Secoisolariciresinol diglucoside attenuates neuroinflammation and cognitive impairment in female Alzheimer's disease mice via modulating gut microbiota metabolism and GPER/CREB/BDNF pathway

Abstract

Background: Gender is a significant risk factor for late-onset Alzheimer's disease (AD), often attributed to the decline of estrogen. The plant estrogen secoisolariciresinol diglucoside (SDG) has demonstrated anti-inflammatory and neuroprotective effects. However, the protective effects and mechanisms of SDG in female AD remain unclear.

Methods: Ten-month-old female APPswe/PSEN1dE9 (APP/PS1) transgenic mice were treated with SDG to assess its potential ameliorative effects on cognitive impairments in a female AD model through a series of behavioral and biochemical experiments. Serum levels of gut microbial metabolites enterodiol (END) and enterolactone (ENL) were quantified using HPLC-MS. Correlation analysis and broad-spectrum antibiotic cocktail (ABx) treatment were employed to demonstrate the involvement of END and ENL in SDG's cognitive improvement effects in female APP/PS1 mice. Additionally, an acute neuroinflammation model was constructed in three-month-old C57BL/6J mice treated with lipopolysaccharide (LPS) and subjected to i.c.v. injection of G15, an inhibitor of G protein-coupled estrogen receptor (GPER), to investigate the mediating role of the estrogen receptor GPER in the cognitive benefits conferred by SDG.

Results: SDG administration resulted in significant improvements in spatial, recognition, and working memory in female APP/PS1 mice. Neuroprotective effects were observed, including enhanced expression of CREB/BDNF and PSD-95, reduced β-amyloid (Aβ) deposition, and decreased levels of TNF-α, IL-6, and IL-10. SDG also altered gut microbiota composition, increasing serum levels of END and ENL. Correlation analysis indicated significant associations between END, ENL, cognitive performance, hippocampal Aβ-related protein mRNA expression, and cortical neuroinflammatory cytokine levels. The removal of gut microbiota inhibited END and ENL production and eliminated the neuroprotective effects of SDG. Furthermore, GPER was found to mediate the inhibitory effects of SDG on neuroinflammatory responses.

Conclusion: These findings suggest that SDG promotes the production of gut microbial metabolites END and ENL, which inhibit cerebral β-amyloid deposition, activate GPER to enhance CREB/BDNF signaling pathways, and suppress neuroinflammatory responses. Consequently, SDG exerts neuroprotective effects and ameliorates cognitive impairments associated with AD in female mice.

Keywords: Alzheimer’s disease; Cognitive impairment; GPER; Gut microbiota; Neuroinflammation; Secoisolariciresinol diglucoside.

Fig. 1

SDG alleviated cognitive impairment in female APP/PS1 mice. (A) Experimental schedule of the SDG intervention in female APP/PS1 mice (n = 6–10). (B) Body weight gain during the 8-week intervention (n = 6–10). (C) The average weekly food intake and (D) water intake of each mouse (n = 6–10). (E) Escape latency change during the MWM test training days (n = 6–10, *p < 0.05, **p < 0.01, compared to the WT + Vehicle group, #p < 0.05, ##p < 0.01, compared to the APP/PS1 + Vehicle group). (F) Escape latency and (G) average speed on the testing day (n = 6–10). (H) Preference index in the NOR test (n = 6–10). (I) Spontaneous alternation (%) in the Y-maze test (n = 6–10). Data are presented as mean ± SEM and were analyzed using two-way ANOVA with Tukey’s test. *p < 0.05, **p < 0.01, n.s., no significance

Fig. 2

SDG promoted the expression of synapse-related protein and BDNF. (A) Representative images of PSD-95 immunofluorescence staining in the hippocampal DG and cortex.( B) and (C) Quantification of PSD-95 positive area based on immunofluorescence staining sections by ImageJ software (n = 3). (D) Representative images of BDNF immunofluorescence staining in the hippocampal DG and cortex. (E) and (F) Quantification of BDNF positive area based on immunofluorescence staining sections by ImageJ software (n = 3). (G) and (H) The mRNA expression levels of PSD-95 and BDNF in the hippocampus region (n = 6–10). (I) and (J) Protein levels of CREB and p-CREB in the cortex (n = 3). Data are presented as mean ± SEM and were analyzed using two-way ANOVA with Tukey’s test. *p < 0.05, **p < 0.01, n.s., no significance

Fig. 3

SDG reduced Aβ deposition and neuroinflammatory response. (A) Representative images of Aβ plaques immunofluorescence staining in the hippocampal DG and cortex. (B) and (C) Quantification of Aβ deposition area based on immunofluorescence staining sections by ImageJ software (n = 3). (D) – (F) The mRNA expression levels of APP, PS1, and BACE1 in the hippocampus region (n = 6–10). (G) Representative images of IBA-1 immunofluorescence staining in the hippocampal DG and cortex.( H) and( I) Quantification of the number of IBA-1 positive cells based on immunofluorescence staining sections by ImageJ software (n = 3). (J) – (L) The levels of TNF-α, IL-6, and IL-10 in the cortex (n = 6–10). Data are presented as mean ± SEM and were analyzed using two-way ANOVA with Tukey’s test. *p < 0.05, **p < 0.01, n.s., no significance

Fig. 4

The effect of SDG on the gut microbiota composition. (A) Venn diagrams illustrating the discrepancy of OTUs. (B) – (C) Beta diversity index box (unweighted unifrac and weighted unifrac). The five lines from bottom to top are minimum, first quartile, median, third quartile, and maximum. (D) The result of Partial least squares discrimination analysis (PLS-DA) of each group. (E) Volcano plot of differential Relative abundance of OTUs in APP/PS1 + Vehicle and APP/PS1 + SDG group (grey represents not significant; green represents a significant Log2FC value; blue represents a significant p value; and red represents significant Log2FC value and p value). On the right side of the volcano plot, the top 3 OTUs (in blue font) with significantly increased and the top 10 OTUs (in red font) with significantly decreased were listed. The default p-value threshold is 0.05, and the default FC threshold is 2.0

Fig. 5

The gut microbiota-dependent SDG metabolism. (A) and (B) The levels of END and ENL in the serum. (C) Correlation Analysis between the levels of END and ENL in the serum and other biochemical index. In the matrix, the size and color of the circles indicate the degree of correlation (blue represents a positive correlation, and red represents a negative correlation). The calculation method for correlation coefficient is Pearson. *p < 0.05, **p < 0.01, and non-significance is not indicated

Fig. 6

The cognitive improvement effect of SDG depended on the existence of gut microbiota. (A) Experimental schedule of the SDG intervention after the ABx treatment in female APP/PS1 mice (n = 5). (B) The 16 S rDNA copies in the feces (n = 5). (C) Body weight gain during the 8-week intervention (n = 5). (D) The average weekly food intake and (E) water intake of each mouse (n = 5). (F) Escape latency change during the BM test training days (n = 5, #p < 0.05, ##p < 0.01, compared to the APP/PS1 group, &p < 0.05, &&p < 0.01, compared to the APP/PS1 + SDG group). (G) Head exploration latency and (H) Number of head explorations on the testing day (n = 5). (I) Preference index in the Novel object recognition test (n = 5). (J) Spontaneous alternation (%) in the Y-maze test (n = 5). (K) and (L) The levels of END and ENL in the serum. Data are presented as mean ± SEM and were analyzed using one-way ANOVA with Tukey’s test. *p < 0.05, **p < 0.01, n.s., no significance

Fig. 7

The promoting effect of SDG on PSD-95 and BDNF expression depended on gut microbiota existence. (A) Representative images of PSD-95 immunofluorescence staining in the hippocampal DG and cortex. (B) and (C) Quantification of PSD-95 positive area based on immunofluorescence staining sections by ImageJ software (n = 3). (D) Representative images of BDNF immunofluorescence staining in the hippocampal DG and cortex. (E) and (F) Quantification of BDNF positive area based on immunofluorescence staining sections by ImageJ software (n = 3). G) and H) Protein levels of CREB and p-CREB in the cortex (n = 4). Data are presented as mean ± SEM and were analyzed using one-way ANOVA with Tukey’s test. *p < 0.05, **p < 0.01, n.s., no significance

Fig. 8

The inhibitory effect of SDG on Aβ deposition and neuroinflammation depended on gut microbiota existence. (A) Representative images of Aβ plaques immunofluorescence staining in the hippocampal DG and cortex. (B) and (C) Quantification of Aβ deposition area based on immunofluorescence staining sections by ImageJ software (n = 3). (D) Representative images of IBA-1 immunofluorescence staining in the hippocampal DG and cortex. (E) and (F) Quantification of the number of IBA-1 positive cells based on immunofluorescence staining sections by ImageJ software (n = 3). (G) – (J) The levels of TNF-α, IL-6, IL-1β, and IL-10 in the cortex (n = 5). Data are presented as mean ± SEM and were analyzed using one-way ANOVA with Tukey’s test. *p < 0.05, **p < 0.01, n.s., no significance

Fig. 9

The activation of GPER was involved in the preventive effect of SDG on LPS-induced neuroinflammation. (A) Experimental schedule of the of SDG intervention on LPS induced neuroinflammation mouse model after G15 treatment (n = 7). (B-E) The mRNA expression levels of TNF-α, IL-6, IL-1β, and IL-10 in the hippocampus (n = 7). (F) and (G) The mRNA expression levels of PSD-95 and BDNF in the hippocampus (n = 7). Data are presented as mean ± SEM and were analyzed using one-way ANOVA with Tukey’s test. *p < 0.05, **p < 0.01, n.s., no significance

Fig. 10

SDG attenuated cognitive impairment in female APP/PS1 mice SDG is metabolized by gut microbiota to produce END and ENL, which in turn enhance the expression levels of PSD-95 and BDNF, and inhibit neuroinflammatory responses through GPER receptors, and reduce Aβ deposition to enhance the cognitive ability, such as spatial memory, recognition memory, and working memory in the female APP/PS1 mice