Gut Microbiome and Serum Metabolome Profiles of Capsaicin with Cognitive Benefits in APP/PS1 Mice

Capsaicin, a natural bioactive component, has been reported to improve cognition and ameliorate the pathology of Alzheimer’s disease (AD). Studies have linked AD to alterations in gut microbiota composition and serum metabolites. In the present study, we examined the alterations in serum metabolome and gut microbiome in APPswe/PS1dE9 (APP/PS1) mice treated with capsaicin. Capsaicin treatments resulted in a significant increase in the abundance of Akkermansia, Faecalibaculum, Unclassified_f_Atopobiaceae, and Gordonibacter and a significant decrease in the abundance of Adlercreutzia, Peptococcaceae, Alistipes, Oscillibacter and Erysipelatoclostridium. Furthermore, the species Akkermansia muciniphila (A. muciniphila) was significantly enriched in capsaicin-treated APP/PS1 mice (p = 0.0002). Serum metabolomic analysis showed that capsaicin-treated APP/PS1 mice had a significant higher level of tryptophan (Trp) metabolism and a significantly lower level of lipid metabolism compared with vehicle-treated mice. Capsaicin altered serum metabolites, including Kynurenine (Kyn), 5-Hydroxy-L-tryptophan (5-HIT), 5-Hydroxyindoleacetic acid (5-HIAA), indoxylsulfuric acid, lysophosphatidyl cholines (LysoPCs), and lysophosphatidyl ethanolamine (LysoPE). Significant correlations were observed between the gut bacteria and serum metabolite. With regard to the increased abundance of A. muciniphila and the ensuing rise in tryptophan metabolites, our data show that capsaicin alters both the gut microbiota and blood metabolites. By altering the gut microbiome and serum metabolome, a diet high in capsaicin may reduce the incidence and development of AD.


Introduction
Alzheimer's disease (AD), known as a progressive age-related neurodegenerative disorder, affects over 50 million people worldwide. The number of patients with AD is predicted to increase to 152 million in 2050 [1]. The aggregations of extracellularly β-amyloid (Aβ) plaques and hyperphosphorylated tau proteins as intracellularly neurofibrillary tangles are the featured neuropathological hallmarks [2]. AD patients suffer a decline in memory, cognitive abilities, and behavior, often for many years, which seriously affects patients, families, and the public health system [3]. Although drugs interventions alleviate or reverse the elusively aforementioned AD symptoms, there is no curative or disease-modifying treatment for AD. Prevalent methods for reducing the risk of AD include enhancing physical activity, reducing obesity, and choosing balanced diets.
Numerous studies have proven the relationship between the gut microbiota and the occurrence and development of AD [4]. Qian et al. reported the role of gut microbiota in AD via inflammatory pathways, because there existed a decreasing diversity of gut microbiota in AD patients. Specially, Firmicutes and Actinobacteria decreased, while Bacteroidetes

Behavioral Tests
The Morris water maze test was selected to evaluate spatial memory formation and retention [21][22][23]. In brief, a platform was submerged 1 cm below the latex white water surface into a round tank (120 cm in diameter and 50 cm in depth). Four different visual cues were placed on the wall around the pool. The water temperature was kept at 20 ± 2 • C. The procedure included six training days and one probe trial test day. During the training sessions, all mice could swim freely lasting for 60 s or get onto the platform. Otherwise, the researchers placed them on platform and kept them on it for 10 s. During test day, the platform was removed, and the animals were permitted to swim freely for 60 s. All data were acquired and analyzed using the ANY-maze (Stoelting, Chicago, IL, USA) video tracking system.
The Y maze test assessed spatial working and reference memory in mice. The Y-shaped maze had three light-colored arms (start arm, other arm and novel arm) orientated at 120 • angles from each other. During training trials, the researchers placed mice into the start arm and allowed them to wander within two arms for 10 min without the novel arm. After a 1 h interval, the novel arm opened, and the same test mouse was allowed to explore all arms for 5 min. All data were acquired and analyzed using the SuperMaze video tracking system. The number of times the mouse entered to the novel arm and other two arms was recorded.

Fecal Microbiome Preprocessing and Data Analysis
Sample collection, DNA extraction, and 16S ribosomal ribonucleic acid (rRNA) ampliconbased sequencing were included. Briefly, at the end of the experiment, pellet samples were collected. Bacterial genomic DNA was extracted from each fecal sample using the E.Z.N.A. ®  Microbiome diversity analyses were performed using QIIME 2 (version 2020.2). The operational taxonomic unit (OTU) table for all samples generated in QIIME 2 was used to assess differences in the relative abundance of microbial genera. Alpha diversity and beta diversity were separately evaluated by the Mothur metric and the Bray-Curtis metric. Moreover, statistical significance was assessed using the Adonis function.

Serum Metabolomics Analyses
Serum samples were cleaned up with methanol protein precipitation and characterized with ultra-high pressure liquid chromatography coupled with high resolution mass spectrometry (UHPLC-MS/MS). Samples measuring 100 µL were mixed with 400 µL methanol-water (4:1, v/v) and homogenized for 30 s. The solution was ultrasonically extracted on ice for 30 min (5 • C, 40 KHz) and stored at −20 • C for 30 min; then, the solution was centrifuged at 13,000 rpm for 10 min at 4 • C.

Statistics
All data are expressed as the mean ± standard error of the mean (SEM) and were analyzed using GraphPad Prism Software for Windows. The unpaired two-tailed Student t-test and one-way ANOVA followed by Tukey's multiple comparison test were used for differences between two groups and among more than two groups, respectively. Correlations were analyzed using Spearman's correlation. The minimum significance value was considered as p < 0.05.

Capsaicin Improved Cognitive Deficts in APP/PS1 Mice
Following 4 months of either a capsaicin-enriched or vehicle diet ( Figure 1A), all mice had their cognitive functions evaluated by using two neurobehavioral tests; the Morris water maze (MWM) test and Y maze test. We assessed the long-term working memory using the MWM test. It was shown that the escape latency of all groups decreased gradually for the duration of the 6-day acquisition, which declined more quickly than vehicle-treated APP/PS1 mice, and was similar to WT mice ( Figure 1B). There was a significant effect between capsaicin-treated APP/PS1 mice and vehicle-treated animals on day 6 (p < 0.05) ( Figure 1C), and capsaicin-treated APP/PS1animals needed a decrease in latency in order to load the platform ( Figure 1C,D). On probe trial day 7, capsaicin-treated APP/PS1 mice needed more time to reach the escape platform in the target quadrant during the acquisition period, and they crossed a higher number of platform areas than vehicle-treated animals (p < 0.05) ( Figure 1E,F). Above all, it can be concluded that capsaicin-treated APP/PS1 mice demonstrated a decline in the escape latency, a significant preference in the target quadrant, and a higher number of platform area crosses compared to vehicle-treated APP/PS1 mice. To assess short-term working memory, the Y maze test was performed in all groups. The result revealed that capsaicin-treated APP/PS1 animals had a higher occurrence number of entering the novel arm compared to vehicle-treated animals (p < 0.05) ( Figure 1G). Capsaicin-treated APP/PS1 mice preferred the novel arm significantly more than vehicletreated APP/PS1 animals. Overall, these data confirmed that capsaicin-rich diets can rescue impairments and cognitive disorders in APP/PS1 mice.  The Y maze test was used to assess spatial working memory in all animals. All data are the mean ± SEM and the minimum significance value was considered as p < 0.05. * indicates significant difference. To assess short-term working memory, the Y maze test was performed in all groups. The result revealed that capsaicin-treated APP/PS1 animals had a higher occurrence number of entering the novel arm compared to vehicle-treated animals (p < 0.05) ( Figure 1G). Capsaicin-treated APP/PS1 mice preferred the novel arm significantly more than vehicletreated APP/PS1 animals. Overall, these data confirmed that capsaicin-rich diets can rescue impairments and cognitive disorders in APP/PS1 mice.

Capsaicin Reduced the Deposit of Amyloid Plaque in the Hippocampus and Cortex of APP/PS1 Mice
The aggregation of amyloid plaque in the brain tissues is the pathogenesis of AD [24]. We performed Congo red staining to assess the deposits of amyloid plaque in the hippocampus and cortex of all animals. APP/PS1 mice had a significantly higher amyloid plaque deposits both in the hippocampus and cortex than WT animals (Figure 2A), and the capsaicin diet significantly reduced Aβ aggregations in the hippocampus and cortex. Specifically, capsaicin reduced the total Aβ deposits by 92.85% and 83.62% in the hippocampus and cortex of APP/PS1animals, respectively ( Figure 2B,C).

Capsaicin Increased the Relative Abundance of Phylum Verrucomicrobiata, Genus Akkermansia, and Species A. Muciniphila in APP/PS1 Mice
To determine whether capsaicin had an effect on the gut microbiota, the 16S sequencing processes for fecal samples were performed. Principal component analysis (PCA) showed a clear difference between the microbial communities of capsaicin-treated and vehicle-treated APP/PS1 mice ( Figure 3A). Different alpha-diversity indices, including Shannon (p = 0.0413), Simpson (p = 0.0295), Chao (p = 0.4616) and Ace (p = 0.3999), indicated that capsaicin significantly decreased gut bacterial alpha diversity, which was related with the antibacterial activity of capsaicin ( Figure S1A). Comparing the relative abundances of the bacterial communities between capsaicin-treated and vehicle-treated APP/PS1 mice, we found changes in the bacterial phylum levels ( Figure 3B). The capsaicintreated APP/PS1 mice harbored a higher relative abundance of Verrucomicrobiota than vehicle-treated animals (p < 0.0001). Moreover, we also observed that capsaicin diet increased the relative abundance of bacterial genera, including Akkermansia, Faecalibaculum, Unclassified_f_Atopobiaceae, Gordonibacter and decreased the relative abundance of bacterial genera, including Adlercreutzia, Peptococcaceae, Alistipes, Oscillibacter, Erysipelatoclostridium ( Figure 3C). The abundance of phylum Verrucomicrobiota, genus Akkermansia, and species A. muciniphila (all of which comprise Gram-negative bacterial groups) in the APP/PS1_CP group significantly increased compared to the APP/PS1_CTRL group (p = 0.0002) ( Figure 3D). The results implied that capsaicin has a profound effect on the composition of microbiota.
Studies have found that the relative abundance of A. muciniphila in APP/PS1 mice decreased with age [25]. Moreover, recent evidence suggests that supplementation with A. muciniphila obviously reduced amyloid aggregation in the cerebral cortex and improved cognitive deficits and in AD mice [26]. In the present study, the higher abundance of species A. muciniphila in APP/PS1_CP versus APP/PS1_CTRL animals is significant ( Figure 3D), which is in agreement with previous data.

Capsaicin Upregulated Tryptophan Metabolism and Downregulated Lipid Metabolism in APP/PS1 Mice
Serum metabolites link the microbiome to its host by regulating metabolism [27]. Therefore, serum metabolome processes were performed using LC/MS-MS. The PCA analysis showed that serum metabolic profiles in capsaicin-treated APP/PS1 mice were significantly different from vehicle-treated APP/PS1 mice ( Figure 4A). Forty-two metabolites were specifically altered in the serum of the APP/PS1_CP group compared with the APP/PS1_CTRL group ( Figure 4B).  Results are mean ± SEM. The minimum significance value was considered as p < 0.05.
The altered metabolites were involved in various metabolic pathways. Multiple previous studies showed that short-chain fatty acids, bile acids, and tryptophan (Trp) metabolites are the three main metabolic pathways related to host-microbiota interactions [28]. Amino acid metabolism and lipid metabolism were the main pathways between APP/PS1_CP mice and APP/PS1_CTRL mice in this study ( Figure 4C). Specifically, capsaicin upregulated amino acid metabolism and downregulated lipid metabolism in APP/PS1 mice.
Focusing on overlapping metabolites, we found four metabolites linked with tryptophan metabolism, which is the essential aromatic amino acid. The relative abundances of four metabolites Kyn (p = 0.0043), 5-HIT (p = 0.0115), 5-HIAA (p = 0.0277), and indoxyl-sulfuric acid (p = 0.0281) increased significantly in APP/PS1_CP mice compared with APP/PS1_CTRL mice ( Figure 4D Studies have found that the relative abundance of A. muciniphila in APP/PS1 mice decreased with age [25]. Moreover, recent evidence suggests that supplementation with A. muciniphila obviously reduced amyloid aggregation in the cerebral cortex and improved cognitive deficits and in AD mice [26]. In the present study, the higher abundance of species A. muciniphila in APP/PS1_CP versus APP/PS1_CTRL animals is significant ( Figure 3D), which is in agreement with previous data.

Capsaicin Upregulated Tryptophan Metabolism and Downregulated Lipid Metabolism in APP/PS1 Mice
Serum metabolites link the microbiome to its host by regulating metabolism [27]. Therefore, serum metabolome processes were performed using LC/MS-MS. The PCA analysis showed that serum metabolic profiles in capsaicin-treated APP/PS1 mice were significantly different from vehicle-treated APP/PS1 mice ( Figure 4A). Forty-two metabo-

The Correlation between Gut Microbiota and Serum Metabolites
Spearman's correlation analyses between gut bacteria and serum metabolites were performed to investigate the links between both sides. At the phylum level, we observed that Verrucomicrobiota significantly changed microbiota in capsaicin-treated APP/PS1 mice (p = 0.0002) ( Figure 5A    At the genus level, metabolic association heatmap analyses revealed correlations between the levels of gut microbiota and serum metabolites ( Figure 5B). Akkermansia, Unclassified_f_Atopobiaceae, and Faecalibaculum, which were the significantly increased microbiota in capsaicin-treated APP/PS1 mice compared to vehicle-treated APP/PS1 mice ( Figure 2C), displayed positive correlations with Trp metabolites and negative correlations with lipid metabolites.
Akkermansia had a positive correlation with metabolites such as 5-HIAA, Kyn, 5-HIT and indoxylsulfuric acid, but it had a negative correlation with metabolites such as LysoPC

Discussion
Numerous studies have discovered that interactions between the microbiome's metabolites and hosts influenced how the gut microbiome and microbial metabolites regulated brain function and behavior, which may have an impact on the development of AD [29][30][31]. For example, GF mice displayed the impairment of immune responses and the pathogenesis of AD by modulating the number of cells and the maturity of microglia, which resulted in impaired spatial and working memory [32,33]. Antibiotics, such as streptozotocin and ampicillin, can damage the balance of gut bacteria and worsen the development of AD [34]. The administration of ampicillin in rats disrupted the gut microbiota, elevated serum corticosterone, and, thus, impaired spatial memory [33]. APP/PS1 transgenic mice treated with a cocktail of antibiotics (ABX) had increased levels of neuroinflammatory and cytokine-related genes [35]. Therefore, the adjustment of gut microbiota composition maybe a considerable treatment for AD by supplying with probiotics or designing individual diets.
The dietary interventions have a close association with the occurrence of AD by regulating gut microbiota [35]. For instance, dietary ω-3 PUFAs significantly balanced gut microbiota compositions in mice and reduced the risk of AD [36,37]. Coffee drinkers had a lower occurrence of AD compared to individuals with persons drinking no or little coffee, and this is because the fiber and polyphenols in coffee beans regulated the number and compositions of gut microbiota [38,39]. The daily intake of dietary fruits and vegetables balances the gut microbiota, and this may play important roles in AD control [40,41]. The pungent capsaicin molecule is one of the bioactive products in chili pepper, and it is consumed when ingesting vegetables and spices. In this study, the dietary consumption of capsaicin significantly increased the relative abundance of phylum Verrucomicrobiota, genus Akkermansia, and species A. muciniphila in APP/PS1 mice and ameliorated cognitive deficits (Figures 1 and 3). muciniphila was isolated from the human intestine and characterized before two decades. Multiple studies linked numerous diseases with either a lack or decreased abundance of this bacterium [42,43]. Although some studies have observed that AD patients had a higher percentage of A. muciniphila [44,45], intervention experiments clearly reported beneficial effects of this bacterium in the pathology of AD [26]. Alzheimer's disease is a disorder and Aβ plaques are pathological hallmarks. By contrast, clinical studies report the protective effects of A. muciniphila. Moreover, capsaicin-enriched diets significantly increased the relative abundance of A. muciniphila in APP/PS1 mice ( Figure 3D). On the basis of promising initial studies, A. muciniphila contributed to tryptophan secretion, and increased A. muciniphila abundance was correlated with an improved metabolic profile [46].
The upregulated metabolic factors of Trp crossed the blood-brain barrier (BBB) and provided neuronal protection, such as plaques clearness and cognitive benefits [47].
The metabolism of Trp is one of three currently most studied categories of metabolites in the interconnection between the host and its microbiome [28]. Amino acid metabolism and lipid metabolism were two main metabolism pathways in the serum metabolic profile ( Figure 4C). Moreover, Peptococcaceae and Alistipes had decreased relative abundances in capsaicin-treated mice. Thus, amino acids were the main factors relative to metabolism. As shown in Figure 6, Kyn, 5-HTA, 5-HIAA and indoxylsulfuric acid, which are generated from the three pathways of Trp metabolism in the gastrointestinal tract, had significantly higher level than capsaicin-free control mice. This result suggests that the capsaicin-enriched diet could improve Trp metabolism in APP/PS1 mice. Multiple previous studies indicated that almost all free Trps were involved in the Kyn pathway (KP) of Trp metabolism [47]. Moreover, nearly 60% of Kyns, transported across the blood-brain barrier, are distributed in the central nervous system (CNS). According to Kyns pathways in the CNS, astrocytes are equipped to secrete kynurenic acid (Kyna), which provides neuronal protection. On the basis of behavioral tests and the β-amyloid clearance of brain tissues (Figures 1 and 2), the neuroprotective Kyna was the main metabolic factor of Kyn, which was utilized by astrocytes in the brain tissues. The metabolism of Trp is one of three currently most studied categories of metabolites in the interconnection between the host and its microbiome [28]. Amino acid metabolism and lipid metabolism were two main metabolism pathways in the serum metabolic profile ( Figure 4C). Moreover, Peptococcaceae and Alistipes had decreased relative abundances in capsaicin-treated mice. Thus, amino acids were the main factors relative to metabolism. As shown in Figure 6, Kyn, 5-HTA, 5-HIAA and indoxylsulfuric acid, which are generated from the three pathways of Trp metabolism in the gastrointestinal tract, had significantly higher level than capsaicin-free control mice. This result suggests that the capsaicin-enriched diet could improve Trp metabolism in APP/PS1 mice. Multiple previous studies indicated that almost all free Trps were involved in the Kyn pathway (KP) of Trp metabolism [47]. Moreover, nearly 60% of Kyns, transported across the blood-brain barrier, are distributed in the central nervous system (CNS). According to Kyns pathways in the CNS, astrocytes are equipped to secrete kynurenic acid (Kyna), which provides neuronal protection. On the basis of behavioral tests and the β-amyloid clearance of brain tissues (Figures 1 and 2), the neuroprotective Kyna was the main metabolic factor of Kyn, which was utilized by astrocytes in the brain tissues. Figure 6. The interaction between the microbiota and the microbiota-gut-brain axis in APP/PS1 mice and capsaicin intervention. Dietary capsaicin can directly change gut microbiota compositions and significantly enhance the relative abundance of A. muciniphila, which contributed to Trp secretion. Almost all free Trps are involved in the Kyn pathway (KP) related to Trp degradation and the gut microbiota influence the kynurenine-producing indoleamine-2,3-dioxygenase (IDO) pathway. Kynurenine amino transferase in astrocytic cells can transfer from Kyn to kynurenic acid (KA), which regulates cognition and behavior using three receptors, including the α7-nicotinic receptor (α7nAChR), N-methyl-d-aspartate receptors (NMDARs), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) [28]. The peripheral production of 5-HT and 5-HTP by enterochromaffin (EC) cells is also affected by the gut microbiota. 5-HTP, transported across the blood-brain barrier, is further metabolized into 5-HT, which exhibits neurobiological functions using 5-HT receptors in AD [48]. Gut microbial tryptophanase converts Trp into indole, and then it enters the host portal circulation and is transferred into indoxylsulfuric acid in the liver [49]. The red arrows represent the higher abundance of gut microbiota and increased serum metabolites in capsaicin-treated APP/PS1 mice. Figure 6. The interaction between the microbiota and the microbiota-gut-brain axis in APP/PS1 mice and capsaicin intervention. Dietary capsaicin can directly change gut microbiota compositions and significantly enhance the relative abundance of A. muciniphila, which contributed to Trp secretion. Almost all free Trps are involved in the Kyn pathway (KP) related to Trp degradation and the gut microbiota influence the kynurenine-producing indoleamine-2,3-dioxygenase (IDO) pathway. Kynurenine amino transferase in astrocytic cells can transfer from Kyn to kynurenic acid (KA), which regulates cognition and behavior using three receptors, including the α7-nicotinic receptor (α7nAChR), N-methyl-d-aspartate receptors (NMDARs), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) [28]. The peripheral production of 5-HT and 5-HTP by enterochromaffin (EC) cells is also affected by the gut microbiota. 5-HTP, transported across the blood-brain barrier, is further metabolized into 5-HT, which exhibits neurobiological functions using 5-HT receptors in AD [48]. Gut microbial tryptophanase converts Trp into indole, and then it enters the host portal circulation and is transferred into indoxylsulfuric acid in the liver [49]. The red arrows represent the higher abundance of gut microbiota and increased serum metabolites in capsaicin-treated APP/PS1 mice.
Long-chain fatty acid is inversely associated with the pathogenesis of obesity [50]. Obesity, strongly related to AD in aging, is the most common metabolic disorder. Metabolic disorders caused by brain-tissue atrophy, reduced gray and white matter volume, alteration in the electrical characteristics of nerve tissue, a reduction in hippocampal neurogenesis and impairment in the proliferation and differentiation of neuroprogenitor cells (NPCs) into neurons, neuronal death, decreased synaptic plasticity, and impairment of BBB integrity induce cognitive impairment and AD [51]. Dietary capsaicin activated its receptor, transient receptor potential vanilloid 1 (TRPV1), and converted adipose tissues from white to brown, which contributed to reducing obesity [52]. Capsaicin has been reported its anti-obesity effect on high fat diet-fed mice by altering gut microbiota compositions, increasing the relevant abundance of genus Akkermansia [53]. Furthermore, numerous studies identified a close association between A. muciniphila and obesity. The daily intervention of pasteurized A. muciniphila reduced food energy efficiency and mitigated diet-induced obesity in diet-induced obese mice [54]. A. muciniphila regulated L-aspartate metabolism and improved fatty liver associated with metabolic dysfunction [55]. A. muciniphila mitigated metabolism-induced inflammation and prevented obesity-related atherosclerosis by in Apoe -/mice [56]. In this research study, we observed that capsaicin significantly increased the relative abundance of A. muciniphila and decreased the level of long-chain fatty acids, such as LysoPC (17:0) Figure S2). Our results are consistent with those of previous studies.

Conclusions
In this study, we observed that capsaicin rescued the behavioral cognitive deficits of APP/PS1 mice. Congo red staining showed that capsaicin decreased β-amyloid deposits of the brain tissues in APP/PS1 mice. Gut microbiota profiling indicated that the capsaicin-treated mice harbored higher relative abundances of Akkermansia, Faecalibaculum, Unclassified_f_Atopobiaceae, and Gordonibacter, while the vehicle-treated APP/PS1 mice harbored higher relative abundances of Adlercreutzia, Peptococcaceae, Alistipes, Oscillibacter, and Erysipelatoclostridium. Notably, A. muciniphila was observed to be significantly increased in the capsaicin-treated mice. The metabolome profiling of serum samples showed that capsaicin-treated mice had a higher level of Trp metabolism and a lower level of lipid metabolism compared to vehicle-treated APP/PS1 mice. The evidence of A. muciniphila and Trp metabolites highlighted that capsaicin-enriched diets may mitigate the incidence and development of AD by changing gut microbiome and serum metabolome, and A. muciniphila and Trp metabolism may be possible approaches for ameliorating AD.