铜对大鼠肝细胞凋亡的影响以及姜黄素的保护作用

Relationship between copper injury and apoptosis and the effect of curcumin on copper-injured BRL cells

摘要:

目的：探讨铜对大鼠肝细胞（BRL细胞）凋亡的诱导作用及其机制，研究姜黄素对BRL细胞铜损伤时的影响。方法：对硫酸铜（CuSO4）和姜黄素干预培养的BRL细胞，采用荧光探针DCFH-DA进行活性氧(ROS)测定，MTT法检测细胞活力，Hoechst染色和Annexin V-FITC/PI流式细胞术检测细胞凋亡。Western blot法检测磷酸化应激激活的蛋白激酶（JNK/SAPK）蛋白水平。结果：铜处理后6 h BRL细胞ROS和凋亡率达到高峰，分别为711.70±68.33，（45.08±1.87）%，铜处理后24 h JNK磷酸化率上升至（63.36±2.24）%，与正常对照组相比差异有显著性（P<0.01）；姜黄素组ROS、凋亡百分率和JNK磷酸化率均较相应对照组下降(P<0.01)。结论：一定浓度的铜可以诱导BRL细胞的凋亡且与活性氧的产生有关；姜黄素对铜损伤BRL的保护作用与抗氧化和抑制磷酸化JNK的表达有关。［中国当代儿科杂志，2007，9（6）：567-570］

Abstract:

OBJECTIVE: To study the relationship between copper-induced apoptosis and reactive oxygen species (ROS) in BRL cells and the effect of curcumin, a plant-derived polyphenol, on copper-injured BRL cells. METHODS: BRL cells were treated with CuSO4 (100 μmol/L) or curcumin + CuSO4. The BRL cells without any treatment were used as controls. Flow cytometry was applied to detect the production of ROS with fluorescent probe DCFH-DA. MTT colorimetry was used to evaluate cell activity. Apoptosis was measured using Hoechst 33258 staining and Annexin V-FITC and propidiumiodide (PI) staining. JNK/SAPK protein level was detected using Western blot. RESULTS: ROS levels (711.70±68.33 vs 87.22±7.58) and apoptosis rate (45.08±1.87% vs 8.23±2.56%) of BRL cells reached to a peak after 6 hrs of CuSO4 treatment, which were significantly higher than controls (P<0.01). JNK/SAPK levels increased significantly after 6 hrs of CuSO4 treatment and peaked at 24 hrs of CuSO4 treatment compared with controls (P<0.01). Curcumin pretreatment decreased significantly ROS and JNK/SAPK levels as well as the apoptosis rate when compared with the CuSO4-treated alone group (P<0.01).ConclusionsCopper may induce apoptosis of BRL cells. ROS participated in apoptosis induced by copper. Curcumin produced protections on copper-injured BRL cells possibly by anti-oxidation and inhibition of p-JNK expression.［Chin J Contemp Pediatr, 2007, 9 (6):567-570］