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

目的研究藏红花素通过影响Ca^(2+)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Ca^(2+)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Ca^(2+)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Ca^(2+)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。

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 and1 mmol/L for 24 h or48 h,respectively,to establish apoptosis model of RGCs.Afterwards,crocin of different doses(0.1,1.0and 3.0μmol/L)was used to treat the glutamate-induced RGCs for 12 h;then cell apoptosis was detected by Annexin VFITC/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 to0.1 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 0.1,1.0and 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 extracellular calcium influx,inhibited the expression of calcium-dependent proteins Calpain1 and CaMKⅡ.Moreover,crocin at the dose of 1.0μmol/L also increased mitochondrial membrane potential,suppressed the expressions of Caspase-3 and Caspase-9,and elevated Bcl-2/Bax ratio.ConclusionCrocin inhibits glutamate-induced apoptosis of retinal ganglion cells through suppressing extracellular calcium influx,thereby blocking calcium-dependent and mitochondria-dependent apoptosis signaling pathways.