Crocin inhibits glutamate-induced apoptosis of retinal ganglion cells

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 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 0.1 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 Calpain1 and CaMKII. 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. Conclusion: Crocin inhibits glutamate-induced apoptosis of retinal ganglion cells through suppressing extracellular calcium influx, thereby blocking calcium-dependent and mitochondria-dependent apoptosis signaling pathways.