FK506通过抑制钙调神经磷酸酶活性上调星形胶质细胞EAAT1/2表达控制海马区癫痫发作及神经损伤

陈昌领; 刘瑛; 江婷; 熊英; 汪朝云; 张晴; 冯占辉; 叶兰

doi:10.16016/j.2097-0927.202512048

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FK506通过抑制钙调神经磷酸酶活性上调星形胶质细胞EAAT1/2表达控制海马区癫痫发作及神经损伤

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- FK506通过抑制钙调神经磷酸酶活性上调星形胶质细胞EAAT1/2表达控制海马区癫痫发作及神经损伤
- FK506 suppresses hippocampal seizures and neuroinjury by upregulating astrocytic EAAT1/2 expression via inhibition of calcineurin activity
- 陆军军医大学学报 2026年48卷第10期页码：1313-1325
- 作者机构：
  
  1.贵州医科大学基础医学院药理学教研室，机能学实验室，贵州贵阳
  2.贵州医科大学附属医院神经内科，贵州贵阳
  3.贵州省人民医院神经内科，贵州贵阳
- 作者简介：
  
  叶兰，E-mail：frogyl266@163.com
- 基金信息：
  
  国家自然科学基金项目地区科学基金项目(82360266);贵州省科技计划项目（黔科合基础-ZK［2023］一般395,324,黔科合基础-MS［2025］548）
- DOI：10.16016/j.2097-0927.202512048
  中图分类号： R742.1;R966;R979.5
- 收稿：2025-12-10，
  
  修回：2026-01-12，
  
  录用：2026-01-16，
  
  纸质出版：2026-05-30
- 稿件说明：
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陈昌领，刘瑛，江婷，等.FK506通过抑制钙调神经磷酸酶活性上调星形胶质细胞EAAT1/2表达控制海马区癫痫发作及神经损伤［J］.陆军军医大学学报，2026，48（10）：1313-1325.DOI：10.16016/j.2097-0927.202512048.

CHEN C L，LIU Y，JIANG T，et al.FK506 suppresses hippocampal seizures and neuroinjury by upregulating astrocytic EAAT1/2 expression via inhibition of calcineurin activity［J］.J Army Med Univ，2026，48（10）：1313-1325.DOI：10.16016/j.2097-0927.202512048.
陈昌领，刘瑛，江婷，等.FK506通过抑制钙调神经磷酸酶活性上调星形胶质细胞EAAT1/2表达控制海马区癫痫发作及神经损伤［J］.陆军军医大学学报，2026，48（10）：1313-1325.DOI：10.16016/j.2097-0927.202512048. DOI：

CHEN C L，LIU Y，JIANG T，et al.FK506 suppresses hippocampal seizures and neuroinjury by upregulating astrocytic EAAT1/2 expression via inhibition of calcineurin activity［J］.J Army Med Univ，2026，48（10）：1313-1325.DOI：10.16016/j.2097-0927.202512048. DOI：

摘要

目的

钙调神经磷酸酶（calcineurin，CaN）被认为可能参与海马星形胶质细胞兴奋性氨基酸转运蛋白1（excitatory amino acid transporter 1，EAAT1）和兴奋性氨基酸转运蛋白2（excitatory amino acid transporter 2，EAAT2）表达的调控，本研究旨在探究在红藻氨酸（kainic acid，KA）诱导的癫痫模型中他克莫司（tacrolimus，FK506）是否通过上调海马星形胶质细胞EAAT1和EAAT2的表达从而减少癫痫的发生。

方法

采用来源于GEO数据库人类和小鼠脑scRNA-seq数据集GSE190453、GSE241349分析EAAT1、EAAT2、CaN、GFAP和NeuN的细胞类型分布，采用KA癫痫小鼠海马星形胶质细胞RNA-seq数据集GSE237321分析EAAT1、EAAT2、CaN和GFAP表达特征。实验动物为108只SPF级6~8周龄C57BL/6J小鼠（体质量19~23 g，雌雄各半）。为评估低剂量的FK506对癫痫小鼠的皮层放电和EAAT1、EAAT2的调节作用，本研究首先进行低剂量的FK506干预实验，采用随机数字表法将12只小鼠分为Control组、KA组和FK506组（

=4），FK506组造模前24 h及1 h单次腹腔注射1 mg/kg的FK506，KA组向小鼠两侧海马分别注射0.5 μL KA（0.5 μg/μL）造模，KA造模48 h后评估给药对皮层放电及EAAT1、EAAT2的mRNA表达的影响。为确定EAAT1、EAAT2、CaN和GFAP的蛋白的时间表达特征以及合适的取材时间，本研究利用随机数字表法将36只小鼠分为Control组、6 h、1 d、3 d、5 d和7 d组（

=6），经KA造模后收集对应时间点的海马组织，用于Western blotting实验检测各蛋白指标的变化。为评估不同剂量的FK506的疗效，将60只小鼠分为Control组、KA组、FK506 1 mg/kg组、FK506 2 mg/kg组、FK506 4 mg/kg组（

=10），各FK506治疗组于KA造模前24 h及1 h腹腔注射对应剂量的FK506，KA造模后连续3 d 腹腔注射对应剂量的FK506。KA造模后第5天进行旷场实验以评估小鼠运动状态，实验结束后每组收集6只小鼠的海马组织，用于Western blotting实验检测各组EAAT1、EAAT2、CaN和GFAP的蛋白表达，各组其余小鼠采用HE染色观察海马神经元病理改变，采用免疫荧光染色法检测EAAT1、EAAT2与GFAP的共定位情况。

结果

scRNA-seq数据分析结果显示，EAAT1、EAAT2 特异性定位于星形胶质细胞，CaN 同样在星形胶质细胞中存在表达。小鼠海马星形胶质细胞RNA-seq数据分析结果表明，相较于PBS和AAV组，KA组EAAT1、EAAT2 mRNA水平在KA诱导4 d后显著下调（

0.01），GFAP和CaN显著上调（

0.05）。小鼠海马组织切片免疫荧光结果表明，EAAT1和EAAT2与星形胶质细胞标志物GFAP共定位，与Control组相比，KA组GFAP荧光强度显著增加（

0.05）。Western blotting结果显示，与Control组比较，5 d组EAAT1和EAAT2显著下调（

0.01）；GFAP水平在KA处理后6 h开始升高，3 d时显著增加（

0.05）；CaN全长蛋白水平自3 d起下降（

0.01），而45 kDa CaN活性片段在3 d和5 d时显著增加。表明CaN在癫痫发生早期就已经被激活，可能参与EAAT1和EAAT2蛋白表达的调控。造模前给予1 mg/kg FK506可改善KA小鼠大脑皮层异常放电，并部分缓解KA诱导的EAAT1和EAAT2 mRNA下调（

0.05）。造模后连续给予不同剂量FK506干预可减轻海马CA1、CA3区神经元损伤及小鼠运动障碍。Western blotting显示，不同剂量FK506可抑制KA诱导的EAAT1和EAAT2表达下调（

0.01），抑制CaN全长蛋白下调及45 kDa CaN活性片段产生（

0.01），同时部分抑制GFAP上调（

0.01），并显著抑制NeuN下调（

0.05），表明不同剂量的FK506具有神经保护作用。

结论

在KA诱导的癫痫小鼠模型中，FK506通过抑制CaN活性片段的产生，上调EAAT1和EAAT2的表达，从而减轻癫痫诱导的神经损伤。

Abstract

Objective

Calcineurin (CaN) is implicated in regulating hippocampal astrocytic excitatory amino acid transporter 1 (EAAT1) and 2 (EAAT2) expression. This study investigates whether tacrolimus (FK506) reduces epileptogenesis by upregulating EAAT1 and EAAT2 in hippocampal astrocytes in a kainic acid (KA)-induced epilepsy model.

Methods

The cellular distribution of EAAT1

EAAT2

CaN

GFAP

and NeuN was analyzed using human and mouse brain single cell RNA sequencing (scRNA seq) datasets (GSE190453

GSE241349) from the GEO database. Hippocampal astrocyte RNA seq data from KA‑induced epileptic mice (GSE237321) were used to examine the expression patterns of EAAT1

EAAT2

CaN

and GFAP. A total of 108 specific pathogen free (SPF) C57BL/6J mice aged 6 to 8 weeks (body weight 19 to 23 g

equal numbers of males and females) were used. To evaluate the effect of low dose FK506 on cortical discharges and the regulation of EAAT1 and EAAT2

12 mice were randomly divided into Control

and FK506 groups(

=4). The FK506 group received a single intraperitoneal injection of 1 mg/kg FK506 24 h and 1 h before modeling; the KA group was modeled by bilateral hippocampal injection of 0.5 μL KA (0.5 μg/μL). Cortical discharges an

d hippocampal EAAT1 and EAAT2 mRNA levels were assessed 48 h after KA injection. To determine the temporal expression profiles of EAAT1

EAAT2

CaN

and GFAP proteins and identify appropriate sampling time points

36 mice were randomly allocated to Control

6 h

1 d

3 d

5 d

and 7 d groups(

=6). Hippocampal tissues were collected at the corresponding time points after KA administration for Western blotting. To evaluate the efficacy of different FK506 doses

60 mice were divided into Control

FK506 1 mg/kg

FK506 2 mg/kg

and FK506 4 mg/kg groups (

=10). FK506 treatment groups received corresponding doses (intraperitoneal injection) 24 h and 1 h before KA modeling

followed by once daily injections for 3 consecutive days after modeling. On day 5 after modeling

open field testing was performed to assess locomotor activity. Hippocampal tissues from 6 mice per group were used for Western blotting of EAAT1

EAAT2

CaN

and GFAP; the remaining mice were used for HE staining to observe neuronal pathology and immunofluorescence staining to examine the colocalization of EAAT1 and EAAT2 with GFAP.

Results

scRNA‑seq analysis revealed that EAAT1 and EAAT2 were specifically localized in astrocytes

and CaN was also expressed in astrocytes. Hippocampal astrocyte RNA seq data showed that compared with PBS and AAV groups

EAAT1 and EAAT2 mRNA levels were significantly downregulated 4 d after KA induction

while GFAP and CaN were significantly upregulated. Immunofluorescence of mouse hippocampal sections indicated that EAAT1 and EAAT2 co‑localized with the astrocyte marker GFAP

and GFAP fluorescence intensity was significantly increased in the KA group versus the Control group. Western blotting demonstrated that EAAT1 and EAAT2 were significantly downregulated in the 5 d group compared with the Control group; GFAP levels began to rise 6 h after KA treatment and were significantly increased at 3 d; full length CaN protein levels decreased from 3 d onward

whereas the 45 kDa CaN active fragment was significantly increased at 3 d and 5 d

suggesting that CaN is activated early in epileptogenesis and may participate in regulating EAAT1 and EAAT2 protein expression. Pretreatment with 1 mg/kg FK506 ameliorated abnormal cortical discharges in KA mice and partially alleviated KA induced downregulation of EAAT1 and EAAT2 mRNA. Continuous post modeling intervention with different doses of FK506 reduced neuronal damage in hippocampal CA1 and CA3 regions and improved motor deficits. Western blotting showed that different doses of FK506 inhibited KA‑induced downregulation of EAAT1 and EAAT2 expression

suppressed the decrease in full length CaN and the generation of the 45 kDa CaN active fragment

partially inhibited GFAP upregulation

and significantly mitigated NeuN downregulation

indicating that FK506 exerts neuroprotective effects in a dose dependent manner.

Conclusion

In the KA induced epileptic mouse model

FK506 upregulates the expression of EAAT1 and EAAT2 in hippocampal astrocytes by inhibiting the production of the CaN active fragment

thereby attenuating epilepsy‑induced neuronal damage.

关键词

Keywords

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