A Probiotic Lactobacillus gasseri Alleviates Escherichia coli-Induced Cognitive Impairment and Depression in Mice by Regulating IL-1β Expression and Gut Microbiota

Excessive expression of interleukin (IL)-1β in the brain causes depression and cognitive dysfunction. Herein, we investigated the effect of Lactobacillus gasseri NK109, which suppressed IL-1β expression in activated macrophages, on Escherichia coli K1-induced cognitive impairment and depression in mice. Germ-free and specific pathogen-free mice with neuropsychiatric disorders were prepared by oral gavage of K1. NK109 alleviated K1-induced cognition-impaired and depressive behaviors, decreased the expression of IL-1β and populations of NF-κB+/Iba1+ and IL-1R+ cells, and increased the K1-suppressed population of BDNF+/NeuN+ cells in the hippocampus. However, its effects were partially attenuated by celiac vagotomy. NK109 treatment mitigated K1-induced colitis and gut dysbiosis. Tyndallized NK109, even if lysed, alleviated cognitive impairment and depression. In conclusion, NK109 alleviated neuropsychiatric disorders and colitis by modulating IL-1β expression, gut microbiota, and vagus nerve-mediated gut–brain signaling.


Introduction
The brain bidirectionally communicates with the gut microbiota/gastrointestinal tract via the hypothalamus-pituitary-adrenal (HPA) and microbiota-gut-brain (MGB) axes [1,2]. Excessive exposure to stresses such as social defeat, pathogens, and immobilization enhances the secretion of adrenal hormones, such as cortisol, and immune cytokines, such as interleukin (IL)-1β and IL-6 through activation of the HPA axis, resulting in gut microbiota alteration, depression, and cognitive impairment [3,4]. The overgrowth of endogenous or exogenous Escherichia coli in the intestine by stressors such as antibiotics causes gut microbiota alteration and collapses gut immune homeostasis, resulting in the occurrence of gut inflammation, depression, and cognitive impairment through the induction of IL-1β and corticosterone production [5,6]. Excessive IL-1β expression in the hippocampus causes psychiatric disorders such as depression [7,8]. Therefore, suppressing IL-1β expression may be beneficial for the therapy of neuropsychiatric disorders.
Probiotics are live microbes that promote health benefits [9]. Of these, Lactobacilli are repeatedly found in yogurt, kimchi, cheese, and mammalian gut bacteria. They revive altered microbiota [10,11], regulate the The mice with cognitive impairment and depression were prepared, as reported previously [5,6]. First, to examine the effect of NK109 on the generation of neuropsychiatric disorder in germ-free mice, the mice were separated randomly into vehicle-treated (CoV), Escherichia coli K1-treated (EcV), and NK109-treated groups (ENK) in K1-treated mice. Each group consisted of four mice. The EcV and ENK groups were gavaged orally with the Escherichia coli K1 (1 × 10 7 CFUs, suspended in 100 µL of saline) daily for 5 days. The CoV group was treated with the vehicle (saline) instead of the K1 suspension. From the next day, the ENK group was orally administered NK109 (1 × 10 9 CFUs/mouse/day) daily for 5 days. The CoV and EcV groups were orally treated with saline instead of the test agents.
Second, to examine the effect of NK109 on the cognitive impairment and depression in the SPF mice, the mice were randomly separated into CoV, EcV, and ENK groups. Each group consisted of six mice. The EcV and ENK groups were orally treated with the K1 suspension (1 × 10 9 CFUs, in 100 µL of saline) daily for 5 days. The CoV group was administered the vehicle instead of the K1 suspension. From the next day, the ENK group was orally administered NK109 (1 × 10 9 CFUs/mouse/day) daily for 5 days. The CoV and EcV groups were orally gavaged with the vehicle.
Third, to understand the effect of NK109 on the vagus nerve-mediated gut-brain signaling, we prepared the SPF mice with celiac vagotomy, as reported previously [20]. The mice were separated into vehicle-treated group in sham mice (CoV), Escherichia coli K1-treated (vEcV) and NK109-treated groups in K1-treated mice with vagotomy (vENK) vehicle-treated group in sham mice (CoV), Escherichia coli K1-treated (vEcV) and NK109-treated groups in K1-treated mice with vagotomy (vENK). Each group consisted of six mice. The vCoV, vEc, and vENK groups were vagotomized. The vEcV and vENK groups were orally gavaged with K1 (1 × 10 9 CFUs, suspended in 100 µL of saline) daily for 5 days. The vCoV group was administered with the vehicle instead of the K1 suspension. The vENK group was orally administered NK109 (1 × 10 9 CFUs/mouse/day) daily for 5 day from 24 h after the final gavage of K1. The vEcV group was orally administered the vehicle instead of NK109.
Depressive and cognitive behaviors were carried out 24 h after the final administration of the test agents. The mice were then killed 18 h after the final behavioral tasks. The blood, colon, and hippocampus were removed and stored at −80 • C until used in experiments.

Behavioral Tasks
For the assay of cognitive behaviors, the Y-maze task was carried out in a three-arm horizontal maze, which was 3 cm wide and 40 cm long with 12 cm-high walls [6,21]. A mouse was initially placed within one arm. The sequence and number of arm entries were then monitored for 8 min.
A spontaneous alternation was indicated as entries into all three arms on consecutive choices and calculated as the ratio (%) of spontaneous to possible alternations.
For the assay of depression-like behaviors, the forced swimming test (FST) and TST were carried out, as reported previously [6]. The TST was carried out on the edge of a table above 30 cm for 5 min. When the mice did not move and hanged passively, they were declared to be immobile. The FST was carried out in a round transparent plastic jar (20 × 40 cm 3 ) filled with fresh water (25 • C) to a height of 25 cm for 5 min [6]. When the mice remained floating in the water without struggling, they were declared to be immobile.

Myeloperoxidase Activity Assay, Immunoblotting, and ELISA
Myeloperoxidase activity was measured in colon homogenate supernatant, as reported previously [14]. BDNF, CREB, p-CREB, p65, p-p65, and β-action expression levels were determined in the supernatants of the tissue homogenates by immunoblotting, as reported previously [16]. Corticosterone, IL-6, and IL-1β levels were determined in the supernatant of the colon and hippocampus homogenate and plasma using commercial ELISA kits [16].

Immunohistochemistry
For the immunohistochemistry analysis, the mice were perfused transcardially with 4% paraformaldehyde. Hippocampi and colons were removed, post-fixed with 4% paraformaldehyde, immersed in 30% sucrose solution, frozen, and sectioned using a cryostat, as reported previously [20].  [6]. Cell nuclei were stained with DAPI. The immunostained section was observed with a confocal microscope.

Microbiota Sequencing
Genomic DNA of mouse fecal bacteria was extracted from fresh mouse stools using a QIAamp DNA stool mini kit, as reported previously [6,19]. Genomic DNA amplification was carried out using barcoded primers, which targeted the V4 region of the bacterial 16S rRNA gene. Each amplicon sequencing was carried out by using Illumina iSeq 100 (San Diego, CA, USA). The pyrosequencing reads were deposited in the NCBI's short read archive under accession number PRJNA622393.

Statistical Analysis
Experimental data are indicated as mean ± standard deviation (SD) and analyzed by Graph-Pad Prism 8 (GraphPad Software Inc., San Diego, CA, USA). The significance was analyzed using the Nutrients 2020, 12, 3441 5 of 14 unpaired t-test (p < 0.05). All p-values for the experimental data in the present study are indicated in Table S1.

Results and Discussion
To examine whether IL-1β expression-suppressing probiotics could alleviate cognition impairment and depression, we selected IL-1β expression-inhibiting probiotics from a human fecal bacterial strain collection. Of the tested probiotics, NK109 strongly suppressed K1-induced IL-1β expression and NF-κB activation in macrophage cells ( Figure 1A,B). NK109 also suppressed IL-1β expression and NF-κB activation in LPS-stimulated macrophage cells ( Figure S2). NK109 was identified as Lactobacillus gasseri on the basis of the results of gram staining, 16S rDNA sequencing, and API 50 CHL kit.

Statistical Analysis
Experimental data are indicated as mean ± standard deviation (SD) and analyzed by Graph-Pad Prism 8 (GraphPad Software Inc., San Diego, CA, USA). The significance was analyzed using the unpaired t-test (p < 0.05). All p-values for the experimental data in the present study are indicated in Table S1.

Results and Discussion
To examine whether IL-1β expression-suppressing probiotics could alleviate cognition impairment and depression, we selected IL-1β expression-inhibiting probiotics from a human fecal bacterial strain collection. Of the tested probiotics, NK109 strongly suppressed K1-induced IL-1β expression and NF-κB activation in macrophage cells ( Figure 1A,B). NK109 also suppressed IL-1β expression and NF-κB activation in LPS-stimulated macrophage cells ( Figure S2). NK109 was identified as Lactobacillus gasseri on the basis of the results of gram staining, 16S rDNA sequencing, and API 50 CHL kit. Next, we examined the effect of NK109 at a dose of 1 × 10 7 CFUs/mouse/day on K1-induced depression in the germ-free mice ( Figure 2A). Exposure to K1 significantly increased the depressionlike behaviors in the TST to 257.1% of the control group. Oral gavage of NK109 alleviated K1-induced depressive behaviors to 141.1% of the control group. Treatment with NK109 also decreased K1induced IL-1β expression, NF-κB + /Iba1 + (activated microglia) and IL-1R + cell populations, and NF-κB activation in the hippocampus, while the K1-suppressed BDNF + /NeuN + cell population and BDNF expression increased ( Figure 2B-F). Furthermore, NK109 treatment decreased K1-induced myeloperoxidase activity, the IL-1β expression, the NF-κB + /CD11c + cell population, and NF-κB activation in the colon ( Figure 2G-J). Next, we examined the effect of NK109 at a dose of 1 × 10 7 CFUs/mouse/day on K1-induced depression in the germ-free mice ( Figure 2A). Exposure to K1 significantly increased the depression-like behaviors in the TST to 257.1% of the control group. Oral gavage of NK109 alleviated K1-induced depressive behaviors to 141.1% of the control group. Treatment with NK109 also decreased K1-induced IL-1β expression, NF-κB + /Iba1 + (activated microglia) and IL-1R + cell populations, and NF-κB activation in the hippocampus, while the K1-suppressed BDNF + /NeuN + cell population and BDNF expression increased ( Figure 2B-F). Furthermore, NK109 treatment decreased K1-induced myeloperoxidase activity, the IL-1β expression, the NF-κB + /CD11c + cell population, and NF-κB activation in the colon ( Figure 2G-J).
To prepare the mice with psychiatric disorders, we orally gavaged K1 at dosages of 1 × 10 7 , 1 × 10 8 , and 1 × 10 9 CFUs/mouse/day in the SPF mice and measured the depression-like behavior in the TST (data not shown), as previously reported [6]. Oral gavage of K1 at a dose of 1 × 10 9 CFUs/mouse/day most strongly caused depression. Therefore, we investigated the effect of NK109 on the occurrence of cognitive impairment and depression in the SPF mice with K1 (orally gavaged at a dose of 1 × 10 9 CFUs/mouse/day)-induced psychiatric disorders ( Figure 3). Exposure to K1 caused cognitive impairment in the Y-maze task to 82.6% of the control group ( Figure 3A). However, oral gavage of NK109 (1 × 10 9 CFUs/mouse/day) alleviated K1-induced cognitive impairment in the Y-maze task to 109.6% of the control group. Exposure to K1 also significantly increased depressive behaviors in the TST and FST to 139.4% and 298.4% of the control group, respectively ( Figure 3B,C). Oral administration of NK109 reduced the K1-increased immobility time in the TST and FST to 81.0% and 148.9% of the control group, respectively. However, oral administration of NK109 did not affect the cognitive function in the control mice ( Figure S1). K1 treatment increased the activation of NF-κB and the expression of IL-1β and IL-6, as well as reduced the expression of BDNF in the hippocampus ( Figure 3D,E). However, oral gavage of NK109 decreased the K1-induced NF-κB activation, IL-1β and IL-6 levels, and NF-κB + /Iba1 + and IL-1R + cell populations, as well as increased the K1-suppressed BDNF + /NeuN + cell population, CREB phosphorylation, and BDNF expression in the hippocampus ( Figure 3D-I). Furthermore, oral gavage of NK109 reduced the K1-induced IL-6 and corticosterone levels in the blood Nutrients 2020, 12, 3441 6 of 14 ( Figure 3J,K). Furthermore, to understand the vagus nerve-mediated effect of NK109 on K1-induced cognitive impairment and depression, we examined its effects in the celiac vagotomy-treated mice. Vagotomy itself did not cause significant memory impairment or depression in the Y-maze task or TST, respectively ( Figure S3). K1 treatment caused cognitive impairment-and depression-like behaviors. Oral administration of NK109 weakly, but not significantly, alleviated K1-induced cognitive impairment in the Y-maze task and depression in the TST ( Figure 3L,M). NK109 also increased hippocampal CREB phosphorylation and BDNF expression ( Figure 3N). The ameliorating activity of NK109 against cognitive impairment and depression was lower in the vagotomy-operated mice than in the vagotomy-untreated mice. Escherichia coli K1-treated (EcV) and K1/NK109-treated (ENK) groups were exposed to K1 (1 × 10 9 CFUs/mouse/day) daily for 5 days and thereafter treated with NK109 (for ENK, 1 × 10 9 CFUs/mouse/day) or the vehicle (for EcV) for 5 days. The control group (CoV) was treated with saline instead of K1 and NK109. Data values were are as mean ± SD (n = 4). # p < 0.05 vs. CoV. * p < 0.05 vs. EcV. Dapi, 4′, 6-diamidino-2-phenylindole dihydrochloride; Iba1, ionized calcium-binding adapter molecule 1; NF-κB, nuclear factor-κB; IL-1R, interleukin 1 receptor; IL-1β, interleukin 1β; CD11c, a type I transmembrane protein.
Next, we investigated the effect of NK109 on the K1-induced gut microbiota alteration in the mice with K1-induced cognitive impairment and depression ( Figure 3H-J, Figure S3, and Tables S2-S4). Oral gavage of K1 significantly increased the populations of Proteobacteria and Cyanobacteria, while the population of Bacteroidetes decreased. K1 treatment did not affect α-diversity, including estimated operational taxonomic unit (OTU) richness and Shannon's diversity index, compared to those in the control mice, while K1 treatment significantly shifted the β-diversity, as shown by the principal coordinate analysis (PCoA) based on Jansen-Shannon. Oral administration of NK109 decreased the K1-induced β-diversity change, while the α-diversity was not affected. NK109 treatment decreased the K1-induced populations of Proteobacteria and Cyanobacteria and increased the K1-suppressed population of Bacteroidetes. Oral administration of NK109 increased the K1-suppressed populations of Bacteroidaceae and Muribaculaceae in the family level and Bacteroides, Muribaculaceae_uc, and PAC001063_g at the genus level and Bacteroides coccae and PAC00163_g_uc at the species level. However, NK109 treatment decreased the K1-induced population of Christensenellaceae, Desulfovibrionaceae, and Rhodospirillaceae in the family and Alloprevotella, FR888536_g, Helicobacter, and LARJ_g at the genus level and LARJ_g, FJ880724_s, and the Helicobacter rodentium group at the species level.
Next, to evaluate the biological difference between the live and tyndallized probiotics, we tyndallized NK109, lysed by ultrasonication, centrifuged, and examined the effects of tNK109 and its supernatant (NC) and precipitate fractions (NM) on K1-induced cognitive impairment and depression in the SPF mice ( Figure 5). Their treatment significantly alleviated K1-suppressed spontaneous alteration in the Y-maze task and the K1-increased immobility time in the TST and FST (Figure 5A-C). Of the tested tNK109, NC, and NM, tNK109 and NM more strongly alleviated K1-induced cognitive impairment and depression than did NC. They also suppressed K1-induced hippocampal IL-1β and IL-6 expression, NF-κB + /Iba1 + and IL-1R + cell populations, and NF-κB activation, as well as increased the K1-suppressed hippocampal BDNF + /NeuN + cell population and BDNF expression ( Figure 5D-I). K1-induced IL-1β and corticosterone levels decreased in the blood by oral administration of tNK109, NC, or NM ( Figure 5J,K). Treatment with tNK109 or NM also suppressed the K1-induced colitis markers: They reduced colon shortening and NF-κB activation, as well as decreased myeloperoxidase activity, IL-1β, IL-6, and TNF-α levels, and the NF-κB + /CD11c + cell population ( Figure 6A-G).
The prevalence of psychiatric disorders is significantly higher in patients with inflammatory bowel disease (IBD) than in healthy people [22]. Exposure to stresses, including immobilization, causes psychiatric disorders with colitis and gut dysbiosis via the HPA axis activation [3,4]. Gut dysbiosis and inflammation induce the occurrence of psychiatric disorders through activation of the MGB axis [5,21,22]. Exposure of SPF mice to stressors causes gut dysbiosis: They increase the Proteobacteria population in the gut microbiota [5,16]. A gut commensal Lactobacillus johnsonii alleviates anxiety-like behaviors with gut dysbiosis in mice [5,8]. The brain bidirectionally communicates to the gut microbiota through the HPA and MGB axes [1,2].
In the present study, exposure to Escherichia coli K1 significantly caused psychiatric disorders, including depression and memory impairment, and IBD-like colitis in the SPF or germ-free mice, as reported previously [5,6]. Exposure to Escherichia coli (at a dose of 1 × 10 7 CFUs/day/mouse) caused depression more strongly in the germ-free mice than in the SPF mice. The occurrence of depression-like behaviors by exposure of the germ-free mice to Escherichia coli at a dose of 1 × 10 7 CFUs/day/mouse was similar to that in the SPF mice exposed to Escherichia coli at a dose of 1 × 10 9 CFUs/day/mouse. This suggests that normal gut microbiota may defend the colonization of pathogenic microbes such as Escherichia coli in the gastrointestinal tract. We also found that K1 caused neuroinflammation and colitis: It increased the expression of IL-1β and IL-1 receptor (IL-1R) in the hippocampus and IL-1β expression in the colon. The overgrowth of gut commensal Escherichia coli by immobilization stress, a colitis inducer, or oral gavage of Escherichia coli causes colitis and neuroinflammation: Escherichia coli induces the expression of IL-1β and IL-6 in the hippocampus and colon [5]. Stress significantly induces the expression of IL-1β in the hippocampus and the administration of IL-1R antagonist alleviates depression-like behaviors [7]. An anti-inflammatory therapy in patients with IBD alleviates cognitive function [23]. These findings suggest that K1 may cause psychiatric disorders by enhancing the expression of pro-inflammatory cytokines, particularly IL-1β, and their receptors, such as IL-1R, in the hippocampus, and the suppression of IL-1β expression can alleviate psychiatric disorders. Nutrients 2020, 12, x FOR PEER REVIEW 10 of 14  (b) their intensities) populations. Effects on the blood corticosterone (J) and IL-6 levels (K), assessed by ELISA. Escherichia coli K1-treated (EcV), tyndallized NK109-treated (EtN), NK109 sonicate supernatant fraction-treated (ENC) groups were exposed to K1 and thereafter treated with test agents (EcV, vehicle; EtN, tNK109; ENC, supernatant fraction (NC) of NK109 (1 × 10 9 CFU) lysate/mouse/day; ENM, precipitate fraction (NM) of NK109 (1 × 10 9 CFU) lysate/mouse/day) daily for 5 days. Control group (CoV) was treated with saline instead of K1 and NK109. Data values were described as mean ± SD (n = 6). # p < 0.05 vs. CoV. * p < 0.05 vs. EcV.
The prevalence of psychiatric disorders is significantly higher in patients with inflammatory bowel disease (IBD) than in healthy people [22]. Exposure to stresses, including immobilization, causes psychiatric disorders with colitis and gut dysbiosis via the HPA axis activation [3,4]. Gut dysbiosis and inflammation induce the occurrence of psychiatric disorders through activation of the MGB axis [5,21,22]. Exposure of SPF mice to stressors causes gut dysbiosis: They increase the Proteobacteria population in the gut microbiota [5,16]. A gut commensal Lactobacillus johnsonii alleviates anxiety-like behaviors with gut dysbiosis in mice [5,8]. The brain bidirectionally communicates to the gut microbiota through the HPA and MGB axes [1,2].
In the present study, exposure to Escherichia coli K1 significantly caused psychiatric disorders, including depression and memory impairment, and IBD-like colitis in the SPF or germ-free mice, as reported previously [5,6]. Exposure to Escherichia coli (at a dose of 1 × 10 7 CFUs/day/mouse) caused depression more strongly in the germ-free mice than in the SPF mice. The occurrence of depressionlike behaviors by exposure of the germ-free mice to Escherichia coli at a dose of 1 × 10 7 and ENM groups were exposed to K1 (1 × 10 9 CFUs/mouse/day) daily for 5 days and thereafter treated with the test agents (EcV, vehicle; EtN, 1 × 10 9 CFUs/mouse/day of tNK109; ENC, supernatant fraction (NC) of NK109 (1 × 10 9 CFUs) lysate/mouse/day; ENM, precipitate fraction (NM) of NK109 (1 × 10 9 CFUs) lysate/mouse/day) daily for 5 days. The control group (CoV) was treated with the vehicle instead of K1 and NK109. Data values are described as mean ± SD (n = 6). # p < 0.05 vs. CoV. * p < 0.05 vs. EcV.
We also found that oral gavage of NK109, which significantly inhibited IL-1β expression in K1-stimulated macrophages, alleviated K1-induced cognition impairment and depression in the mice. Furthermore, NK109 suppressed hippocampal inflammation in the K1-treated mice: It decreased NF-κB activation and IL-1β expression in the hippocampus, while the IL-1R + and NF-κB + /Iba1 + cell (activated microglia) populations increased. NK109 alleviated K1-induced colitis: It suppressed the expression of IL-1β and activation of NF-κB in the colon. These results suggest that NK109 can mitigate neuroinflammation and colitis by inhibiting Il-1β expression through the regulation of the NF-κB signaling pathway, resulting in the amelioration of cognition impairment and depression.
NK109 treatment increased K1-suppressed BDNF expression and the BDNF+/NeuN+ cell population in the hippocampus. NK109 also suppressed K1-induced expression of IL-6, which may be associated in the occurrence of neuropsychiatric disorders [24], and corticosterone in the blood. Neuroinflammation suppresses BDNF expression in the brain of mice [25]. Hippocampal BDNF expression is associated with the outbreak of cognitive impairment and depression [25][26][27]. These findings suggest that NK109 may alleviate cognitive impairment and depression by regulating NF-κB-mediated BDNF expression.
Moreover, the ameliorating effect of NK109 against K1-induced cognitive impairment and depression was partially attenuated by celiac vagotomy. Vagotomy suppresses the ameliorating effect of Lactobacillus rhamnosus against anxiety and depression [28]. These findings suggest that NK109 may alleviate neuropsychiatric disorders by modulating the immune response and vagus nerve-mediated gut-brain signaling through the regulation of the pro-inflammatory cytokine expression and gut bacteria byproducts.
NK109 attenuated K1-induced gut microbiota alteration: It suppressed the Proteobacteria population. Excessive growth of Proteobacteria, particularly Enterobacteriaceae, causes colitis, resulting in the occurrence of neuroinflammation. NK109 alleviated K1-induced colitis and neuroinflammation and colitis with gut dysbiosis in the mice, leading to the attenuation of cognitive impairment and depression. Bifidobacterium breve strain A1 improves Aβ protein-induced cognition impairment and neuroinflammation in mice [29]. Lactobacillus plantarum C29 alleviates the cognition impairment in aged mice and patients with cognitive decline by inducing BDNF expression [27,28]. Lactobacillus mucosae NK41 significantly mitigates Escherichia coli-induced neuropsychiatric disorders, neuroinflammation, and colitis in rodents [6]. These findings suggest that NK109 can alleviate the generation of cognitive impairment and depression by suppressing neuroinflammation and colitis through the modulation of gut microbiota.
tNK109 and its lysates NC and NM alleviated K1-induced depression and cognitive impairment with colitis in the mice by regulating NF-κB-mediated BDNF and IL-1β expression. Their effects were comparable to those of live NK109, suggesting that NK109, even if killed and lysed, can regulate NF-κB-mediated BDNF and IL-1β expression by the modulation of gut dysbiosis, resulting in the amelioration of neuropsychiatric disorders.

Conclusions
NK109 significantly alleviated Escherichia coli K1-induced cognitive impairment-and depression-like behaviors in germ-free and SPF mice by regulating the immune response through NF-κB-involved BDNF expression, IL-1β expression, and vagus nerve-mediated gut-brain signaling. NK109 treatment also mitigated Escherichia coli-induced colitis and gut dysbiosis. tNK109 and its lysates NC and NM also alleviated cognitive impairment-and depression-like behaviors and colitis. Finally, the attenuation of gut dysbiosis and IL-1β expression by beneficial bacteria, including NK109, can alleviate neuropsychiatric disorders such as cognitive impairment and depression.
Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/12/11/3441/s1; Figure S1. NK109 alleviated K1-induced cognitive impairment and depression in mice; Figure S2. Effects of NK109 on the LPS-induced IL-1β and NF-κB activation in macrophages; Figure S3. The difference in the cognitive impairment and depression-like behaviors between mice with and without celiac vagotomy; Table S1. p-values for the experimental data in the present study. Table S2. Effect of NK109 on the gut microbiota composition at the family level in EC-treated mice. Table S3. Effect of NK109 on the gut microbiota composition at the genus level in EC-treated mice. Table S4. Effect of NK109 on the gut microbiota composition at the species level in EC-treated mice.