Animal study

Preventive Effect of Saccharomyces boulardii on Memory

Impairment Induced by Lipopolysaccharide in Rats

Figure 1. S. boulardii mitigates the LPS-induced impairments in recognition memory. The exploration time for targets during the familiarization phase (A) and the preference indices for the new targets in 90 min (short-term memory (STM)) (B) and 24 h (long-term memory (LTM)) (C) after the familiarization phase are expressed as mean ± standard error of the mean (SEM). *P < 0.05 and **P < 0.01, N = 10. Lipopolysaccharide (LPS).

deposition,17 and cognitive impairment18,19 via the overactivation of glial cells and enhanced proinflammatory cytokine levels.

Inflammasomes are a complex of cytosolic proteins that facilitate the activation of inflammatory mediators.20 Among inflammasomes, the nucleotide oligomerization domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) inflammasome

■ INTRODUCTION

Gut microbiota is a group of microbes that live in the digestive tract, including bacteria and fungi. The gut−brain axis, a bidirectional linkage between the brain and gut microbiota, regulates the homeostasis in the intestinal and central nervous systems.1,2 Studies have confirmed the beneficial effects of the intestinal microbiota on brain function, behavior,3−5 memory, and cognition.6

Consistent with these reports, a link has been

plays a vital role in regulating immunological responses and maintaining intestinal homeostasis and gut

intestinal permeability and the blood−brain barrier via the secretion of amyloid and lipopolysaccharide (LPS). The subsequent inflammatory response leads to neuroinflammation and neuronal death in patients with AD.12

Neuroinflammation contributes to the etiology of neurodegenerative diseases such as AD.13,14 AD is characterized by Aβ accumulation and neurofibrillary tangles, resulting in cognitive impairment and memory dysfunction.15 Neuroinflammation plays a critical role in neuronal injury,16 amyloid

In vitro and in vivo studies have shown that the LPS, a toxin derived from gram-negative bacteria, can induce amyloidogenesis and upregulate inflammatory cytokines, thereby leading to neuroinflammation and memory deficits.24−26 Recent studies have revealed that systemic LPS exposure is a suitable animal model for investigating neuroinflammation, neuronal dysfunction, and memory deficits.27,28,26,29 Furthermore, LPSinduced animal models exhibit considerable alterations in the gut microbiota structure, which can be adjusted by probioticbased treatments.30

Probiotics are living microbes, such as bacteria or fungi, that modulate the composition of the gut microbiota and exert various effects on the human body.31 Recent investigations have proposed probiotics as therapeutics that can ameliorate AD-related brain dysfunction and behavioral disorders owing to their ability to restore intestinal microbiota composition.32,33 The preventive effect of different probiotics, including Lactobacillus helveticus, Lactobacillus plantarum, Streptococcus thermophilus, and Bifidobacterium longum, has been reported in AD-related memory dysfunction, Aβ accumulation, and neuroinflammation.28,34,35 Saccharomyces boulardii is a yeast probiotic exhibiting beneficial effects in various disorders, ranging from inflammatory gastrointestinal diseases to CNSrelated disorders.36−40 S. boulardii can exhibit anti-inflammatory effects by lowering proinflammatory mediators via nuclear factor κB (NF-κB), mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK) 1/2, and p38 signaling pathways.36

In this study, we evaluated the protective effects of S. boulardii on LPS-induced memory deficits in rats. Memory impairment was investigated using the novel object recognition (NOR) task. Serum levels of inflammatory cytokines, including IL-1β, IL-6, and tumor necrosis factor-α (TNF-α), were measured using enzyme-linked immunosorbent assay (ELISA) kits. Western blotting was performed to determine hippocampal protein levels of NLRP3 and caspase-1. In addition, Congo red staining was performed to examine Aβ deposition in hippocampal tissue specimens.

RESULTS

S. boulardii Prevented LPS-Induced Memory Impairment. To examine the impact of S. boulardii on LPS-induced memory impairment, we assessed short-term memory (STM) and long-term memory (LTM) using the NOR task. During the familiarization phase of the NOR task, we detected no considerable differences in exploration time for each target in each experimental group (Figure 1A).

The results of the two-way ANOVA revealed a significant main effect of LPS (F(1,29) = 6.873, P = 0.0138) and treatment (F(1,29) = 5.583, P = 0.0251) on STM. Moreover, STM was reduced in LPS-treated rats when compared with control animals, indicating STM impairment following LPS administration (P < 0.05, Figure 1B); this value did not differ between the Cnt and LPS + S groups. S. boulardii supplementation before LPS injection considerably increased STM in the LPS + S group when compared with that in the LPS group (P < 0.05, Figure 1B).

Moreover, we noted a significant main effect of LPS (F(1,32) = 8.051, P = 0.0078) and treatment (F(1,32) = 12.44, P = 0.0013) on LTM. LTM was decreased in LPS-induced animals than that in control rats, indicating LPS-induced LTM dysfunction (P < 0.05, Figure 1C). In addition, there was no difference between the Cnt and LPS + S groups. Treatment with S. boulardii prevented the reduction in LTM in the LPS + S group when compared with that in the LPS group (P < 0.01,

Figure 1C).

S. boulardii Reduced the LPS-Induced Elevated Serum Levels of Inflammatory Cytokines. We measured inflammatory cytokines, including IL-1β, IL-6, and TNF-α, to

Figure 2. S. boulardii decreases the LPS-induced elevated serum levels of inflammatory cytokines. The serum levels of IL-1β (A), IL-6 (B), and TNF-α (C) were evaluated by ELISA. Data are expressed as mean ± standard error of the mean (SEM). *P < 0.05 and **P < 0.01, N = 7.

Interleukin-1β (IL-1β); interleukin-6 (IL-6); lipopolysaccharide (LPS); tumor necrosis factor-α (TNF-α).

determine the anti-inflammatory effects of S. boulardii. Serum levels of IL-1β, IL-6, and TNF-α were affected by LPS (F(1,11) = 8.416, P = 0.0144; F(1,12) = 14.05, P = 0.0028; and F(1,11) = 21.57, P = 0.0007, respectively) and treatment (F(1,11) = 24.39, P = 0.0004; F(1,12) = 7.092, P = 0.0207; and F(1,11) = 28.25, P = 0.0002, respectively). Serum concentrations of IL-1β, IL-6, and TNF-α were significantly increased in the LPS-treated animals when compared with those in the Cnt group (P < 0.05, P < 0.01, and P < 0.05, respectively; Figure 2A−C). In addition, no significant differences were observed between the Cnt and LPS + S groups. Compared with the LPS group, treatment with S. boulardii significantly decreased the LPS-induced increase in serum IL-1β, IL-6, and TNF-α levels in the LPS + S group (P < 0.01, P < 0.05, and P < 0.01, respectively; Figure 2A−C).

S. boulardii Reduced the LPS-Induced Upregulation of NLRP3 and Caspase-1 Proteins in the Hippocampus. In addition, our results demonstrated a considerable main effect of LPS (F(1,12) = 6.562, P = 0.0249 and F(1,11) = 7.165, P

= 0.0215) and treatment (F(1,12) = 13.92, P = 0.0029 and F(1,11) = 16.71, P = 0.0018) on hippocampal levels of NLRP3 and caspase-1, respectively. LPS administration increased hippocampal protein levels of NLRP3 and caspase-1 when compared with that in control animals (P < 0.05, Figure 3A,B). There was no difference between the Cnt and LPS + S groups. Treatment with S. boulardii markedly reduced protein levels of NLRP3 and caspase-1 in the LPS + S group when compared with those in the LPS-treated animals (P < 0.01, Figure 3A,B).

S. boulardii Inhibited LPS-Induced Aβ Deposition. In LPS-treated rats, Congo red staining revealed Aβ deposits in hippocampal CA1 and CA3 regions. The LPS + S group exhibited few Aβ deposits, indicating that S. boulardii could effectively lower LPS-induced Aβ accumulation. The S and Cnt groups exhibited no Aβ deposits (Figure 4).

■ DISCUSSION

In this study, we observed that the probiotic S. boulardii ameliorated LPS-induced recognition memory impairment in rats. Our findings, for the first time, revealed that the antiinflammatory effects of S. boulardii are mediated via modulation of the NLRP3 inflammasome. Furthermore, we, for the first time, reported that S. boulardii could effectively reduce Aβ deposition in hippocampal CA1 and CA3 regions in LPS-treated rats. These results confirm our theory that probiotic S. boulardii supplementation can attenuate neuroinflammation, Aβ accumulation, and memory deficits induced by LPS, as shown in Figure 5.

Recent studies have indicated that intraperitoneally administered LPS enhances plasma and hippocampal levels of inflammatory cytokines, including TNF-α, IL-1β, neuroinflammation, Aβ accumulation, and subsequent memory deficits.41,28,42,43,25 Consistently

our findings revealed that LPS induces memory impairment, as determined using the NOR task. Moreover, LPS administration considerably elevated serum levels of IL-1β, IL-6, and TNF-α, as well as hippocampal levels of NLRP3 and caspase-1. Furthermore, LPS promoted Aβ accumulation in hippocampal CA1 and CA3 regions. Accordingly, LPS administration can lead to memory dysfunction by inducing inflammatory responses and amyloidogenesis.

Accumulated evidence has demonstrated the preventive role of probiotics in cognitive and memory dysfunction through the gut−brain axis. Xiao et al. (2020) have shown that the longterm treatment with probiotics, including B. longum, S. thermophilus, and Lactobacillus species, can alleviate memory impairment in lead-exposed rats by restoring the disrupted gut microbiota composition.35 Lactobacillus paracasei, L. plantarum, and S. thermophilus were found to improve learning and memory dysfunction in D-galactose-treated mice by inhibiting neuronal apoptosis and brain injury.34 Furthermore, the probiotic Lactobacillus johnsonii BS15 was shown to mitigate fluoride-induced recognition memory deficits by restoring gut microbiota and reducing intestinal permeability and inflammatory cytokines TNF-α and IL-1β in mice.44 In addition, Mohammadi et al. (2019) have revealed that L. helveticus and B. longum can attenuate memory deficits, as well as serum and hippocampal levels of TNF-α and IL-1β in LPS-treated rats.28 A human study has reported the safety and efficacy of the probiotic Bifidobacterium breve in ameliorating memory functions in elderly individuals with suspected mild cognitive impairment.45 Recently, Song et al. (2022) have reported that the cognitive deficit induced by D-galactose/aluminum chloride (AlCl3) could be reversed by the anti-inflammatory effect of probiotic Bacillus coagulans JA845 in mice. B. coagulans reportedly reduces serum levels of inflammatory cytokines by regulating the Nrf2/HO-1 and MyD88/TRAF6/NF-κB pathways.46 Interestingly, our findings also confirm the existence of a connection between brain function and intestinal microbiota. The current results revealed that S. boulardii supplementation could alleviate memory impairment and decrease serum levels of IL-1β, IL-6, and TNF-α in LPS-treated rats. In addition, our results presented a decrease in hippocampal levels of NLRP3 and caspase-1 in the LPS model. Thus, the anti-inflammatory effect of S. boulardii may be mediated by inhibiting the NLRP3 inflammasome. Similarly, Avolio et al. (2019) have shown that S. thermophilus and Lactobacillus species can attenuate anxietylike behaviors by modulating inflammatory responses in the brains of hamsters exposed to unpredictable chronic mild stress. The probiotics were found to exert an anti-inflammatory effect by inhibiting the NLRP3 inflammasome.47

Our findings also revealed that modifying the gut microbiota using S. boulardii attenuated LPS-induced Aβ deposits in the rat hippocampus. This result is consistent with that reported by Bonfili et al. (2017). Using the 3xTg-AD mouse model, the authors reported the preventive effect of the SLAB51 probiotic formulation on recognition memory deficits and Aβ accumulation by modulating the gut microbiota composition.32 Similarly, the probiotic B. coagulans JA845 can ameliorate cognitive decline and reduce Aβ deposits in the hippocampus of the D-galactose/AlCl3-induced AD mouse model.46 In addition, Abdelhamid et al. (2022) have reported that the probiotic B. breve can attenuate memory dysfunction and reduce Aβ production in APP knock-in mice.11 Given the

promising strategy to prevent or treat AD. Recently, Sarkar et al. (2021) have demonstrated the protective effects of S. boulardii on antibiotic-mediated gut dysbiosis in mice. Reportedly, S. boulardii inhibits antibioticinduced intestinal and brain inflammation and prevents hippocampal neuronal damage and cognitive impairment.38

■ CONCLUSIONS

The present research demonstrated the preventive effects of S. boulardii on LPS-induced memory dysfunction in rats. Administration of probiotic S. boulardii ameliorated LPSinduced neuroinflammation, Aβ deposition, and subsequent memory impairment. These S. boulardii-mediated neuroprotective effects are probably mediated by modulating the gut microbiota. Collectively, these results demonstrate the substantial role of the gut−brain axis in regulating AD-related diseases. Our results could help design preventive or therapeutic strategies against AD. Further studies are needed to clarify the additional mechanisms involved in the neuroprotective effects of S. boulardii.

MATERIALS AND METHODS

Animals. Adult male Wistar rats (200−220 g) were obtained from the breeding colony of the Shahid Beheshti University of Medical Sciences, Tehran, Iran. The rats were maintained under standard laboratory conditions, a 12/12 h light/dark cycle, and a constant room temperature (21 ± 2 °C). Food and water were provided ad libitum. The rats were randomly divided into four groups (N = 10 each). Experiments were carried out between 8 a.m. and 1 p.m. All experiments were approved by the Ethics Committee of Shahid Beheshti University of Medical Sciences (IR.SBMU.MSP.REC.1399.450) and performed, according to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.

Drugs. The present study employed the probiotic capsule DAILYEAST (Zist Takhmir Company, Tehran, Iran). Each capsule contained 250 mg of the active yeast S. boulardii (equivalent to 1010 colony-forming units [CFU]) and dissolved in saline before administration. LPS (Escherichia coli 055:B5, Sigma) was dissolved in phosphate-buffered saline immediately before injection.

■ EXPERIMENTAL METHOD

(1) The control group (Cnt) was administered saline by gavage and intraperitoneal (i.p.) injection; (2) the LPS group was administered saline by gavage and LPS by i.p. injection; (3) the LPS + S group was administered S. boulardii by gavage and LPS by i.p. injection; and (4) the S group was administered S. boulardii by gavage and saline by intraperitoneal injection. Saline or S. boulardii (250 mg/rat) was orally administered for 4 weeks. From day 14, LPS (0.25 mg/kg/day) was administered daily via i.p. injection for 9 days (days 14−22). On day 25, animals were subjected to the NOR task (N = 10). The day after the completion of the NOR task, rats were anesthetized with ketamine/xylazine (i.p. injection, 60/6 mg/kg), and blood samples were collected for ELISA (N = 7). Animals were subsequently sacrificed by decapitation, and the hippocampi were quickly harvested and stored at −80 °C until assessment. Following transcardial perfusion, rats were anesthetized with ketamine/xylazine and fixed in 4% paraformaldehyde (N = 3). Brains were removed, cut in half sagittally, immersed in 4% paraformaldehyde for 48 h, and then embedded in paraffin for Congo red staining.48 Figure 6 illustrates the experimental procedure of the study.

NOR Task. On 25th day of the study, the animals were subjected to the NOR task for 4 days. The animal recognition memory was assessed using the NOR paradigm based on the propensity of the animal to interact with a new target more than that with a familiar one.49 Animals were habituated to the arena for 2 days. Each animal was placed in a 40 × 40 × 50 cm3 box and allowed to explore the empty arena for 10 min. The familiarization phase was completed on the third day of the experiment. Animals were exposed to two identical targets (A1 and A2) in a familiar arena for 3 min. After 90 min, a new target (B) was substituted with a familiar target (A2), and short-term memory (STM) was examined by allowing the animals to explore targets for 3 min. On the fourth day, another new target (C) was substituted for target B, and animals were permitted to individually explore targets for 3 min to measure their long-term memory (LTM). All targets were plastic lego with distinct shapes and similar sizes. Animal behavior was video recorded, and the total time spent exploring each target was reported by an experimenter blinded to treatment conditions. Sniffing or touching targets with the nose is considered an exploratory action. To eliminate rats′ odors or residues, both targets and the arena were cleaned with 70% ethanol between the trials. The new target preference index percentage was calculated as follows:50

STM: the ratio of exploration time for a new target (B) to the total exploration time for both targets (A + B) × 100.

LTM: the ratio of exploration time for a new target (C) to the total exploration time for both targets (A + C) × 100.

Enzyme-Linked Immunosorbent Assay. For serum samples to perform ELISA, blood samples were centrifuged at 3500 rpm for 15 min. Serum levels of IL-1β (RLB00, R&D Systems), IL-6 (R6000B, R&D Systems), and TNF-α (DY510-05, R&D Systems) were measured by ELISA kits, according to the manufacturing protocols. The serum concentration of each cytokine was measured based on a standard curve, presented as pg/mL ± standard error of the mean (SEM).

Western Blotting. Briefly, hippocampi were homogenized in a lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 0.25% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 1 mM ethylenediaminetetraacetic acid [EDTA], 0.1% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride [PMSF]; pH 8.0) using a microhomogenizing system (Micro Smash MS-100) at 4 °C and 3000 rpm. Tissue lysates were centrifuged at 12,000 rpm for 20 min at 4 °C, and the supernatants were collected. The protein concentration of each supernatant was measured using the Bradford assay, and the same amount of each sample (40 μg) was loaded on 12% SDS gel electrophoresis (SDS-PAGE) and separated, according to the molecular weight. Subsequently, the protein samples were transferred to poly(vinylidene difluoride) (PVDF) membranes. Then, the membranes were immersed in a blocking solution (5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween-20) for 1 h at room temperature to inhibit the nonspecific binding of antibodies to the membrane surface proteins. The blots were then incubated with primary antibodies against caspase-1 (1:1000, NBP145433, NOVUS) and NLRP3 (1:1000, b263899, Abcam) at 4 °C overnight. The blots were washed and incubated with a secondary antibody (horseradish peroxidase [HRP]-conjugated goat antirabbit immunoglobulin [Ig] G H&L, 1:7000, ab6721, Abcam) for 1 h at room temperature. An ECL detection system (ECL kit, Bio-Rad) was used to visualize the protein bands on X-ray films. ImageJ software (National Institutes of Health, Bethesda, MD) was used to quantify protein expression levels. β-Actin (1:3000, ab8227, Abcam) was used as an internal control to normalize results.

Congo Red Staining. Aβ deposits in the hippocampal tissue were examined using a Congo red staining kit (Asiapajohesh, Amol, Iran), according to the manufacturer′s instructions. Briefly, 7 μm thick brain sections were deparaffinized with xylene and then hydrated. Subsequently, a Congo red solution was used to stain tissue sections for 60 min, rinsed under running tap water, and dipped in lithium carbonate. Sections were finally counterstained in hematoxylin, rinsed under running tap water, dehydrated in 90 and 100% alcohol, and cleared in xylene. The stained slides were mounted with mounting medium, captured using a microscope (Nikon Eclipse E600), and analyzed for changes among groups.

Statistical Analysis. Data were analyzed using GraphPad Prism version 8.0.2 (GraphPad Software, Inc., La Jolla, CA). The interaction between the drug treatment and the LPS model was examined using a two-way analysis of variance (ANOVA), followed by Tukey′s post hoc test for multiple comparisons. During the familiarization phase of the NOR test, differences in the exploration time for targets in each experimental group were examined using a two-tailed paired t-test. P < 0.05 was established as statistically significant. Data are presented as the mean ± SEM.

■ ASSOCIATED CONTENT

*sı Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschemneuro.2c00500.

Data underlying this study are available in the published article; NLRP3 and caspase-1 protein levels in the hippocampal tissue were examined using Western blotting; Aβ deposits in hippocampal CA1 and CA3 regions were detected using Congo red staining (PDF)

■ AUTHOR INFORMATION

Corresponding Authors

Marjan Nassiri-Asl − Department of Pharmacology, School of

Medicine, Shahid Beheshti University of Medical Sciences,

Tehran 19839-63113, Iran; Neurobiology Research Center,

Shahid Beheshti University of Medical Sciences, Tehran

19839-69364, Iran; orcid.org/0000-0003-3701-0758; Phone: +98212243996; Email: marjannassiriasl@ sbmu.ac.ir

Saeed Karima − Department of Clinical Biochemistry, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran 19839-63113, Iran; Phone: +982196661028;

Email: saeed.karima@sbmu.ac.ir; Fax: +982196661029

Authors

Fatemeh Babaei − Department of Clinical Biochemistry,

School of Medicine, Shahid Beheshti University of Medical

Sciences, Tehran 19839-63113, Iran

Mohammadreza Mirzababaei − Department of Clinical

Biochemistry, School of Medicine, Kermanshah University of

Medical Sciences, Kermanshah 6715847141, Iran

Leila Dargahi − Neuroscience Research Center, Shahid

Beheshti University of Medical Sciences, Tehran 19839-

63113, Iran

Zahra Shahsavari − Department of Clinical Biochemistry,

School of Medicine, Shahid Beheshti University of Medical

Sciences, Tehran 19839-63113, Iran

Complete contact information is available at: https://pubs.acs.org/10.1021/acschemneuro.2c00500

Author Contributions

F.B. carried out the experiment, wrote and revised the manuscript, and analyzed the data. M.M. wrote the original draft and drew the figures. L.D. and Z.S. reviewed and edited the manuscript. M.N.A. and S.K. designed the experiment, verified the analytical methods, supervised the study, and reviewed and revised the manuscript.

Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

The authors thank and appreciate Neurobiology and Neuroscience Research Centers, Shahid Beheshti University of Medical Sciences, for scientific and technical aid. This study was performed as a part of F.B’s Ph.D. thesis.

■ ABBREVIATIONS

LPS, lipopolysaccharide; CFU, colony-forming unit; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α; Aβ, amyloidβ; NLRP3, NOD-like receptor family pyrin domain containing 3; ASC, adaptor apoptosis speck-like protein; AD, Alzheimer′s disease; NOR, novel object recognition test; i.p., intraperitoneal; STM, short-term memory; LTM, long-term memory; CA1/3, cornu ammonis 1/3; Cnt, control; NF-κB, nuclear factor κB; ERK, extracellular signal-regulated kinase