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

doi:10.12122/j.issn.1673-4254.2025.03.08

Abstract

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

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.

Figures/Tables 5

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.

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.

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.

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.

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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