基于lncRNA-UCA1/miR-187/MAPK8通路探讨加味柴胡疏肝汤治疗癫痫作用机制

作者	单位
李瑞暖;谢苗莹;曾榕;黄柳瑛;黄鹏宇;于晓东;李明芬;周艳英;梁霜;罗玉珍;刁丽梅	1.广西中医药防治医学分子生物重点实验室，广西中医药大学研究生院（南宁 530001）,2.广西中医药大学第一附属医院脑病一区（南宁 530023）

摘要点击次数: 272

全文下载次数: 76

中文摘要:

目的观察加味柴胡疏肝汤（MCSF）对癫痫小鼠行为学的影响及其对长链非编码 RNA 尿路上皮癌相关基因 1（lncRNA-UCA1）、微小RNA 187（miR-187）和丝裂原激活蛋白激酶 8（MAPK8）的调控作用。方法将80只小鼠随机分为正常组、模型组（盐酸匹罗卡品180 mg/kg）、MCSF组[7 g/（kg·d）]、MCSF[7 g/（kg·d）]+miR-187 模拟剂组（2 μL）、MCSF[7 g/（kg·d）]+miR-187 抑制剂组（2 μL）、MCSF[7 g/（kg·d）]+lncRNA-UCA1 模拟剂组（2 μL）、MCSF[7 g/（kg·d）]+lncRNA-UCA1 抑制剂组（2 μL）、卡马西平（CBZ）组[30 mg/（kg·d）]。采用匹罗卡品（180 mg/kg）诱导建立癫痫小鼠模型，抑制及过表达lncRNA-UCA1（2 μL）和miR-187（2 μL）后，给予对应药物：MCSF[7 g/（kg·d）]、卡马西平混悬液[30 mg/（kg·d）]或生理盐水[20 mL/（kg·d）]灌胃2周，观察小鼠一般情况；行脑电图和水迷宫行为学实验；苏木精-伊红（HE）染色法观察造模前后海马病理学变化；逆转录-聚合酶链反应（RT-qPCR）检测小鼠海马lncRNA-UCA1、miR-187和MAPK8 mRNA表达量；蛋白质免疫印迹法检测小鼠海马MAPK8及磷酸化水平变化。结果与正常组比较，模型组小鼠体重减少，脑电图可观察到明显连续性痫性放电波，平台逃避潜伏期明显延长，穿越平台次数和目标象限停留时间减少（P<0.01，P<0.05），海马组织神经元疏松紊乱，模型组miR-187的表达水平降低，lncRNA-UCA1和MAPK8基因表达水平升高（P<0.01），p-MAPK8蛋白表达量明显增加（P<0.01）；与模型组比较，给药组干预2周后，小鼠体重增加，各组脑电波痫性放电波频率和强度明显减少，MCSF组、MCSF+miR-187 模拟剂组、MCSF+lncRNA-UCA1 抑制剂组和CBZ组平台逃避潜伏期缩短，穿越平台次数增多（P<0.01，P<0.05），海马组织miR-187的表达水平升高，lncRNA-UCA1和MAPK8基因表达水平降低（P<0.01），p-MAPK8蛋白表达量均明显减少（P<0.01），MCSF+miR-187 抑制剂组lncRNA-UCA1基因水平表达量降低（P<0.05）；MCSF+lncRNA-UCA1 模拟剂组miR-187升高（P<0.05）。结论 MCSF可以减少癫痫小鼠痫性波，改善小鼠癫痫行为，发挥抗癫痫作用，其作用机制可能与通过抑制lncRNA-UCA1表达从而激活miR-187，降低MAPK8水平有关。

英文摘要:

Objective To observe the effects of Modified Chaihu Shugan Formula（MCSF）on the behavior of epileptic mice and their regulatory effects on long noncoding RNA-UCA1（lncRNA-UCA1），microRNA-187（miR-187） and recombinant mitogen activated protein kinase 8（MAPK8）. Methods Totally 80 mice were randomly divided into the normal group，model group（pilocarpine hydrochloride 180 mg/kg），MCSF group（7 g·kg-1·d-1），MCSF（7 g·kg-1·d-1）+miR-187 mimic group（2 μL），MCSF（7 g·kg-1·d-1）+miR-187 inhibitor group（2 μL），MCSF（7 g·kg-1·d-1）+lncRNA-UCA1 mimic group（2 μL），MCSF（7 g·kg-1·d-1）+lncRNA-UCA1 inhibitor group（2 μL），CBZ group（7 g·kg-1·d-1）.The mouse model of epilepsy was induced by pilocarpine（180 mg/kg）.After inhibition and overexpression of lncRNA-UCA1（2 μL）and miR-187（2 μL），mice were treated with MCSF（7 g·kg-1·d-1），carbamazepine suspension（30 mg·kg-1·d-1） or normal saline （20 mL·kg-1·d-1） by gavage for 2 weeks.The general state of mice was observed. EEG and Morris water maze test were conducted；HE staining was used to observe the pathological changes of hippocampus before and after modeling. The expression levels of lncRNA-UCA1，miR-187 and MAPK8 mRNA in hippocampus of mice were detected by RT-qPCR. Western Blot was used to detect the changes of MAPK8 and phosphorylation levels in hippocampus of mice. Results Compared with the normal group，the mice in the model group showed a significant reduction in body weight，a significant continuous epileptic discharge in EEG，a significant prolongation of the platform escape latency，a reduction in the number of crossing the platform and the residence time of the target quadrant（P<0.01，P<0.05），and a reduction in the expression of miR-187 in the model group. The expression levels of lncRNA-UCA1 and MAPK8 genes and the expression of p-MAPK8 protein were significantly increased（P<0.01）. Compared with the model group，after two weeks of treatment，the weight of the mice in the drug group increased，the frequency and intensity of epileptic waves in each group decreased significantly，the platform escape latency was shortened，and the number of platform crossings was increased in the MCSF group，MCSF+miR-187 mimic group，MCSF+lncRNA-UCA1 inhibitor group and CBZ group（P<0.01，P<0.05）. Compared with the model group，the expression level of miR-187 in the hippocampus was increased，the expression levels of lncRNA-UCA1 and MAPK8 genes were decreased（P<0.01），and the expression level of p-MAPK8 protein was significantly decreased（P<0.01）. The expression level of lncRNA-UCA1 gene was decreased in MCSF+miR-187 inhibitor group（P<0.05）. The expression of miR-187 was increased in MCSF+lncRNA-UCA1 mimic group（P<0.05）. Conclusions MCSF can reduce epileptic waves in epileptic mice，improve epileptic behavior in mice，and play an anti-epileptic role. Its mechanism may be related to the activation of miR-187 and the reduction of MAPK8 level by inhibiting the expression of lncRNA-UCA1.

关闭