缝隙连接蛋白Cx43抑制剂对癫痫大鼠认知功能的影响及其机制研究

Effect of gap junction protein Cx43 inhibitor on cognitive function and its possible mechanism in epileptic rats

目的探讨缝隙连接蛋白Cx43抑制剂甘珀酸(CBX)对癫痫大鼠认知功能的影响及其可能的机制。方法120只Wistar大鼠按随机数字表法分为假手术组、癫痫组、癫痫+溶剂组、癫痫+CBX组,每组30只。后3组大鼠海马内注射海人酸(KA)制备颞叶癫痫模型。癫痫+CBX组大鼠造模前30 min腹腔注射CBX(20 mg/kg),癫痫+溶剂组大鼠造模前30 min腹腔注射等量生理盐水。造模后6、12、24 h采用Western blotting检测大鼠海马CA3区磷酸化(p)-Cx43和微管相关蛋白轻链3(LC3)的表达,造模后24 h采用免疫组化染色检测大鼠海马CA3区p-Cx43和LC3的定位及其阳性细胞的吸光度值,造模后30 d采用Morris水迷宫实验检测大鼠空间学习记忆能力的变化。结果Western blotting检测结果显示,与假手术组比较,癫痫组、癫痫+溶剂组大鼠造模后6 h、12 h、24 h海马CA3区p-Cx43、LC3蛋白的表达均增加;与癫痫组、癫痫+溶剂组比较,癫痫+CBX组大鼠各时间点海马CA3区p-Cx43、LC3蛋白的表达均降低,差异均有统计学意义(P<0.05)。免疫组化染色检测结果显示,p-Cx43定位于海马星形胶质细胞的细胞膜和细胞质中,LC3定位于海马神经元胞质内。与假手术组比较,癫痫组、癫痫+溶剂组大鼠海马CA3区p-Cx43、LC3阳性细胞的吸光度值增加;与癫痫组、癫痫+溶剂组比较,癫痫+CBX组大鼠海马CA3区p-Cx43、LC3阳性细胞的吸光度值降低,差异均有统计学意义(P<0.05)。Morris水迷宫实验结果显示,与假手术组比较,癫痫组、癫痫+溶剂组大鼠的逃避潜伏期明显延长;与癫痫组、癫痫+溶剂组比较,癫痫+CBX组大鼠的逃避潜伏期明显缩短,差异有统计学意义(P<0.05)。结论CBX通过抑制癫痫大鼠海马CA3区星形胶质细胞p-Cx43蛋白的表达,减弱神经元自噬,从而减轻其认知功能损害。

Objective To investigate the effect of gap junction protein Cx43 inhibitor carbenoxolone(CBX)on cognitive function and its possible mechanism in epileptic rats.Methods One hundred and twenty Wistar rats were randomly divided into sham-operated group,epilepsy group,epilepsy+solvent group,and epilepsy+CBX group(n=30).The models of temporal lobe epilepsy in the later three groups were prepared by injection of kainic acid in the hippocampus.Intraperitoneal injection of CBX(20 mg/kg)or equal amount of normal saline were given to the rats in the epilepsy+CBX group and epilepsy+solvent group 30 min before modeling.Western blotting was used to detect the protein expressions of phosphorylated(p)-Cx43 and microtubule associated protein light chain 3(LC3)in the hippocampus 6,12,and 24 h after modeling;the protein localization of p-Cx43 and LC3 in the hippocampus and optical density of their positive cells were detected by immunohistochemistry 24 h after modeling;the learning and memory abilities of rats were tested by Morris water maze experiment 30 d after modeling.Results Western blotting results showed that as compared with those in the sham-operated group,p-CX43 and LC3 protein expressions in the hippocampal CA3 regions of epilepsy group and epilepsy+solvent group were significantly increased at 6,12 and 24 h after modeling(P<0.05);as compared with the epilepsy group and epilepsy+solvent group,the epilepsy+CBX group had statistically decreased p-CX43 and LC3 protein expressions in the hippocampal CA3 regions at each time point(P<0.05).Immunohistochemical staining showed that p-CX43 was localized at the cell membrane and cytoplasm of hippocampal astrocytes;LC3 was located at the cytoplasm of hippocampal neurons.As compared with those in the sham-operated group,the optical density values of p-CX43 and LC3 positive cells in hippocampal CA3 regions of epilepsy group and epilepsy+solvent group were increased(P<0.05).As compared with those in the epilepsy group and the epilepsy+solvent group,the optical density values of p-CX43 and LC3 positive cells in the hippocampal CA3 regions of the epilepsy+CBX group were significantly decreased(P<0.05).Morris water maze test results showed that as compared with that in the sham-operated group,the escape latency in the epilepsy group and epilepsy+solvent group was significantly prolonged(P<0.05);as compared with that in the epilepsy group and epilepsy+solvent group,the latency in the epilepsy+CBX group was significantly shortened(P<0.05).Conclusion CBX can weaken the neuronal autophagy and reduce the damage to cognitive function by inhibiting the p-Cx43 protein expression in the astrocytes of the hippocampal CA3 regions.