Yang Zi-Jian, Huang Jun-Yao, Gao Ye-Fei, Huang Jun-Yao, Huang Wen-Ji, Huang Jun-Yao, Xu Shi-Lei, Huang Jun-Yao, Jin Jing-Jing, Huang Jun-Yao, Wan Ye-Dong, Huang Jun-Yao, Yan Ming, Huang Jun-Yao, Mao Hong-Jiao, Huang Jun-Yao, Zhang Yun, Huang Jun-Yao
College of Medicine, Shaoxing University, Shaoxing 312000.
School of Automation, Biomedical Engineering Department, Hangzhou Dianzi University, Hangzhou 310018, China.
Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2019 Nov;35(6):563-568. doi: 10.12047/j.cjap.5845.2019.124.
To study the effects of bergapten (BP) on damages of osteocytes MLO-Y4 induced by tricalcium phosphate (TCP) wear particles and its mechanism. ;Methods: MLO-Y4 cells were treated with TCP wear particles for 48 h to establish the model of osteocytes injuries in vitro. The MLO-Y4 cells were divided into the following five groups: control group, TCP wear particles treated (0.1 mg/ml) group, bergapten (1, 5 and 20 μmol/L) treated groups. MTT assay and Calcein-AM staining were used to determine the viability of MLO-Y4 cells; Hoechst 33342 staining and the flow cytometry were applied to detect the apoptosis of MLO-Y4; real-time PCR was performed to examine the mRNA levels of dentin matrix protein1 (DMP-1), sclerostin (SOST) and fibroblast growth factor23 (FGF23); Western blot was performed to examine protein expressions of glucose-regulated protein 78 (GRP78), protein kinase R-like ER kinase (PERK) phospho-PERK (p-PERK), eukaryotic initiation factor 2α (eIF2α), phospho-eIF2α (p-eIF2α), activating transcription factor 4 (AFT4), C/EBP homologous protein (CHOP) and caspase-3 in MLO-Y4 cells. ;Results: Compared with control group, the MLO-Y4 viability and DMP-1 mRNA level in TCP group were decreased significantly (P＜0.05), while the percentage of apoptosis and mRNA levels of SOST and FGF23 were obviously increased (P＜0.05), and protein expressions of GRP78, AFT4, CHOP, p-PERK/PERK and p-eIF2α/eIF2α were up-regulated significantly in MLO-Y4 cells (P＜0.05). Compared with TCP group, the damages of MLO-Y4 and cell apoptosis in bergapten treated groups were decrease obviously (P＜0.05), the expressions of GRP78, AFT4, CHOP, p-PERK/PERK and p-eIF2α/eIF2α were down-regulated remarkably (P＜0.05). ;Conclusion: Bergapten can inhibit osteocytes damages induced by TCP wear particles, which may be related to reducing ER stress and PERK pathway activation.
研究补骨脂素（BP）对磷酸三钙（TCP）磨损颗粒诱导的骨细胞MLO-Y4损伤的影响及其机制。方法：用TCP磨损颗粒处理MLO-Y4细胞48小时，以建立体外骨细胞损伤模型。将MLO-Y4细胞分为以下五组：对照组、TCP磨损颗粒处理（0.1mg/ml）组、补骨脂素（1、5和20μmol/L）处理组。采用MTT法和钙黄绿素-AM染色法测定MLO-Y4细胞的活力；采用Hoechst 33342染色法和流式细胞术检测MLO-Y4细胞的凋亡情况；采用实时定量PCR检测牙本质基质蛋白1（DMP-1）、硬化蛋白（SOST）和成纤维细胞生长因子23（FGF23）的mRNA水平；采用蛋白质免疫印迹法检测MLO-Y4细胞中葡萄糖调节蛋白78（GRP78）、蛋白激酶R样内质网激酶（PERK）、磷酸化PERK（p-PERK）、真核起始因子2α（eIF2α）、磷酸化eIF2α（p-eIF2α）、激活转录因子4（AFT4）、C/EBP同源蛋白（CHOP）和半胱天冬酶-3的蛋白表达。结果：与对照组相比，TCP组MLO-Y4细胞活力和DMP-1 mRNA水平显著降低（P＜0.05），而凋亡率以及SOST和FGF23的mRNA水平明显升高（P＜0.05），MLO-Y4细胞中GRP78、AFT4、CHOP、p-PERK/PERK和p-eIF2α/eIF2α的蛋白表达显著上调（P＜0.05）。与TCP组相比，补骨脂素处理组MLO-Y4细胞损伤和细胞凋亡明显减少（P＜0.05），GRP78、AFT4、CHOP、p-PERK/PERK和p-eIF2α/eIF2α的表达显著下调（P＜0.05）。结论：补骨脂素可抑制TCP磨损颗粒诱导的骨细胞损伤，这可能与减轻内质网应激和PERK通路激活有关。
