| Calcium overload induced by hypoxia resulting in neuronal death had been extensively detected in a majority of laboratories. One of the important pathways which mediate the calcium overload is a non-selective cation channel-TRPM7. Except for mediating the calcium influx, TRPM7 had been reported to maintain the magnesium homeostasis in physiological states. So when TRPM7 channel is activated by hypoxia to induce the calcium overload in neurons, the question here is whether magnesium is also moving into the intracellular space through TRPM7 to form the "magnesium overload". Using Mg2+ fluorescent probe Mag-fura-2 to detect intracellular magnesium concentration ([Mg2+]i) and flame atomic absorption spectrometry to test extracellular magnesium concentration ([Mg2+]o), the changing profile of intracellular magnesium in cultured hippocampal neurons was investigated in two ischemia models established: oxygen-glucose deprivation/re-oxygen (OGD/RO) and acute chemical ischemia(CI); Next silencing TRPM7 with shRNA to downregulation the expression of TRPM7, or using innibitors of TRPM7 channel to attenuate the channel function in neurons to detect whether TRPM7 attends the magnesium dynamic change in OGD/CI. The results showed that:(1) Neuronal [Mg2+]i increased 1.51 fold than basal lever (0.49±0.005 mM, n=72) after OGD lhour (1h). The increase kept on for 15min、30min 60 min within re-oxygen.Compared with pre-hypoxia, the [Mg2+]i of these three time point had significant difference (P<0.05). The increase recovered to normal after cells were re-oxygened for 6h or 12h, having no abvious difference with pre-hypoxia. Conversely, [Mg2+]i in neurons was surprisingly below the basal level after cells were 24h re-oxygened, having significant difference with pre-hypoxia (P<0.05). Removing extracellular magnesium did not affect the baseline intensity of mag-fura-2, but attenuated the increase of [Mg2+]i by 40.88% from 0.71±0.01 mM to 0.62±0.02 mM (OGD1h groups with and without magnesium had significant difference,P<0.05) which was also above the basal [Mg2+]i level(campared to normal group,both of the OGDlh groups with and without magnesium had significant difference,P<0.05). The results mean that oxygen-glucose deprivation for 1h could induce the intracellular magnesium overload which may relate with extracellular magnesium in hippocampal neurons.(2) Using KCN to establish chemical ischemia model, it shoewed that the [Mg2+]i peak immediately appeared in 10 seconds but recovered to pre-ischemia level in less than 20 seconds. taking magnesium away from the extracellular liquid before 3 mM KCN was added, the basal level of [Mg2+]i was not altered but the Rincrease of [Mg2+]i was reduced by 39.52% from 101.39%±5.74% to 61.32%±3.50%. These two groups had significant difference(P<0.05). These results here suggest CI caused intracellular magnesium overload which may partially from the extracellular magnesium in hippocampal neurons.(3) Magnesium concentration contained in the extracellular liquid was detected during the different time course(15min、30min、60min、6h、12h、24h) after OGD 1h using flame atomic absorption spectrometry. The results showed that [Mg2+]o decreased from 15.29±0.25μg/mL to 13.72±0.21μg/mL after OGD 1h. These two groups had significant difference (P<0.05). When cells were re-oxygened for 15 min following OGD 1h, [Mg2+]o returned to the basal level which did not correspond with the outcome of [Mg2+]i·[Mg2+]o maintained in the basal level for 12h within reperfusion. Matching with the [Mg2+]o changing, [Mg2+]o increased to 18.25+0.38μg/mL after cells were 24 h re-oxygened. These two groups had significant difference (P<0.05). [Mg2+]o was increased from 13.53±0.20μg/mL to 14.46±0.17μg/mL in the addition of 2-APB, the non-specific inhibitor of TRPM7 channels before OGD 1h (P<0.05). But the [Mg2+]o of 2-APB+OGD group was still lower than pre-OGD group. These two groups had significant difference (P<0.05). These results here suggest OGD 1h caused extracellular magnesium to transmembrane into intracellular space possibly partially through the TRPM7 channels in hippocampal neurons.(4) Using shRNA to knock down the expression of TRPM7 channels, [Mg2+]i in hippocampal neurons was detected after OGD 1h or CI. The results showed that silencing TRPM7 reduced not only the basal [Mg2+]i but also the increased [Mg2+]i caused by OGD 1h. In physiological states, [Mg2+]i in neurons whose TRPM7 channels were knocked down by shRNA (0.43±0.01 mM)) was lower than normal or control nerurons (0.51±0.02 mM). These two groups had significant difference(P<0.05); Compared to control neurons, the increased [Mg2+]i caused by OGD 1h in TRPM7 knocked down neurons was reduced by 24.64% from 0.72±0.02 mM to 0.57±0.02 mM. They had significant difference(P<0.05). The effect of down-regulation of TRPM7 on basal or increased [Mg2+]i in CI is similar to that in OGD 1h. The results suggest that TRPM7 channels play an important role in the magnesium ion homeostasis and [Mg2+]i increase induced by OGD 1h/CI in rat hippocampal neurons.(5) Consistent with an involvement of TRPM7 channels, addition of Gd3+ and 2-aminoethoxydiphenyl borate (2-APB), non-specific inhibitors of TRPM7 channels, compared to addition of DMSO, attenuated CI-induced [Mg2+]i increase by 20.68% and 19.9% respectively. They both had significant difference (P<0.05) with DMSO group.These two drugs did not influence the baseline of [Mg2+]i (P>0.05) The effect of TRPM7 inhibitors was also detected in OGD model. Adding 2-APB, the basal [Mg2+]i was not altered but the increased [Mg2+]i caused by OGD 1h was reduced by 18.67% from 0.72±0.01mM to 0.68±0.01 mM. These two groups had significant difference(P<0.05). These results implied that TRPM7 is involved in intracellular magnesium accumulation induced by OGD 1h or CI in neurons. |
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