Jujuboside A Hippocampal Neurons And Gaba Receptor Gene Expression

Jujuboside A (JuA) is a main component of jujubogenin extracted from the seed of Ziziphus jujuba Mill var spinosa (Bunge) Hu ex H F Chou (Ziziphus), which is widely used in Chinese traditional medicine for the treatment of insomnia and anxiety symptoms. A number of previous studies showed that JuA was a non-competitive inhibitor of CaM and had an inhibitory effect on the activities of mice. These studies suggested JuA as an inhibitor on the central nervous system (CNS), especially on hippocampus. Hippocampus is an important component of limbic system, with abundance and concentrated neurons, it possesses relatively simple and high sequence samdwish and the neurons are independent distribution, and have an important actions of sleep, learning, memory, feeling and autonomic function. In our past study, we have proved JuA had evidently inhibitory effect on mice spontaneous activity, and suggested that the JuA could mediate sedation and hypnosis.γ-aminobutyric acid (GABA) is an important inhibitory neuron transmitters in CNS, its transmission was mediated by GABA receptors which were located on neuron membrane and mediated pres-synaptic inhibition and post-synaptic inhibition in the brain. GABA receptors were closely associated with many nerve diseases, it is include epilepsy, alcoholism, insomnia and Parkinson's disease, etc. GABAA receptors were consanguineously associated with the sedation and hypnosis of benzodiazepine drugs, and some receptors associated with sedation and hypnosis were highly expressed during postnatal development on rat hippocampal neurons. Our prophase study primary proved the induction of sedation and hypnosis by JuA. However, these reports did not explain the function of JuA on hippocampus clearly, whether JuA induced the sedation and hypnosis though the action of GABA receptors like benzodiazepines, and the possible inhibitory molecular mechanism of JuA on hippocampal neurons was still unclear. So, we used the technology of modern biomedicine to study the mechanism of the sedation and hypnosis induced by JuA. First of all, the procedures of neuron culture in previous reports were optimized and the newborn rat hippocampal neurons were cultured in vitro. Then the Microtubule-associated protein 2(MAP2) as Marker was used to identified the cultured neurons by immunohistochemistry and immunoflutorescence. The results showed that the cultures were MAP2 masculine cells. It suggested that the cultures were highly purified hippocampal neurons and could be used in the followed study of JuA.Secondly, the newborn rat hippocampal neurons by JuA were investigated, and the influence of hippocampal neurons by JuA was observed. First, many typical morphological characteristics of hippocampal neurons under phase-contrast microscope were observed. Then we used MTT reduction assay to detect the survival rate of cultured hippocampal neurons induced by JuA: different doses of JuA(0.05g/L and 0.01g/L) were cultured with hippocampal neurons for 7 days, and the rate of MTT were increased by two doses JuA , compared with control. The results showed that the survival rate of hippocampal neurons by JuA were obviously higher than control (p<0.05). It was shown that 0.05g/L and 0.1g/L JuA could promote the growth of cultured hippocampal neurons, obviously increased the survival rate of hippocampal neurons, and presented some relativity of dosage. The results suggested that the two doses JuA in our study might promote and protect the growth of the cultured newborn rat hippocampal neurons in vitro.Finaly, the possible molecular mechanisms of sedation and hypnosis of JuA were primary invesigated. The cultured newborn rat hippocampal neurons were further investigated, the influence of JuA on GABA receptors mRNA expression level on hippocampal neurons by was detected by the method of semi-quantitative RT-PCR, and compared with diazepam. The results were shown to us, 0.1g/L dose JuA induced similar increase of GABA receptors(α1,α5 and R1) gene and decrease ofβ2 subunit mRNA expression compared with 10uM diazepam after 24 hours treatment; 0.1g/L dose JuA also induced decrease of GABA receptor gene(α1,β2) but no significant changes inα5 and R1 subunit mRNA after 72 hours treatment, meanwhile diazepam induced decrease of GABA receptor(α1,α5,β2 and R1) gene expression after 72 hours treatment. However, 0.05g/L dose JuA induced increase of GABA receptors(α1,α5,β2 and R1) gene expression after both 24 hours and 72 hours treatment. The result suggested that 0.1g/L dose JuA has a similar molecular mechanism of sedative-hypnotic activity after short-term treatment and induction of tolerance-dependence after long-term treatment, compared with diazepam. But 0.05g/L dose JuA has a different molecular mechanism of sedative-hypnotic activity without significant induction of tolerance-dependence compared with diazepam after short and long-term treatment. Therefore, the results suggested that 0.1g/L dose and 0.05g/L dose JuA all could induce sedation and hypnosis, but 0.05g/L dose JuA might be more applicable for insomnia therapy in clinic because of its no significant molecular mechanism of tolerance and dependence induction.