百日咳毒素减轻脑缺血后神经元钙内流的实验研究

Pertussis toxin alleviates neuronal calcium influx after ischemic stroke: An experimental study

摘要:

目的 探讨百日咳毒素（PTx）对缺血性脑卒中后神经元的保护作用及其可能的机制。方法 将C57BL6小鼠用大脑中动脉闭塞（MCAO）法建立卒中模型，随机分为两组，每组12只鼠。实验组卒中后予PTx 1000 ng溶于1 ml生理盐水腹腔注射干预；对照组予1 ml生理盐水腹腔注射。24 h后行TTC染色检测梗死面积，并行免疫组化检测细胞凋亡。在体外培养原代神经元，用谷氨酸兴奋刺激模拟卒中后神经元损伤模型。用MTT及乳酸脱氢酶（LDH）释放实验检测PTx对谷氨酸兴奋刺激后神经元的存活和损伤情况。然后，检测PTx对谷氨酸诱导神经元钙离子内流的影响。结果 PTx治疗可减小卒中后小鼠脑梗死面积，使之由（51±11）%降至（34±8）%（P<0.05）。免疫组化发现PTx可使脑内Caspase-3阳性细胞数由（677.7±117.8）个/mm²减少至（297.5±83.6）个/mm²（P<0.05），且较少细胞凋亡。体外结果提示，PTx可增加谷氨酸刺激后神经元的存活率，使MTT吸光值由（0.618±0.06）提升至（1.1±0.12）（P<0.05）；同时，PTx还可减少LDH的释放，使吸光值由（1.31±0.11）降低至（0.76±0.08）（P<0.05）。PTx可减缓和减少钙离子进入神经元，经PTx治疗后钙内流可由基础值的5倍降低至基础值的2~3倍，且钙离子内流达到半峰值浓度的时间也由（18.5±2.5）s延长至（85.4±10.2）s。结论 百日咳毒素可减少卒中后钙离子内流到神经元，继而减少神经元损伤，减小梗死面积。

Abstract:

Objective To investigate the protective effect of pertussis toxin (PTx) on neurons after ischemic stroke and possible mechanisms.Methods C57BL6 mice were used to establish a model of stroke by middle cerebral artery occlusion (MCAO) and were then randomly divided into experimental group and control group, with 12 mice in each group. The mice in the experimental group were given intraperitoneal injection of PTx 1000 ng dissolved in 1 ml normal saline after stroke, and those in the control group were given intraperitoneal injection of 1 ml normal saline. TTC staining was performed 24 hours later to measure infarct area, and immunohistochemistry was used to measure neuronal apoptosis. Primary neurons were cultured in vitro and glutamate stimulation was performed to mimic neuronal injury after stroke. MTT assay and lactate dehydrogenase (LDH) release assay were used to observe the influence of PTx on the survival and injury of neurons after glutamate stimulation, and then the influence of PTx on glutamate-induced neuronal calcium influx was examined.Results PTx treatment reduced cerebral infarct area from 51%±11% to 34%±8% in mice after stroke (P<0.05). Immunohistochemistry showed that PTx reduced the number of caspase-3-positive cells from 677.7±117.8 cells/mm2 in the control group to 297.5±83.6 cells/mm2 in the experimental group (P<0.05) and reduced cell apoptosis. In vitro results showed that PTx increased the viability of neurons after glutamate stimulation, and MTT absorbance was increased from 0.618±0.06 to 1.1±0.12 (P<0.05); at the same time, PTx reduced the release of LDH, and the absorbance of LDH was reduced from 1.31±0.11 to 0.76±0.08 (P<0.05). PTx slowed down and reduced calcium influx into neurons, and after PTx treatment, calcium influx was reduced from 5 times to 2-3 times of the baseline value; the time to half-peak concentration was increased from 18.5±2.5 s to 85.4±10.2 s.Conclusions PTx can reduce calcium influx into neurons after stroke and thus alleviate neuronal injury and reduce infarct area.