摘要： 目的 探讨自噬在葛根素调节hFOB1.19人成骨细胞分化过程中的作用。方法 将成骨细胞分为A组（成骨分化诱导组）、B组（A组+10^-8 mol/L葛根素3 h）和C组[B组+5 mmol/L自噬抑制剂3-甲基嘌呤（3-MA）1 h]。采用碱性磷酸酶（ALP）和茜素红染色检测成骨细胞的分化及钙化；透射电镜检测成骨细胞内自噬小体的改变情况；免疫荧光染色检测自噬微管相关蛋白轻链3-Ⅱ（LC3-Ⅱ）在细胞内的分布情况；Western blotting检测成骨细胞内成骨分化和自噬相关蛋白的表达情况。结果 ALP和茜素红染色显示，葛根素（10^-8 mol/L）能够明显促进成骨细胞的分化，加入3-MA后葛根素的促成骨细胞分化作用受到抑制；葛根素促进成骨细胞自噬小体增加，3-MA抑制自噬小体增加；免疫荧光显示葛根素促进LC3-Ⅱ在细胞核表面的聚集，加入3-MA后，这种现象被抑制；葛根素促进成骨分化蛋白矮小相关转录因子2（RUNX 2）、骨保护素（OPG）和自噬相关蛋白LC3-Ⅱ、BECLIN 1的表达，3-MA抑制LC3-Ⅱ和BECLIN 1的表达。结论 葛根素通过自噬调节成骨细胞分化。

自噬在葛根素促成骨细胞分化调节中的作用

于冬冬¹, 赵丹阳², 杨鸫祥¹

1. 辽宁中医药大学附属医院骨科, 沈阳 110032;
2. 沈阳市第一人民医院神经内科, 沈阳 110041

收稿日期:2020-10-14出版日期:2021-09-30发布日期:2021-09-18
通讯作者:杨鸫祥E-mail:ydx18940166713@163.com
作者简介:于冬冬(1983-),男,副主任医师,博士.
基金资助:国家自然科学基金（81973719）；辽宁省教育厅科学技术研究项目（L201921）；沈阳市科学技术计划（20-205-4-040）；辽宁中医药大学中医脏象理论及应用教育部重点实验室开放基金（zyzx1908）


关键词: 葛根素, 自噬, 成骨细胞
Abstract: Objective To explore the role of autophagy in regulating hFOB1.19 human osteoblast differentiation by puerarin. Methods These experiments incorporated three different conditions:A) basic osteogenic differentiation-inducing conditions,B) conditions in A + 10^-8 mol/L puerarin for 3 h,and C) conditions in B + 5 mmol/L autophagy inhibitor 3-methyladenine (3-MA) for 1 h. Alkaline phosphatase (ALP) and Alizarin red staining were used to detect osteoblast differentiation and calcification. Transmission electron microscopy was used to detect changes in autophagosomes in osteoblasts. Immunofluorescence was used to detect subcellular distribution of the key autophagy regulatory protein microtubule-associated protein light chain 3-Ⅱ (LC3-Ⅱ) in osteoblasts. Western blotting was used to detect expression of osteogenic differentiation and autophagy related proteins in osteoblasts. Results ALP and alizarin red staining showed that puerarin (10^-8 mol/L) promoted osteoblast differentiation,and 3-MA inhibited osteoblast differentiation. Puerarin also promoted an increase in autophagosomes in osteoblasts,whereas 3-MA inhibited the puerarin-induced increase in autophagosomes. Immunofluorescence showed that puerarin promoted LC3-Ⅱ aggregation on the nuclear surface,which was inhibited by addition of 3-MA. Puerarin promoted RUNX 2,OPG,LC3-Ⅱand BECLIN 1 expression,while 3-MA inhibited expression of these proteins. Conclusion Puerarin regulates osteoblast differentiation through autophagy.
Key words: puerarin, autophagy, osteoblast
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