氨基咪唑-4-甲酰胺核苷酸对破骨细胞自噬和分化的影响

doi:10.11843/j.issn.0366-6964.2019.11.019

摘要/Abstract

摘要： 为了探究氨基咪唑-4-甲酰胺核苷酸（AICAR）对破骨细胞自噬及分化能力的影响，以BALB/c小鼠骨髓巨噬细胞诱导分化形成的破骨细胞为研究对象，添加0.5 mmol·mL^-1AICAR（AMPK激活剂）处理4 h后，抗酒石酸酸性磷酸酶（TRAP）染色鉴定破骨细胞；CCK-8法检测破骨细胞存活率；高内涵系统分析TRAP⁺细胞比率、面积、荧光强度及圆度；蛋白免疫印迹（Western blot）和荧光定量聚合酶链式反应方法（qRT-PCR）检测AMPK、NFATc1、c-Fos、CTSK、TRAP、（LC3-Ⅱ）、ATG5、Beclin1、p62的mRNA和蛋白水平；激光共聚焦显微镜观察EGFP-pmCherry-LC3荧光聚点。结果显示，通过30 ng·mL^-1M-CSF和60 ng·mL^-1RANKL诱导分化4 d的细胞TRAP染色后大部分呈阳性且细胞核数目≥3。AICAR处理后，与对照组相比，细胞存活率无显著差异（P>0.05），但细胞面积、TRAP⁺（细胞核数目≥3）细胞率、TRAP荧光强度显著下降（P<0.05）。AICAR组破骨细胞c-Fos、TRAP（P<0.01）、p62（P<0.05）的mRNA水平显著下降，LC3、ATG5的mRNA水平显著升高（P<0.05）。AICAR处理致AMPKα蛋白磷酸化水平极显著升高（P<0.01），NFATc1、c-Fos、CTSK、p62（P<0.01）和TRAP（P<0.05）蛋白表达量显著下调，LC3-Ⅱ、ATG5和Beclin1蛋白表达水平极显著上调（P<0.01）。转染LC3质粒结果显示AICAR处理后LC3黄色及红色荧光聚点数目增多，绿色荧光聚点数目减少。结果表明0.5 mmol·mL^-1AICAR作用4 h可激活破骨细胞AMPKα，增强破骨细胞的自噬水平，但抑制了破骨细胞的分化。

Abstract: The aim of this study was to analyze the effects of 5-amino-imidazole-4-carboxamide nucleotide (AICAR) on autophagy and differentiation in osteoclasts. Bone marrow macrophages were derived from the tibia and femur of BALB/c mice and induced by 30 ng·mL^-1 M-CSF and 60 ng·mL^-1RANKL for 4 days. After 4 days differentiation, the cells were identified by tartrate resistant acid phophatase (TRAP) staining. The osteoclasts were treated with 0.5 mmol·mL^-1AICAR, AMPK activator for 4 h. Cell area, TRAP⁺ number, roundness and fluorescence intensity were analyzed by High Content Screening (HCS) and cell viability was detected by Cell Counting Kit-8 (CCK-8). Protein and mRNA expression levels of autophagy and differentiation-related proteins were detected by Western blot and qRT-PCR,respectively. After transfection of the EGFP-pmCherry-LC3 plasmid, the change of endogenous LC3 puncta was analyzed by confocal immunohistochemistry. Results from CCK-8 test indicated that the cell viability was not significantly affected by AICAR. The ratio, area and intensity of TRAP⁺cells were significantly lower than those of control group (P<0.05). The results of qRT-PCR showed that AICAR treatment significantly decreased the mRNA transcription level of c-Fos, TRAP (P<0.01) and p62 (P<0.05), and increased LC3, ATG5 mRNA transcription level (P<0.05). Data from Western blot showed that protein expression rate of p-AMPKα/AMPKα was significantly increased (P<0.01), and the protein expression rate of TRAP (P<0.05) were significantly dowm-regulated, and the protein expression rate of NFATc1, c-Fos, CTSK, p62 were extremely significantly down-regulated (P<0.01), while the protein expression level of ATG5, Beclin1, LC3-Ⅱ were extremely significantly increased (P<0.01). The results of EGFP-pmCherry-LC3 plasmid transfection showed that the yellow (EGFP+pmCherry) and red (pmCherry) puncta increased, while the green (EGFP) puncta decreased in osteoclasts after AICAR treatment. All the results suggest that AICAR could inhibit the differentiation of osteoclasts by inducing autophagy via AMPKα activation.

中图分类号:

S852.23

陈苗苗, 仝锡帅, 郑嘉铭, 赵鸿雁, 顾建红, 刘宗平. 氨基咪唑-4-甲酰胺核苷酸对破骨细胞自噬和分化的影响[J]. 畜牧兽医学报, 2019, 50(11): 2339-2347.

CHEN Miaomiao, TONG Xishuai, ZHENG Jiaming, ZHAO Hongyan, GU Jianhong, LIU Zongping. Effects of 5-amino-imidazole-4-carboxamide Nucleotide on Autophagy and Differentiation in Osteoclasts[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(11): 2339-2347.

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