Dietary fructose-induced gut dysbiosis promotes mouse hippocampal neuroinflammation: a benefit of short-chain fatty acids

Abstract

Background: Western-style diets arouse neuroinflammation and impair emotional and cognitive behavior in humans and animals. Our previous study showed that a high-fructose diet caused the hippocampal neuroinflammatory response and neuronal loss in animals, but the underlying mechanisms remained elusive. Here, alterations in the gut microbiota and intestinal epithelial barrier were investigated as the causes of hippocampal neuroinflammation induced by high-fructose diet.

Results: A high-fructose diet caused the hippocampal neuroinflammatory response, reactive gliosis, and neuronal loss in C57BL/6N mice. Depletion of the gut microbiota using broad-spectrum antibiotics suppressed the hippocampal neuroinflammatory response in fructose-fed mice, but these animals still exhibited neuronal loss. Gut microbiota compositional alteration, short-chain fatty acids (SCFAs) reduction, intestinal epithelial barrier impairment, NOD-like receptor family pyrin domain-containing 6 (NLRP6) inflammasome dysfunction, high levels of serum endotoxin, and FITC-dextran were observed in fructose-fed mice. Of note, SCFAs, as well as pioglitazone (a selective peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist), shaped the gut microbiota and ameliorated intestinal epithelial barrier impairment and NLRP6 inflammasome dysfunction in fructose-fed mice. Moreover, SCFAs-mediated NLRP6 inflammasome activation was inhibited by histamine (a bacterial metabolite) in ex vivo colonic explants and suppressed in murine CT26 colon carcinoma cells transfected with NLRP6 siRNA. However, pioglitazone and GW9662 (a PPAR-γ antagonist) exerted no impact on SCFAs-mediated NLRP6 inflammasome activation in ex vivo colonic explants, suggesting that SCFAs may stimulate NLRP6 inflammasome independently of PPAR-γ activation. SCFAs and pioglitazone prevented fructose-induced hippocampal neuroinflammatory response and neuronal loss in mice. Additionally, SCFAs activated colonic NLRP6 inflammasome and increased DCX⁺ newborn neurons in the hippocampal DG of control mice.

Conclusions: Our findings reveal that gut dysbiosis is a critical factor for a high-fructose diet-induced hippocampal neuroinflammation in C57BL/6N mice possibly mediated by impairing intestinal epithelial barrier. Mechanistically, the defective colonic NLRP6 inflammasome is responsible for intestinal epithelial barrier impairment. SCFAs can stimulate NLRP6 inflammasome and ameliorate the impairment of intestinal epithelial barrier, resulting in the protection against a high-fructose diet-induced hippocampal neuroinflammation and neuronal loss. This study addresses a gap in the understanding of neuronal injury associated with Western-style diets. A new intervention strategy for reducing the risk of neurodegenerative diseases through SCFAs supplementation or dietary fiber consumption is emphasized.

Keywords: Gut dysbiosis; NLRP6 inflammasome; Neuroinflammation; Neuronal loss; Short-chain fatty acids.

Fig. 1

Depletion of the gut microbiota inhibits hippocampal neuroinflammation but fails to reverse the neuronal loss in high-fructose diet-fed C57BL/6N mice. a TNF-α, IL-1β, and IL-6 mRNA levels in the hippocampus (n = 6). b Representative immunofluorescence images and quantitative analysis of Iba-1-positive cells (red), DCX-positive cells (green), NeuN-positive cells (red), and GFAP-positive cells (green) with nuclear counterstain (blue) in the hippocampal DG. Bars, 25 μm. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant difference. C control group, F₁ eight-week fructose-fed group, AB antibiotics-treated group

Fig. 2

A high-fructose diet induces gut dysbiosis, SCFAs reduction, and colonic epithelial barrier impairment in C57BL/6N mice. a PCoA based on the relative abundance of bacterial OTU, b Chao1 diversity indexes of bacterial community, c relative abundance of bacterial phyla, and d SCFA concentrations in fecal samples (n = 7). e Colon histopathology (bars, 50 μm). f Endotoxin and FITC-dextran levels in serum (n = 8). g Representative transmission electron micrographs of colon epithelial cells (bars, 1 μm). Arrows indicate gap junctions between two neighbored cells. Asterisks indicate the mitochondria in epithelial cells. h Immunoblot analysis for protein levels of Muc2, occludin, and ZO-1 in colon tissue (n = 6). Quantification: band intensity normalized to β-actin. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant difference. C control group, F₁ eight-week fructose-fed group, AB antibiotics-treated group

Fig. 3

SCFAs and pioglitazone shape gut dysbiosis and improve intestinal epithelial barrier impairment in high-fructose diet-fed C57BL/6N mice. a PCoA based on the relative abundance of bacterial OTU, b Chao1 diversity indexes of bacterial community, and c relative abundance of bacterial phyla in fecal samples (n = 7). d Colon histopathology (bars, 50 μm). e Endotoxin levels in serum (n = 8). f Representative transmission electron micrographs of colonic epithelial cells (bars, 1 μm). Arrows indicate gap junctions between two neighbored cells. Asterisks indicate the mitochondria in epithelial cells. g Immunoblot analysis for protein levels of Muc2, occludin, and ZO-1 in colon tissue (n = 6). Quantification: band intensity normalized to β-actin. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant difference. C control group, F₂ 12-week fructose-fed group, S SCFAs-treated group, P pioglitazone-treated group

Fig. 4

A high-fructose diet causes colonic NLRP6 inflammasome dysfunction in C57BL/6N mice, which is ameliorated by SCFAs. Immunoblot analysis of NLRP6, NLRP3, and caspase-1 P10/P45 and IL-18 production (a, b) in colonic tissues of mice (n = 6), c cultured ex vivo colonic explants (n = 6), and d cultured mouse colon CT26 cells from three independent experiments. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant difference. C control group, F₁ eight-week fructose-fed group, F₂ 12-week fructose-fed group, AB antibiotics-treated group, S SCFAs-treated group, P pioglitazone-treated group, H histamine-treated group, G GW9662-treated group

Fig. 5

SCFAs and pioglitazone inhibit hippocampal neuroinflammation and protect against neuronal loss in high-fructose diet-fed C57BL/6N mice. a TNF-α, IL-1β, and IL-6 mRNA levels in the hippocampus (n = 6). b Representative immunofluorescence images and quantitative analysis of Iba-1-positive cells (red), DCX-positive cells (green), NeuN-positive cells (red), and GFAP-positive cells (green) with nuclear counterstain (blue) in the hippocampal DG. Bars, 25 μm. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant difference. C control group, F₂ 12-week fructose-fed group, S SCFAs-treated group, P pioglitazone-treated group