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

Abstract

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.

CLC Number:

S852.23

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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Effects of 5-amino-imidazole-4-carboxamide Nucleotide on Autophagy and Differentiation in Osteoclasts

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