摘要
目的研究不同浓度白藜芦醇(RSV)对破骨细胞分化的影响及自噬在其中的作用。方法RANKL诱导RAW264.7细胞分化过程中,加入不同浓度(0、0.1、0.5、1、5及10μmol/L)RSV,CCK-8检测干预后12、24、48、72 h时的细胞活力;TRAP染色观察破骨细胞分化程度。加或不加入3-甲基嘌呤(3-MA)抑制自噬,RT-PCR检测破骨分化相关标志物TRAP、MMP-9、CTSK和自噬相关标志物LC3、Beclin-1、P62的mRNA表达情况;Western blot检测自噬相关蛋白LC3II/I、Beclin-1、P62的表达情况。结果RANKL诱导分化过程中,细胞增殖活力提高,加入0.1~10μmol/L的RSV,细胞活力先上升后下降,在0.5μmol/L时达到最大;0.1μmol/L和0.5μmol/L的RSV能提高TRAP染色阳性的破骨细胞数和TRAP、MMP-9、CTSK、LC3、Beclin-1、P62的mRNA表达,自噬相关蛋白LC3II/I和Beclin-1也增加,P62的蛋白表达则减少;而1~10μmol/L RSV随浓度升高相关mRNA及蛋白LC3II/I和Beclin-1的表达减少,P62的蛋白表达增加;加入3-MA后,相关mRNA及蛋白LC3II/I和Beclin-1的表达减少,P62的蛋白表达增加。结论RSV浓度在0.1~10μmol/L范围内,破骨细胞分化和自噬水平先升高后降低,抑制自噬可以抑制破骨细胞的分化。白藜芦醇影响破骨细胞分化可能部分是通过调节自噬发挥作用。
Objective To investigate the effects of resveratrol(RSV)at different concentrations on osteoclast differentiation and the role of autophagy.Methods RAW264.7 cells were added with RANKL and RSV of different concentrations(0,0.1,0.5,1,5 and 10μmol/L).Cell viability was measured by CCK-8 at 12,24,48 and 72 hours after the intervention.Osteoclast differentiation was observed by TRAP staining.With or without autophagy inhibitor 3-Methyladenine,RT-PCR was used to detect the expression of osteoclast differentiation related markers including TRAP,MMP-9,CTSK and autophagy related markers LC3,Beclin-1 and P62.Western Blot was used to detect the expression of autophagy related proteins LC3II/I,Beclin-1 and P62.Results In the process of RANKL induced differentiation,the cell proliferation activity increased,and the cell proliferation activity first increased and then decreased with the addition of 0.1-10μmol/L RSV,reaching the maximum at 0.5μmol/L.RSV with 0.1μmol/L and 0.5μmol/L increased TRAP positive osteoclast number and mRNA expression of CTSK,MMP9,TRAP,LC3,P62 and Beclin-1,autophagy related protein LC3II/I and beclin-1 expression were also increased,and P62 expression was decreased.With the increase of RSV concentration 1~10μmol/L,the number of osteoclasts and the expression of related mRNA and protein LC3II/I and beclin-1 decreased,and the expression of protein P62 increased.3-Methyladenin inhibited the expression of related mRNA and protein LC3II/I and beclin-1,and the expression of protein P62 was increased.Conclusion In the range of 0.1-10μmol/L resveratrol concentration,the level of osteoclast differentiation and autophagy first increased and then decreased.Inhibition of autophagy can inhibit the differentiation of osteoclasts.The effect of resveratrol on osteoclast differentiation may be partly mediated by autophagy.
作者
张倩
韩婕
汤旭磊
ZHANG Qian;HAN Jie;TANG Xunlei(Department of Endocrinology,The First Hospital of Lanzhou University,Lanzhou 730000,China)
出处
《中国骨质疏松杂志》
CAS
CSCD
北大核心
2020年第4期564-569,594,共7页
Chinese Journal of Osteoporosis
基金
甘肃省省级科技计划(144FKCA075)。
