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Paeoniflorin ameliorates interferon-alpha-induced neuroinflammation and depressive-like behaviors in mice - PubMed

  • ️Sun Jan 01 2017

. 2017 Jan 31;8(5):8264-8282.

doi: 10.18632/oncotarget.14160.

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Paeoniflorin ameliorates interferon-alpha-induced neuroinflammation and depressive-like behaviors in mice

Jianwei Li et al. Oncotarget. 2017.

Abstract

Long-term treatment with high-dose Interferon-alpha (IFN-α) has resulted in depression in 30-50% of the patients. Paeoniflorin may ameliorate the IFN-α-induced depression; however, the underlying mechanism is less studied. Here, we investigated the prophylactic antidepressant and anti-neuroinflammatory effects of paeoniflorin on the behaviors and specific emotion-related regions of the brain in mice with IFN-α-induced depression. A series of behavior assessments were conducted to identify the depressive state after subcutaneously IFN-α injections and with or without intragastrically paeoniflorin administration in C57BL/6J mice. Levels of many inflammatory-related cytokines in serum, mPFC, vHi and amygdala were determined by cytokine array analysis. Furthermore, microglia and astrocyte activation in these three regions were evaluated by immunohistochemistry. We found that the mice which were subcutaneously injected IFN-α 15×106 IU/kg for 4 successive weeks to mimic an IFN-α-induced depression model had distinct inflammatory changes in the amygdala. Interestingly, 4-week 20 mg/kg or 40 mg/kg paeoniflorin pretreatments reversed the depressive-like behaviors and the abnormal inflammatory cytokine levels in the serum, mPFC, vHi and amygdala. These cytokines were not limited to the commonly reported IL-6, IL-1β and TNF-α, but also IL-9, IL-10, IL-12, and MCP-1. Besides, the increased density of microglia in IFN-α-treated mice was reversed by paeoniflorin in these three brain areas. Taken together, our data suggest that paeoniflorin can reverse the long-term, high-dose IFN-α-induced depressive-like behaviors that were associated with local distinct neuroinflammation in the mPFC, vHi and particularly the amygdala. Paeoniflorin might have a preventive therapeutic potential in IFN-α-induced depression.

Keywords: amygdala; depression; interferon; neuroinflammation; paeoniflorin.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1. Identification of the interferon (IFN)-α dosage required to establish a model of IFN-α-induced depression in mice

Experimental design. A. The effects of four different doses of IFN-α on depressive-like behaviors in mice. B–G. After the 28day injections of IFN-α, mice were subjected to the open field test (B–D), the sucrose preference test (E), the tail suspension test (F), and the forced swimming test (G). The total distance covered, central time, and numbers of vertical movements were measured in the open field test to identify the locomotive activities of IFN-α-treated mice. The sucrose preference (%) was detected to reflect anhedonia, the core symptom in depression. The immobility times in the tail suspension test and forced swimming test were examined to evaluate despair behavior. The results of these tests were quantified and compared among the groups. n=10 mice per group. *p< 0.05, **p< 0.01, ***p< 0.001. Error bars: means ± SEM.

Figure 2
Figure 2. Paeoniflorin reversed interferon (IFN)-α-induced depressive-like behaviors in vivo

Experimental design. A. The antidepressant effects of dose-dependent paeoniflorin on IFN-α treated mice for 4weeks. B–H. Mice were pretreated with paeoniflorin 30 min before IFN-α injection for 4weeks. After the 28day IFN-α injection, the mice were subjected to the open field test to evaluate locomotive activity by total distance covered (B), central time (C), and number of vertical movements (D). After 2 weeks of IFN-α administration in the middle period of study, the sucrose preference test (E) was performed to identify whether paeoniflorin or escitalopram had already had an effect; neither was found to have a significant antidepressant effect at this time. After 4 weeks, the sucrose preference test (F), the tail suspension test (G), and the forced-swimming test (H) produced significantly different results in paeoniflorin-treated mice, particularly in the high-dose group, as well as with escitalopram, a commonly used agent for treating depression. n=10 mice per group. *p< 0.05,**p< 0.01,***p< 0.001, compared with the saline-treated group; #p< 0.05, ##p< 0.01, ###p< 0.001 compared with the IFN-α-treated group. Error bars: means±SEM.

Figure 3
Figure 3. Anti-neuroinflammatory effects of paeoniflorin in serum

The mice were sacrificed after 4weeks of treatment with paeoniflorin and behavioral tests. The serum was quickly removed and the levels of a series of inflammation-related cytokines and chemokines were analyzed using cytokine array analysis. The most effective dose of paeoniflorin in reversing depressive-like behaviors in interferon-α-treated mice, 40 mg/kg, showed the changed from IFN-α reversed in the serum. n=4 mice per group. *p<0.05, **p<0.01, ***p<0.001 compared with the saline-treated group; #p<0.05, ##p < 0.01, ###p<0.001 compared with the interferon-α-treated group. Error bars: means ± SEM.

Figure 4
Figure 4. Anti-neuroinflammatory effects of paeoniflorin in the medial prefrontal cortex (mPFC)

The mice were sacrificed after 4weeks of treatment with paeoniflorin and behavioral tests. The medial prefrontal cortex was quickly removed and the levels of a series of inflammation-related cytokines and chemokines were analyzed using cytokine array analysis. The most effective dose of paeoniflorin in reversing depressive-like behaviors in interferon-α-treated mice, 40 mg/kg, showed anti-neuroinflammatory effects in the emotion-related mPFC that were, in some instances, similar to those seen with escitalopram. n=4 mice per group. *p< 0.05, **p< 0.01, ***p< 0.001 compared with the saline-treated group; #p< 0.05, ##p < 0.01, ###p< 0.001 compared with the interferon-α-treated group. Error bars: means±SEM.

Figure 5
Figure 5. Anti-neuroinflammatory effects of paeoniflorin in the ventral hippocampus (vHi)

The mice were sacrificed after 4 weeks of treatment with paeoniflorin and behavioral tests. The ventral hippocampus was quickly removed and the levels of a series of inflammation-related cytokines and chemokines were analyzed using cytokine array analysis. The most effective dose of paeoniflorin in reversing depressive-like behaviors in interferon-α-treated mice, 40 mg/kg, showed some anti-neuroinflammatory effects in the emotion-associated vHi, in part similar to those seen with escitalopram. n=4 mice per group. *p< 0.05, **p< 0.01, ***p< 0.001 compared with the saline-treated group; #p < 0.05, ##p < 0.01, ###p<0.001 compared with the interferon-α-treated group. Error bars: means±SEM.

Figure 6
Figure 6. Anti-neuroinflammatory effects of paeoniflorin in the amygdala

The mice were sacrificed after 4 weeks of treatment with paeoniflorin and behavioral tests. The amygdala was quickly removed and the levels of a series of inflammation-related cytokines and chemokines were analyzed using cytokine array analysis. The most effective dose of paeoniflorin in reversing depressive-like behaviors in interferon-α-treated mice, 40 mg/kg, showed some anti-neuroinflammatory effects in the emotion-associated amygdala, in part similar to those seen with escitalopram. n=4 mice per group. *p< 0.05, **p< 0.01, ***p< 0.001 compared with saline-treated group; #p< 0.05, ##p< 0.01, ###p< 0.001 compared with the interferon-α-treated group. Error bars: means ± SEM.

Figure 7
Figure 7. Paeoniflorin reduced interferon (IFN)-α-induced neuroinflammation in the medial prefrontal cortex

Immunofluorescence co-staining of Iba1(red) and GFAP (green) after pretreatment with paeoniflorin (40 mg/kg i.g. daily for 4 weeks) and systemic IFN-α administration (15×106 IU/kg s.c.) for 4 weeks. Nuclei are counterstained with DAPI (blue). A. Quantification of the number of activated Iba1-and GFAP-positive cells after IFN-α treatment using an observer-independent unbiased stereology approach. B, C. Immunoreactivity was evaluated for the microglial marker Iba1 and the astrocytic marker GFAP in the region of interest. The elevated activated microglia in the medial prefrontal cortex of IFN-α-injected mice were reduced by both paeoniflorin and escitalopram (10 mg/kg). Astrocytes were not significantly affected by IFN-α in this region. The region of interest (the medial prefrontal cortex) is shown in the stereotaxic atlas of the mouse brain. D. Representative confocal microphotographs. Upper images: Iba1+GFAP+DAPI staining with an original magnification of × 100. Lower images:Iba1+GFAP+DAPI staining with an original magnification of × 400. Data are mean values ± SEM, evaluated by one-way analysis of variance followed by Tukey's post hoc tests (n=5 animals per group). *p< 0.05, **p< 0.01, ***p< 0.001 compared with the saline-treated group; #p< 0.05, ##p< 0.01, ###p< 0.001 compared with the IFN-α-treated group. Scale bar, 100 μm.

Figure 8
Figure 8. Paeoniflorin reduced interferon (IFN)-α-induced neuroinflammation in the ventral hippocampus

Immunofluorescenceco-staining of Iba1(red) and GFAP (green) after pretreatment with paeoniflorin (40 mg/kg i.g. daily for 4weeks) and systemic IFN-α administration (15×106 IU/kg s.c.) for 4 weeks. Nuclei are counterstained with DAPI (blue). A. Quantification of the number of activated Iba1-and GFAP-positive cells after IFN-α treatment using an observer-independent unbiased stereology approach. B, C. Immunoreactivity was evaluated for the microglial marker Iba1 and the astrocytic marker GFAP in the region of interest. The elevated activated microglia and astrocytes in the ventral hippocampus of IFN-α-injected mice were reduced by both paeoniflorin and escitalopram (10 mg/kg). The region of interest (the ventral hippocampus) is shown in the stereotaxic atlas of the mouse brain. D. Representative confocal microphotographs. Upper images: Iba1+GFAP+DAPI staining with an original magnification of ×100. Lower images:Iba1+GFAP+DAPI staining with an original magnification of ×400. Data are mean values ± SEM, evaluated by one-way analysis of variance followed by Tukey's post hoc tests (n=5 animals per group). *p< 0.05, **p< 0.01, ***p< 0.001 compared with the saline-treated group; #p< 0.05, ##p< 0.01, ###p< 0.001 compared with the IFN-α-treated group. Scale bar, 100 μm.

Figure 9
Figure 9. Paeoniflorin reduced interferon (IFN)-α-induced neuroinflammation in the amygdala

Immunofluorescence co-staining of Iba1(red) and GFAP (green) after pretreatment with paeoniflorin (40 mg/kg i.g. daily for 4 weeks) and systemic IFN-α administration (15×106 IU/kg s.c.) for 4 weeks. Nuclei are counterstained with DAPI (blue). A. Quantification of the number of activated Iba1-and GFAP-positive cells after IFN-α treatment using an observer-independent unbiased stereology approach. B, C. Immunoreactivity was evaluated for the microglial marker Iba1 and the astrocytic marker GFAP in the region of interest. The elevated activated microglia in the amygdala of IFN-injected mouse were reduced by both paeoniflorin and escitalopram (10 mg/kg), although escitalopram was still associated with significantly increased levels compared with the saline-treated group. In addition, the volume of astrocytes was increased by IFN-α in this region. The region of interest (the amygdala) is shown in the stereotaxic atlas of the mouse brain. D. Representative confocal microphotographs. Upper images: Iba1+GFAP+DAPI staining with an original magnification of × 100. Lower images: Iba1+GFAP+DAPI staining with an original magnification of × 400. Data are mean values ± SEM, evaluated by one-way analysis of variance followed by Tukey's post hoc tests (n=5 animals per group). *p< 0.05, **p< 0.01, ***p< 0.001 compared with the saline-treated group; #p< 0.05, ##p< 0.01, ###p< 0.001 compared with the IFN-α-treated group. Scale bar, 100 μm.

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