenin on the expression of genes and proteins related to c-Fos/NFATc1 and MAPKs signaling pathways by polymerase chain reaction(PCR)technology and Western-blot technology(Western-blot);4.Via Corning microplate And cytoskeleton(actin microfilament,F-ACTIN)green fluorescent staining reagent to detect the effect of different doses of arctigenin on osteoclast bone resorption;5.Statistical analysis by SPSS 20.0,using Mann-Whitney’s U test to determine Significant difference;and p<0.05 considered the difference to be statistically significant.Results: 1.In this experiment,osteoclasts were obtained by inducing the established rat bone marrow-derived macrophages to differentiate into osteoclasts;2.Using the actin loop experiment and XTT experiment methods,it was concluded that In the analysis,arctigenin can limit the bone resorption activity of osteoclasts in vitro within the concentration range of 0.3μM-30μM.Arctigenin has no toxic effect on osteoclasts at the various concentrations set in this experimental study(that is,within the range of0.3μM-30μM),and can limit the differentiation composition of osteoclasts in a dose-dependent manner,and Compared with the control group,there is a significant difference;3.In the experimental results of detecting osteoclast bone resorption in Corning microplates,the arctigenin can be obvious within the concentration range of0.3μM-30μM set in this experimental study To reduce the area of ? ? femoral pits,the statistical difference between the experimental group and the control group is more significant(p<0.05),and with the increase of the dose,its inhibitory effect is more significant.Similar to the Corning microplate detection experiment,in the cytoskeleton(actin microfilament;F-ACTIN)detection experiment,compared with the control group,the experimental group added with arctigenin can significantly affect the amount of arctigenin.The statistical difference between the experimental group and the control group is more significant.At the same time,the experimental results also show that the greater the concentration of arctigenin,the more significant the inhibitory effect;4.In this experimental study,In the concentration range of the0.3μM-30μM,arctigenin can inhibit the differentiation and formation of osteoclasts by inhibiting the phosphorylation of MAPKs signaling pathway;5.Arctigenin can inhibit RANKL at each concentration set in this experimental study The signal transduction pathway induces the activity of c-Fos and NFATc1 expression to achieve the effect of inhibiting osteoclast differentiation.Conclusion: Arctigenin can inhibit the differentiation and formation of osteoclasts in vitro and the functional activity of osteoclasts in the concentration range set in this experimental study.In addition,it is also proved by this experiment that arctigenin is effective for osteoclasts.When cell differentiation is restricted,the phosphorylation of MAPKs signal transduction channels can be used to restrict the formation of cells,thereby reducing the activity of the transcription factors NFATc1 and c-Fos,which express specific regulators.This experiment will help to develop and find new drug targets as the basis for the treatment of related bone diseases caused by the pathological overexpression of osteoclast activity,and the potential for diagnosis and treatment of anti-osteoporosis and reducing the occurrence of fragility fractures.Sexually broadens the orientation. |
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