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

周琴 张勤 李光乾 周素芽

●论 著

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

周琴 张勤 李光乾 周素芽

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-κB/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-кB/p65的表达，且较A组明显升高（P＜0.05或0.01）。C组较B4组NF-κB/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-κB/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-κB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。

惊厥 凋亡 Toll样受体4 NF-κB 半胱氨酸蛋白酶-3 吡咯烷二硫代氨基甲酸盐

【 Abstract】 Objective To investigate the effects of pirrolidine dithiocarbamate(PDTC)on expression of TLR4,NF-кB, 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-κB/p65 protein was detected by immunohistochemistry(IHC),the expression of TLR4 and caspase-3 mRNAwas 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-кB/p65 was observed in nuclei of hippocampal neurons in group B,compared with group A (P＜0.05),and the protein expression of NF-кB/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 CAlofgroup B were more than those ofgroup 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-кB 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 ofrats with pilocarpine-induced seizures.These results suggest that TLR4/NF-κB may have protective effect on the development ofthe hippocampus apoptosis caused by status convulsion.

有研究发现，持续惊厥发作后将导致以海马区为主的选择性脑损伤，而神经细胞的死亡分为坏死与凋亡，坏死是被动的突发性细胞肿胀与溶解过程，凋亡则属于主动程序性细胞死亡[1]。……