5-羟色胺受体6在苔藓纤维发芽中的作用：在毛果芸香碱诱导的慢性癫痫大鼠中激活Fyn和磷酸化细胞外信号调节激酶1/2
The Role of 5-HTR6 in Mossy Fiber Sprouting: Activating Fyn and p-ERK1/2 in Pilocarpine-Induced Chronic Epileptic Rats.
作者信息
Lin Wanhui, Huang Wenli, Chen Shenggen, Lin Mingxing, Huang Qingyu, Huang Huapin
机构信息
Department of Neurology, Fujian Medical University Union Hospital, Fuzhou, China.
Department of Geriatrics, Fujian Medical University Union Hospital, Fuzhou, China.
出版信息
Cell Physiol Biochem. 2017;42(1):231-241. doi: 10.1159/000477322. Epub 2017 May 25.
OBJECTIVE
Our primary objective is to verify whether 5-HTR6 is involved in the development of mossy fiber sprouting (MFS), and to determine how the progression of MFS is affected by 5-HTR6.
METHODS
A total of 90 male adult Sprague-Dawley rats were allocated into either the control group (n=36) or the epileptic group (n=54). Status epilepticus (SE) of rats was induced by the intraperitoneal (i.p.) injection of LiCl-pilocarpine. We conducted our experiments in two stages. The first stage involves equally dividing 36 epileptic rats into three groups with treatments of none, 5-HTR6 antagonist SB-27104 (SB) and vehicle DMSO. Then behavior and electroencephalogram (EEG) of rats were monitored by video-EEG. The second stage involves dividing 126 epileptic rats into seven groups with treatments of none, 10% DMSO, SB (100 µg/kg), Fyn antagonist PP2 (50 µg/kg), p-ERK1/2 antagonist PD-98059 (30 µg/kg), SB (100 µg/ kg) + PP2 (50 µg/kg); SB (100 µg/kg) + PD-98059 (30 µg/kg). We also treated 18 rats in the control group of the first stage with 100 µg/kg 5-HTR6 agonist WAY-181187 (WAY). MFS of rats was detected through the approach of Timm's staining. Finally, expressions of 5-HTR6, Fyn, p-ERK1/2 and GAP-3 were qualified and semi-quantified via western blotting or RT-PCR.
RESULTS
Induction of SE could stimulate formation of MFS and increased GAP-43 expressions. Expressions of 5-HTR6, Fyn and p-ERK1/2 were also up-regulated with increasing time after establishment of SE models. The development of MFS was remarkably inhibited by SB, PP2 and PD. Compared to the single antagonist, such an inhibitory effect was enhanced by SB+PD or SB+PP. Moreover, treatment of healthy rats with WAY would contribute to up-regulated Fyn and p-ERK1/2 expressions, as well as development of MFS (P < 0.05). Suppression of Fyn triggered a down-regulating trend of p-ERK1/2 (P < 0.05), however, suppressed p-ERK1/2 did not have such a significant effect on Fyn expression.
CONCLUSION
HTR6 may affect the progression of MFS by activating both p-ERK1/2 and Fyn, which further modulate the expression of GAP-43.
目的
我们的主要目的是验证5-羟色胺受体6（5-HTR6）是否参与苔藓纤维发芽（MFS）的发展，并确定5-HTR6如何影响MFS的进展。
方法
将90只成年雄性Sprague-Dawley大鼠分为对照组（n = 36）或癫痫组（n = 54）。通过腹腔注射氯化锂-匹罗卡品诱导大鼠癫痫持续状态（SE）。我们分两个阶段进行实验。第一阶段将36只癫痫大鼠平均分为三组，分别给予无处理、5-HTR6拮抗剂SB-27104（SB）和溶剂二甲基亚砜（DMSO）。然后通过视频脑电图监测大鼠的行为和脑电图（EEG）。第二阶段将126只癫痫大鼠分为七组，分别给予无处理、10% DMSO、SB（100μg/kg）、Fyn拮抗剂PP2（50μg/kg）、p-细胞外信号调节激酶1/2（p-ERK1/2）拮抗剂PD-98059（30μg/kg）、SB（100μg/kg）+PP2（50μg/kg）；SB（100μg/kg）+PD-98059（30μg/kg）。我们还用100μg/kg 5-HTR6激动剂WAY-181187（WAY）处理了第一阶段对照组的18只大鼠。通过Timm染色法检测大鼠的MFS。最后，通过蛋白质印迹法或逆转录-聚合酶链反应（RT-PCR）对5-HTR6、Fyn、p-ERK1/2和生长相关蛋白-3（GAP-3）的表达进行定性和半定量分析。
结果
SE的诱导可刺激MFS的形成并增加GAP-43的表达。随着SE模型建立后时间的增加，5-HTR6、Fyn和p-ERK1/2的表达也上调。SB、PP2和PD显著抑制了MFS的发展。与单一拮抗剂相比，SB+PD或SB+PP增强了这种抑制作用。用WAY处理健康大鼠会导致Fyn和p-ERK1/2表达上调以及MFS的发展（P<0.05）。抑制Fyn会引发p-ERK1/2的下调趋势（P<0.05），然而，抑制p-ERK1/2对Fyn表达没有如此显著的影响。
结论
5-HTR6可能通过激活p-ERK1/2和Fyn来影响MFS的进展，进而调节GAP-43的表达。
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