6-姜酚肟对PC12细胞的抗凋亡保护作用和促分化作用
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摘要
目的:建立谷氨酸诱导PC12细胞凋亡的模型,探讨6-姜酚肟对谷氨酸诱导PC12细胞凋亡的影响;建立NGF诱导PC12细胞分化的模型,探讨6-姜酚肟对NGF诱导PC12细胞分化的影响。
方法:体外培养PC12细胞,倒置显微镜下观察活细胞形态。用谷氨酸造成细胞损伤,MTT法检测细胞活力,流式细胞术和Hoechst 33258 DNA染色法检测细胞凋亡,比较各种浓度的6-姜酚肟对PC12细胞凋亡的影响;用NGF诱导PC12细胞分化,倒置显微镜观察6-姜酚肟和NGF对PC12细胞分化和突起生长的影响并拍照;采用免疫荧光技术检测各组PC12细胞中突触素的反应的阳性强度。
结果:(1)经2.50～20.0 mmol/L谷氨酸处理24 h后,PC12细胞的活力比对照组明显降低,细胞存活率随谷氨酸浓度的升高而降低。谷氨酸对PC12细胞活力的半数影响浓度(IC_(50))约为5.00 mmol/L。(2)在一定浓度范围内,6-姜酚肟能保护PC12细胞免受谷氨酸的影响,但是在较高浓度时6-姜酚肟对细胞有一定的毒性。经不同浓度的6-姜酚肟(3.13,6.25,12.50,25.00μg/mL)预处理后,PC12细胞活力明显提高,其保护作用随其浓度降低而提高。当浓度为6.25μg/mL时效果最好,细胞存活率达到75.20%(P＜0.01),当浓度为3.13μg/mL时对细胞的保护能力有所下降,细胞存活率为70.24%(P＜0.01)。(3)谷氨酸能诱导PC12细胞凋亡,5.00 mmol/L的谷氨酸处理24 h后细胞凋亡率为20.10%。经6-姜酚肟(3.13,6.25,12.50,25.00μg/mL)预处理后,细胞凋亡率明显降低,分别为3.50%(P＜0.01),2.80%(P＜0.01),9.60%(P＜0.01),17.7%(P＜0.05)。(4)NGF能诱导PC12细胞分化,10μg/L与50μg/L的NGF作用PC12细胞48 h后,分化细胞的百分比和阳性突起数目与对照组相比均明显增加(P＜0.01),50μg/L的NGF作用效果强于10μg/L的NGF。6-姜酚肟和10μg/LNGF共同孵育PC12细胞48h后,细胞的分化情况与50μg/L的6-姜酚肟单独作用相似。(5)6-姜酚肟单独作用于PC12细胞时,免疫荧光突触素染色阴性。6-姜酚肟和10μg/L NGF共同作用时的荧光强度与50μg/L NGF单独作用时的荧光强度相似,两者无明显差别(P＞0.05)。
结论:(1)谷氨酸能诱导PC12细胞凋亡,较低浓度的6-姜酚肟能有效对抗Glu诱导的PC12细胞凋亡而保护细胞,其中6.25μg/mL的6-姜酚肟效果最好。
(2)NGF能诱导PC12细胞向神经元样细胞分化。6-姜酚肟单独作用不能引起PC12细胞分化,但它能使低浓度NGF诱导PC12细胞分化的能力增强,使之达到高浓度NGF的效果。
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 IC_(50)(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.
方法:体外培养PC12细胞,倒置显微镜下观察活细胞形态。用谷氨酸造成细胞损伤,MTT法检测细胞活力,流式细胞术和Hoechst 33258 DNA染色法检测细胞凋亡,比较各种浓度的6-姜酚肟对PC12细胞凋亡的影响;用NGF诱导PC12细胞分化,倒置显微镜观察6-姜酚肟和NGF对PC12细胞分化和突起生长的影响并拍照;采用免疫荧光技术检测各组PC12细胞中突触素的反应的阳性强度。
结果:(1)经2.50～20.0 mmol/L谷氨酸处理24 h后,PC12细胞的活力比对照组明显降低,细胞存活率随谷氨酸浓度的升高而降低。谷氨酸对PC12细胞活力的半数影响浓度(IC_(50))约为5.00 mmol/L。(2)在一定浓度范围内,6-姜酚肟能保护PC12细胞免受谷氨酸的影响,但是在较高浓度时6-姜酚肟对细胞有一定的毒性。经不同浓度的6-姜酚肟(3.13,6.25,12.50,25.00μg/mL)预处理后,PC12细胞活力明显提高,其保护作用随其浓度降低而提高。当浓度为6.25μg/mL时效果最好,细胞存活率达到75.20%(P＜0.01),当浓度为3.13μg/mL时对细胞的保护能力有所下降,细胞存活率为70.24%(P＜0.01)。(3)谷氨酸能诱导PC12细胞凋亡,5.00 mmol/L的谷氨酸处理24 h后细胞凋亡率为20.10%。经6-姜酚肟(3.13,6.25,12.50,25.00μg/mL)预处理后,细胞凋亡率明显降低,分别为3.50%(P＜0.01),2.80%(P＜0.01),9.60%(P＜0.01),17.7%(P＜0.05)。(4)NGF能诱导PC12细胞分化,10μg/L与50μg/L的NGF作用PC12细胞48 h后,分化细胞的百分比和阳性突起数目与对照组相比均明显增加(P＜0.01),50μg/L的NGF作用效果强于10μg/L的NGF。6-姜酚肟和10μg/LNGF共同孵育PC12细胞48h后,细胞的分化情况与50μg/L的6-姜酚肟单独作用相似。(5)6-姜酚肟单独作用于PC12细胞时,免疫荧光突触素染色阴性。6-姜酚肟和10μg/L NGF共同作用时的荧光强度与50μg/L NGF单独作用时的荧光强度相似,两者无明显差别(P＞0.05)。
结论:(1)谷氨酸能诱导PC12细胞凋亡,较低浓度的6-姜酚肟能有效对抗Glu诱导的PC12细胞凋亡而保护细胞,其中6.25μg/mL的6-姜酚肟效果最好。
(2)NGF能诱导PC12细胞向神经元样细胞分化。6-姜酚肟单独作用不能引起PC12细胞分化,但它能使低浓度NGF诱导PC12细胞分化的能力增强,使之达到高浓度NGF的效果。
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 IC_(50)(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.
引文
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