Protective Effects Of Dipfluzine Against The Injury Of Hippocampal Neuron Induced By Glutamate In Rats

Dipfluzine (Dip), a novel calcium antagonist, was first synthesized by School of Pharmacy, Hebei Medical University. It is lipophilic, which enable to cross the blood-brain-barrier and achieve effective drug concentration in the cerebrospinal fluid. Dip could inhibit calcium influx and maintain intracellular calcium homeostasis. It could improve cerebral microcirculation and assure supply of energy and nutrition in cells. It could improve the amnesia induced by sodium nitrite and sodium pentobarbital in mice. From all these we can see that Dip may become a good protector to cerebrum. In order to go on studying the effects of Dip, we tested for its neuroprotective efficacy against glutamate excitotoxicity. If it has the protective effects, we continue to explore the mechanism of action. The projects and results were as follow: 1 Protective effects of Dip against the injury of cultured hippocampal neuron induced by glutamate in rats Objective: To culture hippocampal neuron and choose the best growing states. To study the effects of glutamate on cultured hippocampal neuron of rats and the protective effects of Dip. Methods: Newly born rats were used. Hippocampal tissue were dissected from the brain and produced cell suspension by digesting, flushing, suspending. Cultures were plated into 24-well and 96-well culture plates. Neurons were observed the soma area in different periods (24h, 3d, 7d, 14d and 21d) by microscopy and choosed the best grown status. The neuronal cells were cultured for one week, and then exposed to 0.1mmol/L, 0.5mmol/L and 1mmol/L glutamate respectively for 10 min in HEPES buffer containing (in millimoles) 100NaCl, 2.0KCl, 2.5CaCl₂, 1.0MgSO₄, 1.0NaH₂PO₄, 4.2NaHCO₃, 12.5HEPES, and 10.0glucose. Cultures were washed tow times with HEPES buffer and replaced with fresh NBM. Cultures were returned to the culture incubator and allowed to incubate for 24 hr prior to MTT measurement and chose the model group.Three different concentrations of Dip(0.1μmol/L, 1μmol/L and 10μmol/L)were added to cultured hippocampal neurons, which were damaged by glutamate and incubated together. After 24 hours, the lactate dehydrogenase release (LDH %) were assayed by the lactate dehydrogenase Kit and MTT (absorbance) were assayed by Fluostar Results: The hippocampal neurons grew quickly in the first three days. From day 3 to day 14 the hippocampal neurons grew steadily. After 21 days, the hippocampal neurons began to shrink. The soma area began to decrease. The enations began tolight and dissociation. One-week-old neuron cultures were used for experimental analyses.Glutamate could cause injury to the hippocampal neurons. The MTT absorbance of 0.1mmol/L glutamate , 0.5mmol/L glutamate and 1mmol/L glutamate (0.80±0.08, 0.77±0.10 and 0.67±0.09), were all reduced significantly, compared with the blank group (1.02±0.07, p<0.01). We chose the 1mmol/L glutamate as the model group, which caused the injury markedly.Dipfluzine could protect the injured hippocampal neuron of rats induced by glutamate.The LDH% in 1mmol/L glutamate group (34.2±1.9) was far higher than that in blank group (17.4±18.5, p<0.01). Both 1μmol/L Dip (22.3±1.6, p<0.01) and 10μmol/L Dip (20.3±2.0, p<0.01) decreased the LDH%. The MTT absorbance value of 1mmol/L glutamate (0.62±0.05), was reduced significantly, which compared with the value of blank group (0.80±0.09, p<0.01). The absorbance value of 10μmol/L Dip (0.75±0.10, p<0.05), was increased significantly. Conclusion: The hippocampal neurons grew quickly in the first three days and got a steady grown status in two weeks. One-week-old neuron cultures were used for experimental analyses.Glutamate could cause injury to the hippocampal neurons. Dip had protective effects against the injury of cultured hippocampal neuron induced by glutamate in rats. 2 Protective effects of Dip on the intracellular calcium overload in hippocampal neuron of rats induced by glutamateAim: To study the protective effects of Dipfluzine on the intracellular calcium overload in cultured and acutely dissociated hippocampal neuron of rats induced by glutamate Methods: Three different concentrations of Dip(0.1μmol/L, 1μmol/L and 10μmol/L)were added to cultured hippocampal neurons which were damaged by glutamate and incubated together. After 24 hours, the level of intracellular free calcium fluorescence intensity were assayed by LSCM. Acutely dissociated hippocampal neurons were prepared from normal SD rats aged 7-10 days and randomly divided into 3 group (1)Control group (2)Model group(1mmol/L glutamate) (3)Protect group(10 μmol/L Dip). Calcium fluorescence indicator Fura-2/AM was used to measure intraneuronal free calcium content ([Ca²⁺]_i) by Fluostar. Results: Clutamate (1mmol/L) could cause the level of intracellular free calcium fluorescence intensity(174.08±21.47) increased markedly in cultured hippocampal neurons, which was compared with the intensity of blank group(63±7, p<0.01). Both 1μmol/L Dip(93±10, p<0.01), 10μmol/L Dip(79±6, p<0.01) decreased the intensities.Clutamate (1mmol/L) could cause the level of intracellular free calcium fluorescence intensity (711±96) increased markedly in acutely dissociated hippocampal neurons, which was compared with the intensity of blank group (158±38, p<0.01). 10μmol/L Dip (314±42, p<0.01) decreased the intensity Conclusion: Glutamate could increase intracellular free...