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Activation of N-methyl-d-aspartic acid (NMDA) receptor plays an important role in neuronal apoptosis induced by cerebral ischemia but the underlying mechanisms are still unclear. The present study examined the neuroprotection of three chloride blockers in an in vitro cell model of cerebral ischemia established by treatment of cultured rat hippocampal neurons with NMDA. Hoechst 33258 staining and MTT assay were used to detect neuronal apoptosis and cell viability, respectively. The neuroprotective effects of chloride channel blockers on the cell viability and neuronal apoptosis were only observed when the blockers were applied before NMDA exposure. In comparison with DIDS, SITS showed more potent protective effect in a dose-dependent manner, whereas NPPB showed no significant neuroprotective effect. The results demonstrate that pretreatment with both SITS and DIDS have protective effect against neuronal apoptosis, which is achieved by blocking both NMDA receptor and chloride channel.

To investigate the effect of liquid extract of Lishi No.5 formula in protecting the neurons from excitatory amino acid injury.Cultured neonatal SD rat hippocampal neurons were treated with 300 micromol/L NMDA and 5 micromol/L glycine for 10 min, and the conditioned culture medium was replaced by normal culture medium. After cell culture for 18 h, MTT assay and Hoechst33258 staining were performed to examine the cell survival rate and cell apoptosis, respectively. Intracellular free calcium concentration was assayed by image analysis of the calcium signals with confocal microscope and Fluo-3/AM indicator.The survival rate of the cultured cell was significantly decreased in response to treatment with 300 micromol/L NMDA and 5 micromol/L glycine, but the effects were obviously reversed by treatment with 0.5 mg/ml liquid extract from Lishi No.5 formula. Intracellular free calcium concentration was significantly increased by 10 micromol/L NMDA, which was remarkably inhibited by the liquid extract. The effects of NMDA on intracellular free calcium was abolished by pretreatment of the cells with MK-801 for 10 min, whereas the liquid extract significantly lowered the antagonizing effect of MK-801.Lishi No.5 formula protects the neurons from toxicity by excitatory amino acids possibly by alleviating intracellular free calcium overload induced by these amino acids.