氧化应激参与调控磷酸三钙磨损颗粒诱导假体周围骨细胞凋亡和自噬

Oxidative Stress Contributes to Apoptosis and Autophagy of Calavria Osteocytes Induced by Tricalcium Phosphate Wear Particles

目的:研究氧化应激在磷酸三钙(TCP)磨损颗粒诱导假体周围骨细胞凋亡和自噬中的作用。方法:36只雄性ICR小鼠随机分为3组:正常组、TCP磨损颗粒(TCP)组和N-乙酰半胱氨酸(NAC)组,每组12只。TCP组和NAC组小鼠采用TCP磨损颗粒30 mg置于小鼠颅骨中缝骨膜外缝合皮肤构建小鼠颅骨溶解模型,NAC组小鼠于术后第2天颅顶皮下注射抗氧化剂NAC(1.0 mg/kg),2天1次。2周后处死动物取血清和颅骨。应用HE染色观察小鼠假体周围骨细胞活性;ELISA法检测血清中牙本质基质蛋白1(DMP-1)和硬化蛋白(SOST)等骨细胞特征蛋白水平;TUNEL和DCFH-DA染色经流式细胞术检测假体周围骨细胞凋亡和活性氧(ROS)含量的变化;化学比色法检测假体周围骨细胞中丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性;Western blot法检测小鼠假体周围骨细胞自噬关键分子Beclin-1和微管相关蛋白1轻链3(LC-3)等蛋白的表达变化。结果:与正常组比较,TCP组小鼠假体周围骨细胞死亡、功能损伤及骨细胞凋亡显著增加(P<0.05),骨细胞中MDA和ROS含量明显升高,而SOD活性明显降低(P<0.05),自噬相关蛋白Beclin-1和LC-3表达显著上调,LC-3I向LC-3II转换明显增加(P<0.05);与TCP组比较,NAC组假体周围骨细胞死亡、凋亡及自噬的活化均被明显抑制(P<0.05)。结论:氧化应激参与调控TCP磨损颗粒诱导的假体周围骨细胞凋亡和自噬。

Objective To observe whether the oxidative stress contributes to the apoptosis and autoph-agy of calvaria osteocytes induced by tricalcium phosphate（TCP） wear particles in mice. Methods Thir-ty-six ICR male mice were randomly divided into a normal control group,a TCP wear particle（TCP）group and an N-acetyl-L-cysteine（NAC） group. A murine calvarial model of osteolysis was establishedby implantation 30 mg of TCP wear particles onto the surface of the bilateral parietal bones followingthe removal of the periosteum. On the 2 nd day after the successful modeling,the NAC group was local-ly injected antioxidant NAC（1.0 mg/kg） onto the calvaria under the periosteum every three days for 2 weeks. Then,the mice were sacrificed to obtain blood and the calvaria. The hematoxylin and eosinstaining and enzyme-linked immunosorbent assay were used to observe the viability of calvaria osteo-cytes and serum levels of the dentin matrix protein 1（DMP-1） and sclerostin（SOST）. The flow cytometry was performed to detect the apoptosis and reactive oxygen species（ROS） of calvaria osteocytes us-ing the terminal-deoxynucleoitidyl transferase mediated nick end labeling（TUNEL） staining and 2,7-di-chlorodihydrofluorescein diacetate（DCFH-DA） staining respectively. Moreover,the chemical colorimetrywas applied to examine the malondialdehyde（MDA） level and the superoxide dismutase（SOD） activity,while the Western blot was used to examine the protein’s expression of Beclin-1 and LC-3 in calvar-ia osteocytes. Results Compared with the control group,there was a significant increase in the percent-age of death and apoptosis of osteocytes and levels of MDA and ROS in calvaria osteocytes,as well asa significant up-regulation of autophagy biomarkers such as Beclin-1,LC-3 and the conversion of LC3-II from LC3-I,but a significant decrease in the SOD activity of the TCP group（P〈0.05 for all）. Com-pared with the TCP group,the death,apoptosis and activation of autophagy were all inhibited significant-ly in the NAC group（P〈0.05）. Conclusion Oxidative stress contributes to the apoptosis and autopha-gy of calavria osteocytes induced by TCP wear particles.