PERK-eIF2α-ATF4信号通路参与调控磷酸三钙磨损颗粒诱导的假体周围骨溶解

doi:10.16098/j.issn.0529-1356.2018.01.016

摘要/Abstract

摘要：

目的探讨蛋白激酶R样内质网激酶（PERK）-真核细胞起始因子2α（eIF2α）-活化转录因子4（ATF4）介导的内质网应激通路在磷酸三钙（TCP）磨损颗粒诱导假体周围骨溶解中的作用。方法取雄性ICR小鼠30只，随机分为3组：假手术组（sham，n=10）、TCP磨损颗粒组（模型组，n=10）和salubrinal干预组(SAL，n=10)。采用TCP磨损颗粒诱导小鼠颅骨溶解模型，于术后第2天颅顶局部注射SAL（1 mg/kg），每隔2 d 1次，持续干预2周。实验结束后处死动物取颅骨和外周血。Western blotting法检测TCP磨损颗粒植入部位周围骨组织中内质网应激分子伴侣葡萄糖调节蛋白78（GRP78）、C/EBP同源蛋白（CHOP）表达水平及PERK-eIF2α-ATF4信号通路的活化情况；HE染色和抗酒石酸酸性磷酸酶（TRAP）染色观察SAL对假体周围骨溶解和破骨细胞形成的影响；Real-time PCR检测SAL对破骨细胞活化相关基因抗酒石酸酸性磷酸酶（TRAP）、基质金属蛋白酶-9（MMP-9）和 c-fos的mRNA水平；ELISA法检测SAL对血清中肿瘤坏死因子α（TNF-α）、白细胞介素1β（IL-1β）和前列腺素E₂（PGE₂）水平的影响。结果 TCP磨损颗粒可诱导假体周围骨组织发生内质网应激反应，同时激活PERK-eIF2α-ATF4信号通路，表现为TCP磨损颗粒组小鼠颅骨组织中内质网应激通路蛋白GRP78、CHOP和磷酸化PERK（p-PERK）、磷酸化eIF2α（p-eIF2α）和ATF4蛋白表达均显著上调，而PERK和eIF2α蛋白明显下调（P<0.05）；而颅顶局部注射eIF2α特异性抑制剂SAL可明显阻止TCP磨损颗粒诱导假体周围破骨细胞形成和骨溶解，减少TRAP、MMP-9和cathepsin K 的mRNA水平（P<0.05）；同时抑制TNF-α、PGE2和IL-1β等炎症因子的产生（P<0.05）。结论 PERK-eIF2α-ATF4介导的内质网应激通路参与调控TCP磨损颗粒诱导的小鼠颅骨溶解；抑制该信号通路可减轻TCP磨损颗粒诱导的假体周围骨溶解和关节松动。

关键词: 磷酸三钙磨损颗粒, 骨溶解, 破骨细胞, PERK-eIF2α-ATF4信号通路, 抗酒石酸酸性磷酸酶染色, 免疫印迹法, 实时定量聚合酶链反应, 小鼠

Abstract:

Objective To explore whether protein kinase R-like ER kinase(PERK)-eukaryotic translation initator factor 2(eIF2α)-activating transcription factor 4(ATF4) signalling pathway is involved in periprosthetic osteolysis induced by tricalcium phosphate（TCP）wear particles in mice. Methods Thirty male ICR mice were randomly divided into sham group (n=10), TCP group (n=10) and salurinal (SAL) group (n=10). A murine calvarial model of osteolysis was established by 30 mg of TCP wear particles implantation onto the surface of bilateral parietal bones following removal of the periosteum. From the second postoperative day, SAL (1 mg/kg) was locally injected to the calvarium under the periosteum three times a week. After 2 weeks, the calvaria and serum were obtained. Western blotting was used to examine the expression of ER stress marker glucoseregulated protein 78（GRP78）and C/EBP homologous protein (CHOP), together with activation of PERK-eIF2α-ATF4 signalling pathway. Tartrate resistant acid phosphatase（TRAP）staining, HE staining and Real-time PCR were performed to observe the effects of SAL on TCP wear particles-induced osteolysis, osteoclastogenesis and mRNA levels of osteoclastogenic genes including TRAP, cathepsin K and c-Fos in the calvaria, respectively. Enzyme-linked immumsorbent as ay (ELISA) was performed to determine the effect of SAL on serum level of tumor necrosis factor-α (TNF-α), prostaglandin E₂ (PGE₂) and interleukin-1 beta (IL-1β). Results TCP wear particles triggered remarkable ER stress responses in the mouse calvaria, which was confirmed by up-regulation of ER stress markers GRP78 and C/EBP homologous protein (CHOP), and activation of protein kinase RNA-like ER kinase-eIF2α-activating transcription factor 4 pathway (including phosphorylation of both protein kinase RNA-like ER kinase and eIF2α and elevated level of activating transcription factor 4) (P<0.05). Block of the ER stress response with the eIF2α specific inhibitor salubrinal markedly prevented TCP wear particles-induced osteoclastogenesis and osteolysis, and suppressed the increase in mRNA level of osteoclastogenic genes such as TRAP, MMP-9 and c-fos in the mouse calvaria (P<0.05). In addition, SAL significantly inhibited TCP wear particles-induced release of TNF-α, PGE2 and IL-1β (P<0.05). Conclusion PERK-eIF2α-ATF4 pathway is involved in the calvarial osteolysis induced by TCP wear particle in mice, and inhibition of this signalling pathway reduces periprosthetic osteolysis and prostheses caused by TCP wear debris.

Key words:

TCP wear particle, Osteolysis, Osteoclast, PERK-eIF2α-ATF4 pathway, Tartrate resistant acid phosphatas staining, Western blotting, Real-time PCR, Mouse

陈岳良张云楼倩萍王成龙杜康慧沈琪华毛红娇. PERK-eIF2α-ATF4信号通路参与调控磷酸三钙磨损颗粒诱导的假体周围骨溶解[J]. 解剖学报, 2018, 49(1): 98-103.

CHEN Yue-liang ZHANG Yun LOU Qian-ping WANG Cheng-long DU Kang-hui SHEN Qi-hua MAO Hong-jiao. PERK-eIF2α-ATF4 signaling pathway is involved in periprosthetic osteolysis induced by tricalcium phosphate wear particles in mice[J]. Acta Anatomica Sinica, 2018, 49(1): 98-103.

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