目的 探讨右美托咪定预处理对异氟醚引起新生大鼠脑发育毒性的保护作用及与坍塌反应调节蛋白-2的关系。 方法 60只出生后7 d的SD大鼠,随机分为对照组,右美托咪定预处理对照组,异氟醚组以及异氟醚复合不同剂量右美托咪定(25, 50和75 μg·kg-1)预处理组。对照组吸入空气,异氟醚组吸入0.75%异氟醚6 h。在麻醉前20 min 右美托咪定预处理组腹腔内注射不同剂量的右美托咪定,其他组注射150 μL生理盐水。麻醉结束后即刻蛋白质印迹法检测海马激活型caspase-3、磷酸化坍塌反应调节蛋白-2(p-CRMP2)以及坍塌反应调节蛋白-2蛋白表达变化(n=6)。麻醉结束后2 h,TUNEL荧光染色检测海马CA1区神经细胞凋亡(n=4)。结果 异氟醚组海马CA1区TUNEL阳性细胞数 较对照组 增加了434.99%(P<0.001),右美托咪定75 μg·kg-1预处理 降低了异氟醚诱导的TUNEL阳性细胞数71.87%(P<0.001)。异氟醚组激活型caspase-3表达比对照组增加90.20%(P<0.001),右美托咪定25,50和75 μg·kg-1预处理减少caspase-3的表达分别是30.54%,53.71%(P<0.05)和96.71%(P<0.001)。异氟醚增加了新生大鼠海马p-坍塌反应调节蛋白-2/坍塌反应调节蛋白-2比值119.41%(P<0.001),没有改变总坍塌反应调节蛋白-2的表达;右美托咪定50和75 μg·kg-1预处理降低异氟醚诱导的p-坍塌反应调节蛋白-2/坍塌反应调节蛋白-2比值增加分别为63.75%(P<0.01)和77.70%(P<0.01),同时分别增加了坍塌反应调节蛋白-2的表达44.30%(P<0.05)和62.13%(P<0.01)。结论 右美托咪定预处理能通过减少海马细胞凋亡来减轻异氟醚对新生大鼠的脑毒性作用,抑制坍塌反应调节蛋白-2磷酸化和增加坍塌反应调节蛋白-2的表达可能是保护作用的机制之一,为婴幼儿临床麻醉用药的选择提供参考。
Abstract
ObjectiveTo investigate the effects of dexmedetomidine(Dex) preconditioning on neuroapoptosis in hippocampus induced by isoflurane and the expression of CRMP2 proteins in neonatal rats. Methods Sixty neonatal rats at postnatal day 7 were assigned randomly into control group, Dex preconditioning control group, isoflurane group and isoflurane with Dex preconditioning groups which at dose of 25, 50 and 75 μg·kg-1. Rats in groups of isoflurane or control were exposed 0.75% isoflurane or air for 6 h. Rats in groups of Dex preconditioning were injected intraperitoneally with different doses of Dex 20 min before exposure and rats in other groups were injected intraperitoneally with 150 μL saline. At the end of exposure, the hippocampus of some rats were separated and proteins expression of cleaved caspase-3, phospho-CRMP2 (p-CRMP2), CRMP2 were detected by Western blot (n=6); other rats were perfused and their brains were embedded by paraffin, the neuronal apoptosis in hippocampus CA1 was detected by TUNEL staining 2 h after exposure (n=4). Results The number of TUNEL positive cells in hippocampus CA1 of rats in isoflurane group was increased by 434.99% (P<0.001) when compared with control group , Dex at dose of 75 μg·kg-1 significantly inhibited the increase of isoflurane-induced TUNEL positive cells by 71.87% (P<0.001). The expression of cleaved caspase-3 protein in isoflurane group increased by 90.20% (P<0.001) compared with control group, while Dex at dose of 25, 50 and 75 μg·kg-1 reversed isoflurane-induced increase of cleaved caspase-3 by 30.54% and 53.71%(P<0.05)and 96.71% (P<0.001)separately. Isoflurane increase the ratio of p-CRMP2/CRMP2 in the hippocampus by 119.41% (P<0.001), and didn’t influence the expression of CRMP2; while Dex at dose of 50 and 75 μg·kg-1 inhibited isoflurane-induced increase of p-CRMP2/CRMP2 ratio by 63.75% (P<0.01) and 77.70% (P<0.01) and increased the expression of CRMP2 by 44.30% (P<0.05) and 62.13% (P<0.01). Conclusion Dex Attenuates isoflurane-induced neurodegeneration through inhibitory effect of hippocampal neuroapoptosis in neonatal rats, decrease the ratio of p-CRMP2/CRMP2 and increase the expression of CRMP2 may be one of the mechanisms for neuroprotection and this Conclusion may provide the theoretic foundation for reasonable selection of anaesthetics.
关键词
异氟醚 /
右美托咪啶 /
海马 /
坍塌反应调节蛋白-2
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Key words
isoflurane /
dexmedetomidine /
hippocampus /
collapsin response mediator protein-2
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中图分类号：
R965
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参考文献
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基金
基金项目:国家青年自然科学基金资助项目(30700787/C03030301); 广东省自然科学基金面上项目(S2011010004558)
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