藏红花素抑制谷氨酸盐诱导的视网膜神经节细胞凋亡

摘要

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 0.1 mmol/L and 1 mmol/L for 24 h or 48 h,respectively,to establish apoptosis model of RGCs.Afterwards,crocin of different doses (0.1,1.0 and 3.0 μmol/L) was used to treat the glutamate-induced RGCs for 12 h;then cell apoptosis was detected by Annexin V-FITC/PI staining.The intracellular calcium concentration was determined by FIuo-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 0.1 mmol/L of glutamate for 24 h did not significantly change (P＞ 0.05).However,apoptosis rate of the cells reached (43.050 ± 2.616) ％ when the stimulation time lasted for 48 h and showed a significant increase (P＜0.01).Treatment with higher-dose glutamate (1 mmol/L) significantly increased apoptosis of RGCs at 24 h (46.450±1.061)％ and 48 h (45.500±3.253)％ compared with the controls (P＜0.01).RGCs were induced by 1 mmol/L of glutamate for 12 h,followed by the treatment with crocin at concentrations of 0.1,1.0 and 3.0 μmol/L,respectively.Each dose of crocin could significantly inhibit cell apoptosis in the dose-dependent manner (P＜0.01).In addition,crocin at 1.0 μmol/L blocked glutamate-induced extracellular calcium influx,inhibited the expression of calcium-dependent proteins Calpainl and CaMK Ⅱ.Moreover,crocin at the dose of 1.0 μmol/L also increased mitochondrial membrane potential,suppressed the expressions of Caspase-3 and Caspase-9,and elevated Bcl-2/Bax ratio.Cornclusion Crocin inhibits glutamate-induced apoptosis of retinal ganglion cells through suppressing extracellular calcium influx,thereby blocking calcium-dependent and mitochondria-dependent apoptosis signaling pathways.%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、1 mmol/L的谷氨酸盐刺激RGCs 24、48 h,建立RGCs凋亡模型,并用0.1、1.0、3.0 μmol/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 0.1 mmol/L谷氨酸盐刺激24 h,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48 h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(1 mmol/L)刺激24、48 h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1 mmol/L谷氨酸盐刺激RGCs 12 h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.