c-Jun在胆红素脑病幼鼠海马神经细胞凋亡中的作用

Effect of c -dun on hippocampal neuron cell apoptosis in neonatal mice injected with bilirubin encephalopathy

目的探讨c－Jun在胆红素脑病幼鼠海马神经细胞凋亡中的作用。方法选取7日龄新生SD大鼠，经小脑延髓池注射胆红素溶液（10 g/L），注射后6 h、12 h断头取脑，重氮反应法检测海马胆红素水平，Nissl染色观察海马神经元的病理变化；Western blot和免疫荧光染色检测c－Jun氨基末端激酶（JNK）、c－Jun及p－c－Jun的表达；ELISA实验和荧光分光光度法测定凋亡相关因子Caspase－3和Caspase－6。结果小脑延髓池注射胆红素后幼鼠出现不同程度的异常行为学表现，6 h和12 h后海马胆红素水平较对照组显著升高[（44.00±13.00）%（P〈0.05）和（96.86±9.00）%（P〈0.01）]，24 h后海马神经元脱失，Nissl染色浅淡。随着海马胆红素水平增高，JNK蛋白表达水平（2.41±0.15）较对照组（1.00±0.09）增加1.4倍（P〈0.05），其下游靶分子c－Jun和p－c－Jun表达水平（1.43±0.16，2.16±0.23）也分别较对照组（1.00±0.13，1.00±0.11）上升0.43倍和1.16倍（P均〈0.05）。免疫荧光染色结果显示，胆红素注射6 h后海马及大脑皮质c－Jun和p－c－Jun阳性细胞增多。凋亡相关分子Caspase－3和Caspase－6活性在注射胆红素12 h后开始上升，24 h后分别较对照组增加2.08倍和0.28倍（P均〈0.05）。 结论c－Jun蛋白磷酸化在促进胆红素诱发的幼鼠海马神经细胞凋亡中发挥重要作用。

Objective To investigate the effect of c - dun on hippocampal neuron apoptosis in neonatal mice with bilirubin encephalopathy. Methods Seven - day - old newborn SD rats were selected and injected bilirubin solu- tion （ 10 g/L） into the cerebellum medulla oblongata pool. The bihrubin level in hippocampus was tested at 6 h and 12 h post- injection and neurons were labeled via Nissl staining;c -dun amino -terminal kinase （JNK） , c -dun and p - c - dun level in hippocampus were measured by Western blot and immunofluorescence staining. The expression le- vels of Caspase - 3 and Caspase - 6 were analyzed by enzyme - linked immunosondent assay and fluorescence spectro- photometry. Results Apart from a certain degree of abnormal behaviors following injection, a sharp rise of （44.00±13.00 ） % （ P 〈 0.05 ） and （96.86±9.00） % （ P 〈 0.01 ） in hippocampal bilirubin respectively at 6 h and 12 h post - injection. Hippocampal neuron lost and Nissl staining swallowed gradually after 24 h. At the time of 6 h following injec- ting, c - dun amino - terminal kinase （ JNK ） , c - dun and p - c - dun protein level in hippocampus was separately elevated byl.4（2.41±0.15 vs 1.00±0.09）,0.43 （1.43±0.16 vs 1.00±0.09） and 1.16 （2.16±0.23 vs 1.00±0.09 ） fold（ all P 〈 0.05 ） , compared with the control group. Immunofluoreseent staining showed that c - Jun and p -c -Jnn positive cells in hippoeampus and cortex all increased after 6 h injection of bilirnbin. At the same time ,the ac- tivity of apoptosis - associated factors downstream Caspase - 3 and Caspase - 6 initially obviously raised at 12 h post - in- jection,respectively increased by 2.08 and 0.28 fold of control group after 24 h（ all P 〈 0.05 ）. Conclusion The phos- phorylation of c - Jun may act as an important activator of hippocampal neuron apoptosis in the bilirubin encephalopathy.