羟基积雪草苷对创伤性脑损伤后海马区NR2B表达与认知功能的影响

Effect of madecassoside on hippocampal NR2B expression and cognitive function in rat traumatic brain injury model

目的探讨创伤性脑损伤(TBI)后羟基积雪草苷(MC)对海马区N-甲基-D-天冬氨酸受体亚单位NR2B(NR2B)表达的调节作用,及其对认知功能的影响。方法由西安交通大学医学部实验动物中心提供健康雄性SD大鼠116只,按随机数字表法均分为对照组(Con组)、脑创伤组(TBI组)、脑创伤+积雪草苷治疗组(TBI+MC组)、对照+积雪草苷治疗组(Con+MC组),每组29只。伤后12 h、1 d、3 d、7 d、14 d和21 d,Western blotting检测各组海马区NR2B蛋白表达水平。伤后21 d,免疫组织化学染色观察各组海马区NR2B阳性细胞数量。伤后21~25 d,Morris水迷宫实验检测各组大鼠逃避潜伏期、平台象限游泳时间与穿越平台次数,评估认知功能。正态分布的计量资料采用均数±标准差(Mean±SD)表示,组间比较采用单因素方差和LSD-t分析。结果TBI组海马区NR2B蛋白表达量伤后12 h、1 d和3 d分别为3.31±0.28、2.17±0.31和1.96±0.31,三组间差异具有统计学意义(P<0.05),并且与Con组相比均显著增加(P<0.05);伤后7 d表达量为0.93±0.13,与Con组相比差异无统计学意义(P>0.05);伤后14 d和21 d表达量分别为0.45±0.04和0.21±0.04,两组间差异具有统计学意义(P<0.05),并且与Con组相比均显著降低(P<0.05)。TBI组NR2B免疫组化染色阳性细胞数量(33.26±9.71)个,Con组为(86.62±17.05)个,两组相比差异具有统计学意义(P<0.05)。TBI组大鼠水迷宫逃避潜伏期与Con组相比显著增加,TBI组大鼠穿越平台次数和平台象限游泳时间与Con组相比显著减少,两组差异具有统计学意义(P<0.05)。TBI+MC组海马区NR2B蛋白表达量在伤后12 h、1 d、3 d分别为2.37±0.34、1.38±0.22和1.14±0.16,三组间差异具有统计学意义(P<0.05),并且与TBI组相比均显著减少(P<0.05);伤后7 d表达量为0.97±0.06,与TBI组相比差异无统计学意义(P>0.05);伤后14 d和21 d表达量分别为0.86±0.08和0.52±0.06,两组间差异具有统计学意义(P<0.05),并且与TBI组相比均显著增加(P<0.05)。TBI+MC组海马区NR2B免疫组化染色阳性细胞数量(69.08±12.24)个,TBI组为(33.26±9.71)个,两组相比差异具有统计学意义(P<0.05)。TBI+MC组大鼠水迷宫潜伏期与TBI组相比显著缩短,TBI+MC组大鼠穿越平台次数和平台象限游泳时间与TBI组相比显著增加,两组间差异具有统计学意义(P<0.05)。结论MC对TBI后海马区NR2B表达的具有时程调节作用,即TBI早期MC可抑制海马区NR2B表达异常上调,TBI晚期MC可阻止NR2B表达过度下降,对改善脑创伤后认知功能障碍具有重要作用。

Objective To investigate the Effect of madecassoside(MC)on hippocampal NR2B expression and cognitive function following traumatic brain injury(TBI).Methods One hundred and sixteen SD rats were provided by the experimental animal center of Medical College in Xi′an Jiao Tong University.Rats were randomly divided into four groups(n=29)as following:Control group(Con group),TBI group,MC treated TBI rats group(TBI+MC group)and MC treated control rats group(Con+MC group).NR2B protein levels in hippocampus were detected by Western-blot at 12 h,1 d,3 d,7 d,14 d and 21 d post trauma.Hippocampal NR2B positive cells were studied by immunohistochemical(IHC)staining at 21 d post trauma.Cognitive functions of rats were evaluated by Morris water maze(MWM)test from 21 d to 25 d post trauma.Results Expression of hippocampal NR2B in TBI group at 12 h,1 d and 3 d post injury were 3.31±0.28,2.17±0.31 and 1.96±0.31,which presented statistical difference among the three time-points(P<0.05)and were significantly increased compared to Con group(P<0.05).However,there was no difference of NR2B level between TBI group(0.93±0.13)and Con group(P>0.05)at 7 d post injury.In addition,NR2B expression in TBI group at 14 d and 21 d post injury were 0.45±0.04 and 0.21±0.04,which were much lower than that in Con group.IHC staining revealed that the number of hippocampal NR2B positive cells in TBI group(33.26±9.71)were lesser than that of Con group(86.62±17.05).Increased Escape latency,decreased platform crossing times and target quadrant duration were found in TBI group compared with Con group(P<0.05).Hippocampal NR2B expression in TBI+MC group at 12 h,1 d and 3 d post injury were 2.37±0.34,1.38±0.22 and 1.14±0.16.Difference among the three time-points exhibited statistical significance(P<0.05).In addition,they were much lower than that in TBI group at the same time-points(P<0.05),but no difference was found between TBI+MC group(0.97±0.06)and TBI group at 7 d post injury(P>0.05).Moreover,NR2B expression in TBI+MC group at 14 d and 21 d post injury were 0.86±0.08 and 0.52±0.06,which were much higher than that in TBI group(P<0.05).IHC staining showed the number of hippocampal NR2B positive cells in TBI+MC group(69.08±12.24)were much more than that of TBI group(33.26±9.71).A decrease of escape latency,an increase of platform crossing times and target quadrant duration were found in TBI+MC group compared to TBI group(P<0.05).Conclusion MC treatment could inhibit the up-regulation of NR2B in hippocampus at early period of TBI and prevent the down-regulation of NR2B at advanced stage of TBI,which led to a remarkable improvement for the cognitive dysfunction caused by TBI.