Department of Orthopaedic Surgery, The First People's Hospital of Wenling, Chuan'an Nan Road NO 333, Wenling, 317500, Zhejiang, China.
J Orthop Surg Res. 2023 Oct 8;18(1):761. doi: 10.1186/s13018-023-04250-1.
The aim of this study was to investigate whether Osteonectin/Secreted protein acidic and rich in cysteine (ON/SPARC) had a two-way dose-dependent regulatory effect on osteoblast mineralization and its molecular mechanism.
Initially, different concentrations of ON were added in osteoblasts, and the gene of bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN) and alkaline phosphatase (ALP) were detected using reverse-transcription quantitative polymerase chain reaction (RT-PCR). Secondly, based on the above results, the Optima and inhibitory concentration of ON for osteoblast mineralization were determined and regrouped, the Control group was also set up, and the gene detections of Collagen 1 (Col 1), Discoidin domain receptor 2 (DDR2) and p38 mitogen‑activated protein kinase were added using RT-PCR. In the third stage of the experiment, osteoblasts were pretreated with 0.4Mm ethyl-3,4-dihydroxybenzoate (DHB) (a specific inhibitor of collagen synthesis) for 3 h before adding the optima SPARC, the gene and protein expressions of OCN, OPN, BSP, ALP, DDR2, ALP, Col 1, DDR2 and P38 were detected by RT‑qPCR and western blot analysis, and the mineralized nodules were observed by alizarin red staining.
The results showed that the expression of OCN, OPN, BSP, ALP, DDR2, ALP, Col 1, DDR2 and P38 genes and proteins in osteoblasts were significantly enhanced by 1 ug/ml ON, 100 ug/ml ON or 1 ug/ml ON added with 3,4 DHB significantly inhibited the expressions of DDR2, P38 and the above-mentioned mineralization indexes, and significantly reduced the formation of mineralized nodules.
This study suggested that ON had a bidirectional dose-dependent regulatory effect on osteoblast mineralization, and the activation of P38 pathway by collagen binding to DDR2 was also an important molecular mechanism.
本研究旨在探讨骨连接蛋白/富含半胱氨酸酸性分泌蛋白（ON/SPARC）对成骨细胞矿化是否具有双向剂量依赖性调节作用及其分子机制。
首先，在成骨细胞中加入不同浓度的 ON，采用反转录定量聚合酶链反应（RT-PCR）检测骨涎蛋白（BSP）、骨钙素（OCN）、骨桥蛋白（OPN）和碱性磷酸酶（ALP）的基因。其次，根据上述结果，确定并分组 ON 对成骨细胞矿化的最佳和抑制浓度，同时设置对照组，并采用 RT-PCR 检测Ⅰ型胶原（Col 1）、盘状结构域受体 2（DDR2）和 p38 丝裂原活化蛋白激酶的基因。在实验的第三阶段，在加入最佳浓度的 SPARC 之前，用 0.4mM 乙基-3,4-二羟基苯甲酸（DHB）（胶原合成的特异性抑制剂）预处理成骨细胞 3h，采用 RT-qPCR 和 Western blot 分析检测 OCN、OPN、BSP、ALP、DDR2、Col 1、DDR2 和 P38 的基因和蛋白表达情况，并用茜素红染色观察矿化结节。
结果显示，1ug/mlON、100ug/mlON 或加入 3,4-DHB 的 1ug/mlON 均可显著增强成骨细胞中 OCN、OPN、BSP、ALP、DDR2、ALP、Col 1、DDR2 和 P38 基因和蛋白的表达，而加入 3,4-DHB 可显著抑制 DDR2、P38 及上述矿化指标的表达，显著减少矿化结节的形成。
本研究表明，ON 对成骨细胞矿化具有双向剂量依赖性调节作用，而胶原与 DDR2 结合激活 P38 通路也是一个重要的分子机制。
