芍药苷干预成骨细胞凋亡抗骨质疏松作用研究

林紫微; 杨菲; 黄天一; 陈婷婷; 崔杰; 李梦雨; 华永庆

芍药苷干预成骨细胞凋亡抗骨质疏松作用研究

江苏省中药资源产业化过程协同创新中心，江苏省中药药效与安全性评价重点实验室，南京中医药大学药学院，江苏南京 210023

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出版历程
- 刊出日期: 2019-07-10

Study on the Anti-Osteoporosis Effect of Paeoniflorin on Osteoblast Apoptosis and Zebrafish Osteoporosis Model

摘要

摘要: 目的研究芍药苷(PF)对前成骨细胞凋亡的影响及相关机制，通过斑马鱼骨质疏松模型验证其整体效应。方法体外培养前成骨细胞系MC3T3-E1细胞，采用地塞米松（DEX，1 μmol/L）诱导细胞凋亡，观察PF的干预效应。采用MTT法检测细胞活力，流式细胞术检测细胞凋亡率，Western blot法检测抗凋亡蛋白Bcl-2和凋亡蛋白Bax的表达，免疫荧光检测FoxO3a的核易位情况。建立泼尼松龙（Pred，25 μmol/L）诱导的斑马鱼骨质疏松模型，钙黄绿素染色观察PF对Pred干扰的斑马鱼骨骼面积的影响，qPCR检测破骨标志基因抗酒石酸酸性磷酸酶（TRAP）、组织蛋白酶K(CTSK）、基质金属蛋白酶-9(MMP-9）以及成骨细胞标志基因碱性磷酸酶（ALP）、Runt相关转录因子2a（Runx2a）、锌指转录因子7（Sp7）的mRNA变化。结果 PF可缓解DEX对MC3T3-E1的细胞活力的抑制作用，并显著降低细胞凋亡率；对凋亡相关蛋白的检测发现，PF显著促进Bcl-2的蛋白表达，升高Bcl-2/Bax的比率；进一步研究发现PF可显著抑制FoxO3a的入核。与Pred组相比，PF增加了斑马鱼第一椎骨的骨骼面积，抑制了TRAP、CTSK和MMP-9的基因表达，促进了ALP、Runx2a和Sp7的基因表达。结论 PF能够抑制前成骨细胞凋亡，改善斑马鱼骨质疏松，该作用可能与升高Bcl-2/Bax比率及抑制FoxO3a的核易位有关。
- 芍药苷 /
- 前成骨细胞 /
- 凋亡 /
- FoxO3a /
- 斑马鱼 /
- 骨质疏松
Abstract: OBJECTIVE To investigate the effect of paeoniflorin (PF) on the pre-osteoblasts cell apoptosis and its related mechanisms， and to explore the effect of PF on zebrafish osteoporosis. METHODS In vitro， the pre-osteoblast cell line MC3T3-E1 cells were cultured and the dexamethasone (DEX)-induced cell apoptosis model was used to observe the protective effect of PF. The cell viability and cell apoptosis rate were detected by MTT assay and flow cytometry respectively. Western blot was used to detect the expressions of apoptosis-related proteins bcl-2 and Bax. The nuclear translocation of FoxO3a protein was detected by immunofluorescence assay. The prednisolone (Pred)-induced zebrafish osteoporosis model was established to observe the anti-osteoporosis effect of PF. The first vertebrae bone area of zebrafish was observed by calcein staining. qPCR was used to detect the mRNA expressions of osteoclast marker genes tartrate resistant acid phosphatase (TRAP)， cathepsin K (CTSK)， matrix metalloproteinases-9 (MMP-9) and osteoblast marker genes alkaline phosphatase (ALP)， runt-associated transcription factor 2a (Runx2a)， Sp7 transcription factor (Sp7). RESULTS PF alleviated the inhibitory effect of DEX on the MC3T3-E1 cell viability and reduced the cell apoptosis rate. Detection of apoptosis-related proteins revealed that PF significantly promoted Bcl-2 protein expression and increased the ratio of Bcl-2/Bax. PF also significantly inhibited the nuclear translocation of FoxO3a protein. Compared with the Pred group， PF increased the first vertebra bone area of zebrafish， inhibited the mRNA expressions of TRAP， CTSK and MMP-9， and promoted the mRNA expressions of ALP， Runx2a and Sp7. CONCLUSION PF inhibits pre-osteoblast apoptosis and alleviates zebrafish osteoporosis， which may be related to the increasing of Bcl-2/Bax ratio and inhibition of nuclear translocation of FoxO3a.
- paeoniflorin /
- pre-osteoblast /
- apoptosis /
- FoxO3a /
- zebrafish /
- osteoporosis

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