CAJ | 학술논문

目的探讨二甲双胍对破骨细胞体外分化的影响及其可能机制.方法采用RANKL诱导鼠巨噬细胞系Raw264.7细胞破骨分化模型,给予不同浓度的二甲双胍(400 μmol/L、800 μmol/L和1000μmol/L)和雷帕霉素(100 hmol/L)处理后,通过抗酒石酸酸性磷酸酶(tartrate-resistant Acid Phosphatase,TRAP)染色和破骨细胞骨架结构荧光染色观察破骨细胞数量,骨吸收培养板观察骨陷窝面积,RT-PCR技术检测破骨细胞特异性基因TRAP、组织蛋白酶K、降钙素受体和金属基质蛋白酶-9的表达,ELISA法检测肿瘤坏死因子-α(tumor necrosis factor,TNF-α)表达水平,Western-b1ot检测c-Fos蛋白以及哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin complex l,mTORC1)信号通路下游底物S6K1 Thr389、S6 Ser235/236、4EBP1 Thr37/46的表达及磷酸化水平.结果二甲双胍和雷帕霉素均可使RANKL诱导的破骨细胞数量减少,抑制破骨细胞特异性基因的表达、抑制TNF-α、c-Fos蛋白以及mTORC1信号通路下游底物S6K1 Thr389、S6 Ser235/236、4E-BP1 Thr37/46的磷酸化,且二甲双胍的抑制作用具有浓度依赖性.结论二甲双胍可抑制RANKL诱导的破骨前体细胞分化,其机制可能与抑制TNF-α和c-Fos蛋白的生成,以及抑制mTORC1信号通路激活有关.
목적 탐토이갑쌍고대파골세포체외분화적영향급기가능궤제.방법 채용RANKL유도서거서세포계Raw264.7세포파골분화모형,급여불동농도적이갑쌍고(400 μmol/L、800 μmol/L화1000μmol/L)화뢰파매소(100 hmol/L)처리후,통과항주석산산성린산매(tartrate-resistant Acid Phosphatase,TRAP)염색화파골세포골가결구형광염색관찰파골세포수량,골흡수배양판관찰골함와면적,RT-PCR기술검측파골세포특이성기인TRAP、조직단백매K、강개소수체화금속기질단백매-9적표체,ELISA법검측종류배사인자-α(tumor necrosis factor,TNF-α)표체수평,Western-b1ot검측c-Fos단백이급포유동물뢰파매소파단백(mammalian target of rapamycin complex l,mTORC1)신호통로하유저물S6K1 Thr389、S6 Ser235/236、4EBP1 Thr37/46적표체급린산화수평.결과 이갑쌍고화뢰파매소균가사RANKL유도적파골세포수량감소,억제파골세포특이성기인적표체、억제TNF-α、c-Fos단백이급mTORC1신호통로하유저물S6K1 Thr389、S6 Ser235/236、4E-BP1 Thr37/46적린산화,차이갑쌍고적억제작용구유농도의뢰성.결론 이갑쌍고가억제RANKL유도적파골전체세포분화,기궤제가능여억제TNF-α화c-Fos단백적생성,이급억제mTORC1신호통로격활유관.
Objective To investigate the effects of mefformin on the differentiation of osteoclastas well as relative mechanism.Methods Raw264.7 cells from the murine macrophage cell line was used.Receptor activator of NF-κB ligand (RANKL) was used to stimulate osteoclast differentiation from Raw264.7 cells.Osteoclast differentiation was assessed by tartrate-resistant acid phosphatase (TRAP) and actin fluorescence staining and counting the TRAP-positive cells after exposure to different concentrations of mefformin (0 μmol/L,400 μmol/L,800 μmol/L and 1000 μmol/L) or rapamicin (100 nmol/L) in the presence of 50 ng/ml RANKL for 5 days.Bone-resorbing activity was evaluated by BD BioCoatTM OsteologicTM Bone Cell Culture System.The expression of osteoclast-specific genes like TRAP,capthesin K,calcitonin receptor (CTR) and matrix metalloproteinase (MMP-9) was evaluated by RT-PCR.The expression of tumor necrosis factor-α(TNF-ct) S6K1Thr389,S6 Ser235/236,4E-BP1Thr37/46 and c-Fos protein was evaluated by ELISA kit and Western blot analysis,respectively.Results Mefformin dose-dependently inhibited RANKL-stimulated osteoclasts differentiation in Raw264.7 cell culture,as manifested by decrease of TRAP-positive multinucleated cells and pit erosion area,down-regulation of TRAP,cathepsin K,CTR and MMP-9 mRNA and reduction of TNF-α and c-Fos protein expression.Further study revealed that RANKL activated mTOR complex 1(mTORC1) signaling,while mefformin impaired RANKL-stimulated mTORC1 signaling.Rapamycin,an mTORCl-specific inhibitor and immunosuppressive macrolides could also prevent RANKL-induced osteoclast differentiation and bone resorption in vitro.Conclusion Mefformin inhibits osteoclastogenesis in vitro,which may due to reduction of TNF-α and c-Fos protein expression,and mTORC1 signaling is involved in this process.