Kim Kyeong-Min, Son Hyo-Eun, Lim Young-Ju, Jang Won-Gu
Department of Biotechnology, School of Engineering, Daegu University, Gyeongbuk 38453, Republic of Korea; Research Institute of Anti-Aging, Daegu University, Gyeongbuk 38453, Republic of Korea.
Department of Biotechnology, School of Engineering, Daegu University, Gyeongbuk 38453, Republic of Korea; Research Institute of Anti-Aging, Daegu University, Gyeongbuk 38453, Republic of Korea.
Acta Histochem. 2023 Oct;125(7):152095. doi: 10.1016/j.acthis.2023.152095. Epub 2023 Sep 25.
Topiramate [2,3:4,5-bis-o-(1-methylethylidene) β-D-fructo-pyranose sulfamate; TPM] is one of the most used new-generation antiepileptic drugs. It has been reported to regulate the differentiation of human bone cells. However, the molecular mechanism of TPM in osteoblast differentiation is not fully elucidated. In the present study, we examined the effect of TPM on osteogenic differentiation of C3H10T1/2, MC3T3-E1, primary mouse calvarial cells, and primary bone marrow stem cells (BMSCs). Primary cells were isolated from mice calvaria and bone marrow respectively. Expression of the osteogenic gene was determined by RT-PCR. The osteogenic protein levels were measured by Western blot analysis. Alkaline phosphatase (ALP) staining experiment was performed to evaluate ALP activity. Alizarin red s (ARS) staining was performed to measure zebrafish caudal fin regeneration. Treatment of TPM up-regulated the osteogenic genes including distal-less homeobox 5 (Dlx5) and runt-related transcription factor 2 (Runx2). In addition, TPM also increased the Dlx5 and Runx2 protein levels, Smad1/5/9 phosphorylation, and alkaline phosphatase (ALP) activity. Furthermore, TPM activated AMPK, and inhibition of AMPK decreased TPM-induced osteogenic differentiation. In the zebrafish model, osteogenic effect of TPM was identified. TPM was increased amputated caudal fin rays of zebrafish. These results demonstrate that TPM enhances osteogenic differentiation via AMPK-mediated Smad1/5/9 phosphorylation.
托吡酯[2,3:4,5-双-o-(1-甲基亚乙基)-β-D-果糖吡喃糖氨基磺酸盐；TPM]是最常用的新一代抗癫痫药物之一。据报道，它可调节人骨细胞的分化。然而，TPM在成骨细胞分化中的分子机制尚未完全阐明。在本研究中，我们检测了TPM对C3H10T1/2、MC3T3-E1、原代小鼠颅骨细胞和原代骨髓干细胞（BMSC）成骨分化的影响。原代细胞分别从小鼠颅骨和骨髓中分离。通过RT-PCR测定成骨基因的表达。通过蛋白质免疫印迹分析测量成骨蛋白水平。进行碱性磷酸酶（ALP）染色实验以评估ALP活性。进行茜素红S（ARS）染色以测量斑马鱼尾鳍再生。TPM处理上调了包括远端同源盒5（Dlx5）和 runt相关转录因子2（Runx2）在内的成骨基因。此外，TPM还增加了Dlx5和Runx2蛋白水平、Smad1/5/9磷酸化以及碱性磷酸酶（ALP）活性。此外，TPM激活了AMPK，抑制AMPK可降低TPM诱导的成骨分化。在斑马鱼模型中，确定了TPM的成骨作用。TPM增加了斑马鱼截断的尾鳍射线。这些结果表明，TPM通过AMPK介导的Smad1/5/9磷酸化增强成骨分化。
