豚鼠脑缺血再灌注后听皮层内质网应激相关因子的表达及意义

摘要

Objective To investigate the expression of GRP78 and caspase-12 on the auditory cortex and to study the effects of endoplasmic reticulum stress in the auditory cortex neuron apoptosis after the brain ischemia reperfusion injury in guinea pig.Methods Fifty healthy male guinea pigs were selected and randomly divided into 5 groups which were hormal group group A(reperfusion for 6 hours),group B(reperfusion for 12 hours),group C (reperfusion for 24 hours),group D(reperfusion for 72 hours).The cerebral ischemia reperfusion injury model was produced by the occlusion of bilateral common carotid arteries.The guinea pigs were sacrificed at reperfusion of 6, 12,24,and 72 hours respectively after they received ABR tests.The pathological changes were observed by HE and the levels of GRP78 and caspase-12 protein were detected by immunohistochemistry and Western blot.Results The hearing thresholds increased gradually from the normal group to group B,and decreased gradually from group C to group D,but the thresholds of group D were still higher than that of the normal group.HE staining showed that the neurons in the normal control group were arranged in order,the cytoplasm was abundant,large and round.The cells were stained clearly.After reperfusion,the number of neurons in each time point was decreased,the nucleus presented atrophic,fragmented,the disappeared.The expression of GRP78 and caspase-12 protein in normal con-trol group was only a trace or a small amount by immunohistochemistry and Western blot.The expression of GRP78 and caspase-12 began to inerease.The expression of GRP78 reached the peak at reperfusion of 12 hours and de-creased gradually.There were significant statistic differences between each group comparison.The expression of caspase-12 reached the peak at reperfusion of 24 hours,then decreased gradually.There was no statistic difference between group A and group B.There were significant statistic differences anong other groups.Conclusion Endo-plasmic reticulum stress(ERS)may be induced by brain ischemia reperfusion injury,and can increase the expression of GRP78 and caspase-12.GRP78 and caspase-12 participate in the process of neuron apoptosis on auditory cor-tex caused by ERS.%目的：观察豚鼠脑缺血再灌注后葡萄糖结合蛋白（glucose－regulated protein，GRP78）、半胱氨酸天冬氨酸蛋白酶－12（caspase－12）在听皮层的表达，探讨内质网应激（endoplasmic reticulum stress，ERS）在脑缺血再灌注后听皮层神经元凋亡中的作用。方法选取健康豚鼠50只，随机分为5组：正常对照组、A组（缺血再灌注6 h）、B组（缺血再灌注12 h）、C组（缺血再灌注24 h）、D组（缺血再灌注72 h）。采用夹闭双侧颈总动脉的方法建立脑缺血再灌注损伤模型，分别在脑缺血再灌注后6、12、24、72 h行听性脑干反应（ABR）测试后处死各组豚鼠，应用 HE染色法观察听皮层神经元病理变化，免疫组化染色及 Western－blot 方法检测各组GRP78、caspase－12的表达。结果从正常对照组到B组豚鼠ABR反应阈值逐渐增加，从C 组到D组又逐渐下降，但D组仍比正常对照组高（P＜0．05）。HE染色示正常对照组听皮层神经元排列整齐，胞浆丰富，胞核大而圆，染色清晰；A、B、C、D组听皮层神经元均有不同程度的数量减少，且体积萎缩，胞核固缩，碎裂，核仁消失。免疫组化染色及 Western－blot示正常对照组 GRP78、caspase－12蛋白仅有微量或少量表达，缺血再灌注后6 h，二者表达开始上调，至12 h GRP78表达达到高峰，12 h后逐渐降低，各组之间差异均有统计学意义（P＜0．05）。caspase－12表达24 h达到峰值，后逐渐下降，缺血再灌注6 h组与12 h组间差异无统计学意义，其余各组间差异均有统计学意义（P＜0．05）。结论脑缺血再灌注损伤可诱发内质网应激，使GRP78、caspase－12表达增加，GRP78、caspase－12可能参与了ERS介导的听皮层神经元细胞凋亡过程。