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中国应用生理学杂志 ›› 2019, Vol. 35 ›› Issue (6): 563-568.doi: 10.12047/j.cjap.5845.2019.124

• 研究论文 • 上一篇    下一篇

佛手苷内酯对磷酸三钙磨损颗粒诱导骨细胞损伤的影响及机制*

杨子键1, 黄君瑶1, 高烨飞1, 黄文吉1, 徐世磊1, 金晶晶1, 万烨东1, 严明2, 毛红娇1, 张云1△   

  1. 1. 绍兴文理学院医学院基础医学部, 浙江 绍兴 312000;
    2. 杭州电子科技大学自动化学院生物医学工程系, 浙江 杭州 310018
  • 收稿日期:2019-03-14 出版日期:2019-11-28 发布日期:2020-04-02
  • 通讯作者: 国家自然科学基金资助项目(81700936);浙江省自然科学基金资助项目(LY17H060007);杭州电子科技大学教育教学改革研究资助项目(YBJG201843,《生物统计学》混合式教学模式改革实践);绍兴市大学生科技创新项目(SXSDC201809);浙江省大学生科技创新活动计划(2018R432005)

Effects of bergapten on damages of osteocytes MLO-Y4 induced by TCP wear particles and its mechanism

YANG Zi-jian1, HUANG Jun-yao1, GAO Ye-fei1, HUANG Wen-ji1, XU Shi-lei1, JIN Jing-jing1, WAN Ye-dong1, YAN Ming2, MAO Hong-jiao1, ZHANG Yun1△   

  1. 1. College of Medicine, Shaoxing University, Shaoxing 312000;
    2. School of Automation, Biomedical Engineering Department, Hangzhou Dianzi University, Hangzhou 310018, China
  • Received:2019-03-14 Online:2019-11-28 Published:2020-04-02

摘要: 目的:研究佛手苷内酯(BP)对磷酸三钙(TCP)磨损颗粒诱导骨细胞损伤的影响,并阐明其可能作用机制。方法:将TCP磨损颗粒与小鼠骨细胞MLO-Y4细胞共孵育48 h建立骨细胞体外损伤模型,随机分为正常对照(Control)组、TCP磨损颗粒(TCP,0.1 mg/ml)组、佛手苷内酯(1 μmol/L)组、佛手苷内酯(5 μmol/L)组和佛手苷内酯(20 μmol/L)组。MTT法和Calcein-AM染色检测各组骨细胞活性和形态改变;Hoechst 33342染色和流式细胞术分析各组骨细胞凋亡情况;实时荧光定量PCR检测各组骨细胞特征蛋白牙本质基质蛋白-1(DMP-1)、骨硬化蛋白(SOST)、成纤维细胞生长因子23(FGF23)的mRNA水平;Western blot法检测各组骨细胞中内质网应激标志蛋白葡萄糖调节蛋白78(GRP78)、蛋白激酶R样内质网激酶(PERK)、磷酸化PERK(p-PERK)、真核细胞翻译起始因子2α (eIF2α)、磷酸化eIF2α(p-eIF2α)、活性转录因子(ATF4)和 C/EBP 同源蛋白(CHOP)等的表达及caspase-3的活化变化。结果:与Control组比较,TCP组骨细胞的活性和DMP-1的mRNA水平显著降低(P<0.05),骨细胞凋亡率及SOST、FGF23的mRNA水平显著增加(P<0.05),GRP78、ATF4和CHOP等蛋白质表达、p-PERK/PERK值和p-eIF2α/eIF2α值显著升高;与TCP组比较,佛手苷内酯组骨细胞损伤明显减轻,骨细胞凋亡率显著减少(P< 0.05),GRP78、ATF4和CHOP等蛋白质表达、p-PERK/PERK值和p-eIF2α/PERK值也明显下降(P<0.05)。结论:佛手苷内酯可明显抑制TCP磨损颗粒所致的骨细胞损伤,其机制可能与减弱TCP磨损颗粒诱导的内质网应激反应及PERK通路的活化密切相关。

关键词: 佛手苷内酯, 磷酸三钙磨损颗粒, 骨细胞, 凋亡, 内质网应激

Abstract: Objective: 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.

Key words: bergapten, TCP wear particles, osteocytes, apoptosis, ER stress

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