ABSTRACT
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.
ABSTRACT
GABA (-aminobutyric acid) is one of the important neurotransmitters in the central nervous system of mammals and its action is variable according to the maturation phases of neurons. The neurons of early cultural days (less than 7 days) have been used for a developing neuronal model, while the neurons of later days (over 3 weeks) used for a mature model. This study was performed to investigate the electrophysiological property of GABAergic synapses in the hippocampal neurons cultured for 10 to 14 days which are considered to be transitional period between the developing and the mature phases. Membrane potential was depolarized and a inward transmembrane current was induced by 20 M GABA infusion. Frequency and amplitude of spontaneous postsynaptic currents (PSCs) were inhibited during the GABA infusion, but decay time constant was not affected significantly. In most hippocampal neurons, no GABAergic PSCs were observed during the administration of 0.5 M TTX, 50 M APV and 10 M CNQX. In the neurons counting 25% approximately, however, small persisted PSCs showed the existence of GABAergic synapses which were blocked by 10 M bicuculine. As the functional property of isolated GABAergic synapses, amplitude of GABAergic PSCs were diminished, and decay time constants and rising times were prolonged during the 20 M GABA infusion in all recorded neurons. In conclusion, approximately 25% of the hippocmpal neurons cultured 10 to 14 days used GABA as well as glutamate as a neurotransmitter. It seems that the GABAergic synapses composed of functionally homogenous GABAA receptors act as inhibitory modulator of the excitatory signal transmission.