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. 2020 Feb 17;17(1):61.
doi: 10.1186/s12974-020-01744-1.

Zerumbone ameliorates behavioral impairments and neuropathology in transgenic APP/PS1 mice by suppressing MAPK signaling

Lei Li  1 , Xiang-Hui Wu  1 , Xiao-Jing Zhao  1 , Lu Xu  1   2 , Cai-Long Pan  1   2 , Zhi-Yuan Zhang  3   4   5   6
Affiliations

Zerumbone ameliorates behavioral impairments and neuropathology in transgenic APP/PS1 mice by suppressing MAPK signaling

Lei Li et al. J Neuroinflammation. .

Abstract

Background: Alzheimer's disease (AD) is a major clinical problem, but there is a distinct lack of effective therapeutic drugs for this disease. We investigated the potential therapeutic effects of zerumbone, a subtropical ginger sesquiterpene, in transgenic APP/PS1 mice, rodent models of AD which exhibit cerebral amyloidosis and neuroinflammation.

Methods: The N9 microglial cell line and primary microglial cells were cultured to investigate the effects of zerumbone on microglia. APP/PS1 mice were treated with zerumbone, and non-cognitive and cognitive behavioral impairments were assessed and compared between the treatment and control groups. The animals were then sacrificed, and tissues were collected for further analysis. The potential therapeutic mechanism of zerumbone and the signaling pathways involved were also investigated by RT-PCR, western blot, nitric oxide detection, enzyme-linked immunosorbent assay, immunohistochemistry, immunofluorescence, and flow cytometry analysis.

Results: Zerumbone suppressed the expression of pro-inflammatory cytokines and induced a switch in microglial phenotype from the classic inflammatory phenotype to the alternative anti-inflammatory phenotype by inhibiting the mitogen-activated protein kinase (MAPK)/nuclear factor-kappa B signaling pathway in vitro. After a treatment period of 20 days, zerumbone significantly ameliorated deficits in both non-cognitive and cognitive behaviors in transgenic APP/PS1 mice. Zerumbone significantly reduced β-amyloid deposition and attenuated pro-inflammatory microglial activation in the cortex and hippocampus. Interestingly, zerumbone significantly increased the proportion of anti-inflammatory microglia among all activated microglia, potentially contributing to reduced β-amyloid deposition by enhancing phagocytosis. Meanwhile, zerumbone also reduced the expression of key molecules of the MAPK pathway, such as p38 and extracellular signal-regulated kinase.

Conclusions: Overall, zerumbone effectively ameliorated behavioral impairments, attenuated neuroinflammation, and reduced β-amyloid deposition in transgenic APP/PS1 mice. Zerumbone exhibited substantial anti-inflammatory activity in microglial cells and induced a phenotypic switch in microglia from the pro-inflammatory phenotype to the anti-inflammatory phenotype by inhibiting the MAPK signaling pathway, which may play an important role in its neuroprotective effects. Our results suggest that zerumbone is a potential therapeutic agent for human neuroinflammatory and neurodegenerative diseases, in particular AD.

Keywords: APP/PS1 transgenic mouse; Alzheimer’s disease; Cerebral amyloidosis; MAPK signaling pathway; Neuroinflammation; Zerumbone.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Molecular structure of zerumbone
Fig. 2
Fig. 2
Zerumbone reduces inflammation and promotes the phenotypic conversion of microglia in vitro. The murine N9 microglial cell line and primary microglia isolated from newborn mice were stimulated with lipopolysaccharide (1 μg/ml) or β-amyloid (Aβ; 10 μM), and incubated with or without zerumbone (1, 3, or 10 μg/ml) for 24 h. a Bar graph showing the nitric oxide (NO) concentration in N9 cells. Zerumbone treatment significantly reduced NO production. b Bar graph showing the expression levels of indicated genes including interleukin-1β (IL-1β), interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), CD206, interleukin-10 (IL-10), and arginase-1 (ARG-1) in N9 cells. Zerumbone treatment significantly decreased the mRNA levels of IL-1β, IL-6, iNOS, and TNF-α, and increased the mRNA levels of CD206, IL-10, and ARG-1 in a dose-dependent manner. c The purity of primary microglia was identified using flow cytometry, and CD11b+/CD45low cells were confirmed as microglia. d, e Cell counting kit-8 (CCK8) experiments showed that neither Aβ nor different doses of zerumbone affected cell number or viability. f Bar graph showing the NO concentration in primary microglia cells. Zerumbone treatment significantly reduced NO production. g Bar graph showing the expression levels of indicated genes including IL-1β, IL-6, iNOS, TNF-α, CD206, IL-10, and ARG-1 in primary microglia. Zerumbone treatment significantly decreased the mRNA levels of IL-1β, IL-6, iNOS, and TNF-α, and increased the mRNA levels of CD206, IL-10, and ARG-1 in a dose-dependent manner. h Bar graph showing the concentrations of cytokines (IL-1β, TNF-α, and IL-10) in the cell culture supernatants of primary microglia. Zerumbone treatment significantly decreased the levels of IL-1β and TNF-α, and increased the levels of IL-10. i Fluorescence-activated cell sorting (FACS) results and a bar graph showing that zerumbone increased the phagocytosis of Aβ in microglia cells. Data are presented as median and interquartile ranges (n = 5). *p < 0.05, **p < 0.01
Fig. 3
Fig. 3
Zerumbone attenuates mitogen-activated protein kinase activation in microglial cells. a Bar graph showing the concentration of prostaglandin E2 (PGE2) in N9 cells. The production of PGE2 was significantly reduced by zerumbone. b Representative western blots and a bar graph showing the protein expression of cyclooxygenase-2 (Cox-2) in N9 cells. The production of Cox-2 was significantly reduced by zerumbone. c Representative western blots and a bar graph showing the protein expression of microsomal prostaglandin E synthase-1 (mPGES-1) in N9 cells. The production of mPGES-1 was significantly reduced by zerumbone. d Representative western blots and a bar graph showing the protein expression levels of p-extracellular signal-regulated kinase (p-ERK), ERK, p-p38, p38, p-IκBα, IκBα, p-p65, and p65 in N9 cells. Zerumbone treatment significantly decreased the levels of p-ERK, p-p38, p-IκBα, and p-p65. e Zerumbone inhibited the translocation of nuclear factor-kappa B (NF-κB) from the cytosol to the nucleus in N9 cells treated with Aβ for 24 h. f The concentrations of cytokines (interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10)) in the cell culture supernatants of N9 microglia. Zerumbone, U0126, SB202190, and BAY 11-7082 blocked the Aβ induced up-regulation of IL-1β and TNF-α production. The Aβ-induced decrease in IL-10 production was also reversed by zerumbone, U0126, SB202190, and BAY 11-7082. Data are presented as median and interquartile ranges (n = 4). Zer1, 3, 10 = zerumbone concentrations of 1, 3, or 10 μg/ml. U, U0126 (an ERK inhibitor); SB, SB202190 (a p38 inhibitor); BAY, BAY 11–7082 (an NF-κB inhibitor). g, h Primary microglia were isolated from the cortex of vehicle- and zerumbone-treated APP/PS1 mice. The microglia isolated from zerumbone-treated mice exhibited decreased protein levels of p-ERK1/2, p-p38, p-IκBα, and p-p65 compared to vehicle-treated mice. Data are presented as median and interquartile ranges (n = 3). *p < 0.05, **p < 0.01
Fig. 4
Fig. 4
Zerumbone alleviates behavioral impairments in APP/PS1 mice. APP/PS1 mice were treated for 20 days with zerumbone (or carboxymethylcellulose (CMC), vehicle) by gavage. a A nest construction assay was conducted involving a 3-point scale. The nest-building scores at day 0 and day 10 were not significantly different between the vehicle- and zerumbone-treated APP/PS1 mice. At day 20, zerumbone-treated mice exhibited significantly higher nest-building scores compared to vehicle-treated APP/PS1 mice. b, c A resident-intruder assay was conducted, and the distances traveled and numbers of interactive events were analyzed. Bar graph showing the distances traveled by vehicle- and zerumbone-treated APP/PS1 mice before and after zerumbone administration for 20 days. The vehicle- and zerumbone-treated mice did not exhibit significant differences in distances traveled at any time point (b). c Bar graph showing the number of interactive events of vehicle- and zerumbone-treated APP/PS1 mice before and after 20 days of treatment. At day 20, zerumbone-treated mice exhibited a significantly higher frequency of interactive behavior. d In the novel object recognition test, the length of time that the mouse spent exploring the novel object was recorded and the recognition index was calculated. The bar graph shows the recognition index of vehicle- or zerumbone-treated wild-type (WT) and APP/PS1 mice. At day 20, zerumbone significantly increased the recognition index of APP/PS1 mice. e Zerumbone ameliorated cognitive deficits in APP/PS1 mice in the hidden platform test of the Morris water maze test. All mice showed shorter escape latency on the fourth day, and APP/PS1 mice showed impaired cognitive function compared to WT mice. Zerumbone-treated APP/PS1 mice showed significant improvements in cognitive function during the 4 days of training compared to the vehicle-treated APP/PS1 mice. Data are presented as median and interquartile ranges (n = 7). *p < 0.05, **p < 0.01
Fig. 5
Fig. 5
Zerumbone decreases Aβ deposition and microglial activation in APP/PS1 mice. Five-month-old APP/PS1–21 mice, five males and two females, were treated for 20 days with zerumbone (25 mg/kg by daily gavage). a–d Representative photomicrographs of β-amyloid (Aβ) immunohistochemical staining and ionized calcium-binding adaptor molecule 1 (Iba-1) immunohistochemical staining in the cortex of vehicle- and zerumbone-treated APP/PS1 mice. Zerumbone reduced Aβ deposition and microglial activation in the cortex of APP/PS1 mice. e–h Representative photomicrographs of Aβ immunohistochemical staining and Iba-1 immunohistochemical staining in the hippocampus of vehicle- and zerumbone-treated APP/PS1 mice. Zerumbone reduced Aβ deposition and microglial activation in the hippocampus of APP/PS1 mice. Scale bar in (a–h) = 100 mm. i Quantification of the numbers of Aβ plaques in the cortex and hippocampus of APP/PS1 mice. j Quantification of the percentage areas of the Aβ staining in the cortex and hippocampus of APP/PS1 mice. k Quantification of the percentage areas of the Iba-1 staining in the cortex and hippocampus of APP/PS1 mice. l Soluble and insoluble Aβ protein levels were measured in half-brain homogenates by ELISA. AD mice treated with Zerumbone decreased the concentration of Aβ in brains significantly. m Representative photomicrographs of glial fibrillary acidic protein (GFAP) immunofluorescence in the hippocampus of vehicle- and zerumbone-treated APP/PS1 mice. Statistical analysis showed no significant differences between the groups. n Shape analyses of two-dimensional somatic projections based on maximum length (L) and projection area (A) in confocal images of Iba-1-immunostained microglia using ImageJ. The somatic shape index (L:A ratio) increases in rod-shaped somata. The L, A, and L:A ratio of microglial somata were measured. o Representative photomicrographs of Aβ and Iba-1 double immunofluorescence in the cortex of vehicle- and zerumbone-treated APP/PS1 mice. Zerumbone treatment increased the phagocytosis of Aβ by Iba-1+ microglia (n = 7). Data are presented as median and interquartile ranges. *p < 0.05
Fig. 6
Fig. 6
Zerumbone increases the expression of CD206 (an anti-inflammatory marker) in the cortex of APP/PS1 mice. Five-month-old APP/PS1-21 mice, five males and two females, were treated for 20 days with zerumbone (25 mg/kg by daily gavage). a Representative photomicrographs of CD206 immunohistochemical staining in the cortex of APP/PS1 mice. b Representative photomicrographs of CD206 and ionized calcium-binding adaptor molecule 1 (Iba-1) double immunohistochemical staining in the cortex of APP/PS1 mice. c Representative photomicrographs of Iba-1 and β-amyloid (Aβ) double immunohistochemical staining in the cortex of APP/PS1 mice. d Representative photomicrographs of CD206 and Aβ double immunohistochemical staining in the cortex of APP/PS1 mice. e Quantification of the percentage of CD206 and Iba-1 in the cortex of vehicle- and zerumbone-treated APP/PS1 mice. The areas of CD206 IR and Iba-1+CD206 double IR in the cortex were slightly increased by zerumbone treatment. f Quantification of the ratio of the area of Iba-1 + CD206 double IR to the area of Iba-1 IR in the cortex of vehicle- and zerumbone-treated APP/PS1 mice. The percentage of Iba-1 + CD206 double staining area to Iba-1 IR was significantly increased by zerumbone treatment. g Quantification of the ratio of the area of Iba-1 + CD206 double IR to the area of Aβ IR in the cortex of vehicle and zerumbone-treated APP/PS1 mice. The ratio of the area of Iba-1 + CD206 double IR to the area of Aβ IR was significantly increased by zerumbone treatment. h Representative photomicrographs of CD206 and Iba-1 immunofluorescence double staining in the cortex of APP/PS1 mice. The percentage areas of CD206 and the ratio of the area of Iba-1 + CD206 double IR to the area of Iba-1 IR were determined (n = 7). *p < 0.05. i Representative immunofluorescence images, and quantitative analysis of synaptophysin in the hippocampal CA3 and in the DG, in the brains of Zerumbone treated AD mice and vehicle-treated AD mice (Scale bars, 100 μm) (n = 5)
Fig. 7
Fig. 7
Schematic drawing depicting the suppression of MAPK signaling in microglia to alleviate AD by zerumbone. In the Alzheimer’s disease (AD) brain, β-amyloid (Aβ) induces the release of interleukin-1β (IL-1β), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS) and activates inflammatory microglia, suppressing Aβ phagocytosis and promoting behavioral impairments (left, red). In this study, zerumbone inhibited the production of prostaglandin E2 (PGE2), cyclooxygenase-2 (Cox-2), and microsomal prostaglandin E synthase-1 (mPGEs-1). It also increased the proportion of anti-inflammatory microglia and ameliorated behavioral impairments and neuropathological changes in transgenic APP/PS1 mice by suppressing mitogen-activated protein kinase (MAPK) signaling (green). The inhibition of p-extracellular signal-related kinase (p-ERK), p-p38, and nuclear factor-kappa B (NF-κB) was a critical mechanism underlying the neuroprotective effect of zerumbone in microglia (right)
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