中国骨质疏松杂志

壮骨止痛胶囊调控自噬相关蛋白对去卵巢大鼠骨髓间充质干细胞增殖与骨向分化的影响

Effect of Zhuanggu Zhitong Capsule regulating autophagy related proteins on proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells in ovariectomized rats

DOI：10.3969/j.issn.1006-7108.2022.01.020

中文关键词: 去势雌性大鼠骨髓间充质干细胞骨向分化自噬相关蛋白

英文关键词:ovariectomized female rats bone marrow mesenchymal stem cells bone differentiation autophagy-related protein

基金项目:湖南省自然科学基金面上项目（2018JJ2297）；湖南省教育厅科学研究项目（19A370、20A385）；湖南中医药大学校级科研基金项目和精准科技扶贫项目（2018XJJJ24)；湖南中医药大学大学生创新创业训练计划（2019-68）

作者	单位
陈沙李荣慧吴结枝曹宝丹田国力刘平安* 张国民*	湖南中医药大学，湖南长沙 410208

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中文摘要:

目的观察壮骨止痛胶囊调控自噬相关蛋白Beclin1、p62、LC3对去卵巢雌性大鼠骨向分化的影响。方法去除大鼠双侧卵巢进行造模，造模成功后分为模型组、壮骨止痛胶囊组、雷帕霉素（RAPA）组和3-甲基腺嘌呤（3-MA）组，另设空白组、假手术组（仅去除卵巢周围相应体积脂肪），每组7只。通过骨髓间充质干细胞（BMMSCs）原代培养测定生长曲线并检测其碱性磷酸酶（ALP）活力，通过免疫组织化学染色法分析股骨Beclin1、LC3及p62蛋白的表达，酶联免疫分析法检测血清PPAR-γ的表达。结果 ①与空白组比较，假手术组各项指标表达差异无统计学意义（P＞0.05）；②与假手术组相比，模型组各项指标均有显著差异（P﹤0.01）；③与模型组相比，壮骨止痛胶囊组、RAPA组和3-MA组三组BMMSCs的增长及ALP活力均显著升高（P﹤0.01），壮骨止痛胶囊组和3-MA组Beclin1和LC3蛋白平均灰度值表达显著降低（P﹤0.01），RAPA组表达显著升高（P﹤0.01）；p62蛋白平均灰度值表达显著升高（P﹤0.01），RAPA组表达显著降低（P﹤0.01），三组血清PPAR-γ值表达显著降低（P﹤0.01）；④与RAPA组相比，壮骨止痛胶囊组和3-MA组PPAR-γ值、BMMSCs增长及ALP活力表达有差异（P﹤0.05），Beclin1和LC3蛋白平均灰度值表达显著降低（P﹤0.01），p62蛋白平均灰度值表达显著升高（P﹤0.01），且壮骨止痛胶囊组和3-MA组各项指标表达差异无统计学意义（P＞0.05）。结论壮骨止痛胶囊通过脂向分化的自噬抑制作用促进成骨。

英文摘要:

Objective To observe the effects of Zhuanggu Zhitong Capsules regulating autophagy-related proteins Beclin1, p62, and LC3 on bone differentiation in ovariectomized female rats.Methods The bilateral ovaries were removed for modeling, and the rats after successful modeling were divided into model group, Zhuanggu Zhitong capsule group, rapamycin (RAPA) group and 3-methyladenine (3-MA) group. In the sham operation group, the corresponding volume of fat around the ovaries was removed, 7 in each group. Primary culture of bone marrow mesenchymal stem cells (BMMSCs) was used to determine the growth curve and the alkaline phosphatase (ALP) activity, immunohistochemical staining method to analyze the expression of femoral Beclin1, LC3 and p62 protein, and enzyme-linked immunoassay to detect serum PPAR-γ expression.Results ① Compared with the blank group, there was no statistically significant difference in the expression of various indicators in the sham operation group(P>0.05); ②Compared with the sham operation group, all indicators of the model group were significantly different (P<0.01); ③Compared with the model group, the growth of BMMSCs and ALP activity in the Zhuanggu Zhitong capsule group, RAPA group and 3-MA group were significantly increased (P<0.01). Beclin1 and LC3 in the Zhuanggu Zhitong capsule group and 3-MA group The average gray value of protein expression was significantly reduced (P<0.01), and the expression of RAPA group was significantly increased (P<0.01); the average gray value of p62 protein expression was significantly increased (P<0.01), and the expression of RAPA group was significantly decreased(P<0.01), the expression of serum PPAR-γ values in the 3 groups was significantly reduced (P<0.01); ④ Compared with the RAPA group, the Zhuanggu Zhitong capsule group and 3-MA group PPAR-γ value, BMMSCs growth and ALP activity expression were different (P<0.05), and the average gray value expression of Beclin1 and LC3 protein was significantly reduced (P<0.01), the average gray value of p62 protein expression was significantly increased(P<0.01), and there was no statistically significant difference in the expression of various indexes between the Zhuanggu Zhitong capsule group and the 3-MA group(P>0.05). Conclusion Zhuanggu Zhitong Capsule promotes osteogenesis through autophagy inhibition of lipid differentiation.

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