Liu Ya-Ke, Yang Hong-Wei, Wang Meng-Hong, Wang Wei, Liu Fan, Yang Hui-Lin
Department of Orthopaedics, The First Affiliated Hospital of Soochow University, Suzhou, China.
Department of Orthopaedics, Nantong Third People's Hospital of Nantong University, Nantong, China.
Orthop Surg. 2016 Nov;8(4):496-502. doi: 10.1111/os.12298.
Complex cobalt-chromium alloys, bearing surfaces of the second-generation metal-on-metal (MoM) hip prostheses, are subject to wear and generate cobalt nanoparticles (CoNPs). CoNPs could reduce cellular viability, activate the mitogen-activated protein kinase (MAPK) pathway and increase cell apoptosis via reactive oxygen species (ROS). However, the detailed mechanisms of ROS functioning on CoNP-mediated signaling molecules and cytotoxicity has not yet been fully demonstrated. The present study investigated the functional role of N-acetylcysteine (NAC) in reversing the activation of ROS signaling pathways triggered by CoNPs in normal mice kidney cells (TCMK-1 cells).
After being pretreated with NAC, TCMK-1 cells were treated with 300-700 μmol/L CoNPs, then, CCK-8 assay was used to verify the survival of TCMK-1 cells. Annexin V/PI staining was performed to investigate the apoptosis of TCMK-1 cells after NAC and different concentrations of CoNP treatments. In addition, western blot was performed to identify the cytokine (p-ERK, p-p38, and p-JNK) expression of the ROS-related MAPK signaling pathway.
Apoptosis rate of TCMK-1 cells was increased obviously after different concentrations of CoNP treatment. However, TCMK-1 cells, pretreated with NAC, exhibited a significantly decreased apoptosis rate. In addition, p-ERK, p-p38, and p-JNK expressions were increased with CoNP treatment, which indicated that CoNPs could activate the MAPK pathway. Interestingly, this entire stimulated phenomenon by CoNPs was reversed with NAC treatment.
These findings indicated that NAC could reverse CoNP-induced cytotoxicity by inhibiting ROS-induced cell death and cytokine expression. To our knowledge, this is the first report that describes how CoNP-induced cytotoxicity in TCMK-1 cells could be attenuated by anti-oxidative agents (NAC), which may function through inhibition of cell death and ROS.
作为第二代金属对金属（MoM）髋关节假体的承载表面，复杂钴铬合金会发生磨损并产生钴纳米颗粒（CoNP）。CoNP可降低细胞活力，激活丝裂原活化蛋白激酶（MAPK）通路，并通过活性氧（ROS）增加细胞凋亡。然而，ROS在CoNP介导的信号分子和细胞毒性作用的详细机制尚未完全阐明。本研究探讨了N-乙酰半胱氨酸（NAC）在逆转正常小鼠肾细胞（TCMK-1细胞）中由CoNP触发的ROS信号通路激活方面的功能作用。
用NAC预处理后，将TCMK-1细胞用300 - 700μmol/L的CoNP处理，然后，使用CCK-8法验证TCMK-1细胞的存活率。进行膜联蛋白V/碘化丙啶（PI）染色以研究NAC和不同浓度CoNP处理后TCMK-1细胞的凋亡情况。此外，进行蛋白质免疫印迹法以鉴定ROS相关MAPK信号通路的细胞因子（磷酸化细胞外信号调节激酶（p-ERK）、磷酸化p38（p-p38）和磷酸化c-Jun氨基末端激酶（p-JNK））表达。
不同浓度CoNP处理后，TCMK-1细胞的凋亡率明显增加。然而，用NAC预处理的TCMK-1细胞凋亡率显著降低。此外，CoNP处理后p-ERK、p-p38和p-JNK表达增加，这表明CoNP可激活MAPK通路。有趣的是，CoNP的整个刺激现象在NAC处理后被逆转。
这些发现表明，NAC可通过抑制ROS诱导的细胞死亡和细胞因子表达来逆转CoNP诱导的细胞毒性。据我们所知，这是第一份描述抗氧化剂（NAC）如何减轻CoNP在TCMK-1细胞中诱导的细胞毒性的报告，其可能通过抑制细胞死亡和ROS发挥作用。
