新生鼠缺氧缺血性脑病蛋白激酶C与三磷酸肌醇及c-fos表达关系探讨

Relationship Among the Changes of Protein Kinase C, Inositol_(1,4,5)-Trisphosphate and c-fos Expression in Hypoxic-Ischemic Encephalopathy of Neonatal Rats

目的 对新生大鼠缺氧缺血性脑病 (HIE)第二信使蛋白激酶C(PKC)和 1,4 ,5 三磷酸肌醇 (IP3 )及c fos基因蛋白表达的关系进行研究。方法 生后 7d龄Wistar大鼠采用结扎右侧颈总动脉后放入含 5 %氧气和 95 %氮气的密闭容器 2 0min的方法制作HIE模型 ,分别于造模后 0 ,4 ,12 ,2 4 ,4 8,72h及 7,14 ,2 1d留取标本。对照组只做假手术 ,时间点与HIE组一致。PKC蛋白定量采用Lowry法 ,活性测定采用γ - 3 2 P催化活性测定法。IP3 测定采用放射受体竞争结合法 ,c fos基因蛋白表达采用免疫组织化学染色法。结果 与对照组比 ,HIE新生鼠脑皮质、海马神经细胞膜PKC活性降低 (P <0 .0 5或 0 .0 1) ,脑皮质神经细胞胞浆PKC活性升高 (P <0 .0 1) ,海马神经细胞胞浆PKC活性变化不大 ;动态观察PKC活性显示上述改变在造模后 72h内明显 ,并持续到 14d ,造模后 2 1d基本恢复正常。与此同时 ,脑皮质、海马神经细胞IP3 含量降低 ,丘脑IP3 含量升高 ,以上改变在 14d基本恢复正常。新生鼠HIE造模后即刻脑组织各部即有不同程度的c fos表达 ,且于缺氧缺血后 4h达高峰 ,以后强度逐渐降低 ,并持续至 72h。脑皮质c fos表达以Ⅱ -Ⅴ层明显 ,海马以CA1和DG区明显 ,CA3区也有表达 ,丘脑c fos表达稍有增加。

Objective To study the role of the changes of second messenger protein kinase C (PKC), inositol 1,4,5 trisphosphate (IP 3) and expression of immediately early gene c fos protein (c fos) in the pathogenesis of hypoxic ischemic encephalopathy (HIE) of neonatal rats. Methods Fifty seven 7 day old Wistar rats were put into a an air tight container with 5% O 2 and 95% nitrogen for 20 minutes after their right common carotid arteries were occluded, and then they were sacrificed at 0, 4, 12, 24, 48 and 72 h, and 7, 14 and 21 d after hypoxic ischemic (HI) insult. The rats experienced only pseudo operation were used as the controls. PKC activity was measured by the incorporation of [γ- 32 P] into a specific substrate peptide in the cytosolic and particulate fraction respectively. IP 3 was determined by the radioreceptor binding assay. C fos expression was detected by immunohistochemical method. Results Compared with the non HI controls, PKC activities in particulate fractions in both cerebral cortex and hippocampus decreased, whereas increased in cytosol in cerebral cortex and remained within the normal range in cytosol in hippocampus at 0, 4, 12, 24, 48 and 72 h, and 7 d after HI. The changes of PKC activities were obvious within 72 hs and lasted to 14 d after HI. A decrease of the IP 3 level in cerebral cortex and hippocampus and an increase of the IP 3 level in thalamus were noted after HI. The changes of the IP 3 level restored to normal on 14 d after HI. The expression of c fos was found in different parts of brain immediately after HI and peaked at 4 h, and thereafter, its density gradually decreased and lasted to 72 h. The expression of c fos was obvious in Ⅱ-Ⅴ layers of cortex, CA1 and DG of hippocampus respectively, and slightly increased in thalamus. Conclusions HI could induced the changes of the second messengers PKC and IP 3. The continuous PKC down regulation and c fos gene expression may take part in neuron injuries during hypoxic ischemic brain injury.