Background
Obesity prevalence is increasing steadily around the world that has a devastating impact on the sustainable health of individuals in the long-term [
1]. Obesity is not only associated with insulin resistance, type 2 diabetes mellitus, cardiovascular disease and steatohepatitis, but can also increases the risk of developing neurodegenerative diseases such as Alzheimer’s disease (AD) [
2,
3]. Evidence shows that obesity and/or high fat feeding are associated with deficits in learning, memory, and executive functioning [
4,
5] and potentially brain atrophy [
6]. However, effective therapeutic manipulation still lacks. Thus, it is urgent to clarify the underlying mechanism of obesity-induced cognitive decline and develop a targeted therapeutic strategy.
Neuroinflammation is proposed to be an important pathophysiological hallmark underlying cognitive decline [
7]. Microglia, being the resident immune cells of the central nervous system, plays an important role in maintaining brain homeostasis and contributes towards brain development under normal conditions. However, excessive microglial activation can mediate cognitive impairment via inducin
g progressive loss of neurons [
8]. Hence, addressing neuroinflammation mediated by microglia bears great promise as a novel treatment strategy to reduce neuronal damage and to foster a permissive environment for further regeneration effort [
9].
Gut microbiota can affect brain plasticity and cognitive function through the gut-brain axis. The gut microbiota maintains host intestinal homeostasis and regulates immunity. It is reported that
Bacteroides fragilis of Bacteriodetes phylum can increase tight junction proteins expression and attenuate intestinal permeability, while
Ruminococcus of Firmicutes phylum can degrade mucus [
10]. In the mice fed by a long-term high-fat (HF) diet, an increase in intestinal permeability allows the translocation of bacteria or bacterial lipopolysaccharide (LPS) into the blood circulation and then induces systemic inflammation. Moreover, these cytokines can infiltrate in the brain via the blood–brain barrier and act on microglia to induce local production of proinflammatory cytokines, thereby triggering neuroinflammation and cognitive impairment. Furthermore, evidence from germ-free, antibiotic-treated and pathogen-free rodents has revealed gut microbiota dysbiosis negatively affect hippocampal neurogenesis and brain development via microglia activation [
11,
12], suggesting a vital role of gut microbiota in cognitive function. Therefore, the gut-brain axis is considered as the potential therapeutic target for HF induced neuroinflammation and cognitive decline [
13‐
16].
Lentinula edodes (
L. edodes) is one of the most popular edible mushrooms in the global market, and (1, 3)/(1, 6)-β-glucan is its major bioactive component. The biological activities of the β-glucan have attracted more attention recently in the medical fields not only due to its nutritional value but also to the possible potential for therapeutic applications [
17]. It has been reported that
L. edodes can be used medicinally for diseases involving hyperlipidemia, hypertension and diabetes [
17]. Interestingly, two studies have reported that the supplementation of
L. edodes derived β-glucan for consecutive 28 days can improve gut microbiome dysbiosis in aged mice [
18], and thereby improve insulin resistance in insulin-deficient type 2 diabetic rats [
19]. Moreover, a recent cross-sectional study has shown that mushroom consumption had reduced the incidence rate of mild cognitive impairment in aged individuals in Singapore [
20]. These findings suggest that the main ingredient of
L. edodes, β-glucan, may have the potential to regulate gut microbiota and gut-brain axis. However, it is unknown if
L. edodes derived β-glucan can prevent microbiota dysbiosis induced by HF diet and thereby improve cognitive impairments via gut-brain axis.
In this study, we designed short-term and long-term experiments of
L. edodes derived β-glucan supplementation to examine acute effects on gut microbiota and chronic effects on cognition and its underlying gut-brain axis. The composition of gut microbiota was examined in a short-term experiment for 7 days prior to body weight alteration. Most research of gut microbiota studies in humans and rodents are chronic studies. For example, studies have shown that chronic HF diet induced microbial dysbiosis with regards to the diversity and composition [
15,
21]. However, the study of gut microbiota after an acute HF diet and intervention is rare. The results of short-term experiment indicate that
L. edodes derived β-glucan supplementation prevented the microbial dysbiosis which appears in the early stage of HF diet feeding, before the onset of significant weight gain and obesity. Furthermore, a similar gut microbiota composition was observed after long-term supplementation of the β-glucan. The chronic high fat diet-fed mice are a commonly used animal model of cognitive impairment reported in the literature. In the long-term experiment, we assessed the effects of dietary
L. edodes β-glucan supplementation by measuring behavioral tests, synaptic ultrastructure, neuroinflammation and brain-derived neurotrophic factor (BDNF) expression in the critical brain region, prefrontal cortex and hippocampus. Moreover, the intestinal parameters, including the colonic mucus thickness and tight junction protein expression, as well as serum endotoxin (LPS) level, were also evaluated. We demonstrated that chronic
L. edodes derived β-glucan supplementation prevented HF diet-induced cognitive deficits with improvement in the gut-brain axis.
Discussion
The present study, with an obese cognitive impairment mice model, demonstrated the beneficial effects of
L.
edodes β-glucan supplementation on the gut microbiota-brain axis and improvement of cognitive decline. We showed that short-term and long-term
L.
edodes β-glucan supplementation alleviated the gut microbial dysbiosis induced by the HF diet. Notably, long-term supplementation with
L.
edodes β-glucan significantly improved the cognitive impairment in HF diet-fed mice, which was supported by the inhibition of microgliosis, alleviation of neuroinflammation and improvement of synaptic ultrastructure. Furthermore, long-term
L.
edodes β-glucan supplementation significantly mitigated the impairment of colonic barrier and inflammation in HF diet-fed mice. Collectively, these data demonstrated that
L.
edodes β-glucan ameliorated the cognitive deficits induced by chronic HF diet, and these neuroprotective effects potentially occurred through the improvement of the colon-microbiota-brain axis. Previously, edible mushrooms have shown beneficial effects on cognition in a cross-sectional study [
20]. Here we reported that the main ingredient of edible mushroom,
L.
edodes β-glucan, improved the gut microbiota-brain axis. Therefore, the beneficial effects of β-glucan may contribute to the ability of mushroom in the improvement of cognitive function, as described previously in the human study [
20].
The gut microbiome has emerged as a major contributor to cognitive health, and it can be remodeled by dietary factors. Recently, our laboratory demonstrated that gut microbiota dysfunction negatively impacts the cognitive impairments induced by HF diet [
15,
16,
35]. Nevertheless, only a few studies focused on the alternation of gut microbiota occur before the onset of body weight gain in this model [
35]. In previous long-term studies for HF diet for 8–22 weeks, it is reported that the proportion of gut bacteria belong to phylum Bacteroidetes is decreased, while phylum Firmicutes is increased in rodents with an increased ratio of Firmicutes to Bacteroidetes [
36]. The alternation of these indexes was also observed in the present study, in which the mice were fed by HF diet for 15 weeks. Moreover, the changes were reversed by the long-term supplementation of
L.
edodes β-glucan. Interestingly, in our short-term HF diet for 7 days, although there was a slight increase in the Firmicutes and decrease in the Bacteroidetes, the alteration of these bacteria did not a reach statically significant difference in the gut microbiota of mice. It suggests that microbiota alteration in the Bacteroidetes and Firmicutes is in progress along with the duration of HF diet feeding. However, in the present study, we found that the mean proportion of Proteobacteria and Actinobacteria at phylum was significantly decreased by the short-term HF diet, suggesting the composition of Proteobacteria and Actinobacteria altered early before the significant change in the Bacteroidetes and Firmicutes. In addition, in the long-term study of HF diet, the richness and diversity of gut microbiota in mice were altered [
3], characterized by decreased Chao index and Shannon index. While in the present short-term study, HF diet feeding did significantly change the richness (Chao index) and diversity (Shannon index). This indicates that the shift in the composition of gut microbiota, especially in Proteobacteria and Actinobacteria, occurred before any changes in microbiome diversity and richness during HF diet feeding. Importantly, we found that supplementation of dietary
L.
edodes β-glucan prevented this shift in microbiota composition, as it significantly increased Bacteroides and decreased Firmicutes. Consistent with our findings, two previous studies have shown a similar capability of
L.
edodes β-glucan to alleviate the gut microbiome dysbiosis in aged mice and insulin deficient type 2 diabetic rats [
18,
19]
. In addition, it has been reported that microbiota belonging to the phylum Bacteroidetes is associated with cognition and neurodegenerative diseases [
37]. For example, in a cross-section study, a lower abundance of Bacteroides at the genus is reported in the gut microbiota of dementia patients [
38]. Interestingly, the consumption of mushrooms has been shown to reduce the risk of mild cognitive impairment in aged individuals [
20]. This study found that the increased abundance of Bacteriodetes phylum following
L.
edodes β-glucan administration. Thus, it is rationally proposed that dietary
L. edodes β-glucan in promoting the abundance of certain members of the bacterial community belongs to the Bacteroidetes phylum, contribute to ameliorate the cognitive impairments induced by HF diet.
Emerging research is revealing that gut microbiota has potent effects on gut permeability and endotoxemia [
27]. The gut barrier consists of semi-permeable mucosal, as well as epithelial cell layers reinforced by tight junction proteins. This barrier serves to regulate nutrient and water entry and prevents the entry of harmful compounds into extra-luminal tissues. HF diet consumption impairs gut permeability, which, in turn, allows for the influx of adverse substances [
39]. A compromised gut barrier makes the intestinal tract potentially vulnerable to the gram-negative bacteria-derived LPS, which upon excess entry into circulation, promotes endotoxemia and systemic inflammation [
16,
40,
41]. According to previous studies, we found that HF diet intake dramatically increased intestinal inflammation and diminished intestinal barrier integrity, which is consistent with the increased level of LPS in the sera of mice. However, long-term
L.
edodes β-glucan supplementation increased colonic mucus thickness, upregulated colonic tight-junction protein occludin levels and lowered the LPS level in sera, indicating that
L.
Edodes alleviated the loss of intestinal barrier integrity induced by HF diet. It is reported that the outer membrane protein of Bacteroidetes can bind to β-glucan [
42]. Moreover, the genome of Bacteroidetes encodes many β-glucan lyases and glycoside hydrolases, which are largely involved in the acquisition and metabolism of β-glucan [
43]. Thus, it is possible that
L.
edodes β-glucan favoured the growth of the β-glucan-degrading Bacteroidetes and its next taxonomic levels observed in our study. In the present study, we found that at the phylum level, the short-term supplementation of
L. edodes β-glucan for 7 days significantly increased the proportion of Bacteroidetes in mice fed with HF diet. Previously, in a chronic study, the effect of barley β-glucan supplementation for 5 weeks increased Bacteroidetes in gut microbiota in humans [
44]. These results suggest that the changes in microbiota, such as Bacteroidetes by the short-term supplementation of β-glucan may maintain after long-term supplementation. In addition, Bacteroidetes has been reported to benefit their host mucus and gut barrier [
10]. Therefore,
L.
edodes β-glucan might be fermented by Bacteroidetes, to provide an energy source for bacteria within the Bacteroidetes phylum. In accordance with this, the glycan production in mucus was significantly increased by
L.
edodes β-glucan supplementation, which thus prevented the epithelial damage induced by the HF diet. This, in turn, might relieve the translocation of bacterial LPS into the blood circulation.
The hippocampus and the cortex, regions implicated in cognitive processing, learning and memory, are particularly vulnerable to inflammation in obesity [
32,
45]. It is reported that LPS from the intestinal tract was increased in the cortex and hippocampus of AD patients [
46], which suggests that the increased gut permeability and hyperndotoxinemia could contribute to cognition decline. Our results showed that
L.
edodes β-glucan administration enhanced the intestinal barrier and resulted in a profound reduction in endotoxinemia, which may contribute to the improvements in cognition we observed by a comprehensive array of behavioral, learning and memory tests. Neuroinflammation is considered to be the link between gut dysbiosis to synaptic and cognitive decline, while it is also one of the key mechanisms underlying various neurodegenerative diseases [
47]. LPS over-exposure by intraperitoneal injection has been reported to induce microglial activation and increased expression of proinflammatory cytokines in the brains of mice
48. Moreover, the gut microbiota directly stimulates the production of the proinflammatory cytokines IL-1β and TNF-α [
49], which have been shown to impair hippocampal-dependent memories in rodents [
50,
51]. In the present study, we found that a long-term HF diet upregulated TNF-α, IL-6 and IL-1β in the PFC and hippocampus, which were attenuated by long-term
L.
edodes β-glucan supplementation, indicative of an anti-neuroinflammatory effect of
L.
edodes β-glucan.
There is accumulating evidence demonstrates the microglia play a vital role in mediating the cognitive dysfunction in neurodegenerative dysfunctions [
52]. Microglia, the resident immune cells of the central nervous system, maintain brain homeostasis and contribute to brain development. However, excessive microglial activation can damage the surrounding healthy neural tissue, and the factors secreted by the dead or dying neurons, in turn, exacerbate the chronic activation of microglia, causing progressive loss of neurons and then cognitive impairment [
8,
52]. This study showed the HF diet promoted the accumulation of microglia in the PFC and hippocampus, which was inhibited by long-term
L.
edodes β-glucan supplementation. Synaptic structure and plasticity are closely correlated with learning and memory functions [
34]. The dysregulation of synaptic formation and plasticity in the hippocampus has been implicated in patients with cognitive impairment and AD [
53]. We herein showed the long-term HF diet disrupted the ultrastructural synaptic architecture in the PFC and hippocampus, which was characterized by decreased PSD thickness and broadened synaptic cleft observed by the TEM technique. Notably, long-term
L.
edodes β-glucan supplementation prevented the damage of ultrastructural synaptic architecture induced by the HF diet. Consistently, we also found that
L.
edodes β-glucan supplementation reversed HF diet-associated decreases in the molecular markers of synaptic plasticity, BDNF and PSD-95 in the PFC and hippocampus. Therefore,
L.
edodes β-glucan supplementation significantly improved the ultrastructure and increased synaptic protein expression, which thus supported the enhancement and maintenance in cognitive function despite chronic HF diet feeding following
L.
edodes β-glucan treatment.
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