Division of Histology and Embryology, Joint Laboratory for Embryonic, Development and Prenatal Medicine, Medical College, Jinan University, Guangzhou, China.
Key Laboratory for Regenerative Medicine of the Ministry of Education, Jinan University, Guangzhou, China.
J Cell Physiol. 2020 Nov;235(11):8626-8639. doi: 10.1002/jcp.29707. Epub 2020 Apr 23.
Folic acid (FA) is routinely supplemented in the food of pregnant women or women planning a pregnancy, but whether FA exerts a positive effect on preventing fetal bone malformation remains obscure. In this study, we first exposed chick embryos with different concentrations of FA (1-10,000 pmol/egg) and studied vertebral mineralization and ossification through alcian blue and alizarin red as well as hematoxylin and eosin staining. Morphological measurements of the thoracic vertebral bodies demonstrated that 100 pmol/egg FA exhibited the tendency of shortening the growth plate, extended the ossification center, and increased the amount of Type I collagen. Second, we suggested that FA treatment promotes osteogenesis by demonstrating increased RUNX family transcription factor 2 (Runx2) and Osterix expressions in MC3T3-E1 and ATDC5 cells. Transforming growth factor-β (TGF-β) signaling was also upregulated by FA exposure, and addition of smad2/3 small interfering RNA knocks down FA-induced increased p-smad2/3, Runx2, and Osterix expression in vitro during chondrogenesis induction. Third, we employed dexamethasone (Dex), exposed chick embryos as an animal model of skeletal developmental retardation, to explore whether FA could rescue the loss of embryonic bone mass. Micro-computed tomography imaging showed that the addition of FA improved the reduction of bone mass in our model. Histological analysis of the vertebral bodies revealed that FA dramatically improved the delayed turnover of the zones of growth plate caused by Dex exposure. Immunofluorescence on the chick embryonic vertebrae and chondrocytes showed that FA supplementation upregulated the expression of TGF-β1, p-smad2/3, and improved Runx2 as well as Osterix expression in the Dex + FA group compared with the Dex group. Lastly, we found that supplementation with TGF-β1 (1 ng/egg) rescued bone mass loss caused by Dex as was also seen in FA exposure. Taken together these results, our data revealed that FA supplementation was able to rescue Dex exposure-induced inhibitive osteogenesis through targeting on the TGF-β signaling pathway.
叶酸（FA）通常被添加到孕妇或计划怀孕的妇女的食物中，但 FA 是否对预防胎儿骨骼畸形有积极作用仍不清楚。在这项研究中，我们首先用不同浓度的 FA（1-10000pmol/卵）暴露鸡胚，并通过阿利新蓝和茜素红以及苏木精和伊红染色研究椎体矿化和骨化。胸椎体的形态学测量表明，100pmol/卵 FA 表现出缩短生长板、延长骨化中心和增加 I 型胶原的趋势。其次，我们表明 FA 处理通过增加 MC3T3-E1 和 ATDC5 细胞中 RUNX 家族转录因子 2（Runx2）和 Osterix 的表达来促进成骨作用。FA 暴露还上调转化生长因子-β（TGF-β）信号通路，并且在用 smad2/3 小干扰 RNA 敲低时，FA 诱导的在体外软骨形成诱导过程中 p-smad2/3、Runx2 和 Osterix 的表达增加。第三，我们使用地塞米松（Dex）作为骨骼发育迟缓的动物模型来研究 FA 是否可以挽救胚胎骨量的损失。微计算机断层扫描成像显示，添加 FA 改善了我们模型中骨量的减少。椎体的组织学分析表明，FA 显著改善了 Dex 暴露引起的生长板区代换延迟。对鸡胚椎体和软骨细胞的免疫荧光显示，与 Dex 组相比，FA 补充剂上调了 TGF-β1、p-smad2/3 的表达，并改善了 Dex+FA 组中 Runx2 和 Osterix 的表达。最后，我们发现补充 TGF-β1（1ng/卵）可挽救 Dex 暴露引起的骨量损失，这与 FA 暴露的结果相似。综上所述，我们的数据表明，FA 补充剂能够通过靶向 TGF-β 信号通路来挽救 Dex 暴露引起的抑制性成骨作用。
