Cai Biao, Ye Shu, Wang Yan, Wang Ting-Ting, Wang Liang, Jiang Ai-Juan, Fang Zheng-Qing, Shen Guo-Ming, Xie Dao-Jun
College of Integrated Chinese and Western Medicine, Anhui University of Chinese Medicine, Hefei 230012, China.
Institute of Integrated Chinese and Western Medicine, Anhui Academy of Chinese Medicine, Hefei 230012, China.
Zhongguo Zhong Yao Za Zhi. 2018 Jun;43(11):2378-2383. doi: 10.19540/j.cnki.cjcmm.20180403.001.
The loss of hippocampal neurons is one of the main pathological features of Alzheimer's disease (AD), which is related to the apoptosis of hippocampal neurons. Huangpu Tongqiao capsule is used for the treatment of AD, but the underlying mechanism is still unclear. This study is to investigate the mechanism of neuroprotective effect of Huangpu Tongqiao capsule in the treatment of AD, through observing the effect of Huangpu Tongqiao capsule containing serum on cell injury of primary cultured hippocampal neurons induced by Aβ₂₅₋₃₅ via inhibiting the cell apoptosis. Primary cultured hippocampal neurons were cultured and identified by MAP-2 immunofluorescence staining, and cell growth state was observed by inverted microscope. The Huangpu Tongqiao capsule containing serum was prepared using the method of serum pharmacology. MTT assays were used to measure the optimum concentration range of Huangpu Tongqiao capsule containing serum, and optimum Aβ concentration for establishing the AD model. After primary cultured hippocampal neurons AD cell model was induced by Aβ₂₅₋₃₅, cell survival rate was detected by MTT, cell apoptosis rate was assayed by flow cytometry, and protein expressions of Bax, Cyt C and caspase-3 were determined by Western blot analysis. The results showed that the primary cultured hippocampal neurons were cultured successfully, and cells grew mature at seventh days; Compared with normal group, the survival rate of hippocampal neurons in AD cell model group was decreased, the apoptosis rate of hippocampal neurons was increased, and the protein expressions of Bax, Cyt C and caspase-3 were increased (<0.05, <0.01); Compared with AD cell model group, the survival rate of hippocampal neurons in Huangpu Tongqiao capsule containing serum group was increased, the apoptosis rate of hippocampal neurons was decreased, and the protein expressions of Bax, Cyt C and caspase-3 were decreased (<0.05, <0.01). These findings suggest that Huangpu Tongqiao capsule containing serum has a neuroprotective effect on cell injury of the primary cultured hippocampal neurons induced by Aβ₂₅₋₃₅, and its effect on the treatment of AD is associated with the inhibition the apoptosis of hippocampal neurons.
海马神经元的丢失是阿尔茨海默病(AD)的主要病理特征之一,这与海马神经元的凋亡有关。黄埔通窍胶囊用于治疗AD,但其潜在机制仍不清楚。本研究旨在通过观察含血清的黄埔通窍胶囊对Aβ₂₅₋₃₅诱导的原代培养海马神经元细胞损伤的影响,探讨其抑制细胞凋亡从而发挥神经保护作用的机制。通过MAP-2免疫荧光染色对原代培养的海马神经元进行培养和鉴定,并用倒置显微镜观察细胞生长状态。采用血清药理学方法制备含血清的黄埔通窍胶囊。用MTT法测定含血清的黄埔通窍胶囊的最佳浓度范围以及建立AD模型的最佳Aβ浓度。用Aβ₂₅₋₃₅诱导原代培养海马神经元建立AD细胞模型后,用MTT法检测细胞存活率,用流式细胞术检测细胞凋亡率,并用蛋白质免疫印迹法检测Bax、Cyt C和caspase-3的蛋白表达。结果显示,原代培养的海马神经元成功培养,细胞在第7天生长成熟;与正常组相比,AD细胞模型组海马神经元存活率降低,海马神经元凋亡率升高,Bax、Cyt C和caspase-3的蛋白表达增加(P<0.05,P<0.01);与AD细胞模型组相比,含血清的黄埔通窍胶囊组海马神经元存活率升高,海马神经元凋亡率降低,Bax、Cyt C和caspase-3的蛋白表达降低(P<0.05,P<0.01)。这些结果表明,含血清的黄埔通窍胶囊对Aβ₂₅₋₃₅诱导的原代培养海马神经元细胞损伤具有神经保护作用,其治疗AD的作用机制可能与抑制海马神经元凋亡有关。