Tang Yi, Liu Lin, Wang Pei, Chen Donglei, Wu Ziqiang, Tang Chunbo
Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, Jiangsu, China.
Department of Dental Implantology, Affiliated Hospital of Stomatology, Nanjing Medical University, Nanjing, Jiangsu, China.
Cell Prolif. 2017 Dec;50(6). doi: 10.1111/cpr.12369. Epub 2017 Aug 23.
Mesenchymal stem cell (MSC)-mediated periodontal tissue regeneration is considered to be a promising method for periodontitis treatment. The molecular mechanism of functional regulation by MSCs remains unclear, thus limiting their application. Our previous study discovered that Periostin (POSTN) promoted the migration and osteogenic differentiation of periodontal ligament mesenchymal stem cells (PDLSCs), but it is still unclear whether POSTN is able to restore the regenerative potential of PDLSCs under inflammatory conditions. In this study, we investigated the effect of POSTN on PDLSCs under inflammatory conditions and its mechanism.
PDLSCs were isolated from periodontal ligament tissue. TNF-α was used at 10 ng/mL to mimic inflammatory conditions. Lentivirus POSTN shRNA was used to knock down POSTN. Recombinant human POSTN (rhPOSTN) was used to stimulate PDLSCs. A scratch assay was used to analyse cell migration. Alkaline phosphatase (ALP) activity, Alizarin Red staining and expression of osteogenesis-related genes were used to investigate the osteogenic differentiation potential. Western blot analysis was used to detect the mitogen-activated protein kinases (MAPK) and AKT signalling pathways.
After a 10 ng/mL TNF-α treatment, knockdown of POSTN impeded scratch closure, inhibited ALP activity and mineralization in vitro, and decreased expression of RUNX2, OSX, OPN and OCN in PDLSCs, while 75 ng/mL rhPOSTN significantly accelerated scratch closure, enhanced ALP activity and mineralization in vitro, and increased expression of RUNX2, OSX, OPN and OCN. In addition, knockdown of POSTN inhibited expression of phosphorylated c-Jun N-terminal kinase (p-JNK), while 75 ng/mL rhPOSTN increased expression of p-JNK in PDLSCs with TNF-α treatment. Furthermore, inhibition of JNK by its inhibitor SP600125 dramatically blocked POSTN-enhanced scratch closure, ALP activity and mineralization in PDLSCs.
Our results revealed that POSTN might promote the migration and osteogenic differentiation potential of PDLSCs via the JNK pathway, providing insight into the mechanism underlying MSC biology under inflammatory conditions and identifying a potential target for improving periodontal tissue regeneration.
间充质干细胞（MSC）介导的牙周组织再生被认为是治疗牙周炎的一种有前景的方法。MSC功能调节的分子机制仍不清楚，从而限制了它们的应用。我们之前的研究发现骨膜蛋白（POSTN）促进了牙周膜间充质干细胞（PDLSCs）的迁移和成骨分化，但尚不清楚POSTN在炎症条件下是否能够恢复PDLSCs的再生潜能。在本研究中，我们研究了POSTN在炎症条件下对PDLSCs的影响及其机制。
从牙周膜组织中分离出PDLSCs。使用10 ng/mL的肿瘤坏死因子-α（TNF-α）模拟炎症条件。使用慢病毒POSTN短发夹RNA（shRNA）敲低POSTN。使用重组人POSTN（rhPOSTN）刺激PDLSCs。采用划痕试验分析细胞迁移。使用碱性磷酸酶（ALP）活性、茜素红染色和成骨相关基因的表达来研究成骨分化潜能。采用蛋白质印迹分析检测丝裂原活化蛋白激酶（MAPK）和AKT信号通路。
在10 ng/mL TNF-α处理后，敲低POSTN阻碍了划痕闭合，抑制了体外ALP活性和矿化，并降低了PDLSCs中RUNX2、OSX、OPN和OCN的表达，而75 ng/mL rhPOSTN显著加速了划痕闭合，增强了体外ALP活性和矿化，并增加了RUNX2、OSX、OPN和OCN的表达。此外，敲低POSTN抑制了磷酸化c-Jun氨基末端激酶（p-JNK）的表达，而75 ng/mL rhPOSTN增加了经TNF-α处理的PDLSCs中p-JNK的表达。此外，其抑制剂SP600125对JNK的抑制显著阻断了POSTN增强的PDLSCs划痕闭合、ALP活性和矿化。
我们的结果表明，POSTN可能通过JNK途径促进PDLSCs的迁移和成骨分化潜能，为深入了解炎症条件下MSC生物学的潜在机制提供了依据，并确定了一个改善牙周组织再生的潜在靶点。
