脑室内注射胰岛素对大鼠全脑缺血后海马CA1区Bcl-2 mRNA变化及其蛋白表达的影响

The effect of intraventricular administration of insulin on expression of Bcl-2 mRNA and protein in rat hippocampal CA1 region following global brain ischemia

目的观察胰岛素对全脑缺血后海马CA1区Bcl-2 mRNA含量变化及其蛋白表达的影响,探讨胰岛素保护作用的机制。方法利用4-VO法制作大鼠全脑缺血模型。造成脑缺血15 min后行再灌注,治疗组于再灌注后即刻经脑室注入1 u胰岛素(10μL,1×105u/L),缺血组则经脑室注入10μL生理盐水。利用反转录PCR(RT-PCR)、免疫组化及Western-blot法分别于全脑缺血后1、3、5 d观察海马CA1区Bcl-2蛋白表达及其mRNA相对含量的变化。结果全脑缺血后1、3、5 d治疗组海马CA1区可见呈阳性表达的Bcl-2蛋白,而缺血组海马CA1区Bcl-2蛋白的表达则呈阴性;Western-blot分析,全脑缺血后1、35、d治疗组海马CA1区Bcl-2蛋白电泳条带密度分别为2 890.791±213.616,3 147.396±243.561和2 594.834±204.736,明显高于缺血组的743.376±84.123,804.637±64.547和637.867±54.394(P<0.01)。全脑缺血后1、35、d缺血组及胰岛素治疗组海马CA1区Bcl-2 mRNA相对含量则未见明显差异。结论全脑缺血后脑室内注入胰岛素可能通过促进海马CA1区神经元Bcl-2基因的翻译途径从而促进Bcl-2蛋白表达,这可能是其对全脑缺血后海马CA1区神经元产生中枢直接保护作用的主要机制之一。

Objective To investigate the mechanism of neuroprotective effect of insulin on hippocampal CA1 region, the relatively content of Bcl-2 mRNA and the expression of Bcl-2 protein in rat hippocampal CA1 region following global brain ischemia were observed. Methods Four-vessel occlusion was used to establish the rat global brain ischemic model. After 15 minutes brain ischemia, 1 unit insulin （10 μL, 1 ×10^5 u/L） was injected into the cerebral ventricular immediately post reperfusion in treated group, while, 10μL NaCl （90 g/L） solution was injected into the cerebral ventricular at the same time in ischemic group. The relatively content of Bcl-2 mRNA and the expression of Bcl-2 protein in hippocampal CA1 region had been detected by reverse transcription polymerase chain reaction （RT-PCR）, immunocytochemistry, and Western-blot, respectively. Results The expression of Bcl-2 protein was negative in ischemic group and was positive in treated group, in 1, 3, 5 day post brain ischemia. While, Western-blotting analysis showed that the density of electrophoretic strip of Bcl-2 protein was 2 890. 791±213. 616, 3 147. 396±243. 561 and 2 594. 834±204. 736 in treated group. It was much higher than that in ischemic group, which was 743. 376± 84. 123, 804. 637± 64. 547 and 637. 867±54. 394, respectively. But, the relatively content of Bcl-2 mRNA had no obvious difference between ischemic group and treated group. Conclusion Intraventricular administration of insulin can improve the expression of Bcl-2 protein by up- regulating the translation of the Bcl-2, and this may be the main point to achieve the neuroprotective effect on hippocamal CA1 region post global brain ischemia.