药学学报, 2020, 55(5): 907-914
引用本文:
陈婷婷, 黄天一, 李梦雨, 崔杰, 华永庆, 许惠琴. 1,2,3,4,6-五没食子酰葡萄糖的骨保护作用与Nrf2/HO-1信号通路的相关性研究[J]. 药学学报, 2020, 55(5): 907-914.
CHEN Ting-ting, HUANG Tian-yi, LI Meng-yu, CUI Jie, HUA Yong-qing, XU Hui-qin. Correlation between bone protection of 1,2,3,4,6-pentyl-O-galloyl-beta-D-glucose and Nrf2/HO-1 signaling pathway[J]. Acta Pharmaceutica Sinica, 2020, 55(5): 907-914.

1,2,3,4,6-五没食子酰葡萄糖的骨保护作用与Nrf2/HO-1信号通路的相关性研究
陈婷婷1,2,3, 黄天一1,2,3, 李梦雨1,2,3, 崔杰1,2,3, 华永庆1,2,3, 许惠琴2,3
1. 江苏省中药资源产业化过程协同创新中心, 江苏 南京 210023;
2. 江苏省中药药效与安全性评价重点实验室, 江苏 南京 210023;
3. 南京中医药大学药学院, 江苏 南京 210023
摘要:
研究1,2,3,4,6-五没食子酰葡萄糖(1,2,3,4,6-pentyl-O-galloyl-beta-D-glucose,PGG)骨保护作用及其抗成骨细胞凋亡相关机制。建立泼尼松龙诱导的斑马鱼骨质疏松模型,钙黄绿素染色观察PGG对斑马鱼骨骼面积的影响。体外培养骨髓间充质干细胞,茜素红染色观察钙化结节数目,qRT-PCR法检测成骨细胞分化相关指标Runt相关转录因子2(Runt-related transcription factor 2,Runx 2)和骨钙蛋白(osteocalcin,OCN)的mRNA水平;体外培养前成骨细胞系MC3T3-E1,过氧化氢(400 μmol·L-1)建立氧化应激模型,观察在该模型下PGG对成骨细胞的作用。采用MTT法检测PGG对成骨细胞活力的影响;Hoechst 33342染色观察PGG对细胞凋亡的作用;Western blot检测PGG对抗凋亡蛋白Bcl-2和促凋亡蛋白Bax、核因子E2相关因子2(nuclear factor erythroid-2-related factor 2,Nrf2)及下游蛋白血红素氧合酶-1(heme oxygenase-1,HO-1)的表达;DCFH-DA荧光染色检测细胞内的活性氧(reactive oxygen species,ROS)水平;JC-1荧光染色检测线粒体膜电位水平。结果显示,与模型组相比,PGG可以显著提高斑马鱼模型椎骨面积;成骨细胞分化第14天时,与空白组相比,PGG组钙化结节数目显著增多,Runx 2、OCN的mRNA水平显著提高;此外,在氧化应激环境下,PGG可以提高成骨细胞活力,显著减少凋亡细胞数,提高Bcl-2/Bax的比率;荧光染色结果显示,PGG降低细胞内ROS荧光密度,提高线粒体膜电位。Western blot实验数据显示,PGG可以促进核内Nrf2蛋白表达,并增强下游蛋白HO-1的表达。综上,PGG可改善斑马鱼骨质疏松,且该作用可能与调控Nrf2/HO-1信号通路改善线粒体功能障碍,抗氧化应激环境下成骨细胞凋亡从而促进骨形成有关。本研究为抗骨质疏松治疗药物的发现提供新的思路与线索。
关键词:   
Correlation between bone protection of 1,2,3,4,6-pentyl-O-galloyl-beta-D-glucose and Nrf2/HO-1 signaling pathway
CHEN Ting-ting1,2,3, HUANG Tian-yi1,2,3, LI Meng-yu1,2,3, CUI Jie1,2,3, HUA Yong-qing1,2,3, XU Hui-qin2,3
1. Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing 210023, China;
2. Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medical, Nanjing 210023, China;
3. School of Pharmacy, Nanjing University of Traditional Chinese Medicine, Nanjing 210023, China
Abstract:
To study the osteoprotective effect of 1,2,3,4,6-pentyl-O-galloyl-beta-D-glucose (PGG) its anti-osteoblast apoptosis related mechanism was investigated. A model of zebrafish osteoporosis induced by prednisolone (Pred, 25 μmol·L-1) was established in vivo, and calcein staining was used to detect the effect of PGG on the bone area of zebrafish. Bone marrow mesenchymal stem cells were cultured in vitro, and the number of calcified nodules was observed by alizarin red staining, and the relevant indexes of osteoblast differentiation runt-related transcription factor 2 (Runx 2), osteocalcin (OCN) mRNA level were detected by qRT-PCR. The osteoblast cell line MC3T3-E1 cells was cultured in vitro, and 400 μmol·L-1 hydrogen peroxide (H2O2) was used to intervene the injury to detect the effect of PGG on osteoblasts under oxidative stress. The effect of PGG on osteoblast activity was detected by MTT assay. The effect of PGG on apoptosis was observed by Hoechst 33342 staining. Western blot was used to detect the expression of Bcl-2, Bax, nuclear factor erythroid-2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). DCFH-DA fluorescence staining for detection of reactive oxygen species (ROS) levels. JC-1 staining was used to detect mitochondrial membrane potential levels. The results showed that PGG could significantly increase the vertebral area of the zebrafish model when compared with the model group. On the 14 th day of osteoblast differentiation, the number of calcified nodules in the PGG group was significantly increased when compared with the control group and the mRNA levels of Runx 2 and OCN were also significantly increased. In addition, under oxidative stress, PGG could increase osteoblast viability, significantly reduce the number of apoptotic cells, and increase the ratio of Bcl-2/Bax. Fluorescence staining results show that PGG decreased intracellular ROS fluorescence density and increased mitochondrial membrane potential. Western blot data showed that PGG could promote the expression of Nrf2 in the nuclear and enhance the expression of downstream protein HO-1. In conclusion, PGG could improve osteoporosis in zebrafish, and this effect may be related to the regulation of Nrf2/HO-1 signaling pathway to improve mitochondrial dysfunction, anti-oxidative stress in osteoblast apoptosis and promote bone formation. This study provides new ideas and clues for the discovery of anti-osteoporosis drugs.
Key words:   
收稿日期: 2019-09-24
DOI: 10.16438/j.0513-4870.2019-0773
基金项目: 国家自然科学基金资助项目(81473390);江苏省中药资源产业化过程协同创新中心开放课题(ZDXM-2-7);江苏省中药药效与安全性评价重点实验室资助项目(JKLPSE201818);江苏省高校优势学科建设项目(PAPD).
通讯作者: 华永庆,Tel:86-25-85811248,E-mail:huayongqing@126.com;许惠琴,Tel:86-25-85811933,E-mail:hqxu309@sina.com
Email: huayongqing@126.com;hqxu309@sina.com
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