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目的研究银杏叶提取物(EGb761)对Aβ淀粉样蛋白(Aβ_(25-35))所致的PC12细胞损伤的保护作用。方法 PC12细胞随机分为5组:正常组、模型组及3个剂量实验组(10,20,40μg·m L~(-1)EGb761)。模型组及实验组经20μmol·L~(-1)Aβ25-35诱导形成PC12细胞损伤模型。以噻唑蓝法及烟酸己可碱染色检测细胞活力及凋亡率;用紫外分光光度法检测天冬氨酸蛋白水解酶(Caspase 3)与Caspase 9活性;用免疫印迹技术分析B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、磷脂酰肌醇3激酶(PI3K)蛋白表达量及蛋白激酶B(AKT)磷酸化水平。结果与模型组的细胞活力(63.52±6.21)%比较,低、中、高3个剂量实验组的细胞活力分别为(75.43±7.90)%,(83.78±8.39)%,(92.95±9.30)%,均显著提高,差异有统计学意义(P<0.01)。与模型组的细胞凋亡率(40.31±4.21)%比较,低、中、高3个剂量实验组的细胞凋亡率分别为(24.89±2.50)%,(15.21±1.63)%,(8.65±0.90)%,3个剂量实验组均显著降低,差异有统计学意义(P<0.01)。与模型组的Caspase 3为(1.36±0.14)、Caspase 9为(1.89±0.15)比较,低、中、高3个剂量实验组中Caspase 3分别为(1.12±0.10),(0.91±0.08),(0.80±0.08)、Caspase 9分别为(1.73±0.17),(1.52±0.14),(1.28±0.13),3个剂量实验组活性皆显著降低,差异有统计学意义(P<0.05)。与模型组的Bax为(1.15±0.12)、Bcl-2为(0.20±0.02)比较,低、中、高3个剂量实验组的Bax分别为(1.04±0.10),(0.88±0.10),(0.42±0.04)、Bcl-2分别为(0.31±0.03),(0.87±0.09),(1.02±0.10),Bax蛋白表达量降低、而Bcl-2蛋白表达量升高,差异有统计学意义(均P<0.05)。与模型组的PI3K为(0.15±0.01)、AKT为(0.10±0.00)比较,低、中、高3个剂量实验组PI3K分别为(0.26±0.03),(0.40±0.04),(0.42±0.04)、AKT分别为(0.19±0.02),(0.34±0.03),(0.35±0.03),实验组的蛋白表达量及磷酸化水平均提高,差异有统计学意义(P<0.01)。结论 EGb761可抑制Aβ_(25-35)诱导的PC12细胞凋亡,与激活PI3K/AKT信号通路有关。
Objective To study the protective effect of Ginkgo biloba extract (EGb761) on PC12 cells induced by Aβ (25-35). Methods PC12 cells were randomly divided into 5 groups: normal group, model group and 3 dose experimental groups (10, 20, 40μg · m L -1 EGb761). The model group and experimental group induced PC12 cells injury induced by 20μmol·L -1 Aβ25-35. Cell viability and apoptosis rate were detected by thiazolyl blue staining and hexofunctional nicotine staining. Caspase 3 and Caspase 9 activities were detected by UV spectrophotometry. The expressions of B lymphocytoma Bcl-2, Bcl-2, PI3K and AKT phosphorylation were detected. Results Compared with the model group (63.52 ± 6.21)%, the cell viability in the low, medium and high dose groups were (75.43 ± 7.90)%, (83.78 ± 8.39)% and (92.95 ± 9.30)%, respectively , Both significantly increased, the difference was statistically significant (P <0.01). Compared with the model group (40.31 ± 4.21)%, the apoptotic rates in the low, middle and high dose groups were (24.89 ± 2.50)%, (15.21 ± 1.63)% and (8.65 ± 0.90)%, the three doses of the experimental group were significantly lower, the difference was statistically significant (P <0.01). Compared with model group, Caspase 3 was (1.36 ± 0.14) and Caspase 9 was (1.89 ± 0.15) respectively. Caspase 3 was (1.12 ± 0.10), (0.91 ± 0.08), (0.80 ± 0.08), Caspase 9 (1.73 ± 0.17), (1.52 ± 0.14) and (1.28 ± 0.13) respectively. The activity of the three experimental groups was significantly decreased (P <0.05). Compared with model group, Bax was (1.15 ± 0.12) and Bcl-2 was (0.20 ± 0.02), Bax was (1.04 ± 0.10), (0.88 ± 0.10), 0.42 ± 0.04), Bcl-2 (0.31 ± 0.03), (0.87 ± 0.09) and (1.02 ± 0.10) respectively. The protein expression of Bax decreased and the expression of Bcl-2 protein increased All P <0.05). Compared with the model group, PI3K was (0.15 ± 0.01) and AKT was (0.10 ± 0.00), PI3K was (0.26 ± 0.03), (0.40 ± 0.04) and (0.42 ± 0.04 ), AKT were (0.19 ± 0.02), (0.34 ± 0.03) and (0.35 ± 0.03) respectively. The protein expression and phosphorylation level in the experimental group were significantly increased (P <0.01). Conclusion EGb761 can inhibit the apoptosis of PC12 cells induced by Aβ 25-35, which is related to the activation of PI3K / AKT signaling pathway.