PI3K/Akt信号通路在磷酸三钙磨损颗粒诱导小鼠颅骨溶解中的作用

摘要

目的:研究PI3K/Akt信号通路在磷酸三钙(tricalcium phosphate,TCP)磨损颗粒诱导小鼠颅骨溶解中的作用.方法:36只雄性ICR小鼠随机分为假手术组(Sham)、模型组(TCP)和LY294002处理组,每组12只.取TCP磨损颗粒30 mg置于颅顶后缝合皮肤构建小鼠颅骨溶解模型.LY294002处理组小鼠于术后第2天颅顶局部注射PI3K抑制剂LY294002(5 mg·kg-1),每周3次;假手术组小鼠颅骨顶仅注射生理盐水,注射时间与LY294002一致.2周后处死动物取骨膜和颅骨.通过Micro-CT分析小鼠颅骨溶解情况、骨密度(bone mineral density,BMD)和骨矿含量(bone mineral content,BMC);HE染色观察颅骨表面炎症反应及破骨细胞(osteoclasts)形成;Real-time PCR检测骨组织中破骨细胞生成标志物抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)、组织蛋白酶K(cathepsin K,CstK)、核因子KB受体活化因子配体(receptor activator for nuclear factor-KB kigand,RANKL)和c-Fos的mRNA水平;ELISA检测骨膜中炎症因子TNF-α、IL-1β和IL-6等水平;Western Blotting检测颅骨组织Akt、p-AktSer473和p-AktThr308等蛋白表达变化.结果:Micro-CT和组织形态学分析结果显示,PI3K抑制剂LY294002能明显抑制TCP磨损颗粒诱导小鼠颅骨溶解和破骨细胞形成(P＜0.05),增加BMD和BMC含量(P＜0.05);下调破骨细胞生成标志物TRAP、CstK、RANKL和c-Fos等mRNA水平(P＜0.05),并抑制炎症因子TNF-α、IL-1β和IL-6的释放(P＜0.05);而且LY294002显著减弱TCP磨损颗粒诱导的Akt信号蛋白活化,导致p-AktS-er473和p-AktThr308等蛋白表达明显下调(P＜0.05).结论:PI3K/Akt信号通路参与调控TCP磨损颗粒诱导的假体周围骨溶解,可作为假体周围骨溶解和关节松动的治疗靶标.%Objective To explore the role of the PI3K/Akt signaling pathway in the calvarial osteolysis induced by TCP wear particles in mice model.Methods Thirty-six male ICR mice were randomly divided into a sham group (n=12),TCP group (n=12)and a LY294002-treated group (n=12).A murine calvarial model of osteolysis was established through implanting 30 mg of TCP particles onto the surface of bilateral parietal bones following the removal of the periosteum.On the second postoperative day,LY294002 (5 mg·kg-1)was locally injected to the calvarium under the periosteum three times a week;mice in the sham group received local injection of normal saline (N.S.)in the calvarium,and the injection time was consistent with that of LY294002.Two weeks later,the calvaria and periostea were obtained after the mice were executed.The calvarial osteolysis,bone mineral density (BMD)and bone mineral content(BMC)were analyzed using Micro-CT,Hematoxylin-Eosin (HE)staining was conducted to observe the inflammatrory response and formation of osteoclasts.Real-time PCR was applied to detect the mRNA level of tartrate-resistant acid phosphatase (TRAP),the marker of osteoclasts formation,cathepsin K (CstK),receptor activator for nuclear factor-κB kigand (RANKL)and c-Fos.The release of tumor necrosis factor-α (TNF-α),interleukin-6 (IL-6)and IL-1β were measured using enzyme-linked immumsorbent assay (ELISA).Results Micro-CT and histological analysis indicated that LY294002,the specific inhibitor of PI3K,significantly prevented TCP wear particles-induced osteolysis and osteoclastogenesis,and increased BMD and BMC in the calvaria of mice.Real-time PCR data revealed LY294002 significantly suppressed the increase in mRNA level of osteoclastogenic genes such as TRAP,CstK,RANKL and c-Fos in the calvaria of TCP wear particles-implanted group.ELISA assay showed that TCP wear particles-induced release of TNF-α,IL-1β and IL-6 was significantly inhibited by LY294002 treatment.Furthermore,LY294002 significantly attenuated TCP wear particles-triggered activation of Akt,and down-regulated the level of p-AktSer473 and p-AktThr308.Conclusion PI3K/Akt signaling pathway contributes to TCP wear particle-induced osteolysis,and can be developed as a new therapeutic target for the prevention and treatment of bone destruction diseases caused by wear debris.