RNA干扰免疫蛋白酶体对大鼠脑缺血再灌注神经炎症反应的影响

Effects of RNA interference targeting immunoproteasome on the neuroinflammatory reaction after cerebral ischemia / reperfusion in a rat model

目的观察RNA干扰免疫蛋白酶体亚基低分子重量蛋白2(LMP2)对大鼠脑缺血再灌注后神经炎症反应的影响。方法线栓法制作SD大鼠大脑中动脉阻塞再灌注(MCAO)模型,脑缺血1 h再灌注72 h。SD大鼠按照体重随机分为假手术组、实验组和对照组,每组10只。实验组和对照组分别在MCAO术前1 h,立体定位脑内注射慢病毒载体LMP2-shRNA及阴性对照shRNA液体。用Western blot法分析LMP2、LMP7、磷酸化核因子-κB p65(NF-κB p65)蛋白表达,用ELISA法测定脑组织白介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)的浓度。结果局灶性脑缺血再灌注后,梗死灶周边的皮层和纹状体区LMP2、NF-κB、IL-1β和TNF-α蛋白表达上调,注射慢病毒LMP2-shRNA载体可显著下调这些蛋白产生,减少脑梗死体积,且未见明显的药物不良反应。结论RNA干扰LMP2表达,可显著下调脑缺血后神经炎症反应,发挥神经保护作用。

Objective To investigate the effects of RNA interference targeting immunoproteasome subunit low molecular weight protein 2（ LMP2） on the neuroinflammatory reaction after cerebral ischemia /reperfusion in a rat model. Methods The middle cerebral artery occlusion（ MCAO） was induced using the intraluminal suture occlusion technique in SD rats. Reperfusion of cerebral blood flow was allowed by gently removing the monofilament after 1 hour ischemia,followed by 72 h reperfusion. Rats were randomly assigned into 3 groups： sham- operated group,experimental group and control group（ n = 10）. The preparations of lentivirus vector targeting LMP2 short hairpin RNA（ shRNA） and carrying scrambled shRNA were infused stereotactically into the ipsilateral hemispheric region 1 h before MCAO in the experimental group and control group,respectively. The expressions of LMP2,phospho- nuclear factor- κB p65（ NF- κB P- p65） protein and levels of interleukin- 1β（ IL- 1β） and tumor necrosis factor- α（ TNF- α） protein in the brain were detected by Western blot and enzyme- linked immunosorbent assay（ ELISA）,respectively. Results The expression of LMP2,NF- κB,IL- 1β and TNF- α increased significantly higher in the cortex and striatum surrounding the infarct core than that in the sham- operation group（ P〈0. 001）. Pretreated with LMP2- shRNA 1 h before MCAOsignificantly decreased the levels of LMP2,NF- κB,IL- 1β and TNF- α protein,more importantly. The shRNA-mediated inhibition of LMP2 efficiently reduced infarction volume and without significant adverse drug reaction.Conclusion The shRNA- mediated inhibition of LMP2 significantly reduces neuroinflammatory reaction and displays neuroprotective effect.