PDTC对惊厥持续状态大鼠海马细胞凋亡及TLR4、caspase-3表达的影响

Effects of PDTC on expression of Toll-like receptor 4,caspase-3 and neuron apoptosis in hippocampus of status convulsion rats

目的观察大鼠惊厥持续状态后海马中Toll样受体4（TLR4）、NF-κB和半胱氨酸蛋白酶-3（caspase-3）的动态表达及神经细胞凋亡的变化,探讨吡咯烷二硫代氨基甲酸盐（PDTC）对惊厥后脑损伤的可能保护机制.方法将106只SD大鼠随机分为0.9％氯化钠组（A组）、SC组（B组）和PDTC组（C组）,B组根据大鼠惊厥后处死时间（4、24、48和72h）,分为B1～B4组,B组和C组大鼠惊厥持续状态模型的制作采用氯化锂-匹罗卡品法,C组在大鼠制模成功后,每天腹腔注射PDTC（100mg/kg）1次,连用3d,于惊厥后72h处死.电镜观察海马超微结构改变;免疫组化法测定大鼠海马NF-κ B/p65的表达;RT-PCR法检测海马TLR4、caspase-3 mRNA表达的动态变化;TUNEL法检测神经细胞凋亡数的变化.结果B组大鼠海马神经元超微结构损伤存在动态变化,72h内病变进行性加重;C组大鼠病理改变较B4组减轻.B1～B4组海马神经元胞核内有不同程度NF-κ B/p65的表达,且较A组明显升高（P＜0.05或0.01）.C组较B4组NF-κ B/p65表达明显下降（P＜0.01）.B1～B4组TLR4、caspase-3 mRNA的表达量均高于A组（P＜0.05或0.01）,且随时间延长逐渐升高,72h达到高峰;C组TLR4、caspase-3 mRNA的表达较B4组明显降低（P＜0.05）.B组在惊厥24h后海马CA1区TUNEL阳性细胞数已明显高于A组（P＜0.01）,72h达高峰,而C组TUNEL阳性细胞数较B4组明显下降（P＜0.01）.结论惊厥持续状态后大鼠海马TLR4、NF-κ B/p65和caspase-3的表达增强.NF-κB抑制剂PDTC可以下调TLR4和caspase-3的表达,并使神经细胞凋亡减轻;提示TLR4/NF-κ B信号通路在惊厥后海马细胞凋亡的发生、发展过程中起促进作用.

Objective To investigate the effects of pirrolidine dithiocarbamate （PDTC） on expression of TLR4, NF- κB, caspase-3 and the change of neuron apoptosis in hippocampus of status convulsion rats. Methods One hundred and six male Sprague-Dawley（SD ）rats were randomly divided into control group （A）, convulsion group （B） and PDTC group （C）. then group B were randomly divided into four subset groups （B1-B4）, which would be executed at 4h, 24h, 48h, 72h after convulsion discontinued. Continuous epilepticus was induced by injecting lithium chloride and pilocarpine, and rats in group C were daily injected with 100mg/kg PDTC 30 min after convulsion discontinued for 3 days. The histopathological changes in hippocampus were observed with electron microscope, NF- κ B/p65 protein was detected by immunohistochemistry （IHC）, the expression of TLR4 and caspase-3 mRNA was detected by RT-PCR. The neuron apoptosis was examined by TUNEL. Results Neuronal injury was observed in group B with electron microscope, and the change was increased gradually after seizure induced; neuronal injury in group C was milder than that in group B at 72h. IHC staining showed that more positive expression of NF-κ B/p65 was observed in nuclei of hippocampal neurons in group B, compared with group A （P〈0.05）, and the protein expression of NF- κ B/p65 in group C was much lower than that in group B4 （P〈0.01）. The mRNA expression of TLR4 and caspase-3 in rat hippocampus of group B was significantly elevated than that in group A （P〈0.05）; and the tendency was increased gradually reaching the peak at 72 h after seizure, and that in group C was much lower than that in B4 group （P〈0.01）. The TUNEL positive cells in hippocampus CAI of group B were more than those of group A at 24h after seizure （P〈0.01） reaching the peak at 72h; andthe TUNEL positive cells in group C were lower than those in B4 group （P〈0.01）. Conclusion The expression of TLR4, NF- κB and caspase-3 increased after SC in rats. PDTC can down-regulate the expression of TLR4 and caspase-3 and inhibit neuronal apoptosis in hippocampus of rats with pilocarpine-induced seizures. These results suggest that TLR4/NF- κB may have protec- tive effect on the development of the hippocampus apoptosis caused by status convulsion.