大鼠弥漫性轴索损伤后高迁移率族蛋白1的动态表达及其对神经细胞凋亡的影响

摘要

Objective To study the dynamic expression and distribution of high mobility group box 1 (HMGB-1)in diffuse axonal injury (DAI)in rats and to clarify its involvement in the inflammatory reaction after DAI in rats,in order to provide new targets for the clinical treatment of DAI.Methods A DAI model was established using a coronal rotation device and evaluated by HE,Glees-Marsland silver staining,and Mallory phosphotungstic acid hematoxylin staining.Immunohistochemistry,Western blot and RT-PCR were used to detect the expression and distribution of HMGB-1 in the cortex of DAI rats at 6 h,1 d,3 d and 7 d.And TUNEL was used to examine the apoptosis of neurons in DAI rats.Results Immunohistochemical results showed that at 6 h and 1 d after DAI,the number of HMGB-1-positive cells decreased,but at 3 and 7 d it began to increase.Western blot also showed that during the early stage after DAI (6 h and 1 d),the level of HMGB-1 protein in the cortex was significantly lower than that in the control group,but at the late stage (3 and 7 d)after DAI it significantly increased compared with that in the control group until 7 d.RT-PCR showed that at 6 h after DAI there was no significant increase in the level of HMGB-1mRNA,but at 1 d there was a slight increase compared with the control group;at 3 and 7 d,it showed an obvious significance.TUNEL staining indicated that the significant neuronal apoptosis appeared as early as 6 h after DAI,and reached the peak at 3 d;it started to decrease at 7 d but still remained at a relatively high level.Conclusion The dynamic expression and distribution of HMGB-1 showed significant changes with the time course after DAI in rats.They decreased at the early stage but increased at the late stage.At the early stage, HMGB-1 is mainly passively released by the necrotic neurons,and at the late stage it may be actively secreted by the active inflammatory cells.HMGB-1 may mediate the post-DAI neural cell apoptosis by inducing the inflammatory reaction.%目的：研究高迁移率族蛋白1(HMGB-1)在弥漫性轴索损伤(DAI)后脑组织内的动态表达及其对神经细胞凋亡的影响,探讨其参与 DAI 后脑组织炎症反应的机制。方法采用大鼠头颅瞬间旋转装置制备大鼠 DAI 模型,通过组织病理学方法评价 DAI 模型的稳定性,采用免疫组化、Western blot、RT-PCR 等方法分别于造模后6 h,1、3、7 d 为时间点,测定 DAI 后 HMGB-1的表达及分布变化,并采用 TUNEL 试剂盒进一步观察 DAI 后神经元的凋亡。结果免疫组化结果显示,在 DAI 后6 h 及1 d,大鼠皮层脑组织中 HMGB-1阳性细胞数呈下降趋势,但是3 d 后开始明显增多；Western blot 结果证实,DAI 后早期(6 h、1 d),皮层脑组织中 HMGB-1蛋白水平较对照组显著降低,然而在 DAI后3 d 出现明显升高,一直持续至第7天；RT-PCR 显示,DAI 后早期(6 h)HMGB-1 mRNA 水平并无明显增加,DAI后1 d 仅有轻度增加,DAI 后3 d HMGB-1 mRNA 水平才开始明显升高。TUNEL 结果显示,在 DAI 后6 h 神经元凋亡已明显增加,并呈持续增加趋势,DAI 后3 d 达到高峰,一直持续至 DAI 后7 d 仍处于较高水平。结论大鼠 DAI后脑组织内 HMGB-1表达呈逐渐升高的动态变化,其脑组织总蛋白水平早期(6 h、1 d)降低,可能由坏死神经元释放及基因转录水平较低所致,而晚期(3、7 d)明显升高,可能是胶质细胞活化诱导基因表达升高所致,HMGB-1与神经元凋亡并无直接相关性,其可能通过诱导炎症反应间接参与 DAI 后的神经元凋亡。