姜黄素对Aβ1-42诱导的细胞损伤和线粒体途径细胞凋亡的抑制作用

摘要

目的 探讨姜黄素对Aβ1-42诱导的细胞损伤和凋亡的抑制作用.方法 采用体外培养的人神经母细胞瘤SH-SY5Y细胞,分为溶剂对照组、Aβ1-4210μmol·L-1损伤组、Aβ1-4210μmol·L-1+姜黄素1,5和10μmol·L-1保护组及姜黄素10μmol·L-1对照组;噻唑蓝(MTT)法测定细胞存活率,酶活性法检测细胞培养液中乳酸脱氢酶(LDH)的含量以考察细胞损伤程度;AnnexinⅤ-FITC/PI染色法测定细胞凋亡;JC-1染色法检测线粒体膜电位变化;比色法测定胱天蛋白酶9和胱天蛋白酶3的活性;Western蛋白印迹法检测胱天蛋白酶3表达.结果 与溶剂对照组比,Aβ1-4210μmol·L-1损伤组细胞存活率显著降低(P<0.01).与Aβ1-4210μmol·L-1损伤组比较,姜黄素5和10μmol·L-1缓解了Aβ1-42诱导的细胞存活率下降(P<0.05),降低了Aβ1-42诱导的乳酸脱氢酶释放水平(P<0.01)和细胞早期及晚期凋亡率(P<0.01).姜黄素抑制了Aβ1-42诱导的细胞线粒体膜电位去极化作用(P<0.01);姜黄素1~10μmol·L-1抑制了Aβ1-42诱导的胱天蛋白酶9及胱天蛋白酶3级联激活作用,且呈浓度依赖性(r=0.990,P<0.01;r=0.996,P<0.01).姜黄素10μmol·L-1对照组以上指标与溶剂对照组无明显差异.结论 姜黄素可能通过升高线粒体膜电位、降低胱天蛋白酶活性抑制Aβ1-42诱导的细胞损伤和线粒体途径细胞凋亡.%OBJECTIVE To investigate the protective effect of curcumin on Aβ1-42 damaged cells. METHODS SH-SY5Y cells were cultured with Aβ1-4210μmol · L-1 in the absence or presence of curcumin 1, 5 or 10 μmol · L-1. Cell viability was assayed by MTT. Cell membrane damage was detected by the concentration of lactate dehydrogenase (LDH) in culture medium. Cell apoptosis was measured by flow cytometry with Annexin Ⅴ-FITC/PI staining. Mitochondrial membrane potential was characterized by fluorescence of JC-1 dye. Enzymatic activity of caspases-9 and-3 was measured by colorimetric assay. Protein expression of caspase-3 was detected by Western blotting. RESULTS Compared with vehicle control, the cell viability, concentration of LDH and both early and late apoptosis in Aβ1-4210 μmol · L-1 damaged group were decreased(P<0.01). However, the cell viability, release of LDH and both early and late apoptosis in curcumin group were promoted compared with that in Aβ1-4210μmol·L-1 damaged group. Curcumin inhibited Aβ1-42-induced depolarization of mitochondrial membrane potential(P<0.01), and attenuated Aβ1-42-induced activation of both caspases9 and caspases3 in a concentration-dependent manner, respectively(r=0.990, P<0.01; r=0.996, P<0.01). There were no significant differences in the above detected indexes between curcumin 10 μmol · L-1 group and vehicle control group. CONCLUSION Curcumin inhibits Aβ1-42-induced cell damage and apoptosis by promoting mitochondrial membrane potential and depressing the activation of caspases.