柴胡疏肝汤通过上调ASIC1蛋白调节NMDAR通道改善大鼠癫痫后的认知障碍

doi:10.12122/j.issn.1673-4254.2025.07.17

摘要/Abstract

摘要：

目的探讨柴胡疏肝汤对氯化锂-匹罗卡品诱导癫痫后认知障碍大鼠认知功能损害的改善作用。方法 SPF级雄性SD大鼠腹腔注射氯化锂-匹罗卡品诱导癫痫后认知障碍模型，空白对照组大鼠腹腔注射生理盐水。将癫痫后认知障碍大鼠随机分为：模型组（生理盐水）、阳性对照组（Donepezil，90 mg·kg^-1·d^-1）、柴胡疏肝汤1组（CHSG-1，2.5 g·kg^-1·d^-1）、柴胡疏肝汤2组（CHSG-2，5 g·kg^-1·d^-1）、柴胡疏肝汤3组（CHSG-3，10 g·kg^-1·d^-1）、柴胡疏肝汤4组（CHSG-4，20 g·kg^-1·d^-1）、柴胡疏肝汤5组（CHSG-5，40 g·kg^-1·d^-1），空白组大鼠（n=12）以生理盐水灌胃给药，各组大鼠连续灌胃给药4周后，Morris水迷宫观察各组大鼠认知行为变化，Western blotting检测大鼠海马区ASIC1、NR1、NR2A、NR2B蛋白表达水平，荧光定量PCR检测大鼠海马区 ASIC1、NR1、NR2A、NR2B的mRNA表达水平。结果柴胡疏肝汤治疗4周后，Morris水迷宫认知行为学结果显示，与模型组相比，CHSG-2和CHSG-3组大鼠逃避潜伏期缩短（P<0.05，P<0.01），CHSG-2和CHSG-3组大鼠目标象限停留时间延长（P<0.05），CHSG-2和CHSG-3组大鼠穿越平台次数增加（P<0.05）；Western blotting结果显示，CHSG-2和CHSG-3组大鼠海马区ASIC1、NR1、NR2A、NR2B蛋白表达增加（P<0.05），与CHSG-3相比较，CHSG-1、CHSG-4和CHSG-5组蛋白表达降低（P<0.01）。PCR结果显示，与模型组相比较，CHSG-2组、CHSG-3组、CHSG-4组及CHSG-5组大鼠海马区ASIC1、NR1、NR2A、NR2B mRNA表达增加（P<0.05）。结论柴胡疏肝汤对癫痫后认知功能损害有一定的治疗作用，可能通过上调ASIC1通道蛋白进而调节抗N-甲基-D-天冬氨酸受体蛋白及亚单位蛋白及基因的表达及通道活性，调节神经元兴奋性及突触可塑性发挥治疗作用；柴胡疏肝汤的治疗作用在一定的剂量范围内显示剂量依赖性，且剂量浓度有一定的安全范围。

关键词: 癫痫, 认知障碍, 从肝论治, 柴胡疏肝汤, 酸敏感离子通道, 抗N-甲基-D-天冬氨酸受体

Abstract:

Objective To explore the therapeutic mechanism of Chaihu Shugan (CHSG) Decoction for improving cognitive impairment in rats with epilepsy induced by lithium chloride and pilocarpine. Methods Male SD rat models of cognitive impairment model after epilepsy induced by intraperitoneal injection with lithium chloride and pilocarpine were randomly divided into 5 groups (n=12) for treatment with daily gavage of saline, donepezil (90 mg/kg), or CHSG Decoction at 2.5, 5.0, 10, 20 and 40 g/kg for 4 consecutive weeks, with 10 rats with intraperitoneal injection with saline as the blank control group. Morris water maze test was used to evaluate cognitive and behavioral changes of the rats after treatment. The mRNA and protein expressions of ASIC1, NR1, NR2A and NR2B in the hippocampus of rats were detected using RT-qPCR and Western blotting. Results Compared with those with saline treatment, the rat models treated with CHSG Decoction at 5 and 10 g/kg showed significantly shortened escape latency and prolonged stay in the target quadrant with increased number of platform crossings in Morris water maze test. CHSG Decoction treatment at the two doses significantly increased ASIC1, NR1, NR2A and NR2B protein expressions in the hippocampus of the rat models, and their mRNA expression levels were all increased significantly after the treatment at the doses above 2.5 g/kg. Conclusion CHSG Decoction can improve cognitive impairment in rats after epilepsy possibly by regulating the expression and channel activity of NMDAR protein and its subunit protein via upregulating ASIC1 to modulate neuronal excitability and synaptic plasticity in the hippocampus.

Key words: epilepsy, cognitive impairment, treatment from Gan, Chaihu Shugan Decoction, acid sensitive ion channel, NMDAR

于云红, 谢炜, 李慧. 柴胡疏肝汤通过上调ASIC1蛋白调节NMDAR通道改善大鼠癫痫后的认知障碍[J]. 南方医科大学学报, 2025, 45(7): 1506-1512.

Yunhong YU, Wei XIE, Hui LI. Chaihu Shugan Decoction improves cognitive impairment after epilepsy in rats by regulating hippocampal NMDAR subunits via upregulating ASIC1[J]. Journal of Southern Medical University, 2025, 45(7): 1506-1512.

图/表 6

表1 反应体系

Tab.1 Reaction system of RT-qPCR

Component	Quantity (μL)
RNase Free d H₂O	10
5×PrimeScript^TM Buffer (for real time)	2
PrimeScript^TMRT Enzyme Mix I	1
Oligo dT Primer (50 μmol/L)	1
Random 6 mers (100 μmol/L)	2
RNA	4
Total	20

表2 引物序列

Tab.2 Primer sequence for RT-qPCR

Gene name	Sequence (5'-3')	Length (bp)
ASIC1	GCAATCTCAATGAGTTCCGCT	107
ASIC1	CTGTGTGTCCGGGATCTCATA
NR1	AGGACTGTCAGTTCATGTGGT	101
NR1	CATCTTCCTCCTCCTCAC
NR2A	GCTCATCCCCAAAGAGTTTCC	99
NR2A	AAGATCCCAAGACCGTCTCTC
NR2B	CAGCAAAGCTCGTTCCCAAA	91
NR2B	GGCGTCTTTTATGGCCACTTC
GAPDH	GTATGACTCTACCCACGGCAAGT	99
GAPDH	TCTCGCTCCTGGAAGATGGT

图1 Morris水迷宫观察各组大鼠认知行为变化

Fig.1 Cognitive and behavioral changes of the rats assessed using Morris water maze test. **P<0.01 vs Control group; #P<0.05, ##P<0.01 vs Model group; &P<0.05, &&P<0.01 vs CHSG-3 group.

图2 Western blotting检测各组大鼠海马区ASIC1、NR1、NR2A、NR2B蛋白表达情况

Fig.2 Expression of ASIC1, NR1, NR2A, and NR2B proteins in the hippocampus of the rats detected by Western blotting. 1: Control group; 2: Model group; 3: CHSG-1 group; 4: CHSG-2 group; 5: CHSG-3 group; 6: CHSG-4 group; 7: CHSG-5 group; 8: Positive control group.

图3 各组大鼠海马区ASIC1、NR1、NR2A、NR2B蛋白表达水平

Fig. 3 Expression levels of ASIC1, NR1, NR2A and NR2B protein in each group. **P<0.01 vs Control group; #P<0.05, ##P<0.01 vs Model group; &P<0.05, &&P<0.01 vs CHSG-3 group.

图4 各组大鼠海马区ASIC1、NR1、NR2A、NR2B的mRNA表达水平

Fig.4 Expression levels of ASIC1, NR1, NR2A and NR2B mRNA in each group. **P<0.01 vs Control group; ##P<0.01 vs Model group.

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