| Ginseng and notoginseng, the roots of Panax species, are well-known herbal medicine. They have been used as traditional medicine in China for thousands of years. The principle active ingredients of ginseng and notoginseng are ginsenosides, which are also called ginseng saponins. Recently, there is accumulated evidence in the literature on the neuroprotective and neurotrophic actions of ginsenoside. For example, ginsenoside can suppress the occurrence of cerebrovascular disease, enhance learning and memory, reduce the detrimental effects of aging processes and so on. Therefore, researches in the field of herbal medicine now focus on abstraction of active ginsenosides and elucidation of their mechanisms.Ginsenoside Rd (GSRd) is a principal member of ginsenosides. In recent years, it has been verified to produce various pharmacological actions, such as anticonvulsive effect, anti-aging effect, regulating immune response, ect. In our previous study, we found that GSRd can attenuate H2O2-induced cytotoxicity in PC12 cell line and inhibit the damage produced by oxygen-glucose deprivation. Moreover, our clinical trial suggested that GSRd may be beneficial to the treatment of acute ischemic stroke. All these results show that GSRd is a kind of neuroprotectant. However, the mechanisms of its neuroprotection are still elusive. Professor Guan first reported that GSRd remarkably inhibits Ca2+ entry through receptor-operated Ca2+ channels (ROCC) and store-operated Ca2+ channels (SOCC) without effects on voltage-dependent Ca2+ channels (VDCC). In addition, their latest research showed that GSRd can reverse basilar hypertrophic remodeling in stroke-pron renovascular hypertensive rats and its underlying mechanism may be associated with inhibitory effects of GSRd on voltage-independent Ca2+ entry but not with VDCC-mediated Ca2+ entry. These researches suggested that GSRd may be a potential Ca2+ channel blocker.Glutamate (Glu) is the most important excitatory amino acid neurotransmitter in the central nervous system (CNS), which is essential to the physiological function of neuronal cells. However, glutamate accumulation and excessive stimulation of glutamate receptors induce intracellular calcium overload, which eventually leads to excitotoxicity. This process is involved into many pathological conditions in CNS, such as stroke, epilepsy and brain trauma. The ionotropic glutamate receptor (iGluR) is one of the typical ligand-gated ion channels. In addition, there are several various VDCC in neuronal cells. But it's not clear that whether GSRd can elicit its neuroprotective effects through influencing these calcium channels. Here we begin from observing the neuroprotection of GSRd against glutamate-induced excitotoxic injury and then discuss the relationship between GSRd and various calcium channels in order to elucidate the underlying mechanisms of its neuroprotection. Experiment 1 The neuroprotection of GSRd against glutamate-induced excitotoxicityObjective: To investigate the neuroprotection of GSRd against glutamate-induced neurotoxicity. Methods: We first adopted the primary culture of rat hippocampal neurons and verified the purity by immunocytochemical staining. Then, on 10-14 days in vitro, neurons were used in our experiment. Hippocampal neurons were exposed to 500μM glutamate for 3 hours. Cell viability was determined by MTT assay. And TUNEL and caspase-3 staining were performed to assess the injury condition of neurons. In addition, using a fluorescent calcium indicator Fluo-4, AM, we measured the intracellular free calcium concentration which was represented by the fluorescent intensity. The effect of GSRd on the intracellular free calcium stimulated by glutamate was examined with a laser scanning confocal microscope. Results: MTT assay revealed that high concentration of glutamate decreased the cell viability in hippocampal neurons to about 50%, while GSRd significantly prevented neurons from glutamate-induced damage. Meanwhile, the TUNEL staining showed that GSRd remarkably diminished the apoptotic cell death, which was consistent with the results of caspase-3 staining. Additionally, high dose glutamate produced a sharp increase of fluorescent intensity, while GSRd markedly reduced the intracellular free calcium concentration, displaying dose-dependent effects. Conclusion: These results suggested that GSRd can enhance the resistance of hippocampal neurons to glutamate-induced exitotoxicity, which may attribute to attenuating calcium overload. Experiment 2 The effects of GSRd on various calcium channelsObjective: To investigate the influence of GSRd on iGluR (especially NMDA receptor), the release of intracellular calcium stores and VDCC. Methods: We adopted the primary culture of rat hippocampal neurons and verified the purity by immunocytochemical staining. Then, on 10-14 days in vitro, neurons were used in our experiment. Using a fluorescent calcium indicator Fluo-4, AM, we measured the intracellular free calcium concentration which was represented by the fluorescent intensity. The effects of GSRd on the intracellular free calcium stimulated by NMDA and KCl were examined with a laser scanning confocal microscope. Finally, we use free-calcium extracellular fluid to observe the effect of GSRd on the release of intracellular calcium stores. Results: High dose NMDA produced an increase of fluorescent intensity, while GSRd markedly reduced the intracellular free calcium concentration. However, in free-calcium extracellular fluid, high dose glutamate induced an abrupt increase and decrease of fluorescent intensity, which may represent a transient calcium release from calcium stores. And GSRd reduced the amplitude of the fluorescent intensity change. In addition, the stimulation of KCl elicited an increase of fluorescent intensity, but GSRd did not influence the peak amplitude. However, the fluorescent intensity decreased as the time passed, which showed significant differences between the two groups. Conclusion: These results indicated that GSRd can inhibit NMDA-induced calcium entry and the release of intracellular calcium stores induced by glutamate. This may be one of the underlying mechanisms of GSRd's neuroprotection.
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