矮壮素对小鼠前成骨细胞系MC3T3-E1骨骼发育相关基因及蛋白的影响及其机制

doi:10.3969/j.issn.1004-616x.2018.03.005

Abstract

Abstract: OBJECTIVE: To investigate the effects of chlorocholine chloride (CCC) on expression of skeletal development-related genes and functional proteins in mouse pre-osteoblast cell line MC3T3-E1. METHODS: MC3T3-E1 cells in logarithmic growth phase were treated with 0,8,40,200,1 000 μg/mL CCC. At different time points (24,48,72 h) after treatment,cytotoxicity was determined by using the MTT assay. At 48 h after treatment,mRNA expression of skeletal and osteogenic development-related genes was detected using qPCR:alkaline phosphatase (ALP),osteocalcin(OCN),growth factor 1 (IGF-1),growth hormone receptor (GHR),RANKL,osteoprotegerin (OPG),macrophage colony-stimulating factor (M-CSF). In addition,expression of functional proteins related to bone development and MAPK signal pathways was determined by Western blot:ALP,GHR,bone morphogenetic protein 2 (BMP2),transcription factor runt-related protein 2 (Runx2). RESULTS: CCC had no significant effect on cytotoxicity of MC3T3-E1 at different time points (24,48,72 h). mRNA expression of ALP,OCN and RANKL in the 1 000 μg/mL groups increased significantly (P < 0.05);the ratio of RANKL and OPG in the 1 000 μg/mL group increased significantly (P < 0.05);mRNA expression of GHR,IGF-1,OPG,M-CSF did not change significantly (P > 0.05). Protein expression of GHR and BMP2 in the 1 000 μg/mL group was significantly decreased,while ALP and RUNX2 did not change significantly. Phosphorylated ERK protein expression decreased and phosphorylated JNK protein expression increased. CONCLUSION: CCC has almost no cytotoxicity on pre-cultured mouse MC3T3-E1 cells,but inhibited the differentiation of MC3T3-E1 cells into mature osteoblasts and promoted osteoclast differentiation. Moreover,involvement of mitochondria on differentiation of pre-mouse osteoblasts might be related to the MAPK pathway. The decrease of phosphorylated ERK and the increase of phosphorylated JNK might have inhibited migration of MC3T3-E1 to mature osteoblasts differentiation of cells,hence their differentiation to become mature osteoblasts.

Key words: chlorocholine chloride, osteoblast, bone development, MAPK pathway

CLC Number:

R994.6

JIA Lixia, ZHANG Qi, HOU Xiaohong, MENG Qinghe, HUANG Yao, ZHOU Wenjuan, HAO Weidong. Effects of chlorocholine chloride on expression of skeletal development-related genes and proteins in MC3T3-E1 cells[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2018, 30(3): 188-193,199.

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Effects of chlorocholine chloride on expression of skeletal development-related genes and proteins in MC3T3-E1 cells

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