| Background and aim:Osteoporosis is becoming a common worldwide health problem which is primarily affect older persons Osteoporosisis defined as a skeletal disorder characterized bythe deterioration of trabecularmicrostructure andcompromised bone strength.With the aging of the population,it has gradually become a serious medical problem or even a social problem.In almost all cases,an imbalance between osteogenesis and bone resorption can be found in patients with osteoporosis,can be expressed as the function of osteoclasts is enhanced and osteogenesis is relatively weakened.Drug therapy is the focus and difficulty in clinical treatment of OP.Although the commonly used anti osteoporosis drugs(such as bisphosphonates)have obvious therapeutic effects,there are still many adverse reactions and complications.So natural Chinese medicine,especially Chinese medicine monomers,has gradually become a new research hotspot in the treatment of osteoporosis.The traditional Chinese medicine monomer,which has the characteristics of pure natural,small side effects and high biological activity,will provide a novel therapeutic method for the treatment of OP.Ginsenoside Rb2 is the most effective component of ginsenoside found in ginseng.Previous studiesshowed that Rb2 can increase bone mass and reduce bone loss in postmenopausal osteoporosis mice.Rb2 exerts protective function in high dose dexamethasone induced mouse bone marrow mesenchymal stem cell apoptosis.Rb2 can promote the proliferation of MC3T3-E1 osteoblast cellsby antagonizing the effect of hydrogen peroxide induced oxidation and enhance the expression of osteoblast-related genes.The effect and mechanism of Rb2 on osteoclast are still largely unknown.Here,we aimed to evaluate the effects and underlying mechanism of Rb2 on osteoclastat the cellular lever.Methods:1.RAW264.7 cells were cultured and passaged at appropriate concentrations,and osteoclast differentiation was induced with the concentration of 100ng/ml in RANKL.2.The primary osteoclasts were harvesting from osteoporotic mice and its differentiationwasinducing by adding M-CSF and RANKL.3.The experiment was divided into four groups in both RAW264.7 cell and primary osteoclast:control group(only RANKL,without Rb2)and experimental groups with different Rb2concentrations(RANKL and 0.1 mu M,1 mu M,10 mu M Rb2).4.Cytotoxicity of Rb2 to cells was evaluated by CCK-8 assay.5.Through TRAP staining and counting the number of osteoclast-like cells,the direct differentiation effect of RB2 was confirmed and compared with that of RANKL.6.The m RNA levels of osteoclast-related genes TRAP and NFATc1 were analyzed by q RT-PCR.7.The protein contents of autophagy-related genes LC3 and Beclin-1 were detected by western-blot.8.The protein content of m TOR was evaluated by western-blot.Results:1.Rb2 showed no significant toxic effect on RAW264.7 cells.2.With the increase of Rb2 concentration,the number of osteoclasts decreased significantly along with the cell volume decreased obviously.Osteoclast count:control group(168.2±22.6),0.1μMgroup(131.0±16.3),P=0.018;1μM group(98.8±17.9),P﹤0.01;10μM group(85.8±17.3),P﹤0.01。3.Compared with the control group,the expression of osteoclast-related genes TRAP and NFATc1 decreased significantly in all Rb2 groups,P < 0.05.4.With the increase of the concentration of Rb2,ratio of LC3 II and LC3 I gradually increased,the expression of Beclin-1 was also a rising trend.5.Rb2 can significantly reduce the expression of m TOR protein content,and the inhibitory effect is directly proportional to the concentrationConclusion:Rb2 can inhibit osteoclast formation and reduce the expression of osteoclast-related genes in vitro.Compared with the control group,autophagy related protein increased significantly,indicating that the role of inhibiting osteoclast differentiation may be achieved by autophagy.The m TOR pathway may play an important role in the regulation of Rb2 on osteoclast. |
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