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

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6h 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24hJNK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。

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（100μmol/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 ）. Conclusions Copper 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.