Lu Zhenhui, Wang Liqin, Pan Hongmei, Lin Xiao, Lin Cuiwu, Liu Buming, Zheng Li, Zhao Jinmin
Cell Physiol Biochem. 2015;37(3):1196-209. doi: 10.1159/000430243. Epub 2015 Sep 30.
The phenotype of chondrocyte is easy to be lost when expanded in vitro by a process defined "dedifferentiation". Traditional growth factors such as transforming growth factor (TGF-β1) are effective in preventing of dedifferentiation, but high costs and loss of activity limited their use. It is of significance to find substitutes which can reduce dedifferentiation and preserve chondrocytes phenotype to ensure sufficient differentiated cells for further study.
We synthesized new type of sulfonamido-based gallates named ZXHA-C and investigated its effect on primary articular chondrocytes of rats. After preliminary screening by cytotoxicity test, ZXHA-C of 1.06 × 10-8, 1.06 × 10-7 and 1.06 × 10-6M were chosen for further studies. Cell proliferation, morphology, viability, GAG synthesis and cartilage specific gene expression were detected. Also the effects of ZXHA-C on Wnt/β-catenin signaling pathway were investigated.
ZXHA-C could significantly promote chondrocytes growth. And it could enhance ECM synthesis by up-regulating expression levels of cartilage specific markers like aggrecan, collagen II and Sox9. Expression of collagen I which marked chondrocytes dedifferentiation was also significantly down-regulated after treated by ZXHA-C. Further exploration of the molecular mechanism indicated that ZXHA-C activated the Wnt/β-catenin signal pathway in chondrocytes, as evidenced by up-regulated gene expression of β-catenin, Wnt-4, cyclin D1 and Frizzled-2 and decreased glycogen synthase kinase 3β (GSK-3β). Among the various concentrations, ZXHA-C of 1.06 × 10-7 M showed the best performance, which was close to positive control (group with TGF-β1).
ZXHA-C might be potential a novel agent for the maintenances of chondrocytes phenotype.
软骨细胞在体外扩增时，其表型容易通过一种被定义为“去分化”的过程而丧失。传统的生长因子如转化生长因子（TGF-β1）在防止去分化方面是有效的，但高成本和活性丧失限制了它们的使用。寻找能够减少去分化并保留软骨细胞表型的替代物，以确保有足够的分化细胞用于进一步研究具有重要意义。
我们合成了一种新型的基于磺酰胺的没食子酸盐，命名为ZXHA-C，并研究了其对大鼠原代关节软骨细胞的影响。通过细胞毒性试验进行初步筛选后，选择1.06×10-8、1.06×10-7和1.06×10-6M的ZXHA-C进行进一步研究。检测细胞增殖、形态、活力、糖胺聚糖合成和软骨特异性基因表达。此外，还研究了ZXHA-C对Wnt/β-连环蛋白信号通路的影响。
ZXHA-C能显著促进软骨细胞生长。它可以通过上调软骨特异性标志物如聚集蛋白聚糖、胶原蛋白II和Sox9的表达水平来增强细胞外基质的合成。经ZXHA-C处理后，标志软骨细胞去分化的胶原蛋白I的表达也显著下调。对分子机制的进一步探索表明，ZXHA-C激活了软骨细胞中的Wnt/β-连环蛋白信号通路，β-连环蛋白、Wnt-4、细胞周期蛋白D1和卷曲蛋白-2的基因表达上调以及糖原合酶激酶3β（GSK-3β）降低证明了这一点。在各种浓度中，1.06×10-7M的ZXHA-C表现最佳，接近阳性对照（TGF-β1组）。
ZXHA-C可能是一种维持软骨细胞表型的潜在新型药物。
