七氟烷暴露促发Caspase-8/GSDME细胞焦亡诱导海马神经元损伤的形态和蛋白变化特征

Characteristics of Morphology and Protein Changes in the Process of Caspase-8/GSDME Induced Hippocampus Neuron Pyroptosis After Sevoflurane Exposure

目的观察七氟烷干预下海马神经元焦亡过程的形态学变化及神经元相关蛋白表达特征。方法海马神经元随机分为对照组和七氟烷组(6、12、24、48 h组)。对照组暴露于21%O2、5%CO23 h,七氟烷组暴露于4.1%七氟烷、21%O2、5%CO23 h,七氟烷4个亚组根据七氟烷暴露3 h后的4个不同观察时间点进行分组。应用显微镜观察神经元形态变化,用尼氏染色检测对照组及48 h组海马神经元细胞内尼氏体个数和突触变化,采用Western blot法检测各组Caspase-8、Caspase-3、功能蛋白D(gasdermin D,GSDMD)、功能蛋白E(gasdermin E,GSDME)和α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体1(α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor,AMPAR1)的表达水平。结果普通显微镜下观察到,6 h组细胞变化不明显;12 h组细胞疏松、水肿和孔洞形成;24 h组见细胞量降低、突触减少、部分细胞膜破裂;48 h组细胞量明显减少并出现大量的凋亡。尼氏染色显示,48 h组大鼠海马神经元排列散乱、神经元数量明显减少、细胞皱缩、胞体缩小、部分树突消失、轴突延长,尼氏体数量较对照组减少,差异有统计学意义(P<0.05)。Western blot法检测显示:Caspase-3表达活性在6、12、24 h组明显高于对照组(P<0.05);Caspase-8表达活性在七氟烷4个组都增加,其中12、24、48 h组与对照组比较,差异有统计学意义(P<0.05);GSDME表达活性在七氟烷4个组都增加,12、24、48 h组与对照组以及6 h组比较,差异均有统计学意义(P<0.05);与对照组比较,GSDMD表达活性在6 h组下降(P<0.05),12 h组增加,24、48 h组恢复正常值,但差异均无统计学意义(P>0.05);与对照组比较,AMPAR1表达活性在24 h组下降至最低,48 h组回升但未恢复至正常水平,差异均有统计学意义(P<0.05)。结论正常的海马神经元表达炎性因子Caspase-3和GSDMD,七氟烷暴露使Caspase-8激活,导致GSDME介导神经元焦亡,并改变海马神经元突触的可塑性。

Objective To observe the morphological changes of hippocampal neurons and the expression characteristics of neuron-relatedproteins under the intervention of sevoflurane induced pyroptosis.Methods Hippocampal neurons were random divided into control group and sevoflurane group(6 h,12 h,24 h,48 h group).Control group exposed to 21%O2,and 5%CO2 for 3 hours.Sevoflurane group exposed to 4.1%sevoflurane,21%O2,and 5%CO2 for 3 hours,sevoflurane group was divided into 4 subgroups according to 4 different times after sevoflurane exposure for 3 hours.The morphological changes of neurons were observed under a microscope,and Nissl staining was used to check the number of Nissl bodies and synaptic changes in the cells on 48 h group and control group.Western blot was used to detect the expression levels of Caspase-8,Caspase-3,GSDMD,GSDME and AMPAR1 receptor.Results We observed with common microscope and discovered that there were not significant morphological changes on 6 h group;on 12 h group,the cells were loose,edema and holes were formed;on 24 h group,the number of cells and synapses decreased,partial cell membrane ruptured were observed;on 48 h group,the number of neurons was significantly reduced and a large number of cells apoptosis appeared.Nissl staining showed that the hippocampal neurons on 48 h group were disorderly arranged,the number of neurons was significantly reduced,cell shrinkage,the cell body was shrunk,some dendrites disappeared,and axons prolonged,compared with control group,the number of Nissl bodies on 48 h group decreased,difference has statistically significant(P<0.05).Western blot analysis showed that compared with control group,the expression activity of Caspase-3 increased significantly on 6,12,24 h group(P<0.05).The expression activity of Caspase-8 increased on sevoflurane 4 groups.Compared with control group,the difference of Caspase-8 was statistically significant on 12,24,48 h group(P<0.05).GSDME expression increased on sevoflurane 4 groups,the difference of GSDME was statistically significant on 12,24,48 h group compared with 6 h group and control group.GSDMD expression activity decreased on 6 h group,increased on 12 h group,then returned to normal on 24,48 h group,but there was not statistically different from the control group(P>0.05).The expression of AMPAR1 receptor declined to the lowest level on 24 h group,and gradually restored on 48 h group,there were statistically differences from the control group(P<0.05).Conclusion Normal hippocampal neurons express inflammatory factors Caspase-3 and GSDMD.Sevoflurane exposure activates Caspase-8,leading GSDME mediated neuron pyroptosis and changes the synaptic plasticity of hippocampal neurons.