不同抗氧化剂对钴纳米粒子毒性的拮抗作用比较

Comparing the antagonistic effects of different antioxidants on the toxicity of cobalt nanoparticles

目的:探讨不同抗氧化剂对钴纳米粒子(CoNPs)诱导的成纤维细胞毒性的保护作用。方法:将Balb/3T3小鼠胚胎成纤维细胞设为空白对照组、CoNPs组、CoNPs+抗坏血酸组(AA组)、CoNPs+乙酰半胱氨酸组(NAC组)、CoNPs+褪黑素组(MT组)以及CoNPs+α-生育酚组(α-tocopherol组)6个组。将不同浓度AA、NAC、MT及α-tocopherol预处理Balb/3T3细胞4 h,再给予CoNPs刺激细胞4 h、24 h及48 h。采用CCK8法检测各组细胞活力,荧光染色技术检测细胞内活性氧(ROS)生成,谷胱甘肽(L-Glutathione,GSH)试剂盒检测细胞内GSH含量,ELISA法检测细胞培养上清液中肿瘤坏死因子α(TNF-α)、白介素-1β(IL-1β)和白介素-6(IL-6)水平。结果:CoNPs刺激Balb/3T3细胞4 h、24 h和48 h后,细胞活力较对照组明显降低,差异均有统计学意义(P<0.05),各抗氧化剂组细胞活力较CoNPs组明显上升,差异均有统计学意义(P<0.05),其中α-tocopherol组细胞活力高于其他抗氧化剂组。CoNPs组ROS生成较对照组明显增加,各抗氧化剂组ROS生成较CoNPs组明显降低,差异均有统计学意义(P<0.05)。CoNPs组细胞培养上清液中TNF-α、IL-1β和IL-6水平较对照组明显增加,各抗氧化剂组上清液中TNF-α,IL-1β和IL-6水平较CoNPs组降低,差异均有统计学意义(P<0.05)。结论:抗氧化剂通过减少钴纳米粒子诱导的成纤维细胞ROS产生,增加细胞内GSH含量,降低炎症因子水平,从而减轻钴纳米粒子细胞毒性,并且α-tocopherol是拮抗CoNPs毒性最有效的抗氧化剂。

Objective: To explore the protective effects of various antioxidants on fibroblast toxicity induced by cobalt nanoparticles(CoNPs). Methods: The Balb/3T3 cell was divided into 6 groups: blank control, CoNPs, CoNPs+AA,CoNPs+NAC, CoNPs+MT, and CoNPs+α-tocopherol.The cell was pretreated with different concentrations of AA, NAC, MT and α-tocopherol for 4 hours, and then stimulated with CoNPs for 4 h,24 h and 48 h. CCK8 was used to detect the cell viability induced by CoNPs and antioxidants.The fluorescent staining was used to detect ROS generation. The ELISA method was used to detect inflammatory cytokines and the GSH level in cells was measured. Results: After CoNPs stimulation, the cell viability decreased significantly than that in the control group(P<0.05);and the cell viability in the different antioxidants groups increased than that in the CoNPs group(P<0.05), and the cell viability was the highest in the α-tocopherol group. Compared with the control group, cellular ROS level increased and GSH content decreased after CoNPs stimulation(P <0.05). However, ROS level decreased and GSH content increased in the antioxidants groups than that in the CoNPs group(P<0.05). Compared with the control group, tumour necrosis factor α(TNF-α), interleukin-1β(IL-1β) and interleukin-6(IL-6) levels in cell supernatant obviously increased after CoNPs stimulation(P <0.05). However, antioxidant pretreatment could significantly decrease levels of inflammatory cytokines(P<0.05). Conclusion: Antioxidants can reduce the cytotoxicity caused by CoNPs by reducing the production of ROS, increasing GSH level and reducing the levels of inflammatory cytokines. α-tocopherol was the most effective antioxidant against the toxicity of CoNPs.