高迁移率族蛋白1拮抗剂BoxA对血管性痴呆大鼠急性期海马区域神经炎症的抑制作用

Anti-neuroinflammatory Role of HMGB1 Inhibitor BoxA in the Hippocampal Area of a Rat Model of Vascular Dementia in the Acute Phase

目的:探究高迁移率族蛋白1(HMGB1)拮抗剂BoxA脑室立体定向注射对双侧颈总动脉闭塞(2VO)后大鼠海马区域神经炎症(血脑屏障通透性、小胶质细胞的激活以及炎症因子水平等)的抑制作用。方法:60只雄性Sprague-Dawley(SD)大鼠分为假手术组(即Sham组)、PBS组以及BoxA组(各组n=20),分别在行2VO(n=40,正常手术)或假手术(n=20,仅暴露不结扎)后,立即用BoxA(10μg在10uL PBS溶液中,n=20)或者等体积PBS(n=20)立体定向注射到左侧侧脑室中。造模3 d后,用HMGB1和NeuN免疫荧光双染,western blot观察HMGB1出核现象,伊文思蓝(EB)染色和脑水含量测量评价血脑屏障(BBB)通透性,Iba1免疫荧光染色观察小胶质细胞活化,定量逆转录聚合酶链反应(RT-PCR)检测炎症细胞因子(白介素1β(Interleukin-1β, IL-1β)和白介素6(In-terleukin-6, IL-6)和肿瘤坏死因子α(Tumor necrosis factor-α, TNF-α))的基因表达水平。结果:相比Sham组,PBS组海马CA1亚区HMGB1阳性细胞核(%)以及HMGB1&NeuN阳性细胞/HMGB1阳性细胞(%)显著下降(P<0.01),而BoxA组以上改变较PBS组部分减少(P<0.05)。PBS组EB渗漏、脑含水量均较Sham组显著增加(P<0.001),而BoxA组以上指标均较PBS组明显减轻(P<0.05)。PBS组Iba1阳性细胞相比Sham组明显增多(P<0.01),且炎症因子(IL-1β、IL-6和TNF-α)表达显著增高(P<0.05);而相比PBS组,BoxA组Iba1阳性细胞表达减少((P<0.05),且炎症因子(IL-1β、IL-6 and TNF-α表达明显减少(P<0.05)。结论:HMGB1抑制剂BoxA立体定向注射能够有效缓解急性期血管性痴呆大鼠海马区域的神经炎症反应。

Objective: To investigate the anti-neuroinflammatory role of stereotactic injection with high mobility group box-1(HMGB1) inhibitor BoxA in the hippocampal area of rats after bilateral common carotid artery occlusion(2VO). Methods: 60 male Sprague-Dawley(SD) rats were divided into the sham group, PBS group and BoxA group(n=20 in each group). 2VO(n=40) or sham operation(n=20, only separation) were performed and BoxA(10 ug in 10 uL PBS) or equal amount of PBS was intraventricularly(to the left one) administered immediately after 2VO surgery. Three days later. HMGB1&NeuN immunostaining and western blot were used to observe the HMGB1 translocation, Evans blue(EB) dying and brain water content were used to measure the blood-brain barrier(BBB) permeability, Iba1 immunostaining was used to observe microglial activation and reverse transcription-polymerase chain reaction(RT-PCR)was used to assess the levels of pro-inflammatory cytokines(Interleukin-1β(IL-1β), Interleukin-6(IL-6) and Tumor necrosis factor-α(TNF-α).Results: Compared with the sham group, there was a significant decrease in HMGB1 positive nucleus(%) and HMGB1&NeuN positive cells/HMGB1 positive cells(%) in the hippocampal CA1 subarea in PBS group(P<0.01). However, compared with the PBS group, the above changes were partially reduced in the BoxA group(P<0.05). In addition, compared with the sham group, PBS group showed significantly increased EB leakage(P<0.001) and increased brain water content(P<0.01). Compared with the PBS group, these changes in the BoxA group were partially adjusted again(P<0.05). Finally, compared with the sham group, the number of iba1-positive cells in the PBS group increased(P<0.01) and the expression of pro-inflammatory factors(IL-1β, IL-6 and TNF-α) increased(P<0.05). Compared with the PBS group, the expression of Iba1 positive cells in BoxA group was decreased(P<0.05), and the expression of proinflammatory factors(IL-1β, IL-6 and TNF-α) were all obviously decreased(P<0.05). Conclusions: BoxA suppresses hippocampal neuroinflammatory responses in the rats with 2VO in the acute phase.