金丝桃素通过调节小鼠小胶质细胞内CN-NFAT钙信号通路改善压力应激诱导的抑郁样行为

doi:10.12122/j.issn.1673-4254.2025.03.08

南方医科大学学报 ›› 2025, Vol. 45 ›› Issue (3): 506-513.doi: 10.12122/j.issn.1673-4254.2025.03.08

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金丝桃素通过调节小鼠小胶质细胞内CN-NFAT钙信号通路改善压力应激诱导的抑郁样行为

高正涛(), 林萍燕, 周炳灿, 陈明恒, 刘尔祺, 雷添翔, 倪慧心, 刘海鑫, 林瑶, 许茜()

福建中医药大学中西医结合学院//中西医结合研究院，福建福州 350122

收稿日期:2024-10-23 出版日期:2025-03-20 发布日期:2025-03-28
通讯作者: 许茜 E-mail:2921438793@qq.com;xmuxuqian@163.com
作者简介:高正涛，本科，E-mail: 2921438793@qq.com
基金资助:
国家自然科学基金青年项目(81904263);福建省自然科学基金面上项目(2024J01745);福建中医药大学中医健康管理学联合省级临床重点专科建设项目（老年病科）(XJG2023018);福建中医药大学校管课题青年项目(X2023010);福建中医药大学校管课题(X2024029);福建省“大学生创新创业训练计划”项目校级立项

Hypericin ameliorates stress-induced depressive-like behaviors in mice by modulating the CN-NFAT calcium signaling pathway in microglia

Zhengtao GAO(), Pingyan LIN, Bingcan ZHOU, Mingheng CHEN, Erqi LIU, Tianxiang LEI, Huixin NI, Haixin LIU, Yao LIN, Qian XU()

College of Integrative Medicine, Academy of Intergrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China

Received:2024-10-23 Online:2025-03-20 Published:2025-03-28
Contact: Qian XU E-mail:2921438793@qq.com;xmuxuqian@163.com
Supported by:
Youth Program of National Natural Science Foundation of China(81904263)

摘要/Abstract

摘要：

目的探究金丝桃素（HY）通过钙调蛋白（CaM）介导引起钙调磷酸酶蛋白（CN）-活化T细胞因子（NFAT）钙信号通路变化调节压力应激引起的抑郁（DP）障碍并研究其机制。方法将C57BL/6J小鼠随机分为正常组（Control组）、DP组和DP+HY组（15只/组），行为学测试旷场实验（PFT）、糖水偏好实验（SPT）、悬尾实验（TST）、黑白场实验（LDB）、新奇食物抑制实验（NSFT），免疫组化检测小鼠脑海马体1区（CA1）酪氨酸羟化酶（TH）阳性表达，ELISA检测小鼠血清5-羟色胺（5-HT）、去甲肾上腺素（NA）水平，Western blotting检测炎症因子肿瘤坏死因子（TNF-α）、白细胞介素-1β（IL-1β）和白细胞介素-2（IL-2）及CN-NFAT通路相关蛋白表达水平。同时，将小胶质细胞（BV-2）分为Control组、脂多糖（LPS）组、LPS+HY组以及钙调磷酸酶抑制剂（CNIS）组，采用免疫荧光法检测离子钙结合接头分子1（IBA-1）阳性表达，ELISA检测细胞上清液5-HT、NA水平，Western blotting检测炎症因子IL-1β、IL-2和TNF-α以及CN-NFAT通路相关蛋白表达水平。结果与Control组相比，DP组小鼠的PFT经过中间区域的次数、SPT糖水消耗率、LDB明暗箱之间的穿梭次数以及NSFT食物的摄食率都降低（P<0.05），TST不动时间增加（P<0.05），TH神经元的数量减少（P<0.05），5-HT和NA的水平减低（P<0.05），促炎细胞因子（TNF-α、IL-1β和IL-2）及CaM-CN-NFAT钙信号通路的蛋白表达水平升高（P<0.05），LPS组的IBA-1表达上升（P<0.05），神经递质水平（NA、5-HT）减少（P<0.05），促炎细胞因子（TNF-α、IL-1β和IL-2）及CaM-CN-NFAT钙信号通路的蛋白表达量升高（P<0.05），与DP组相比，HY组均逆转了上述结果（P<0.05），与LPS组相比，CNIS组与HY组作用相似，对IBA-1表达及神经递质水平（NA、5-HT）和促炎细胞因子（TNF-α、IL-1β和IL-2）及CaM-CN-NFAT钙信号通路的蛋白表达量均有逆转作用（P<0.05）。结论 HY对于压力应激诱导的DP样小鼠和激活态下BV-2的改善作用，可通过靶向降低CN-NFAT钙信号通路表达起有效作用。

关键词: 金丝桃素, 压力应激, CN-NFAT信号通路, 脂多糖, 抑郁

Abstract:

Objective To investigate the role of the calcium/calmodulin (CaM)-mediated activation of calcineurin (CN)-nuclear factor of activated T cells (NFAT) signaling pathway in mediating the regulatory effect of hyperforin (HY) on stress-induced depression-like disorder (DP) in mice. Methods C57BL/6J mice were randomly divided into control group, DP model group, and hyperforin treatment group (n=15). Behavioral changes of the mice were assessed using open field test (OFT), sucrose preference test (SPT), tail suspension test (TST), light/dark box test (LDB), and novel object suppression test (NSFT). Immunohistochemistry was used to detect tyrosine hydroxylase (TH) expression in the CA1 region of the hippocampus, and serum serotonin (5-HT) and norepinephrine (NA) levels were detected with ELISA. Western blotting was used to analyze the expressions of TNF-α, IL-1β, IL-2, and CN-NFAT pathway proteins. In cultured BV-2 microglial cells with lipopolysaccharide (LPS) stimulation, the effects of hyperforin and CN inhibitor (CNIS) on expressions of ionized calcium-binding adapter molecule 1 (IBA-1), 5-HT, NA, inflammatory cytokines and CN-NFAT pathway proteins were examined using immunofluorescence assay, ELISA or Western blotting. Results Compared with the control mice, the mice in DP group showed significantly reduced activity in OFT, decreased sucrose consumption in SPT, reduced shuttle crossing in LDB, and lowered food intake in NSFT with significantly increased immobility in TST. The mice with DP showed significantly decreased TH-positive neurons, lowered 5-HT and NA levels, and increased expressions of TNF-α, IL-1β, IL-2 and CaM-CN-NFAT pathway proteins. In cultured BV-2 cells, LPS stimulation strongly increased cellular IBA-1 expression, decreased the levels of neurotransmitters (5-HT and NA), and increased the levels of inflammatory cytokines and CN-NFAT signaling, and these changes were effectively reversed by treatment with hyperforin or CNIS. Conclusion Hyperforin improves stress-induced depression-like behaviors in mice and activated BV-2 cells by targeting the CN-NFAT signaling pathway.

Key words: hypericin, stress due to pressure, CN-NFAT signaling pathway, lipopolysaccharide, depression

高正涛, 林萍燕, 周炳灿, 陈明恒, 刘尔祺, 雷添翔, 倪慧心, 刘海鑫, 林瑶, 许茜. 金丝桃素通过调节小鼠小胶质细胞内CN-NFAT钙信号通路改善压力应激诱导的抑郁样行为[J]. 南方医科大学学报, 2025, 45(3): 506-513.

Zhengtao GAO, Pingyan LIN, Bingcan ZHOU, Mingheng CHEN, Erqi LIU, Tianxiang LEI, Huixin NI, Haixin LIU, Yao LIN, Qian XU. Hypericin ameliorates stress-induced depressive-like behaviors in mice by modulating the CN-NFAT calcium signaling pathway in microglia[J]. Journal of Southern Medical University, 2025, 45(3): 506-513.

图/表 5

图1 HY对小鼠行为学的影响

Fig.1 Effect of hyperforin (HY) on behaviors of the mice. A: Number of shuttles between the light and dark compartments in light/dark box test. B: Number of crossings in the center zone in open field test. C: Food consumption rate in novel object suppression test. D: Sucrose consumption rate in sucrose preference test. E: Immobility time in tail suspension test. ***P<0.001 vs Control. ###P<0.001, ##P<0.01, #P<0.05 vs DP.

图2 HY对小鼠海马TH神经元及神经递质水平的影响

Fig.2 Effect of HY on hippocampal TH neurons and neurotransmitter levels in mice (Scale bar=50 μm). A: Control group. B: DP group. C: HY group. D: TH expression level. E: 5-HT expression level. F: NA expression level. ***P<0.001 vs Control. ##P<0.01, #P<0.05 vs DP.

图3 HY对促炎细胞因子及CaM-CN-NFAT钙信号通路的影响

Fig.3 Effect of HY on pro-inflammatory cytokines and the CaM-CN-NFAT calcium signaling pathway. A: IL-2 expression level. B: IL-β expression level. C: Protein expression of pro-inflammatory cytokines. D: TNF-α expression level. E: CaM expression level.F: Protein expression of CaM-CN-NFAT calcium signaling pathway. G: CN expression level. H: NFAT1 expression level. I: NFAT4 expression level. ***P<0.001 vs Control. ###P<0.001, ##P<0.01, #P<0.05 vs DP.

图4 HY对BV-2中IBA-1表达及神经递质水平影响

Fig.4 Effect of HY on IBA-1 expression and neurotransmitter levels in BV-2 cells (Scale bar=50 μm). A: IBA-1 fluorescence image. B: IBA-1 expression level. C: 5-HT expression level. D: NA expression level. ***P<0.001 vs Control; ###P<0.001, #P<0.05 vs LPS.

图5 HY对BV-2中促炎细胞因子及CaM-CN-NFAT钙信号通路的影响

Fig.5 Effect of HY on pro-inflammatory cytokines and the CaM-CN-NFAT calcium signaling pathway in BV-2 cells. A: IL-2 expression level. B: IL-β expression level. C: Pro-inflammatory cytokine protein expression. D: TNF-α expression level. E: CaM protein expression. F: Protein levels of the CaM-CN-NFAT calcium signaling pathway. G: CN protein expression. H: NFAT1 protein expression. I: NFAT4 protein expression. ***P<0.001 vs Control; ###P<0.001, ##P<0.01 vs LPS.

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金丝桃素通过调节小鼠小胶质细胞内CN-NFAT钙信号通路改善压力应激诱导的抑郁样行为

Hypericin ameliorates stress-induced depressive-like behaviors in mice by modulating the CN-NFAT calcium signaling pathway in microglia

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