Protective Effect Of 6-gingerol Oxime On Apoptosis And Its Effect Of Promotion On Differentiation In PC12 Cells

Objective:The model of apoptotic PC12 cells induced by Glu was established to investigate the effect of 6-gingerol oxime on Glu-induced apoptosis in PC12 cells.The model of differentiated PC12 cells induced by NGF was set up to study the effect of 6-gingerol oxime on NGF-induced differentiation in PC12 cells.Methods:The apoptosis model was established by treating PC12 cells with Glu in vitro.Cell viability was assayed with MTT method.The cell apoptotic rate was measured by flow cytometry(FCM) and Hoechst 33258 DNA staining method.Compare the effects of 6-gingerol oxime at different concentrations on the apoptosis of PC12 cells.The differentiation model was set up by treating PC12 cells with NGF.Observe the differentiation and neuron outgrowth of PC12 cells treated with 6-gingerol oxime and NGF by inverted microscope. Immuno-fluorescence technique was performed to identify the indensity of positive reaction of synaptophysin in PC12 cells of each group.Results:1) After treated with Glu(2.50～20.0 mmol/L) for 24 h,the viabilities of PC12 cells depressed significantly when compared with control group and the viable cells reduced with the concentration of Glu increased by MTT assay.The IC₅₀(50%inhibiting concentration) of Glu to PC12 cells is 5.00 mmol/L.(2) In a certain rage of concentration,6-gingerol oxime can protect PC12 cells from the injury of Glu,although it showed some toxicity to cell at high concentration. Pretreated with 6-gingerol oxime solution at concentration of 3.13,6.25,12.50 and 25.00μg/mL, respectively,the cell viabilities were all increased significantly.The protective effect of 6-gingerol oxime on cells increased with its concentration decreased.At the dose of 6.25μg/mL, it showed the highest protective effect and the survival rate of PC12 cells reached to 75.20% (P＜0.01),following with 70.24%(P＜0.01) when preincubated with 6-gingerol oxime at 3.13μg/mL.(3) Glu induced apoptosis in PC12 cells,and the apoptotic rate was 20.10%after 24 h of treatment.Pretreated with different doses of 6-gingerol oxime at 3.13,6.25,12.50 and 25.00 μg/mL,the cell apoptotic rate reduced and reached at 3.50%(P＜0.01),2.80%(P＜0.01),9.60% (P＜0.01) and 17.70%(P＜0.05),respectively.(4) NGF induced the differentiation of PC12 cells. After treated with NGF for 48 h,the percentage of differentiated cells and the positive outgrowth of PC12 cells were all increased obviously(P＜0.01),and NGF at the dose of 50μg/L showed stronger effect of the differentiation to cells than that of NGF at 10μg/L.After preincubated with 6-gingerol oxime and NGF(10μg/L) for 48 h,the differentiation of PC12 cells was similar to that of the cells pretreated with NGF(50μg/L) alone.(5) When pretreated with 6-gingerol oxime alone,there was no significant expression of synaptophysin in PC12 cells.The fluorescence indensity of the 6-gingerol oxime and NGF(10μg/L) coincubation group showed no conspicuous difference with that of NGF(50μg/L) treated group(P＞0.05).Conclusions:(1) Glu can induce apoptosis in PC12 cells,while 6-gingerol oxime can protect PC12 cells from the injury by Glu,especially at lower concentration,and 6.25μg/mL of 6-gingerol oxime exerted the best effect.(2) NGF can promote the differentiation of PC12 cells into neurons.6-gingerol oxime can't induce the differentiation of PC12 cells by itself,but it can enhance the ability of NGF at low concentration to induce the differentiation of PC12 cells,and make it reach to the effect of NGF at high concentration.