基于BDNF-Trk B信号通路探讨和血生络方对脑缺血大鼠认知功能的影响及机制研究

Investigated effect of Hexue Shengluo decoction on cognitive function in MCAO rats and mechanism based on BDNF-Trk B signaling pathway

目的:基于BDNF-Trk B信号通路探讨和血生络方对脑缺血大鼠认知功能的影响及其作用机制。方法:成年雄性SD大鼠随机分为假手术组、模型组、和血生络方组和和血生络方+抑制剂组,采用改良Zea-Longa大脑中动脉缺血造模方法建立脑缺血大鼠模型,和血生络方组与和血生络方+抑制剂组给予和血生络方浓缩液灌胃给药18.9 mg/(kg·d),持续14 d,假手术组和模型组给予等量0.9%氯化钠溶液灌胃。采用水迷宫试验评价大鼠认知功能。免疫荧光染色观察Brd U+/Nestin+细胞和Brd U+/DCX+在海马齿状回颗粒细胞下层的表达;RT-PCR法检测齿状回颗粒细胞下层中Nestin、DCX、BDNF及Trk B的基因表达;Western blot检测齿状回颗粒细胞下层中Nestin、DCX、BDNF及Trk B的蛋白表达。结果:水迷宫实验显示,与模型组和和血生络方+抑制剂组比较,和血生络方组大鼠的逃避潜伏期均显著缩短(P<0.05),穿越平台次数显著增加(P<0.05)。免疫荧光结果显示,在PMD3、PMD7和PMD14,与模型组、和血生络方+抑制剂组比较,和血生络方组显著增加了Brd U+/Nestin+细胞和Brd U+/DCX+细胞在齿状回颗粒细胞下层的表达(P<0.05)。RT-PCR结果表明,在PMD3、PMD7和PMD14,与模型组、和血生络方+抑制剂组相比,和血生络方组MCAO大鼠齿状回颗粒细胞下层Nestin、DCX、BDNF及Trk B的mRNA表达水平显著增高(P<0.05)。Western blot结果显示出与RT-PCR结果相似的趋势。结论:和血生络方能够改善脑缺血大鼠的学习、记忆和空间定位能力,其作用机制与通过上调BDNF-Trk B信号通路促进海马齿状回颗粒细胞下层神经再生有关。

Objective:To investigate the effect of Hexue Shengluo decoction on cognitive function of MCAO rats and the mechanism.Methods:Adult male SD rats were randomly divided into sham group,MCAO group,HX group and HX+k252a group.The middle cerebral artery occlusion method was used to induce rats cerebral ischemia model(except the sham group).Rats were given Hexue Shengluo decoction 18.9 mg/(kg•d)by intragastric administration in HX group and HX+k252a group for 14 days.Rats in Sham group and MCAO group were given equivalent normal saline.Water maze test was used to assess the cognitive function of MCAO rats.The expressions of Brd U+/Nestin+cells and Brd U+/DCX+cells in SGZ were shown by immunofluorescence staining;the gene expressions of Nestin,DCX,BDNF and Trk B in SGZ were analyzed by qRT-PCR;the protein expressions of Nestin,DCX,BDNF and Trk B in SGZ were analyzed by Western blot.Results:Compared with MCAO group and HX+k252a group,escape latency of MCAO rats in HX group were significantly shortened(P<0.05).The times of crossing the platform increased significantly(P<0.05).Results of immunofluorescence indicated that on PMD3,PMD7 and PMD14 HX group significantly stimulated the expression of Brd U+/Nestin+cells and Brd U+/DCX+cells in SGZ(P<0.05).On PMD3,PMD7 and PMD14,in HX group,the mRNA expression of Nestin,DCX,BDNF and Trk B in SGZ were significantly higher in comparison with MCAO group and HX+k252a group(P<0.05).The expression of protein indicated a similar trend with the mRNA expression.Conclusion:Hexue Shengluo decoction improved the learning,memory and spatial localization of MCAO rats.The mechanism related to upregulate expression of BDNF-Trk B signaling pathway and stimulated neurogenesis in SGZ.