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J Zhejiang Univ (Med Sci)  2019, Vol. 48 Issue (3): 303-309    DOI: 10.3785/j.issn.1008-9292.2019.06.11
    
Correlation of phosphorylated S6 protein expression in blood and brain tissue in mice and rats with kainic acid-induced seizure
FAN Miao1(),DONG Shuminin1,ZOU Xinyi1,ZHENG Boyuan1,HUANG Yurun1,WANG Jianda2,*(),ZENG Linghui1,*()
1. School of Medicine, Zhejiang University City College, Hangzhou 310015, China
2. Department of Paediatrics, the Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310009, China
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Abstract  

Objective: To determine the correlation of phosphorylated ribosomal S6 protein (P-S6) content in blood and brain tissue in mice and rats with seizure. Methods: Seizure models were induced by intraperitoric injection of kainic acid (KA) in C57BL/mice and SD rats. Flow cytometry was used to detect the content of P-S6 in blood; Western blot was used to detect the expression of P-S6 in brain tissues. The correlation between P-S6 expression in blood and in brain tissue was examine by Pearson analysis, and the correlation between P-S6 expression in blood and the severity of seizure was also observed. Results: Western blotting analysis showed that the expression of P-S6 was significantly increased in peripheral blood and brain tissue in mice 1 h after KA-induced seizure, and the expression levels increased to (1.49±0.45) times (P < 0.05) and (2.55±0.66) times (P < 0.01) of the control group, respectively. Flow cytometry showed that the positive percentage and average fluorescence intensity of P-S6 in the blood of mice increased significantly 1 h after KA-induced seizures (P < 0.01), which was consistent with the expression of P-S6 in brain tissue (r=0.8474, P < 0.01). Flow cytometry showed that the average fluorescence intensity of P-S6 in blood increased from 14.89±9.75 to 52.35±21.72 (P < 0.01) in rats with seizure, which was consistent with the change of P-S6 in brain tissue (r=0.9385, P < 0.01). Rats with higher levels of seizure were of higher levels of P-S6 in peripheral blood. Conclusion: Consistent correlation of P-S6 expression is demonstrated in peripheral blood and in brain tissue after KA-induced seizure, suggesting that the expression of P-S6 in blood can accurately reflect the changes of mTOR signaling pathway in brain tissue.



Key wordsEpilepsy/pathology      Epilepsy/blood      Brain/anatomy &      histology      Protein kinases/physiology      Phosphoprotein phosphatases/metabolism      Flow cytometry      Disease models, animal     
Received: 30 January 2019      Published: 04 September 2019
CLC:  R421.1  
Corresponding Authors: WANG Jianda,ZENG Linghui     E-mail: 15958027903@163.com;2515165@zju.edu.cn;zenglh@zucc.edu.cn
Cite this article:

FAN Miao, DONG Shuminin, ZOU Xinyi, ZHENG Boyuan, HUANG Yurun, WANG Jianda, ZENG Linghui. Correlation of phosphorylated S6 protein expression in blood and brain tissue in mice and rats with kainic acid-induced seizure. J Zhejiang Univ (Med Sci), 2019, 48(3): 303-309.

URL:

http://www.zjujournals.com/med/10.3785/j.issn.1008-9292.2019.06.11     OR     http://www.zjujournals.com/med/Y2019/V48/I3/303


癫痫鼠外周血磷酸化S6蛋白检测及意义

目的: 明确癫痫发作后外周血与脑组织中核糖体磷酸化S6蛋白(P-S6)含量变化的相关性。方法: 取出生后5~6周龄的C57BL/6小鼠30只和SD大鼠22只,采用红藻氨酸腹腔注射诱导癫痫发作。建立P-S6蛋白的流式细胞术检测方法用以检测小鼠及大鼠外周血中mTOR信号通路下游P-S6蛋白的变化,同时采用蛋白质印迹法检测其脑组织中P-S6的变化。采用Pearson相关性分析法分析脑组织与外周血中P-S6蛋白表达的相关性,并对大鼠外周血P-S6蛋白的表达与癫痫发作等级进行相关性分析。结果: 癫痫小鼠外周血和脑组织中P-S6的含量明显增高,其表达量分别升高至对照组的(1.49±0.45)倍(P < 0.05)和(2.55±0.66)倍(P < 0.01);外周血中P-S6的阳性表达率和平均荧光强度均明显升高(均P < 0.01),与脑组织中P-S6蛋白表达具有一致性(r=0.8474,P < 0.01)。大鼠自身致痫前后外周血中P-S6含量明显增加,由14.89±9.75增加至52.35±21.72(P < 0.01),与大鼠脑组织P-S6蛋白表达变化一致(r=0.9385,P < 0.01),且外周血P-S6含量的变化与癫痫发作等级呈正相关。结论: 癫痫鼠外周血mTOR信号通路的变化与脑组织中的变化具有良好的相关性,提示通过检测外周血P-S6的表达水平可准确反映脑组织中mTOR信号通路的变化。


关键词: 癫痫/病理学,  癫痫/血液,  脑/解剖学和组织学,  蛋白激酶类/生理学,  磷蛋白磷酸酶类/代谢,  流式细胞术,  疾病模型, 动物 
Fig 1 Expression of phosphorylated S6 protein in peripheral blood and brain tissue in mice with kainic acid-induced seizure (western blot, n=6)
Fig 2 Expression of phosphorylated S6 protein in peripheral blood of mice with kainic acid-induced seizure(flow cytometry, n=12)
Fig 3 Correlation between the expression of phosphorylated S6 protein in brain tissue and in peripheral blood of mice with kainic acid-induced seizure
Fig 4 Expression of phosphorylated S6 protein in brain tissue and peripheral blood in rats with kainic acid-induced seizure(n=10)
Fig 5 Correlation between the expression of phosphorylated S6 protein in brain tissue and in peripheral blood of rats with kainic acid-induced seizure
Fig 6 Relationship between expression of phosphorylated S6 protein in peripheral blood and seizure severity in rats(n=16)
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