High Salt Elicits Brain Inflammation and Cognitive Dysfunction,Accompanied by Alternations in the Gut Microbiota and Decreased SCFA Production

Abstract

Background:

Excessive salt intake is considered as an important risk factor for cognitive impairment, which might be the consequence of imbalanced intestinal homeostasis.

Objective:

To investigate the effects of dietary salt on the gut microbiota and cognitive performance and the underlying mechanisms.

Methods:

Adult female C57BL/6 mice were maintained on either normal chow (control group, CON) or sodium-rich chow containing 8% NaCl (high-salt diet, HSD) for 8 weeks. Spatial learning and memory ability, short-chain fatty acids (SCFAs) concentrations, gut bacterial flora composition, blood-brain barrier permeability, and proinflammatory cytokine levels and apoptosis in the brain were evaluated.

Results:

The mice fed a HSD for 8 weeks displayed impaired learning and memory abilities. HSD significantly reduced the proportions of Bacteroidetes (S24-7 and Alloprevotella) and Proteobacteria and increased that of Firmicutes (Lachnospiraceae and Ruminococcaceae). SCFA concentrations decreased in the absolute concentrations of acetate, propionate, and butyrate in the fecal samples from the HSD-fed mice. The HSD induced both BBB dysfunction and microglial activation in the mouse brain, and increased the IL-1β, IL-6, and TNF-α expression levels in the cortex. More importantly, the degree of apoptosis was higher in the cortex and hippocampus region of mice fed the HSD, and this effect was accompanied by significantly higher expression of cleaved caspase-3, caspase-3, and caspase-1.

Conclusion:

The HSD directly causes cognitive dysfunction in mice by eliciting an inflammatory environment and triggering apoptosis in the brain, and these effects are accompanied by gut dysbiosis, particularly reduced SCFA production.

Keywords

Blood-brain barrier cognition gut microbiota high-salt diet microglia short-chain fatty acids

INTRODUCTION

High salt is thought to promote cognitive decline via hypertension [1, 2], but excessive salt can also impact brain function without inducing a marked increase in the blood pressure [3, 4]. A recent study showed that a high-salt diet (HSD) reduces the resting blood flow to the brain and caused dementia in mice [5]. In addition, an increase in the gut proportion of Th17 cells, which produce interleukin-17 (IL-17) to induce immune regulation and proinflammatory effects [6, 7], impair the synaptic plasticity, and increase oxidative stress [8], was also found to be involved in HSD-related cognitive dysfunction, which indicates the intricate effects of high salt on cognition.

The human gastrointestinal tract harbors trillions of microorganisms, which include at least 1000 different species of known bacteria with more than 3 million genes, and these microorganisms are termed the gut microbiota (GM) [9]. The GM and its metabolites can modulate not only gastrointestinal function but also enter the circulation to affect the brain. The complex interplay between the GM and the brain is denoted as the “gut-brain axis” [10], and this bidirectional interaction appears to act via neural, endocrine, immune, and humoral links. More than 90% of the important brain neurotransmitter 5-HT is synthesized in the gut [11], and at least 20% of the small molecules in human blood are products of the microbiota [12]. An important set of metabolite of carbohydrates produced by the microbiota in the lower gut, namely, short-chain fatty acids (SCFAs), which are the main energy supply material of the colon and small intestine epithelial cells, modulates microglia development and function through undetermined mechanisms [13, 14].

Because sodium added or inherent to food contributes to nearly 99% of the total sodium intake [9], the effects of dietary salt on the GM and brain function need to be elucidated. A recent study demonstrated that an HSD exerts a certain effect on protein digestion and the composition of the GM [15, 16]. To explore the association between the GM and cognitive performance induced by dietary salt, we used a mouse model fed an HSD for 8 weeks and found cognitive dysfunction, alteration of the GM, decreases in the concentrations of fecal SCFAs and increases in the blood-brain barrier (BBB) permeability.

MATERIAL AND METHODS

Animals and treatments

The adult female C57BL/6 mice (8–10 weeks, 20–24 g) used in the study were purchased from the Nanjing Biomedical Research Institute of Nanjing University and housed in the Animal Care Facility at Guangdong Medical University. The mice were maintained in a pathogen-free environment on a 12 h light/12 h dark cycle. The mice were randomly grouped after one week of acclimatization and were supplied water and chow ad libitum. The mice received normal chow and tap water (control group) or sodium-rich chow containing 8% NaCl (high-salt diet, HSD group). After 8 weeks of treatment, the mice were subjected to the Morris water maze (MWM). The mice were subsequently anaesthetized and killed via the inhalation of isoflurane. Brain tissues were collected and stored at –80°C for further analyses as detailed below. The animal protocol was approved by the Ethics Committee of Guangdong Medical University in accordance with the guidelines of the National Institutes of Health (NIH) on the care and use of animals.

Morris water maze

The MWM task was used to assess the spatial learning and memory ability of the mice as described previously. Briefly, a round pool with a diameter of 1.2 m was filled with water (22±1°C) that was made opaque with a nontoxic white pigment, and a circular platform (10 cm in diameter) was submerged ∼1 cm beneath the surface of the water and placed in the center of one quadrant of the pool. The platform remained in the same position throughout the learning trials and was removed from the pool during the probe test. Several additional maze cues were placed around the pool and remained in the same position throughout the training and testing periods. For training, the mice were subjected to four trials per day for 5 consecutive days (at 30 min intervals). The data obtained each day were averaged across the four trials. A probe trial was conducted on day 6; the hidden platform was removed, and the mice were placed in the quadrant opposite to the target quadrant and allowed to swim for 90 s. The number of platform crossings was scored. During the training period (days 1–5), the time that it took the mice to find the platform was recorded as the escape latency, and an escape latency of 90 s was recorded if the animal did not find the platform. The data were analyzed by two-way repeated-measures ANOVA with the group as a between-subject factor and the trial day as a within-subject factor for the training trials. For the probe trials, the quadrant or platform location was used as the within-subject factor.

DNA extraction from mouse feces and microbiota analysis

Individual fecal pellets from each mouse were collected and pooled in separate autoclaved cages within 30 min. The microbial DNA from the fecal samples was extracted using the QIAamp^® PowerFecal DNA Kit (QIAGEN GmbH, Germany) according to the manufacturer’s recommended protocols. The final DNA concentration and purification were determined using a NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and the DNA quality was checked by 1% agarose gel electrophoresis. The V3–V4 hypervariable regions of the bacterial 16S rRNA gene were amplified using the primers 338F (5’- ACTCCTACGGGAGGCAGCAG-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) and a thermocycler PCR system (GeneAmp 9700, ABI, USA). The PCR programs were as follows: 3 min of denaturation at 95°C; 27 cycles of 30 s at 95°C, 30 s of annealing at 55°C, and 45 s of elongation at 72°C; and a final extension at 72°C for 10 min. The PCRs were performed in triplicate using 20μL reaction volumes consisting of 4μL of 5×FastPfu Buffer, 2μL of 2.5 mM dNTPs, 0.8μL of each primer (5μM), 0.4μL of FastPfu Polymerase, and 10 ng of template DNA. The resulting PCR products were extracted from a 2% agarose gel, further purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified using QuantiFluor™-ST (Promega, USA) according to the manufacturer’s recommended protocol. An Illumina MiSeq was run for 250 cycles to produce paired-end reads. The data were analyzed using the free online Majorbio I-Sanger Cloud Platform (http://www.i-sanger.com).

Histology

From each mouse, the right half of the brain was removed and fixed in 4% buffered formalin. The brain tissue was dehydrated in gradient sucrose solution and then embedded in OCT. For Iba1 immunohistochemistry, 15μm coronal cryosections were prepared and stained with the anti-Iba1 antibody (1 : 100 dilution, cat. no. ab143696, Abcam) for 24 h at 4°C and then with the secondary antibody (Alexa Flour 488-conjugated anti-rabbit IgG, 1 : 800) at RT. For the assessment of apoptotic cells in the mouse cortex and hippocampus, the brain sections were stained with Hoechst 33258 as described previously [17] and according to the manufacturer’s instructions (Beyotime Biotechnology Inc, China).

Fecal SCFA analyses

The fecal concentrations of SCFAs (acetate, propionate, and butyrate) were determined by gas chromatography (GC) as described previously [18]. Briefly, 500 mg of feces was homogenized with 1 mL of phosphate buffer (pH 7.3) and centrifuged at 4°C and 16,000×g for 15 min. The supernatants were filtered through a 0.45μm nylon filter (EMD Millipore). For acidification, an aliquot (200μL) of the supernatant was added to 50μL of 50% (v/v) sulfuric acid. The mixture was vortex and allowed to stand for 2 min, and the organic acids were extracted by adding 0.4 mL of diethyl ether. The supernatants were measured with a GC-2010 gas chromatograph (Shimadzu Japan) equipped with flame ionization using the autosampler AOC-20s/I (Shimadzu Japan), an injected sample volume of 1μL, thermal conductivity detectors, capillary columns and GC ChemStation software. Nitrogen was supplied as the carrier gas at an initial flow rate of 1.3 mL/min. The oven temperature was initially set to 100°C, maintained at 100°C for 0.5 min, increased to 180°C at 8°C/min, maintained at 180°C for 1.0 min, increased to 200°C at 20°C/min, and maintained at 200°C for 5.0 min. The temperatures of the detector and the injection port were set to 250°C and 240°C, respectively. An external standard (Supelco™ WSFA-1 Mix, Supelco Sigma-Aldrich Co., Bellefonte, PA, USA) was used for the quantification of SCFAs. The GC solution Chromatography Data System (Shimadzu Deutschland GmbH) was used for data processing.

Evaluation of BBB disruption

The BBB permeability was determined by measuring the amount of Evans blue dye (EB). The extravasation of EB was performed as described previously [19, 20]. Briefly, 2% EB (3 mL/kg body weight, Sigma, USA) was slowly administered intravenously to the mice in the control and HSD groups. One hour after injection, the mice were perfused with saline to wash any remaining intravascular EB dye. After decapitation, the brain was rapidly removed and dissected to obtain the left and right hemispheres. The left hemisphere was weighed and homogenized in a 2-fold volume of dimethylformamide. After incubation at 60°C for 72 h, the samples were centrifuged for 10 min at 1500 r/min, and the supernatants were used to measure the absorbance of EB using a spectrophotometer (BioTek, USA) at 635 nm. The amount of EB was determined using a standard curve and is expressed as micrograms per gram of brain tissue.

Immunoblotting and western blot analysis

The brain tissues were lysed by ultrasonication in lysis buffer [150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 0.1% sodium deoxycholate, 1% Triton-X-100, 0.1% SDS, 1% Protease Inhibitor Cocktail, and/or Phosphatase Inhibitor (Roche)] and centrifuged. The protein concentrations were determined using the BCA method (Thermo Scientific). Equal volumes of tissue lysates (50μL of protein) were mixed with SDS loading buffer, and the mixtures were boiled, separated on 8–15% SDS polyacrylamide gels and then transferred to PVDF membranes (Millipore). After blocking with 5% nonfat milk in TBS/0.1% Tween-20 (TBST) for 1 h, membranes were incubated with primary antibodies against Iba1 (1 : 1000; ab153696, Abcam), claudin-5 (1 : 500; ab15106, Abcam), caspase-3 (1 : 1000, cat. #9662, Cell Signaling Technology), cleaved caspase-3 (1 : 1000, cat. #9661, Cell Signaling Technology), caspase-1 (1 : 1000, cat. #2225, Cell Signaling Technology), β-tubulin III (1 : 1000, T8578, Sigma) and beta-actin (1 : 1000, cat. #8457, Cell Signaling Technology) overnight at 4°C. The membranes were washed in TBST, incubated with the secondary antibody (1 : 5000 in TBST) for 1 h, and visualized using enhanced chemiluminescence (ECL) (Thermo Fisher Scientific), and the densitometric results were analyzed using ImageJ software. The signal intensity of the proteins of interest was normalized to that of beta-actin or β-tubulin III.

Quantification of cytokines

The concentration of cytokines in mouse cortex lysates was measured by enzyme-linked immunosorbent assay (ELISA) according to the instructions provided by the manufacturer (Beijing 4A Biotech Co., Ltd., China). The absorbance at 450 nm was quantified using an Epoch Microplate Spectrophotometer (BioTek).

Statistical analysis

All the values are expressed as the means±SEMs. The data were analyzed using GraphPad Prism version 6.01 for Windows (GraphPad Software, La Jolla, CA, USA). The data from the MWM and SCFA assays were analyzed by two-way ANOVA followed by Tukey’s post hoc test. The parametric data were analyzed by unpaired t tests. p < 0.05 was considered significant.

RESULTS

HSD impairs the spatial learning and memory ability of mice

The spatial learning and memory ability of the mice were assessed using the MWM test. The amount of time that elapsed until the animal climbed onto the platform to escape the water (escape latency) and the number of platform crossings were recorded and analyzed. The mice fed the normal diet or HSD learned to find the platform, as indicated by a reduction in the escape latency over the 5-day training period (Fig. 1A). As demonstrated by repeated-measures two-way ANOVA, the mice in the HSD group spent more time trying to find the platform than those in the control group, and significant differences were found on days 4 and 5. During the probe trial (day 6), the number of platform crossings obtained for the HSD group was significantly lower than that found for the control group, which suggested a deficit in spatial learning memory. Taken together, the results indicated that the spatial learning and memory abilities of the HSD group were significantly damaged.

Fig. 1

Effects of the high-salt diet (HSD) on the learning and memory ability of mice. These abilities were assessed using the Morris water maze. A) The mice in the HSD group spent more time searching for the platform during the navigation test period than the control mice, as demonstrated by an analysis of the average escape latency calculated each day, and this difference was significant on days 4 and 5. B, C) Total travel distance and average swim speed of the mice on the training days. D) Number of platform crossings during the probe trial. E, F) Representative tracks on day 5 obtained for the mice in the control and HSD groups during the navigation test. n = 7 in each group. The data are expressed as the means±SEMs. *p < 0.05.

HSD induces alterations in the GM

To analyze the effects of the HSD on the microbiota composition, we performed 16S ribosomal (rRNA) gene sequencing of the mouse fecal contents. The sequence analysis of the V3–V4 hypervariable regions of the 16S rRNA gene revealed that the microbiota composition was considerably altered by the consumption of the HSD for 8 weeks. The phylum-level analysis showed that the HSD significantly reduced the proportions of Bacteroidetes (50.53±10.75%) and Proteobacteria (2.96±0.54%) and increased that of Firmicutes (42.77±10.88%) compared with the control levels (70.73±7.52%, 6.69±3.50%, and 19.9±5.81%, respectively, Fig. 2A). The family-level analysis showed that the HSD mice presented significantly reduced sequence proportions of S24-7 and Alloprevotella (phylum Bacteroidetes) and higher sequence proportions of Lachnospiraceae and Ruminococcaceae (phylum Firmicutes; Fig. 2D). More details are available in the Supplementary Material.

Fig. 2

Effects of the high-salt diet (HSD) on the mouse gut microbiota composition. A) Differences between two groups at the phylum level. Four primary phyla were identified. The proportion of Firmicutes was significantly increased in the HSD group, whereas the proportion of Bacteroidetes and Proteobacteria was decreased significantly in this group. B) Linear discriminant analysis (LDA) combined with effect size measurements (LEfSe) at the species level. Differences with p values <0.05 and score ≥2.0 are shown. C) Taxonomic cladogram representing the taxa with differential abundance between the two groups. The bacterial taxa enriched in the normal diet (CON) and HSD groups are shown in green and red, respectively. The size of each dot is proportional to the taxon abundance. D) Relative abundances of S24-7, Alloprevotella, Ruminococcaceae and Lactobacillaceae in the CON and HSD groups. n = 5 in each group. The data are expressed as the mean±SEM. *p < 0.05, **p < 0.01.

HSD decreases the fecal SCFA concentrations in mice

The SCFA levels were measured by GC. The quantitative analysis of the SCFA concentrations in the fecal samples revealed notable decreases in the absolute concentrations of acetate, propionate, and butyrate and a significant reduction in the acetate concentration (Fig. 3A) in the HSD-fed mice compared with the control mice. The lowered fecal SCFA concentrations suggested that the HSD inhibited bacterial fermentation and decreased the SCFA availability in the lower gut.

HSD induces BBB dysfunction in mice

The BBB permeability was determined by measuring the amount of Evans blue dye in the brain 3 h after tail vein injection. Marked increases in the Evans blue content were found in the brain of the HSD-fed mice rain compared with those of the normal diet-fed mice (17.5±4.9 versus 4.6±0.6μg/ml [F_(3,16) = 10.4, p < 0.01], see Fig. 3B). To examine the expression of tight junction proteins in the mouse brain, we measured the expression of claudin-5 by western blotting. The protein level of claudin-5 in the hippocampus of the HSD-fed mice was significantly lower than the level found in the control mouse brain (Fig. 3C).

Fig. 3

Effects of the high-salt diet (HSD) on the fecal short-chain fatty acid (SCFA) contents, blood-brain barrier (BBB) integrity, microglial activation, and caspase expression. A) The HSD significantly decreased the fecal acetate, propionate and butyrate contents (n = 4 in each group). B) The HSD significantly increased the BBB permeability, as indicated by an increased brain Evans blue dye content (CON, n = 3; HSD, n = 4). C, The HSD decreased the claudin-5 expression level and increased the Iba1 expression level in the hippocampus. The expression levels of hippocampus claudin-5 and Iba1 are relative to those of β-actin (n = 5 in each group). D, E) The HSD significantly increased the expression of cleaved caspase-3, caspase-3, and caspase-1 in the cortex (n = 5 in each group). The data are expressed as the means±SEMs. *p < 0.05, **p < 0.01, ***p < 0.001.

HSD increases Iba1 expression in the hippocampus

To examine the activation of microglia in the mouse brain, we measured the expression of Iba1 by western blotting and immunostaining. The western blotting analysis showed increased expression of Iba1 in the hippocampus of the HSD-fed mice, which suggested increased microglia activation (Fig. 3D). The immunostaining images showed a considerable increase in Iba1 in the hippocampus of the HSD-fed mice (Fig. 3E). These data suggested that a high-salt diet induced microglial activation in the mouse brain.

HSD increases apoptosis and the expression of cleaved caspase-3, caspase-3, and caspase-1 in the mouse cortex

To investigate the brain damage induced by the HSD, we first examined whether HSD induces apoptosis in the hippocampus and cortex by Hoechst staining. In the HSD-fed mice, the percentage of apoptotic cells was significantly higher in the area of the cortex adjacent to the hippocampus (Fig. 4A, B). These results provide evidence showing that HSD directly induces brain injury. Furthermore, the expression of cleaved caspase-3, caspase-3 and caspase-1 in the cortex was significantly higher in the cortex of the HSD-fed mice (Fig. 4C, D), which was consistent with the degree of apoptosis in the cortex of HSD-fed mice.

Fig. 4

The high-salt diet (HSD) induced apoptosis in the hippocampus and cortex. The nuclei were stained with Hoechst 33258, and apoptotic cells are characterized by nuclear condensation and diffraction. A, B) Hoechst staining showing HSD-induced apoptosis in the hippocampal CA1 region (A) and cortex (B) adjacent to the hippocampus (scale bars, 100μm; n = 3 in each group). C) The mice fed the HSD exhibited a significantly higher apoptosis rate in the cortex. D) The HSD significantly increased the IL-1β, IL-6, and TNF-α levels in the mouse cortex (n = 7 in each group). The data are expressed as the means±SEMs. *p < 0.05.

HSD increases the IL-1β, IL-6, and TNF-α levels in mouse brains

Because the HSD could induce apoptosis and microglial activation in the brain, we also examined the levels of proinflammatory cytokines in the cortex by ELISA. We found that the levels of IL-1β, IL-6, and TNF-α in the HSD group were significantly higher than those in the control group (Fig. 4E).

DISCUSSION

Our results indicate the following: 1) the HSD elicits considerable transformation in the GM as well as cognitive dysfunction; 2) the HSD directly induces apoptosis in the brain, and this effect is accompanied by increases in the levels of proinflammatory cytokines and caspases; and 3) decreases in fecal SCFA concentrations along with increases in microglia activation and BBB permeability might be attributable to the above-described phenomenon, which is consistent with the accumulating knowledge of the GM and its metabolites. In the present study, brain injury was observed following consumption of the HSD, and this injury was indicated by apoptosis and high protein levels of caspases, IL-1β, IL-6, and TNF-α in the brain (Fig. 4). In addition, the HSD did not influence the body weight and food intake but significantly increased the water intake and urine excretion (Supplementary Figure 1). These results provide a conceptual framework for elucidating the GM alterations and cognitive dysfunction induced by an HSD.

High salt is recognized as one of the most common risk factors for hypertension and cardiovascular disease. One long-term cohort study suggests that a reduction in the sodium intake, particularly in older adults with low physical activity, might further improve the brain health later in life [2]. Another recent trial found that a reduced sodium intake combined with aerobic exercise is associated with improvements in executive function in adults with cognitive impairments but no dementia or risk factors for cardiovascular disease [21]. Accumulating lines of evidence show that high salt can affect cognitive function via multiple mechanisms beyond its impacts on hypertension [2–4 , 22–24]. For example, salt exerts a pro-inflammatory effect and boosts the activation of classical LPS-induced macrophages [7 , 26]. In the present study, we found that the HSD significantly activated microglia, which are the resident macrophages in the brain, and significantly increased the levels of proinflammatory cytokines in the cortex, which could be produced by excessive activated microglia.

The mechanisms through which the HSD activate microglia in the brain remain unclear. Accumulating lines of evidence link GM dysbiosis to several neural diseases, such as autism spectrum disorder [27, 28], major depression [29], Parkinson’s disease [18], Alzheimer’s disease (AD) [30, 31], and multiple sclerosis [32]. A maternal high-fat diet induces social deficits and GM dysbiosis in offspring [33]. Coincidentally, studies using animal models have shown that probiotics, such as Lactobacillus and Bifidobacteria, restore the memory impairments induced by stress [34, 35]. More importantly, the consumption of probiotics for 12 weeks exerts positive effects on cognitive function and some metabolic statuses in patients with AD [36]. A recent study showed that a high-salt intake alters the gut microbiome in mice and a human cohort, particularly by depleting Lactobacillus spp. [6], which indicates that pivotal bacterial genera or probiotics might be therapeutic methods for cognitive dysfunction associated with dietary salt. Therefore, the inflammatory response elicited by redundant dietary salt has been implied in a broad spectrum of neural diseases, and elucidating the triangular relationship among dietary salt, cognition, and GM would be meaningful. A recent study found that dietary salt promotes cognitive impairment through tau phosphorylation [37]. Interestingly, previous studies have found that the level of trimethylamine N-oxide (TMAO) in cerebrospinal fluid is higher in individuals with mild cognitive impairment and AD dementia. TMAO is produced by the GM, and elevated TMAO in cerebrospinal fluid is associated with biomarkers of AD pathology (phosphorylated tau and phosphorylated tau/Aβ₄₂) and neuronal degeneration (total tau and neurofilament light chain protein) [38]. These findings further indicate that the cognitive impairment caused by high salt is closely related to the GM.

The switch in the GM induced by long-term HSD consumption found in this study, such as an increased Firmicute/Bacteroidetes ratio, was similar to that observed in aged mice [41]. Members of the S24-7 and Rikenellaceae families were the most abundant representatives of the Bacteroidetes phylum, the abundance of which was significantly decreased by the HSD in the present study. The abundance of S24-7, a butyrate-producing bacterium, is reduced by a high-fat diet and increased following exercise and in lean mice compared with the levels in obese mice [39]. Several immune-related peptidases are also secreted by members of the S24-7 family [40]. The other genus decreased by the HSD, Alloprevotella, reportedly produces moderate amounts of acetic acid and major amounts of succinic acid as end-products of the fermentation process [41], and species of this genus are through to induce protection against cardiovascular diseases [42]. In contrast, an HSD induces blooms in some of the major constituent groups within Firmicutes, such as Lactobacillaceae, Lachnospiraceae, and Ruminococcaceae. Interestingly, the trends in the alterations in the abundances of Lachnospiraceae NK4A136 and Ruminococcaceae were opposite to those found in a previous study [15].

We also found that the HSD significantly reduced the production of SCFAs in the gut. SCFAs mainly consist of acetic acid, propionic acid and butyric acid, which account for 90% to 95% of the total amount of SCFAs. SCFAs regulate the permeability of the intestinal epithelial barrier and BBB by upregulating and reorganizing tight junctions or by promoting epithelial cell differentiation [43]. Physiological concentrations of SCFAs immediately promote epithelial barrier function in the large intestine [44]. An HSD significantly damages the ischemic brain by increasing BBB disruption due to the loss of tight junctions without any apparent alteration in the blood pressure [4]. Furthermore, SCFAs can enter the brain and promote full maturation of microglia and their anti-inflammatory capabilities [14], which results in the alleviation of BBB damage in germ-free mice by restoring tight junctions [45]. The intravenous or intraperitoneal administration of sodium butyrate facilitates long-term memory consolidation [46] and promotes angiogenesis and neurogenesis [47]. Acetate is known to improve intestinal defense responses mediated by epithelial cells and thereby protects the host from lethal infection [48]. The decreased SCFA concentrations observed in the current study might account for the increase in BBB permeability and the excessive activation of microglia induced by long-term HSD consumption. Moreover, the protein levels of IL-1β, IL-6, and TNF-α were significantly higher in the brains of HSD-fed mice. These results suggest that HSD induces GM dysbiosis and an inflammatory environment in the mouse brain, which ultimately resulted in the loss of learning and memory abilities.

Our results showed that the HSD can affect cognitive function in many ways, including but not limited to reducing the expression of tight junction proteins and increasing the BBB permeability. An increased BBB permeability might allow higher levels of harmful substances, such as bacterial-related LPS and metabolites, to enter the brain, which causes inflammation in the brain. These complex factors contribute to salt-related cognitive impairment. We also found that the HSD reduced the abundances of SCFA-producing bacteria and increased the activation of microglia in the brain. SCFAs exert anti-inflammatory effects, and whether SCFAs can affect the high-salt-induced inflammatory environment requires further exploration. In contrast, more studies are needed to identify the pivotal role of the GM in dietary sodium-associated brain dysfunction.

Conclusion

Our study shows that excessive salt intake directly elicits brain injury and cognitive dysfunction in mice, which might be the consequence of the GM dysbiosis induced by an HSD, as indicated by alterations in the GM composition and decreased fecal SCFA concentrations. Certain bacterial strains could account for the increased BBB permeability, and SCFAs might be targeted to improve high salt-associated cognitive decline, but further investigations are needed to confirm these findings.

AVAILABILITY OF DATA AND MATERIALS

The dataset analyzed in the current study is available from the corresponding author upon request.

Footnotes

ACKNOWLEDGMENTS

We express our appreciation to the staff at Shanghai Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China), for their help with the fecal 16S rRNA gene sequencing and data analysis. The authors wish to acknowledge American Journal Experts () for the critical editing of the English grammar and syntax of the manuscript.

his work was supported by funding from the Natural Science Foundation of China (grant number 81400986), the Science and Technology Planning Project of Zhanjiang (grant number 2018A01021), the Affiliated Hospital of GDMU (grant numbers BJ201515, BJ201612 and LCYJ2018A003), the Characteristic Innovation Project of General Universities in Guangdong Province (grant number 2019KTSCX045), and the Natural Science Foundation of Guangdong Province (grant number 2020A151501284).

Authors’ disclosures available online ().

The supplementary material is available in the electronic version of this article: .

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