辛伐他汀通过 Wnt/β-连环蛋白通路诱导间充质干细胞成骨分化，促进骨折愈合。

Simvastatin induces osteogenic differentiation of MSCs via Wnt/β-catenin pathway to promote fracture healing.

机构信息

Department of Orthopedic, Liaocheng People's Hospital of Shandong, Liaocheng, China.

出版信息

Eur Rev Med Pharmacol Sci. 2018 May;22(9):2896-2905. doi: 10.26355/eurrev_201805_14992.

DOI:10.26355/eurrev_201805_14992

PMID:29771446

Abstract

OBJECTIVE

This study was designed to investigate whether Simvastatin could facilitate osteogenic differentiation of rat marrow mesenchymal stem cells (MSCs) by modulating the Wnt/β-catenin pathway, thus promoting fracture healing.

MATERIALS AND METHODS

MSCs were isolated from rat bone marrow specimens and their purity was identified. The third generation of MSCs was cultured in osteoinduction medium containing simvastatin of gradient concentration, and the highest dose of simvastatin that did not cause cell proliferation was determined by the result of the CCK8 assay. The effects of simvastatin on osteogenic differentiation of MSCs were evaluated by ALP activity, Alizarin red staining, alkaline phosphatase staining and osteoblast-specific gene expression. Finally, Wnt pathway antagonist DKK1 and β-catenin disturbing agent were added to MSCs to detect the ALP activity, Alizarin red staining, alkaline phosphatase staining and osteoblast-specific genes of MSCs respectively, and to evaluate whether simvastatin promoted osteogenic differentiation of MSCs by activating Wnt/β-catenin pathway.

RESULTS

After osteoinduction, simvastatin of 0.3 nmol/L was found to be the highest dose that did not induce the proliferation of MSCs. After treated with 0.3 nmol/L simvastatin for 7 days, the ALP activity of cells and the number of cell calcified nodules significantly increased. Meanwhile, the expression of osteoblast-related genes, including ALP, Runx2, OCN, and OPN, were clearly up-regulated. However, when the MSCs were treated with DKK1 for 7 days, the ALP activity and the expression of osteoblast-related genes, including ALP, Runx2, OCN, and OPN, were found decreased. Simvastatin markedly up-regulated the expression of the β-catenin protein, while transfection of β-catenin shRNA inhibited the expression of osteoblast-related genes including ALP, Runx2, OCN, and OPN.

CONCLUSIONS

Simvastatin can promote the differentiation of rat MSCs into osteoblast-like cells, and its mechanism may be related to the Wnt/β-catenin pathway.

摘要

目的

本研究旨在探讨辛伐他汀是否通过调节 Wnt/β-连环蛋白通路促进大鼠骨髓间充质干细胞（MSCs）的成骨分化，从而促进骨折愈合。

材料与方法

从大鼠骨髓标本中分离 MSCs，鉴定其纯度。将第三代 MSCs 培养在含有梯度浓度辛伐他汀的成骨诱导培养基中，通过 CCK8 检测确定不引起细胞增殖的辛伐他汀最高剂量。通过 ALP 活性、茜素红染色、碱性磷酸酶染色和骨细胞特异性基因表达来评估辛伐他汀对 MSCs 成骨分化的影响。最后，向 MSCs 中加入 Wnt 通路拮抗剂 DKK1 和 β-连环蛋白干扰剂，分别检测 MSCs 的 ALP 活性、茜素红染色、碱性磷酸酶染色和骨细胞特异性基因，以评估辛伐他汀是否通过激活 Wnt/β-连环蛋白通路促进 MSCs 的成骨分化。

结果

经成骨诱导后，发现 0.3 nmol/L 的辛伐他汀是不诱导 MSCs 增殖的最高剂量。用 0.3 nmol/L 辛伐他汀处理 7 天后，细胞的 ALP 活性和细胞钙化结节数量明显增加。同时，骨细胞相关基因，包括 ALP、Runx2、OCN 和 OPN 的表达明显上调。然而，当 MSCs 用 DKK1 处理 7 天时，ALP 活性和骨细胞相关基因，包括 ALP、Runx2、OCN 和 OPN 的表达均下降。辛伐他汀明显上调了β-连环蛋白蛋白的表达，而β-连环蛋白 shRNA 的转染抑制了包括 ALP、Runx2、OCN 和 OPN 在内的骨细胞相关基因的表达。