醒脑静治疗大鼠重型颅脑损伤的作用机制

doi:10.3877/cma.j.issn.1674-0785.2018.06.007

中华临床医师杂志(电子版) ›› 2018, Vol. 12 ›› Issue (06) : 347 -353. doi: 10.3877/cma.j.issn.1674-0785.2018.06.007

所属专题：文献；

基础研究

醒脑静治疗大鼠重型颅脑损伤的作用机制

魏民¹, 孟浩², 董伦¹, 王杏东¹, 严正村¹, 张恒柱¹^,^†(

)

^1. 225001　扬州大学临床医学院神经外科
^2. 100094　北京市科学技术研究院

收稿日期:2018-03-03 出版日期:2018-03-15
通信作者: 张恒柱
基金资助:
江苏省中医药局科技项目(LZ13195); 扬州市社会发展新型技术规范化诊疗项目(YZ2015046); 苏北人民医院院级项目(zucms201712)

AQP4 expression, apoptosis and brain edema in severe traumatic brain injury treated by traditional Chinese medicine and by refreshing method

Min Wei¹, Hao Meng², Lun Dong¹, Xingdong Wang¹, Zhengcun Yan¹, Hengzhu Zhang¹^,^†()

^1. Department of Neurosurgery, Clinical Medical College, Yangzhou University, Yangzhou 225001, China
^2. Beijing Computing Center, Beijing Academy of Science and Technology, Beijing 100094, China

Received:2018-03-03 Published:2018-03-15
Corresponding author: Hengzhu Zhang
About author:
Corresponding author: Zhang Hengzhu, Email: zhanghengzhu@sina.com

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引用本文：

魏民, 孟浩, 董伦, 王杏东, 严正村, 张恒柱. 醒脑静治疗大鼠重型颅脑损伤的作用机制[J]. 中华临床医师杂志(电子版), 2018, 12(06): 347-353.

Min Wei, Hao Meng, Lun Dong, Xingdong Wang, Zhengcun Yan, Hengzhu Zhang. AQP4 expression, apoptosis and brain edema in severe traumatic brain injury treated by traditional Chinese medicine and by refreshing method[J]. Chinese Journal of Clinicians(Electronic Edition), 2018, 12(06): 347-353.

目的

观察醒脑法与传统活血法对重型颅脑损伤大鼠脑组织水肿、凋亡及水通道蛋白4（AQP4）表达的影响，探讨其治疗颅脑损伤的疗效差异及作用机制。

方法

SD大鼠72只，随机分为假手术组（12只），模型组（20只），醒脑静治疗组（20只），传统活血治疗组（20只）。采用改良后Feeney方法建立大鼠重型颅脑损伤模型。分别在24 h，48 h，72 h，7 d 4个时间点，假手术组取3只、余各组取5只测定脑组织含水量、HE染色观察脑组织变化情况，免疫组化方法检测受损区域脑组织AQP4的表达并采用TUNEL法检测凋亡细胞，通过RT-PCR验证治疗后AQP4表达水平。

结果

模型组各时间点脑组织含水量、损伤灶周围细胞凋亡数及AQP4的表达均高于假手术组，差异具有统计学意义（P<0.05）。醒脑静组及传统活血组各时间点脑组织含水量、AQP4表达水平、细胞凋亡水平均比模型组降低，差异具有统计学意义（P<0.05），RT-PCR支持AQP4变化结果。

结论

醒脑静可改善重型颅脑损伤后引起的脑水肿及细胞凋亡，效果优于传统活血疗法。其作用机制可能与抑制AQP4在损伤脑组织中的表达、减轻脑细胞损害有关。

关键词: 重型颅脑损伤, 水通道蛋白4, 醒脑, 水肿

Objective

To investigate the difference in AQP4 expression, apoptosis and brain edema between severe traumatic brain injury treated by refreshing method and by blood circulation activating method.

Methods

Adult Sprague-Dawley rats were randomly divided into a control group (12 rats), a model group (20 rats), a refreshing method group (20 rats), and a blood circulation activating method (20 rats). Severe traumatic brain injury was induced in rats by the improved Feeney′s methods. Brain samples were collected at 24 h, 48 h, 72 h and 7 d for measurement of water content. HE staining was performed to observe the changes in brain tissue. TUNEL method was used to detect apoptotic cells, and immunohistochemical method was used to detect the expression of AQP4 after injury. Besides, RT-PCR was utilized to validate the mRNA expression of AQP4 in different groups.

Results

The water content of brain tissue, the number of apoptotic cells around the focal brain injury and the expression of AQP4 at different times in the model group were significantly higher than those in the control group (P<0.05), while these indexes in the two treatment groups were significantly lower than those in the model group (P<0.05), with the refreshing method being superior to the blood circulation activating method. RT-PCR results suggested that AQP4 mRNA expression was similar to its protein expression pattern.

Conclusion

The changes in edema, apoptotic cells and expression of AQP4 after treatment for severe traumatic brain injury suggested that the refreshing method could improve the outome of severe traumatic brain injury, and it is better than the blood circulation activating method.

Key words: Severe traumatic brain injury, Aquaporins 4, Edema, Refreshing Method

表1 不同时间点大鼠脑组织含水量（%）

组别	鼠数	伤后24 h	伤后48 h	伤后72 h	伤后7 d
假手术组	12	71.51±2.88	71.53±2.97	71.63±2.76	71.57±2.86
模型组	20	80.02±0.54^b	83.42±0.48^b	83.50±0.56^b	80.50±1.49^b
传统活血组	20	79.40±0.72	80.81±0.80^a	80.71±0.81^a	77.97±0.81^a
醒脑静治疗组	20	77.85±0.79^a	80.68±0.90^a	77.42±0.80^ac	75.47±1.44^ac

表1 不同时间点大鼠脑组织含水量（%）

组别	鼠数	伤后24 h	伤后48 h	伤后72 h	伤后7 d
假手术组	12	71.51±2.88	71.53±2.97	71.63±2.76	71.57±2.86
模型组	20	80.02±0.54^b	83.42±0.48^b	83.50±0.56^b	80.50±1.49^b
传统活血组	20	79.40±0.72	80.81±0.80^a	80.71±0.81^a	77.97±0.81^a
醒脑静治疗组	20	77.85±0.79^a	80.68±0.90^a	77.42±0.80^ac	75.47±1.44^ac

图1 脑组织AQP4蛋白表达的变化。A假手术组，可见AQP4蛋白在正常细胞水平上均有表达；B模型组，蛋白表达量在伤后48 h最高，7 d较前明显下降；C传统活血组，蛋白表达峰与醒脑静治疗组均于伤后48 h达到最高，7 d逐渐趋于较低水平；D醒脑静治疗组，7 d蛋白表达量逐渐趋于较低水平。

表2 不同时间点大鼠脑组织AQP4蛋白表达量

组别	鼠数	伤后24 h	伤后48 h	伤后72 h	伤后7 d
假手术组	12	0.43±0.01	0.46±0.01	0.43±0.01	0.44±0.01
模型组	20	0.73±0.01	1.43±0.04	1.21±0.12	0.98±0.08
传统活血组	20	0.75±0.08	1.23±0.10^a	1.05±0.08^a	0.93±0.10
醒脑静治疗组	20	0.54±0.05^ac	0.74±0.04^ac	0.72±0.03^ac	0.61±0.02^ac

表2 不同时间点大鼠脑组织AQP4蛋白表达量

组别	鼠数	伤后24 h	伤后48 h	伤后72 h	伤后7 d
假手术组	12	0.43±0.01	0.46±0.01	0.43±0.01	0.44±0.01
模型组	20	0.73±0.01	1.43±0.04	1.21±0.12	0.98±0.08
传统活血组	20	0.75±0.08	1.23±0.10^a	1.05±0.08^a	0.93±0.10
醒脑静治疗组	20	0.54±0.05^ac	0.74±0.04^ac	0.72±0.03^ac	0.61±0.02^ac

图2 不同组别的脑组织在转录组学水平验证AQP4 mRNA变化。图2a 造模24 h，醒脑静治疗组AQP4 mRNA与模型组表达量有差异性，^aP<0.05；图2b 造模48 h，传统活血组与醒脑静治疗组相对模型组均有差异性，^aP<0.05；图2c 造模72 h，模型组、传统活血组、醒脑静治疗组之间AQP4 mRNA相对表达量差异性明显，^aP<0.05；图2d 造模7 d，AQP4 mRNA相对表达量较前下降，各组差异性明显，^aP<0.05

表3 不同组大鼠神经细胞凋亡数

组别	鼠数	伤后24 h	伤后48 h	伤后72 h	伤后7 d
假手术组	12	4.80±3.93	3.60±2.69	4.20±2.51	3.20±2.78
模型组	20	176.24±10.16	281.80±10.89	224.48±16.56	217.56±15.66
传统活血组	20	155.28±8.53^a	228.44±17.07^a	216.32±12.64^a	191.60±8.81^a
醒脑静治疗组	20	143.52±10.16^ac	197.08±10.55^ac	182.08±7.94^ac	154.52±6.64^ac

表3 不同组大鼠神经细胞凋亡数

组别	鼠数	伤后24 h	伤后48 h	伤后72 h	伤后7 d
假手术组	12	4.80±3.93	3.60±2.69	4.20±2.51	3.20±2.78
模型组	20	176.24±10.16	281.80±10.89	224.48±16.56	217.56±15.66
传统活血组	20	155.28±8.53^a	228.44±17.07^a	216.32±12.64^a	191.60±8.81^a
醒脑静治疗组	20	143.52±10.16^ac	197.08±10.55^ac	182.08±7.94^ac	154.52±6.64^ac

图3 TUNEL法检测不同组大鼠神经细胞凋亡数。A为假手术组，可见凋亡细胞在正常组织上均有表达；B为模型组，凋亡在术后48 h最高，7 d较前明显下降；C为传统活血组，凋亡细胞数于术后48 h达到最高，7 d逐渐恢复正常；D为醒脑静治疗组，7 d凋亡细胞数与假手术组无明显差异

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