氧化应激对磷酸三钙磨损颗粒诱导的假体周围骨溶解的影响及其机制

Effect of oxidative stress on periprosthetic osteolysis induced by TCP wear particles in mouse calvaria and its mechanism

目的:探讨氧化应激对磷酸三钙(TCP)磨损颗粒诱导的假体周围骨溶解的影响及其作用机制。方法:36只雄性ICR小鼠随机分为3组(n=12):假手术(Sham)组、TCP磨损颗粒(TCP)组和N-乙酰-L-半胱氨酸(NAC)组。将TCP磨损颗粒30 mg包埋于小鼠颅骨顶部构建假体周围骨溶解模型,于术后第2天颅顶骨膜局部注射NAC(1.0 mg/kg),隔日1次,持续2周后处死动物采血、取颅骨。抗酒石酸酸性磷酸酶(TRAP)染色观察小鼠颅骨假体周围骨溶解情况; ELISA和化学比色法检测血清中肿瘤坏死因子-α(TNF-α)、白介素-1β(IL-1β)、白介素-6(IL-6)和总抗氧化能力(T-AOC)含量及超氧化物歧化酶(SOD)活性; Western blot检测假体周围骨组织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)和磷酸化eIF2α(p-eIF2α)的表达。结果:与Sham组比较,TCP组小鼠血清TNF-α、IL-1β和IL-6水平及假体周围骨溶解面积显著增加(P<0.05),T-AOC含量和SOD活性明显降低(P<0.05),GRP78蛋白质表达、pPERK/PERK和p-eIF2α/eIF2α值显著升高。与TCP组比较,NAC组小鼠血清TNF-α、IL-1β和IL-6水平及骨溶解面积明显减少(P<0.05),血清T-AOC含量和SOD活性明显增加(P<0.05),GRP78蛋白质表达、p-PERK/PERK和p-eIF2α/eIF2α值明显降低。结论:抑制氧化应激可阻止TCP磨损颗粒诱导的假体周围骨溶解,其机制可能与PERK/eIF2α通路的失活有关。

Objective： To explore the effect of oxidative stress on periprosthetic osteolysis induced by TCP wear particles in mouse calvaria and its mechanism. Methods： Thirty-six male ICR mice were randomly divided into three groups（ n = 12） ： sham group,TCP wear particles（ TCP） group and N-acetyl-L-cysteine（ NAC） group. Aperiprosthetic osteolysis model in mouse was established by implanting 30 mg of TCP wear particles onto the surface of bilateral parietal bones following removal of the periosteum. On the2 nd day post-operation,NAC（ 1.0 mg/kg） was locally injected to the calvarium under the periosteum every other day for 2 weeks.Then,all the mice were sacrificed to obtain blood and the calvaria. Periprosthetic osteolysis in the mouse calvaria was observed by tartrate resistant acid phosphatase（ TRAP） staining; serum levels of tumor necrosis factor-alpha（ TNF-α）,interleukin-1 beta（ IL-1β）,interleukin-6（ IL-6）; total anti-oxidation capacity（ T-AOC） and superoxide dismutase（ SOD） activity were examined by ELISA and chemical colorimetry,respectively; protein levels of glucose-regulated protein 78（ GRP78）,protein kinase R-like ER kinase（ PERK）,phospho-PERK（ p-PERK）,eukaryotic initiation factor 2α（ eIF2α） and phospho-eIF2α（ p-eIF2α） in periprosthetic bone tissue were detected by Western blot. Results： Compared with sham group,serum levels of TNF-α,IL-1β and IL-6,and osteolysis area were increased obviously in TCP group（ P〈0.05）,and serum level of T-AOC and SOD activity were decreased significantly in TCP group（ P〈0.05）,GRP78 expression,the ratio of p-PERK and PERK,p-eIF2α and eIF2α in the mouse calvaria of TCP group were up-regulated markedly. Compared with TCP group,serum levels of TNF-α,IL-1β and IL-6,and osteolysis area were decreased markedly in NAC group（ P〈0.05）,serum level of T-AOC and SOD activity were increased obviously in NAC group（ P〈0.05）,and GRP78 expression,the ratio of p-PERK/PERK and p-eIF2α/eIF2α were obviously down-regulated. Conclusion： Inhibition of oxidative stress can prevent periprosthetic osteolysis induced by TCP wear particles,which may be mediated by inactivation of PERK/eIF2α signaling pathway.