GSK-3βsiRNA对脑出血大鼠血肿周围组织神经元自噬的影响

Effects of the GSK-3β siRNA on neuronal autophagy in brain tissue surrounding hematoma following intracerebral hemorrhage

目的:探讨沉默糖原合成酶激酶-3β(GSK-3β)基因对实验性脑出血(ICH)大鼠脑损伤的神经保护作用。方法:将40只雄性SD大鼠随机分为4组:假手术组(sham)、脑出血组(ICH)、脑出血联合GSK-3β-siRNA处理组(GSK-3β-siRNA)、脑出血联合GSK-3β-siRNA阴性序列处理组(consi GSK-3β)。利用自体动脉血右侧基底节区立体定向注射的方法制备大鼠ICH模型,造模前利用侧脑室注射法给予GSK-3β-siRNA及consi GSK-3β预处理,模型制备成功后72 h采用前肢协调实验检测行为学改变;干湿重法检测大鼠脑组织含水量;real time RT-PCR检测血肿周围脑组织GSK-3βmRNA的表达,免疫组织化学染色观察自噬标志蛋白LC-3的表达;Western Blot检测脑出血大鼠血肿周围组织中GSK-3β、p-GSK-3β(tyr216)、Beclin-1及p62蛋白表达量。结果:与sham组相比,ICH组大鼠行为学评分显著升高(P<0.05),脑组织含水量增加(P<0.05),GSK-3β-siRNA组大鼠行为学评分及脑组织含水量与ICH组和consi GSK-3β组相比明显降低(P<0.05)。与sham组相比,ICH组大鼠血肿周围脑组织中GSK-3β、p-GSK-3β(tyr216)、LC-3及Beclin-1表达显著升高(P<0.05),p62蛋白表达下调(P<0.05);与ICH组和consi GSK-3β组比,GSK-3β-siRNA组大鼠GSK-3β、p-GSK-3β(tyr216)、LC-3及Beclin-1表达进一步增加,p62的表达进一步下调(P<0.05)。结论:siRNA敲低GSK-3β基因的表达对脑出血大鼠诱导的脑损伤具有一定的神经保护作用,其机制可能与增强自噬激活相关。

Objective:To investigate the neuroprotective effects of glycogen synthase kinase-3 beta(GSK-3β)silencing after intracerebral hemorrhage(ICH)in rats.Methods:Forty male SD rats were randomly divided into sham group,ICH group,GSK-3β-siRNA group,and consi GSK-3βgroup.First,we have established a rat model of ICH by the injection of autogeneous blood into the right basal ganglia.Rats were treated with GSK-3β-siRNA and consi GSK-3βvia intracerebroventricular injection before ICH induction,respectively.Forelimb use asymmetry test was used to assess behavioral defect in rats at 72 h post-ICH.The wet and dry weight method was measured to evaluate the brain water content.The transfection efficiency of GSK-3βsiRNA was detected using real time RT-PCR and Western Blot analysis.The location and expression of LC-3 protein was observed by immunohistochemical staining.The protein expression of GSK-3β,p-GSK-3β(tyr216),Beclin-1 and,p62 were determined by Western Blot analysis.Results:The behavioral scores and brain water content in the ICH group is higher than that of rats in the sham group(P<0.05).Compared with the ICH group and the consi GSK-3βgroup,behavioral scores and brain water content at 72 h post-ICH were significantly reduced in the GSK-3β-siRNA group(P<0.05).Compared with the sham group,the protein expression of GSK-3β,p-GSK-3β(tyr216),LC-3 and Beclin-1 were dramatically up-regulated in the region around the hemorrhage(P<0.05),whereas the level of p62 protein was markedly down-regualted in the ICH group(P<0.05).However,compared with the ICH group and the consi GSK-3βgroup,treatment with GSK-3βsiRNA could remarkably reverse ICH-induced these effects as above(P<0.05).Conclusion:Silencing of GSK-3βby siRNA could provide neuroprotective effects against behavior deficits and brain edema following ICH in rats through enhancing ICH-induced neural autophagy.