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.
ABSTRACT
Objective To explore the effect and potential mechanism of suppressors of cytokine signaling 3 (SOCS3) knockout on cell viability and apoptosis of retinal ganglion cells (RGCs) after optic nerve injury.Methods The optic nerve transection was used to construct optic nerve injury model of rats,and RGCs were isolated after optic nerve injury.The experimental animals were divided into optic nerve transection injury (ONT) group and sham-operation (Sham) group.The expression of SOCS3 in RGCs was detected by Western blot and RT-PCR in each group.Subsequently,SOCS3 siRNA was transfected into RGCs of Sham group and ONT group,and the experimental were further subdivided into blank control group,negative control group and SOCS3 silence groups.Cell viability was measured by CCK8 and MTr methods.Apoptosis was detected by Hoechst 33342 staining and flow cytometry.Furthermore,the mTOR siRNA and SOCS3 siRNA were co-transfected into RGCs,and cell viability and apoptosis were detected.Results The expression of SOCS3 was dramatically increased at 3 days after injury in the ONT group when compared with Sham group (P =0.049),and it showed an increased tendency gradually along with the extension of injury time.Compared with the blank control in the ONT group,SOCS3 silence markedly promoted cell viability [(49.47 ± 7.35) ％ vs.(73.24 ± 8.70) ％],reduced cell growth inhibition [(27.25 ±0.75)％ vs.(10.96 ± 1.07)％] and apoptosis [(23.06 ± 1.43)％ vs.(10.65 ± 1.77)％].The result of Hoechst 33342 staining indicated that SOCS3 silence ameliorated the cell apoptosis induced by ONT.In addition,SOCS3 silence significantly improved pS6 expression at 2 weeks after injury,and mTOR and SOCS3 co-silence reduced cell viability,increased cell growth inhibition and apoptosis compared with SOCS3 silence group after injury.Conclusion SOCS3 silence promotes injury-induced cell viability of RGCs and suppresses injury-induced apoptosis of RGCs via up-regulating mTOR activity in the later period of injury.