Crocin inhibits glutamate-induced apoptosis of retinal ganglion cells / 西安交通大学学报(医学版)

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 0.1 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.