藏红花素抑制谷氨酸盐诱导的视网膜神经节细胞凋亡
Crocin inhibits glutamate-induced apoptosis of retinal ganglion cells

摘要：目的 研究藏红花素通过影响Ca2+内流对谷氨酸盐诱导的视网膜神经节细胞(RGCs)凋亡的影响及可能机制。方法 分离大鼠RGCs，以0.1、1mmol/L的谷氨酸盐刺激RGCs 24、48h，建立RGCs凋亡模型，并用0.1、1.0、3.0μmol/L浓度梯度藏红花素分别处理。Annexin V-FITC/PI双标检测细胞凋亡率，Fluo-3/AM荧光标记Ca2+检测胞内钙离子浓度，Western blot检测藏红花素对胞内钙离子介导的凋亡信号分子calpain和CaMKⅡ表达的影响。JC-1荧光染色和Western blot分别检测藏红花素对线粒体膜电位和线粒体凋亡相关信号分子Caspase-3、Caspase-9、Bcl-2/Bax表达的影响。结果 0.1mmol/L谷氨酸盐刺激24h，RGCs细胞凋亡率与对照组差异无统计学意义(P>0.05)；而当刺激48h时，RGCs的凋亡率达到(43.050±2.616)%，差异有统计学意义(P<0.01)。高剂量谷氨酸盐(1mmol/L)刺激24、48h的RGCs凋亡率为(46.450±1.061)%和(45.500±3.253)%，较对照组均显著增加，差异有统计学意义(P<0.01)。用1mmol/L谷氨酸盐刺激RGCs 12h后加入0.1、1.0、3.0μmol/L藏红花素再处理12h，不同浓度藏红花素均可显著抑制细胞凋亡(P<0.01)，且抑制效率具有剂量依赖性。另外，1.0μmol/L藏红花素组的谷氨酸盐诱导的胞外Ca2+内流减少及钙依赖蛋白Calpain1和CaMKⅡ的表达减弱，线粒体膜电位增高，Caspase-3和Caspase-9的表达减少，Bcl-2/Bax表达上调。结论 藏红花素抑制谷氨酸盐诱导的RGCs凋亡，其机制可能与阻止胞外Ca2+内流，抑制钙依赖的凋亡信号通路和线粒体凋亡信号通路有关。
ABSTRACT: Objective To evaluate the effects and underlying mechanisms of crocin on glutamate-induced apoptosis of retinal ganglion cells (RGCs) by affecting extracellular calcium influx. Methods Primary rat retinal ganglion cells were isolated and stimulated with glutamate at concentrations of 0.1mmol/L and 1mmol/L for 24h or 48h, respectively, to establish apoptosis model of RGCs. Afterwards, crocin of different doses (0.1, 1.0 and 3.0μmol/L) was used to treat the glutamate-induced RGCs for 12h; then cell apoptosis was detected by Annexin V-FITC/PI staining. The intracellular calcium concentration was determined by Fluo-3/AM fluorescent labeling. Western blot was used to examine the effect of crocin on Ca2+-mediated apoptotic signal molecules calpain and CaMKII. The mitochondrial membrane potential was detected by JC-1 staining and mitochondrial apoptosis-related signaling molecules Caspase-3, Caspase-9 and Bcl-2/Bax were evaluated by Western blot, respectively. Results In comparison with the untreated controls, the cell apoptosis of RGCs exposed to 0.1mmol/L of glutamate for 24h did not significantly change (P>0.05). However, apoptosis rate of the cells reached (43.050±2.616)% when the stimulation time lasted for 48h and showed a significant increase (P<0.01). Treatment with higher-dose glutamate (1mmol/L) significantly increased apoptosis of RGCs at 24h (46.450±1.061)% and 48h (45.500±3.253)% compared with the controls (P<0.01). RGCs were induced by 1mmol/L of glutamate for 12h, followed by the treatment with crocin at concentrations of 0.1, 1.0 and 3.0μmol/L, respectively. Each dose of crocin could significantly inhibit cell apoptosis in the dose-dependent manner (P<0.01). In addition, crocin at 1.0μmol/L blocked glutamate-induced