食品科学 ›› 2020, Vol. 41 ›› Issue (3): 144-150.doi: 10.7506/spkx1002-6630-20181212-148

• 营养卫生 • 上一篇    下一篇

罗非鱼头磷脂对RANKL诱导RAW264.7细胞分化的抑制作用及其对破骨细胞凋亡作用的机理

马婷,夏光华,李川,申铉日   

  1. (海南大学食品学院,海南 海口 570228)
  • 出版日期:2020-02-15 发布日期:2020-02-26
  • 基金资助:
    “十三五”国家重点研发计划重点专项(2018YFD0901103); 海口市海洋经济创新发展示范城市产业链协同创新类项目(HHCL201804)

Inhibitory Effect of Tilapia Head Phospholipid on RANKL-Induced RAW264.7 Cell Differentiation and Mechanism of Osteoclast Apoptosis Induced by It

MA Ting, XIA Guanghua, LI Chuan, SHEN Xuanri   

  1. (College of Food Science, Hainan University, Haikou 570228, China)
  • Online:2020-02-15 Published:2020-02-26

摘要: 目的:探讨罗非鱼头磷脂(tilapia head phospholipid,TH-PL)对破骨细胞生物活性的影响。方法:首先采用溶剂法提取罗非鱼头总脂,通过硅胶柱层析分离得到TH-PL,采用薄层色谱(thin layer chromatography,TLC)、傅里叶红外变换光谱(Fourier transform infrared spectroscopy,FTIR)和气相色谱分析其性质与部分结构。其次利用核因子κB受体活化因子受体的配体(receptor activator for nuclear factor-κB ligand,RANKL)体外诱导RAW264.7细胞分化为破骨细胞,建立破骨细胞模型,抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)染色法评价TH-PL对RAW264.7细胞向破骨细胞分化的影响,荧光染色法、流式细胞仪测定法和蛋白免疫印迹法检测TH-PL对破骨细胞凋亡的影响。结果:制备的TH-PL占罗非鱼头干质量的1.52%,TLC和FTIR法确定所得样品为TH-PL,且含有丰富的不饱和脂肪酸,约占38%。TRAP染色实验表明,50、100 μg/mL TH-PL显著抑制RAW264.7分化为破骨细胞;进一步实验证明,TH-PL能够引起破骨细胞线粒体跨膜电位下降,导致细胞内caspase-3的蛋白表达量上升,诱导破骨细胞凋亡。结论:体外细胞实验证明,TH-PL具有显著的抑制破骨前体细胞分化和促进破骨细胞凋亡的作用。

关键词: 罗非鱼头磷脂, 破骨细胞, 分化, 凋亡

Abstract: This study was performed to investigate the effect of tilapia head phospholipid (TH-PL) on biological functions in osteoclasts. Total lipids from tilapia head were extracted with organic solvents and separated by silica gel column chromatography to obtain TH-PL. Its properties and structure were partially analyzed by thin layer chromatography (TLC), Fourier transform infrared spectroscopy (FTIR) and gas chromatography. In addition, an osteoclast model was established by inducing the differentiation of RAW264.7 macrophages in vitro with receptor activator for nuclear factor-κB ligand (RANKL). The effect of TH-PL on the differentiation of RAW264.7 macrophages into osteoclasts was explored by tartrate resistant acid phosphatase (TRAP) staining. Fluorescent staining, Western blotting and flow cytometry were used to detect osteoclast apoptosis. The results showed that TH-PL accounted for 1.52% of the dry mass of tilapia heads. TLC and FTIR determined that the as-prepared sample was phospholipid and rich in unsaturated fatty acids, accounting for 38% of the total fatty acids. TRAP staining experiments confirmed that TH-PL at doses of 50 and 100 μg/mL significantly inhibited the differentiation of RAW264.7 macrophages into osteoclasts. Furthermore, TH-PL promoted osteoclast apoptosis by reducing mitochondrial transmembrane potential and increasing the expression of caspase-3 in cells. In conclusion, in vitro cell experiments demonstrated that TH-PL could significantly inhibit osteoclast differentiation and promote osteoclast apoptosis.

Key words: tilapia head phospholipid, osteoclast, differentiation, apoptosis

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