低分子肝素通过促进SUMO2/3蛋白的表达及核转移对脑缺血再灌注大鼠发挥脑保护作用

目的 探讨低分子肝素(low molecular weight heparins,LMWH)对大鼠脑缺血再灌注后NF-κB活化和表达的影响及其可能机制.方法 通过线栓法对大鼠建立大脑中动脉栓塞(middle cerebral artery occlusion,MCAO)模型,将总计60只大鼠分为3组:假手术组(Sham, n=20)、模型组(I/R, n=20)、低分子肝素干预组(LMWH, n=20),并用1.5 mg/kg的低分子肝素液进行干预.观察大鼠脑缺血再灌注后神经功能评分、梗死灶体积,并用Western blot、免疫组化、免疫荧光检测各组IL-1β、NF-κB P65及SUMO2/3蛋白表达的变化.结果 ①神经功能评分结果显示:MCAO后大鼠出现严重的神经功能障碍,经LMWH干预后能明显减轻MCAO导致的神经功能障碍(P<0.01);②大鼠在脑缺血再灌注后出现严重的脑梗死(P<0.01),而LMWH能明显减小梗死体积(P<0.01),尤其能够有效改善MCAO模型梗死敏感区纹状体的损伤程度(P<0.01);③Western blot 检测结果显示:再灌注24 h后I/R组大鼠脑组织中IL-1β、NF-κB P65蛋白表达均高于Sham组(P<0.01),而经LMWH干预后的大鼠IL-1β、NF-κB P65蛋白表达相比I/R组均下降(P<0.01);再灌注3 h后,与Sham组相比,I/R组大鼠脑组织中SUMO2/3蛋白表达增强(P<0.01),而LMWH干预后SUMO2/3蛋白表达进一步增强(P<0.01);④免疫组化和荧光检测结果显示:再灌注3 h后,与Sham组相比,I/R组大鼠脑组织中蛋白SUMO2/3水平增加,出现核内移现象(P<0.01),并且主要发生在神经元细胞内,而LMWH干预后发生SUMO2/3核内移现象的神经元细胞进一步增多(P<0.01).结论 LMWH在大鼠脑缺血再灌注损伤模型中能够促进SUMO2/3表达及核内移,这可能对抑制NF-κB活化减少炎症因子生成产生影响.

Low molecular heparins exert brain protective effect in MCAO rats by promoting nuclear transfer and SUMO2/3 expression

Objective To determine the effect of low molecular weight heparins (LMWH) on NF-κB activation and expression after cerebral ischemia-reperfusion injury (CIRI) in rats and investigate the possible mechanism.Methods Middle cerebral artery occlusion (MCAO) model was established by Longa's method in rats.Then the rats were assigned into 3 groups, that is, sham operation group (Sham, n=20), ischemia reperfusion group (I/R, n=20), LMWH intervention group (LMWH, 1.5 mg/kg, intravenous injection, 5 min prior to reperfusion, n=20).After neurological function was evaluated, the rats were sacrificed in 3 and 24 h after reperfusion, and the brain tissues were harvested for infarct volume.Western blotting, immunohistochemical (IHC) and immunofluorescence (IF) assay were used to detect the expression levels of IL-1β, NF-κB P65 and small ubiquitin-related modifier 2/3 (SUMO2/3).Results ①Neurological function evaluation showed that MCAO resulted in severe neurological dysfunction in the rats, but LMWH intervention obviously attenuated the injury (P<0.01).②Cerebral I/R injury induced severe cerebral infarction (P<0.01), but, LMWH alleviated the symptoms (P<0.01), and the effects were more significant in the striatum region that was sensitive to infarction (P<0.01).③Western blot results showed that the protein levels of IL-1β and NF-κB P65 were significantly higher in I/R group than sham operation group in 24 h after reperfusion (P<0.01), while LMWH intervention obviously attenuated the levels.In 3 h after reperfusion, SUMO2/3 protein level was higher in the I/R group than the sham operation group (P<0.01).But interestingly, LMWH intervention increased the expression level significantly (P<0.01).④IHC and IF results showed that after 3 hours' reperfusion, SUMO2/3 protein level was higher in the I/R group than the sham operation group, and nuclear transfer phenomenon appeared, mainly occurred in the neurons (P<0.01).After LMWH intervention, there were significantly more neurons with SUMO2/3 protein nuclear transfer (P<0.01).Conclusion LMWH can promote the SUMO2/3 protein level and increase nuclear transfer in rats after CIRI, and then possibly influence the NF-κB activation and reduce the production of inflammatory factors.