Ameliorative action of probiotics on the neurotoxicological effect of Aluminium chloride and D-galactose

Ameliorative action of probiotics on the neurotoxicological effect of Aluminium chloride and D-galactose | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ameliorative action of probiotics on the neurotoxicological effect of Aluminium chloride and D-galactose Vaishali Dasriya, Manorama Kumari, Soniya Ranveer, Pradip Behare, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5226459/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 13 You are reading this latest preprint version Abstract This study aimed to investigate the protective effect of Limosilactobacillus fermentum NCDC701 against AlCl 3 and D-galactose (D-gal) -induced Alzheimer rat model. The dose of AlCl 3 (50mg/kg body weight) and D-gal (100mg/kg bodyweight) for 7 days induced oxidative stress such as amyloid plaque deposition, neuroinflammation, and reduction of neurotransmitters in the brain. In contrast, the supplementation of NCDC701 improved the neurotransmitter levels, antioxidants and anti-inflammatory cytokine IL-10, while decreased the amyloid peptide, pro-inflammatory cytokines, as well as LPS in colon and brain. The recovery in amyloid plaque deposition and tissue injury was also confirmed by the histopathological examinations. The supplementation of NCDC701 also improved the decreased expression levels of intestinal tight junction proteins, inhibited the up-regulation of p65, COX-2 and iNOS expression, and improved gut microbiota dysbiosis by increasing the Firmicutes / Bacteroidetes ratio, promoted beneficial microflora ( Oscillospira , Ruminococcus , Verrucomicrobia , Lactobacillus , and Prevotella ), and suppressed the pathogenic microflora ( Spirochaetes , Tenericutes , Prophyromonadaceae , Coprococcus, Clostridium , and Allobaculum ) by increasing the concentration of butyrate and total SCFAs compared to the AlCl 3 and D-gal treated model. The findings of this study provide novel insights into the effect of NCDC701 intervention on the gut–microbiome–brain axis and should aid future understanding of probiotics for improved host health. Limosilactobacillus fermentum NCDC701 Alzheimer gut microbiota neuroinflammation oxidative stress microbiota-gut-brain axis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Cognitive and memory diminishing as well as progressive impairment of daily living activities are the main clinical signs of Alzheimer, a chronic neurodegenerative illness with a complex pathophysiology [ 1 ]. Alzheimer currently has no cure or highly effective treatment due to its multifactorial nature, which results from a combination of individual factors, genetic predisposition, and environmental influences [ 2 ]. The marks of Alzheimer include β-amyloid plaque formation, neurofibrillary tangles, oxidative stress, neuroinflammation, and mitochondrial failure. Additionally, the pathophysiology of Alzheimer has been linked to mitochondrial dysfunction via the production of reactive oxygen species in mitochondria, amyloid plaque deposition activates microglia, which then promotes the production of pro-inflammatory cytokines in the brains, driving neuroinflammation [ 3 ]. Various chemicals, such as pesticides, heavy metals, drugs, aluminium, and D-galactose, can act as neurotoxins and cause neural loss. These chemicals increase the peroxidation of lipid and protein i.e., increased malonaldehyde acrolein, neuroinflammation and apoptosis of neuron cell resulting in decreased neurons [ 4 , 5 ]. The main contributing element to the onset of Alzheimer is the build-up of amyloid peptide (1–42) and (1–40) in the brain.[ 6 ]. Additionally, the disruption of metal homeostasis can cause aluminum toxicity, through increasing the activity of iron, which can cause oxidative damage in Alzheimer pathogenesis [ 7 , 8 ]. The gut microbiota dysbiosis has also been revealed to be associated with the neurological disorders such as Alzheimer's disease [ 9 , 10 ]. The imbalanced gut microbiota can damage the intestinal mucosal barrier, which can activate microglia (i.e., amyloids and lipopolysaccharides) and cause neuro-inflammation in the central nervous system, contributing to neurodegeneration [ 11 , 12 ]. The increased number of pathogenic bacteria increases the development of amyloid plaque formation in the brain [ 13 ]. Inhibiting the pathophysiology associated with amyloid plaque, such as oxidative stress and neuroinflammation, could be a promising approach to slowing the progression of Alzheimer. Several drugs such as Rivastigmine, Galantamine, and Donepezil (acetylcholinesterase inhibitors), Memantine (an N-methyl D-aspartate receptor antagonist), and Benzodiazepine are available in the market for the treatment of Alzheimer's [ 14 ]. However, these drugs have several side effects, which can worsen the condition of Alzheimer's patients [ 15 ]. Therefore, there is a need for a protective therapy with no side effects to improve the condition of Alzheimer's. Probiotics and its bioactive metabolites such as neurotransmitters, gamma-aminobutyric acid (GABA), short-chain fatty acids (SCFAs), and tryptophan has been reported to modulate peripheral and central nervous systems including Alzheimer's disease, autism, and depressive disorder [ 16 , 17 , 18 , 19 , 69 ] in response to the immune system, the vagus nerve, the enteric nervous. Several Lactobacillus species have been reported to demonstrate the significant effects as neuromodulators and neurotransmitters (including monoamines, serotonin, and brain-derived neurotrophic factor) [ 20 ]. Microbial metabolites such as SCFAs, directly alter brain neurological functioning via vagal, endocrine, humoral, and immunological pathways by entering circulation or crossing the blood-brain barrier (BBB) directly activate Treg cells, endocrine cells, and neuronal cells in order to enhance the level of regulatory cytokines that sustain brain functioning [ 21 ]. The detailed in vivo studies to assess the anti-oxidative and anti-inflammatory effects of probiotics in the brain and gut, as well as their influence on the microbiota-gut-brain axis, are required. In previous studies, only a few researchers have done work in the area of a direct relationship between the administration of probiotics and change in alteration of neurotransmitter levels in the host. Further, the efficacy of using Probiotics in neurological disorders is not much studied. So far, indigenous probiotic cultures were not screened and evaluated earlier for anti-Alzheimer properties. This promotes the exploration of the GRAS status of probiotics over side-effects and also screens for a specific activity such as neurotransmitter and short-chain fatty acid production and antioxidant potential. To that end, we developed and tested L. fermentum NCDC701, a SCFA, GABA, and serotonin producing probiotic that could influence the gut-microbiota-brain axis by improving oxidation and inflammation in the brain, strengthening intestinal epithelial barrier integrity, and modulating microbiome composition and SCFA production in the gut. 2. Materials and Methods 2.1. Reagents Limosilactobacillus fermentum NCDC701 was incubated with De Man, Rogosa and Sharpe agar at 37°C for 14 hours. The standard diets were obtained from Small Animal House, NDRI, Karnal. The catalogue numbers of chemical and ELISA kits are added to supplement material S1. 2.2 Quantification of GABA production Sample preparation and quantification of GABA production by Limosilactobacillus fermentum NCDC701 followed by [ 68 ] 2.3. Preparation of Limosilactobacillus fermentum NCDC701 Limosilactobacillus fermentum NCDC701 was collected from the National Collection of Dairy Cultures, ICAR-NDRI, Karnal. The Limosilactobacillus fermentum NCDC701 were grown at 37°C overnight in de Man-Rogosa-Sharpe broth (Himedia). The bacterial cells were collected by centrifugation at 8,000 × g for 10 min, washed three times with phosphate-buffered saline and adjusted to 3×10 8 CFU/ml for oral administration to the rats. 2.4. Animal experimental design 2.4.1 Animal ethics declaration In the pursuit of scientific advancement and the understanding of toxicological impacts, involved following principles regarding the ethical treatment of animals such as ensured their welfare is prioritized in all aspects of our research. The #R princeiple followed such as reduction, replacement, refinement, All proposed studies involved animals that undergo thorough ethical review by an institutional animal care ICAR-NDRI, Karnal and conducted according to the guidelines of the Institutional Animal Ethics Committee in an animal house registered with CPCSEA (1705/GO/Re/SL/13/CPCSEA) to ensure adherence to ethical standards and welfare considerations. We ensured all personnel involved in the care and use of animals are adequately trained and competent in handling and administering procedures to minimize stress and harm to the animals. 2.4.2 Euthanasia method The euthanasia method for rat was the carbon dioxide (CO 2 ) inhalation. The rat was placed in CO 2 chamber for a minute. And supply the 100% CO 2 with the 30–70% displacement rate per minute to existing air chamber. It help to achieve balance gas mixture for rapid unconsciousness with minimal distress to the animals. The active exposure of rat was around 2–3 minutes. 2.4.3 Experimental design In vivo experiments were conducted according to the guidelines of the Institutional Animal Ethics Committee in an animal house registered with CPCSEA (1705/GO/Re/SL/13/CPCSEA) at ICAR-NDRI, Karnal, India. Wistar Albino rats (48 males, 12 weeks old, weighing 155-156.5 g) were obtained from ICAR-NDRI, Karnal, and housed in polycarbonate cages with free access to food and water, and maintained at 24°C and 55–60% humidity on a 12-hour light/dark cycle. After a week of acclimatization, the rats were randomly assigned to six groups (8 animals/ group). Group 1 was maintained on a standard diet and injected intraperitoneally with saline. Group 2, an Alzheimer's model, was injected intraperitoneally with AlCl 3 and D-gal for 7 days. Group 3, a positive control, was injected with AlCl 3 and D-gal (7 days) and orally administered memantine drug (21 days). Group 4 was a co-administered group injected with AlCl 3 and D-gal (7 days) and L. fermentum NCDC701 from the 1st to the 28th day by oral gavage. Group 5 was a prophylactic group that was administered L. fermentum NCDC701 from the 1st day to the 21st day (oral gavage) and last 7 days for AlCl 3 and D-gal intraperitoneal injection. Group 6 was a therapeutic group that was intraperitoneally injected with AlCl 3 and D-gal for the first 7 days and administered L. fermentum NCDC701 for the last 8th to 28th day (oral gavage). Grouping of experimental data and diet plan were mentioned in Table 1 . Table 1 Grouping of experimental rat and diet plan Group No. of rats Diet 1 8 Standard diet (SD) 2 8 SD + (AlCl 3 + D-gal, 7days) i.p.+ Saline by gavage 3 8 SD + (AlCl 3 + D-gal, 7 days) + Memantine (1.5 mg/kg), last 21 days by i.p. 4 8 Co-administrative group : SD + (AlCl 3 + D-gal, 7 days by i.p + Limosilactobacillus fermentum NCDC701, 28 days) by gavage 5 8 Prophylactic group : SD + Limosilactobacillus fermentum NCDC701, first 21 days by i.p + (AlCl 3 + D-gal, last 7 days) by gavage 6 8 Therapeutic group : SD + (AlCL 3 + D-gal, 7 days] by i.p + Limosilactobacillus fermentum NCDC701, last 21 days) by gavage 2.5. Histopathological analysis The brain and colon tissue samples were harvested and fixed in a 10% formalin solution for 12 hours. To remove any remaining moisture, the samples were soaked in xylene. The tissue samples were then cut into 4µm thick sections, fixed in paraffin, and stained with Congo red and haematoxylin and eosin, respectively. Detailed histopathological analysis given in supplementary material S2. 2.6. Biochemical analysis ELISA kits from Biolinkk were used to measure the levels of GABA and serotonin in brain homogenates, quantify β-amyloid peptide 1–42 in brain and serum, assess antioxidant activity including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) in brain and colon homogenates, and detect inflammatory parameters such as IL-6, TNF, and IL-10 in brain and colon homogenates of three mice from each group. The absorbance was determined at 450nm using a microplate reader. The sample and standard preparation method given in supplemented material S3.2.6. Tight junction and inflammatory gene expression by RT-qPCR RT-qPCR was used to analyse the effects of Limosilactobacillus fermentum NCDC701 on gene expression of zonula occludens, occludin, and claudin-1 in colonic tissue, as well as p65, COX-2, and iNOS in both brain and colonic tissues by following the procedure of [ 22 ] given in supplemented material S4. 2.7. Analysis of microbiome The DNA extraction conditions for faecal sample from each group of rats (Group 1–6) was optimized using the MPI Bead Beater (USA) and the Quick-DNA Faecal/Soil Microbe DNA Miniprep Kit (Zymo Research, USA). The concentration and purity of the extracted DNA was assessed using the SYNERGY H1 Hybrid Reader. High-purity faecal DNA samples from each group were pooled together by following the procedure of [ 22 ] given in supplemented material S5. 2.8. Determination of SCFAs from faecal samples SCFAs were extracted from each rat faecal sample in each of the six groups using a modified method [ 23 ]. Detail procedure of sample preparation given in supplement material S6. 2.9. Statistical analysis The results of each experiment were carried out in triplicate and presented as mean ± SEM. When appropriate, analysis of variance (one-way or two-way ANOVA) was performed using GraphPad Prism (version 5), followed by Bartlett’s test to assess the homogeneity of variance and determine significant differences at P < 0.05. Microsoft Office 2010 was used for tabulation and presentation of data. 3. Result 3.1. Effects of aluminium chloride and D-galactose on histopathology 3.1.1. Congo red staining of brain tissue Histopathological observation of brain examined the protective effect of Limosilactobacillus fermentum NCDC701 on the amyloid plaque deposited in brain after the induction of AlCl 3 and D-gal. The brains of the control group had a normal morphology cortical neuron, and intact cell. Amyloid plaque deposition (Red arrow), cerebral amyloid angiopathy (Yellow arrow), and neurofibrillary tangle (Black arrow) were all visible in the AlCl 3 and D-gal induced group. Compared to control groups, the hippocampus and cerebral cortex of rat treated with AlCl 3 and D-gal plus memantine treated displayed few amyloid plaques (Red arrow) and cerebral amyloid angiopathy (Yellow arrow) (Fig. 1). After the administration of L. fermentum NCDC701 these abnormalities (neurofibrillary tangles, neuropil threads, neurotic plaques, and amyloid angiopathy) were reduced and showing normal hippocampus with intact cell (Violet arrow) and no sign of necrosis (Fig. 1). 3.1.2. Haematoxylin and eosin staining of colon Histopathological study demonstrated that the control group had a normal colon mucosa with intact epithelium. In the group of rats treated with AlCl 3 and D-gal, an inflamed and damaged colon was observed. The colon exhibited damaged epithelial integrity (black arrow), inflammatory cell infiltration, and scattered neutrophils (red arrow). The group treated with memantine showed less damaged epithelial integrity and fewer infiltrating leukocytes. The administration of L. fermentum NCDC701 resulted in an improvement in epithelial integrity. A decrease in infiltrating leukocytes and a normal colon mucosa with intact epithelium were observed in the group administered L. fermentum NCDC701 (Fig. 2). 3.2. L. fermentum NCDC701 supplementation & neuromodulatory responses The GABA production of L. fermentum NCDC701 was 99.43 ± 51 mM, comparable higher than another cultures. The administration of L. fermentum NCDC701 was found to modulate the Alzheimer's disease-related changes by reducing the β-amyloid 1–40 and 42 peptide concentration in rat tissue. The administration of L. fermentum NCDC701 resulted in a decrease in β-amyloid 1–40 and 42 peptide concentration in the rat brain and serum, compared to the brain and serum of the AlCl 3 and D-gal-induced rats. Whereas, the positive control group, which received memantine, showed a slight decrease in β-amyloid 1–40 and 42 peptide concentration in brain and serum tissue compared to the L. fermentum NCDC701 treated group (Fig. 3a, b, c and d). The reduction in GABA and serotonin levels was restored by the administration of L. fermentum NCDC701 in rat brain and colon tissue compared to the in brain and colon tissue of AlCl 3 and D-gal induced rats There was no significant differences (P < 0.05) observed in GABA and serotonin levels between the L. fermentum treated groups. The memantine treated group also shown the recovery effect in restoration of GABA and serotonin levels (Fig. 3e, f, g and h). The increase in pro-inflammatory cytokines such as, IL-6 and TNF-α concentration and decreased in the level of anti-inflammatory cytokine IL-10 (P < 0.05), were significantly modulated by the administration of L. fermentum NCDC701 in the brain and colon tissue of the co-administration group, prophylactic group and therapeutic group, compared to the brain and colon tissue of AlCl 3 and D-gal-induced rats (Fig. 3i, j, k, l, m, and n). The AlCl 3 and D-gal group showed a significant decrease (P < 0.05) in SOD, CAT, and GSH-Px, activities in the brain and colon, compared with that in the control group. The supplementation of L. fermentum NCDC701 significantly improved SOD, CAT, and GSH-Px, in the co-administration group, prophylactic group and therapeutic group, compared to the brain and colon tissue of AlCl 3 and D-gal-induced rat (Fig. 3o to t). The levels of SOD, CAT, and GSH-Px did not show a significant change (P > 0.05) in the brain and colon of the L. fermentum NCDC701 administered group. In contrast, the SOD, CAT, and GSH-Px activities of the AlCl 3 and D-gal induced rats were found to be significantly lower (P < 0.05) than those of the control group. Furthermore, the SOD, CAT, and GSH-Px activities in the brain and colon of the rats treated with L. fermentum NCDC701 were increased as compared to the AlCl 3 and D-gal induced rat groups. 3.3. Effects of Limosilactobacillus. fermentum NCDC701 on tight junction and inflammatory response gene expression The mRNA expression of tight junction genes, including Zoccludin-1, Claudin-1, and occludin, was significantly reduced (P < 0.05) in the colonic tissues of the AlCl 3 and D-gal group. However, supplementation with L. fermentum NCDC701 increased the expressions of these genes, demonstrating a protective effect. Memantine (positive control) also improved the expression of these genes, but less than the control group (Fig. 4). Compared to the control group, the AlCl 3 and D-gal groups showed significantly higher expression of p65, COX-2 and iNOS in both colon and brain tissue. However, the administration of L. fermentum NCDC701 and memantine (a positive control) dramatically reduced the enhanced expression (P < 0.05) of p65, COX-2 and iNOS in both brain and colon tissue (Fig. 4). 3.4 Limosilactobacillus fermentum NCDC701 restore gut microbiota of AlCl 3 and D-gal induced rat The V 3 -V 4 region of the 16SrRNA gene, which has high nucleotide diversity and discriminatory power, was sequenced to assess the effect of L. fermentum NCDC701 on gut microbiota composition in an AlCl 3 and D-gal-induced Alzheimer rat model. More details explained in supplement material S7. There were no remarkable differences in shanon index and OTU between the control and of L. fermentum NCDC701 treated groups. However, AlCl 3 /D-gal-induced and memantine treated groups showed lower shanon index and OTU as compared to control (Fig. 5a, b). Supplement material S8 represents the Operational Taxonomic Unit (OTU) and Shannon's Diversity Index values for assessing the impact of L. fermentum NCDC701 on faecal microbial diversity. The relative abundance of taxa shared between groups is taken into consideration via a principal coordinate plot based on weighted UniFrac distance, which accounts for 39.65% of the total variance on the horizontal axis (PCoA1) and 21.09% on the vertical axis (PCoA2) Principal coordinate plot based on unweighted UniFrac distance, which assesses presence or absence, accounts for 33.64% of total variation on the horizontal axis (PCoA1) and 20.65% on the vertical axis (PCoA2) (Fig. 5c, d and ). The bacterial community of different taxonomic level was compared between the faecal sample of different groups of rats, to identify the gut microbiota changes after the administration of L. fermentum NCDC701 to AlCl 3 and D-gal induced Alzheimer rat group. The two major bacterial phyla were Firmicutes (43%), Bacteroidetes (31%) in group 1, while Verrucomicrobia (10.14%) and Proteobacteria (4.4%) in group 2 were observed, including Firmicutes (38.35%), Bacteroidetes (28.80%), Spirochaetes (13%), and Proteobacteria (4.7%) (Fig. 6). The Firmicutes and Bacteroidetes (F/B) ratio were 1.435 for group 1, 1.03 for group 2, 2.57 for group 3, 1.42 for group 4, 1.4 for group 5, and 1.41 for group 6 (Fig. 6) were observed. The administration of L. fermentum NCDC701 and memantine (positive control) improved the decreased F/B ratio induced by AlCl 3 and D-gal. The increased (F/B) ratio was associated with the memantine administrated rat group. The relative abundance of Lactobacillaceae (3.37%), S24-7 (2.64%), and Ruminococcaceae (2.77%) families were reduced in the AlCl 3 and D-gal induced rat groups, but their recovery was observed after administration of L. fermentum NCDC701. On the other hand, the levels of Erysipelotrichaceae (3.36%), Spirochaetaceae (13.69%), Lachnospiraceae (4.31%), Clostridiaceae (6.71%), and Prophyromoadaceae (3.2%) families were found to be elevated in the AlCl 3 and D-gal induced rat groups (Fig. 6). The abundance of genera differed across all groups at the genus level (Fig. 6). The relative abundance of Lactobacillus (3.37%), Ruminococcus (2.77%), and Prevotella (13.51%), was reduced in the AlCl 3 and D-gal-induced Alzheimer model compared to the control and treatment groups. However, after administration of L. fermentum NCDC701 to group 4, 5, and 6, the abundance of Prevotella (20.42, 14.96 and 16.56%), Lactobacillus (15.75, 13.78 and 14.95%), Ruminococcacus (4.54, 3.98 and 3.14%), and Oscillospira (4.25, 6.47 and 4.58%) increased. The Spirochaetaceae genus was found to be highly abundant in the AlCl 3 and D-gal-induced Alzheimer model. 3.5. Analysis of SCFAs after administration of Limosilactobacillus fermentum NCDC701 in the AlCl 3 and D-gal-induced rat Gas-liquid chromatography analysis of faecal samples found significant differences (P < 0.05) in the concentration of SCFAs among all the groups. However, no remarkable differences were observed among the L. fermentum NCDC701 treated samples. The concentration of propionate was significantly increased (P < 0.05) in group 2 (the stressed model, i.e., Alzheimer's model) and group 3 compared to the control group. There were no remarkable differences observed in the concentration of propionate after the administration of L. fermentum NCDC701 in group 4, group 5, and group 6. The total SCFA content in faecal samples of groups 1, 3, 4, 5, and 6 did not show significant differences. The gas-liquid chromatography chromatogram of all groups (Fig. 7). 4. Discussion The results of the present study demonstrated that GABA producing L. fermentum NCDC701 treatment have anti- Alzheimer potential. The mechanism may involve the regulation of the composition of the gut microbiota, modification of the mRNA expression of tight junction and inflammatory genes, regulation of the levels of SCFAs, reduction of the levels of inflammatory and oxidative factors. Most studies have demonstrated that a weekly dose of AlCl 3 (50mg/kg) + D-gal (100mg/kg) can cause plaque formation, decrease in GABA level, abnormal cell morphology, alteration of gut microbiome. GABA is the inhibitory neurotransmitters has been demonstrated that it drastically declines in severe Alzheimer's cases, which may be the reason for the disease's emotional and behavioral symptoms [ 24 ]. Significant declines in serotonin levels in platelets have also been linked to aggressive behavior in Alzheimer's patients [ 25 ]. The injection of AlCl 3 and D-gal has been reported to reduce the expression of Claudin-1 and Occludin proteins [ 26 ]. Studies have reported elevated levels of pro-inflammatory cytokines, such as TNF-α and IL-6 in the liver and kidney [ 27 ], as well as elevated levels of IL-6 in blood and homogenates of the liver, brain, and kidney [ 28 ], with the injection of D-galactose. Moreover, oxidative stress may also be linked to cognitive dysfunction via poor signaling and cell death, which results in neuroinflammation and plaque aggregation. The increase in oxidative stress caused by amyloid plaque development and show neurotoxic effect [ 29 ]. However, many studies have shown that probiotics can regulate the gut microbiota and attenuate Alzheimer related symptoms. The Lactobacillus plantarum MTCC 1325 has been reported to reduce the neurofibrillary tangle, amyloid plaques, and raised acetylcholine levels in the hippocampus and cerebral cortex of D-Galactose-induced Alzheimer animals [ 30 ]. The co-administrative treatment of memantine and L. plantarum showed the reduction in amyloid plaque deposition in APP/PS1 mice of hippocampus region. In contrast alone memantine not able to reduce amyloid plaque deposition as compared to co-administration treatment. [ 67 ]. Lactobacilli, on the other hand, appear to alter memory and plaque formation via regulating pathogenic pathways involved in Alzheimer, such as oxidative stress. Some probiotic studies have shown antioxidative capability of probiotics against oxidative stress by activating the suppressed SIRT1 gene, which codes for antioxidative system [ 31 ]. The activation of the SIRT1 gene has been proven to be neuroprotective. The administration of a mixture of probiotics of 10 10 cfm/ml Bifidobacterium infantis , Lactobacillus rhamnosus , and Lactobacillus reuteri has been reported to lower the level of malondialdehyde acrinols, raised the level of SOD, lower the inflammatory cytokines including TNF-α and IL-1, reduced the amyloid plaque deposition, and improved cognition [ 32 ]. The memantine treatment on APP/PS1 mice did not decrease the proinflammatory cytokine IL-2, IL-17 and TNF-α level as compared to the L. plantarum treatment. Furthermore, the combination of memantine and L. plantarum treatment can overcome with the increased proinflammatory cytokines [ 67 ]. Although the mechanism of lactobacilli upsides on oxidative stress has not been fully determined, there is substantial evidence that probiotics ameliorate oxidative stress by lowering cytokine production. Oxidative stress and inflammation are linked, and free radicals may cause TNF-α production by activating NF-κB [ 33 ]. Lactobacilli have been shown in some studies to increase glutamate-cysteine ligase activity and glutathione synthesis rate; they can also block superoxide anion and hydroxyl radical production, decrease pro-oxidant, and stimulate and reinforce the immune system [ 34 , 35 , 36 , 37 ]. However, this study found the L. fermentum NCDC701 treated group decrease the level of proinflammatory cytokine in contrast increase in anti-inflammatory cytokine to combat the inflammation, as compared to the AlCl 3 and D-gal group by reducing the mRNA expression of p65, COX-2, and iNOS, might be due to the activation of NF-κB. Moreover, the administration of L. fermentum NCDC701 increased the activity of the antioxidants in both the brain and colon, enhancing the enzymatic system's capacity to withstand the harmful effects of free radicals [ 38 ]. Lactobacilli have been shown to support epithelial barrier function and tight junction protein expression in mice with colitis, such as Lactobacillus plantarum NCU116, which significantly increased the expression of Claudin-1, Occludin, and Zoccludin-1 [ 39 ]. The tight junction proteins play a crucial role in maintaining the integrity of the epithelial cell barrier by regulating its permeability through tight cell-cell junctions [ 40 ]. Therefore, regulating the expression and localization of tight junction genes is a potential new therapeutic target for treating such illnesses [ 40 ], and this can help maintain the function of the epithelial cell barrier. Many studies have highlighted the importance of adopting Lactobacilli to enhance barrier integrity. There were substantial differences in microbiota between groups in our study; for example, Lactobacillus, Oscillospora , and Ruminococcus relative abundance were higher in the treatment group as compared to the control and positive control groups. However, inflammatory and oxidative stress conditions caused by Alzheimer's disease may be associated with an increase in Treponema, Coprococcus, Clostridium , and Allobaculum which may lead to neuroinflammatory and oxidative stress via microglia activation [ 41 , 42 , 43 ]. The phylum Verrucomicrobia decreased after the administration of AlCl 3 and D-gal rats compared to the control group. The phylum Elusimicrobia was associated with the control and AlCl 3 and D-gal induced group, but it decreased after treatment with memantine and L. fermentum NCDC701. The microbiota of the depressed rat model faeces employing the Flinders sensitive line is distinct and has relative abundances of Elusimicrobia and Saccharibacteria that were significantly lower than those of the Flinders Resistance lines rat [ 44 ]. Proteobacteria were found to be a potential diseased-related microbial signature, as the administration of AlCl 3 and D-gal increased the level of this phylum in the gut microbiota, and it decreased following treatment with L. fermentum NCDC701, suggesting a protective effect against pathogens [ 45 ]. Studies found that the treatment using the memantine and lactobacilli reduced the TMA and TMAO level in plasma and liver. Increased in TMA and TMAO level indicate the alteration of gut flora. In addition, it also shown that single use of memantine have no more positive effects on TMA and TMAO levels. However, studies shown that combination treatment can modulate the gut microbiome [ 67 ], In this study, L. fermentum NCDC701 improves the gut microbiota which was disrupted by the injection of AlCl 3 and D-gal at the phylum level by reducing pathogenic phyla like Proteobacteria, Tenericutes, Actinobacteria , Spirochaetes , and TM7, and enhancing beneficial microbiota, such as Verrucomicrobia . Low levels of Verrucomicrobia are a sign of an unstable microbial community or gut dysbiosis [ 46 ]. In Alzheimer's patients, there are variations in bacterial abundance across phyla and genera, including a decline in Firmicutes and an elevation in Bacteroidetes [ 47 ]. An increase in the F/B ratio is typically associated with obesity, while a decrease is associated with inflammatory bowel disease (IBD) [ 48 ]. The recovery of the lowered OTU and Shannon index in AlCl 3 and D-gal induced group, after the administration of L. fermentum NCDC701 was consistent with a previous study that found a lower Observed species index, Shannon index, and Observed OTU index in an Alzheimer's model, suggesting decreased species abundance, in contrast to the consistent results achieved in most reports in the literature [ 49 ]. In this study, Prophyromoadaceae family and Coprococcus genus were found only in the AlCl 3 and D-gal induced Alzheimer model (group 2) (3.2%) and was absent in the control and treated groups, implies that Alzheimer's disease is associated with a higher relative abundance of Porphyromonadaceae of the Erysipelotrichaceae family, which are pathogenic and pro-inflammatory members [ 50 , 51 ]. In particular, Coprococcus catus has been found to contribute to the psychological functioning of psychiatric disorders and was linked to the severity of depression [ 52 ]. The elevated relative abundance of Spirochaetaceae AlCl 3 and D-gal induced model, was decreased after the administration of L. fermentum NCDC701.Based on the available data, the link between spirochetes and Alzheimer's is still being evaluated [ 53 ]. Previous data demonstrated that senile plaques in the Alzheimer's brain were formed by Treponema pallidum, a species of spirochetes. The concentration of the Treponema species (genus Spirochaetaceae ) is higher in the AlCl 3 and D-gal-induced Alzheimer model, it is likely that they play a role in plaque formation [ 54 ]. The decreased abundance of Oscillospira , which are beneficial microbes, participating in T-cell differentiation by promoting and maintaining IL-10-producing Treg cells, in the stressed group (Group 2) was recovered with the treatment of L. fermentum NCDC701 [ 55 ]. In the L. fermentum NCDC701-treated groups 4, 5, and 6, the concentration of Prevotella and Lactobacillus species was higher compared to the other groups [ 56 ]. In line with this study, oral administration of the kefir bacterium Lactobacillus. kefiranofaciens to mice concentration of Prevotella, Firmicutes, Bacteroidetes, Lactobacillus , and the total number of bacteria were significantly higher concentrations [ 57 ]. Lactobacilli thus influence the gut microbiota and the synthesized SCFAs content which define human health [ 58 ]. Breakdown of the gut epithelial barrier altered mucosal immune function, and loss of enteric neurons are pathological disorders that have been linked to a reduction in the main butyrate-producing bacteria ( Firmicutes ) in favor of acetate and propionate producers ( Bacteroidetes ), which leads to excessive production of pro-inflammatory cytokines [ 59 ] AlCl 3 and D-gal groups were found to be higher in propionate content in this study. Similar result has been reported in Alzheimer's patients, with increased level of propionic acid compared to a healthy person [ 60 ]. Another study found that the levels of propionic acid (1.35%) and acetic acid (1.25%) were higher in Alzheimer's patients than in the control group [ 61 ]. 16SrRNA sequencing of faecal DNA found a decreased level of Firmicutes and an increased level of Bacteroidetes compared to the other groups. This alteration was linked with a decreased amount of butyrate, which is responsible for the pathophysiology of Alzheimer's disease, compared to the control group. SCFAs, i.e., acetate and propionate, are produced by Bacteroidetes , and butyrate is mainly produced by the Firmicutes [ 62 ]. Acetic acid can cross the blood-brain barrier and modulate brain signals, reducing the permeability of the blood-brain barrier [ 63 ]. The diet supplemented with L. fermentum NCDC701 increased the abundance of genera such as Prevotella spp, Clostridium , Treponema, Oscillospira, Ruminococcus , and Lachnospiraceae that produce SCFAs and associated with the butyrate production that modulate epithelial integrity [ 64 ] The abundant level of SCFAs content could provide a protective effect and benefited in reducing neuro-inflammation by binding to a G-coupled protein receptor that could modulate the anti-inflammatory cytokine pathway [ 65 ]. Previous evidence suggested that the production of SCFAs may mitigate the pathophysiology of Alzheimer. In addition, gut microbiota also modulates neurotransmitter signaling, myelination, long-term potentiation, and synaptic protein expression [ 66 ]. Despite the significant findings suggesting the anti-Alzheimer potential of L. fermentum NCDC701, it is critical to acknowledge the limitations of our study, which included only three rats in each experimental group for biochemical analysis, rather than the standard eight. In spite of financial constraints and other logistical challenges, selecting the use of a reduced sample size enabled us to complete the study within the available resources without compromising the key objectives. While it is acknowledged that a bigger sample size would improve the statistical consistency and generalizability of our findings, the observed effects in the current study serve as a platform for future research with expanded resources. Future research with standard sample size and extensive durations will be required to validate and extend the implications of our findings, ensuring a more comprehensive understanding of L. fermentum NCDC701's therapeutic potential in reducing oxidative stress-related diseases. 5. Conclusion The results of this study indicate that Limosilactobacillus fermentum NCDC701 has potential producer of GABA, SCFAs as well as an antioxidant and anti-inflammatory agent, which could be useful in the development of functional foods with anti-Alzheimer properties. Supplementation with L. fermentum NCDC701 was able to mitigate the undesirable changes induced by AlCl 3 and D-gal, strengthen tight junction gene expression, modulate the gut microbiota in AlCl 3 and D-gal-induced rats, and increase the proportions of the Firmicutes / Bacteroidetes phylum while reducing harmful bacteria. These effects were linked to the protective properties of L. fermentum NCDC701, which suggests its potential use as a modulator of antioxidant enzyme system and anti-inflammatory agent, gut microbiota and short-chain fatty acid enhancer. The findings of this study indicate that L. fermentum NCDC701 could be a useful therapeutic strategy for anti-Alzheimer's properties in both food and pharmaceutical applications. Declarations Conflicts of Interest: Declare conflicts of interest or state “The authors declare no conflict of interest.” Funding: “This research received no external funding” Author Contribution Vaishali Lekchand Dasriya: Investigation, Methodology, Writing - original draft, Data curation; Manorama Kumari: Writing-review & editing, Methodology & Formal analysis; Soniya Ranveer: Methodology & Formal analysis, Pradip Behare: Writing-review & editing & Conceptualization; Shilpa Vij: Writing-review & editing, & Visualization;Anil Kumar Puniya: Writing - review and editing, Supervision, & Project administration. Acknowledgement Authors are thankful to ICAR-NDRI for providing fellowship and research facilities to VD for conducting this doctoral research. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5226459","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":382840488,"identity":"844c5fb8-822c-4b6f-b272-e7521223fb72","order_by":0,"name":"Vaishali Dasriya","email":"","orcid":"","institution":"National Dairy Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Vaishali","middleName":"","lastName":"Dasriya","suffix":""},{"id":382840490,"identity":"69bc40e6-f5a3-4d84-97ea-e070ade33569","order_by":1,"name":"Manorama Kumari","email":"","orcid":"","institution":"National Dairy Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Manorama","middleName":"","lastName":"Kumari","suffix":""},{"id":382840493,"identity":"23da8cb8-f5b3-4e54-81d3-f7c4e6a37f5c","order_by":2,"name":"Soniya Ranveer","email":"","orcid":"","institution":"National Dairy Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Soniya","middleName":"","lastName":"Ranveer","suffix":""},{"id":382840495,"identity":"6f486358-9fed-4db2-a45b-b5c82c2af2a2","order_by":3,"name":"Pradip Behare","email":"","orcid":"","institution":"National Dairy Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Pradip","middleName":"","lastName":"Behare","suffix":""},{"id":382840498,"identity":"a446667c-a317-4a93-9f24-31c11527a034","order_by":4,"name":"Anil Kumar Puniya","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYDACHhBhwMDAD6ITCkjRItkA0mJAtBaQrgNQvQQBf8/ZY9IFBYfljc+vTvzwwIBBnl/sAH4tEmf70qRnGBw23Hbj7WYJoMMMZ85OIGDNeR4zaR6Dw4zbbpzdANKSYHCbgBZ5qBb7zTPObv5BlBaDsz1gLYkb+Hu3EWeL4ZlzydY8BunJM27wbrNIMJAg7Be5M7kHb/P8sbbt7z+7+eaPCht5fmkCWqAR0wwMO7BKCULK4VrqgDF0gBjVo2AUjIJRMBIBAFqEQxOAQjdcAAAAAElFTkSuQmCC","orcid":"","institution":"National Dairy Research Institute","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Anil","middleName":"Kumar","lastName":"Puniya","suffix":""}],"badges":[],"createdAt":"2024-10-08 15:08:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5226459/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5226459/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":70322464,"identity":"d2e4cbe0-621d-4239-a1ea-fe1a88905249","added_by":"auto","created_at":"2024-12-02 06:54:25","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":812530,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCongo red-stained Brain sections\u003c/strong\u003e. \u003cstrong\u003ea\u003c/strong\u003e) Group 1: Control rat group treated with saline show normal hippocampus with intact cell (Violet arrow); \u003cstrong\u003eb\u003c/strong\u003e) Group 2: Rat treated with AlCl\u003csub\u003e3 \u003c/sub\u003eand D-gal showing amyloid plaque deposition (Red arrow), cerebral amyloid angiopathy (Yellow arrow), and neurofibrillary tangle (Black arrow); \u003cstrong\u003ec\u003c/strong\u003e) Group 3: Memantine treated groups shows few amyloid plaques (Red arrow) and cerebral amyloid angiopathy (Yellow arrow); \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 treated rat brain shows normal hippocampus with intact cell (Violet arrow) and no sign of necrosis \u003cstrong\u003ed\u003c/strong\u003e) Group 4: Co-administration treatment, \u003cstrong\u003ee\u003c/strong\u003e) Group 5: Prophylactic treatment and \u003cstrong\u003ef\u003c/strong\u003e) Group 6: Therapeutic treatment.\u003c/p\u003e","description":"","filename":"Manuscript1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5226459/v1/83e0350cf63c3c835802b043.jpg"},{"id":70322460,"identity":"6eb49cb6-0174-457b-b138-4c794d12f785","added_by":"auto","created_at":"2024-12-02 06:54:24","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":945865,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHaematoxylin and eosin (H and E) stained Colon sections.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e) Group 1: Control rat group injected with saline show normal colon mucosa with intact epithelium; \u003cstrong\u003eb\u003c/strong\u003e) Group 2: Rat treated with AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal shows infiltrated leukocytes, scattered neutrophils (Red arrow) and damaged of epithelial integrity (Black arrow); \u003cstrong\u003ec\u003c/strong\u003e) Group 3: Memantine treated groups shows infiltrated leukocytes (Black arrow); \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 treated rat colon shows decreased infiltered leucocyte (Red arrow) and normal colon mucosa with intact epithelium integrity (Black arrow) \u003cstrong\u003ed\u003c/strong\u003e) Group 4: Co-administration treatment, \u003cstrong\u003ee\u003c/strong\u003e) Group 5: Prophylactic treatment and \u003cstrong\u003ef\u003c/strong\u003e) Group 6: Therapeutic treatment.\u003c/p\u003e","description":"","filename":"Manuscript2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5226459/v1/0a33328fe3174ef3ffa9e303.jpg"},{"id":70322760,"identity":"1c7022b6-2a7e-4ba1-997c-f8a2932798d2","added_by":"auto","created_at":"2024-12-02 07:02:28","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":750584,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of \u003cem\u003eLimosilactobacillus. fermentum\u003c/em\u003e NCDC701 on AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal induced rat model. Rat was grouped into control (group 1, n=3) and treatment group (group 2-6, n=8). All group were assessed for neuromodulator response. (a,b) Quantification of β-amyloid 1-40 peptide, (c,d) Quantification of β-amyloid 1-42 peptide, (e,f) Quantification of GABA, (g,h) Quantification of serotonin, (I,j) Quantification of IL-6, (k,l) Quantification of TNF-α, (m,n) Quantification of IL-10, (o,p) Quantification of Superoxide Dismutase (SOD), (q,r) Quantification of catalase and (s,t) Quantification of Glutathione peroxidase (GSH-Px). Data were presented as the mean±SEM (n=3) and analysed using One-way ANOVA with Bonferroni post-test. Values with dissimilar superscript alphabets (a to i) significant differences (P\u0026lt;0.05) among the treatment groups.\u003c/p\u003e","description":"","filename":"Manuscript3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5226459/v1/a8fe1035752d3570cf5e5ee3.jpg"},{"id":70322468,"identity":"186c6a73-93ca-4007-9891-bc555e61c50a","added_by":"auto","created_at":"2024-12-02 06:54:28","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":266034,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 3. Effect of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL. fermentum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e NCDC701 on mRNA inflammatory gene expression in the brain of AlCl\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e and D-gal induced rat.\u003c/strong\u003e A) COX-2 mRNA, B) iNOS mRNA and C) P65 mRNA. Values are means±SEMs. Data were presented as the mean±SEM (n =3). ns-No significant differences were found in the control, and treatment groups. *, **-Significant difference (P\u0026lt;0.05) between control and AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal induced models\u003c/p\u003e","description":"","filename":"Manuscript4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5226459/v1/cf0e4d4d712306bbae04fa4d.jpg"},{"id":70322470,"identity":"27bea1e6-a1aa-4b79-9dec-e0d1050a86aa","added_by":"auto","created_at":"2024-12-02 06:54:29","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":815048,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 4. Effect of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLimosilactobacillus fermentum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e NCDC701 on mRNA tight junction protein in the colon and inflammatory response gene expression in the brain and colon of AlCl\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e and D-gal induced rat.\u003c/strong\u003e a) Claudin mRNA, b) Zoccludin-1 mRNA and c) Occludin mRNA (d,e) COX-2 mRNA, (f,g) iNOS mRNA and (h,i) p65 mRNA. Data were presented as the mean±SEM (n=3) and analysed using One-way ANOVA with Bonferroni post-test. Values with dissimilar superscript alphabets (a to l) indicate significant differences (P\u0026lt;0.05) among the groups.\u003c/p\u003e","description":"","filename":"Manuscript5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5226459/v1/6ffce6c07bdcd66edce1023f.jpg"},{"id":70322761,"identity":"38b5d45f-7460-4370-b034-eb632ea429b4","added_by":"auto","created_at":"2024-12-02 07:02:29","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":170089,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 5. Microbial diversity gut microbiota in experimental rat groups.\u003c/strong\u003e a) Shanon index, b) Observed OTU, c) Principal coordinate plot based on weighted UniFrac distance and d) Principal coordinate plot based on unweighted UniFrac distance.\u003c/p\u003e","description":"","filename":"Manuscript6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5226459/v1/578925c137a62280f97c22c8.jpg"},{"id":70322473,"identity":"9c2f91ea-aa3f-421d-b534-5ffa51ea98ff","added_by":"auto","created_at":"2024-12-02 06:54:29","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":356009,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 6. Compositions of the gut microbiota in the rat experimental groups.\u003c/strong\u003e a) Microbial composition at the phylum level, b) Ratio of \u003cem\u003eFirmicutes\u003c/em\u003e/\u003cem\u003eBacteroidetes\u003c/em\u003e, c)Microbial composition at the family level, and d) Microbial composition at the genus level.\u003c/p\u003e","description":"","filename":"Manuscript7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5226459/v1/aab1051b41e7e368a15570ca.jpg"},{"id":70322467,"identity":"d951c7ec-eb34-4eca-b638-114df1e4d7cf","added_by":"auto","created_at":"2024-12-02 06:54:27","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":269166,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 7. Effects of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL.fermentum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e NCDC701 in SCFAs content of faecal sample of AlCl\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e and D-gal induced rat\u003c/strong\u003e. a) Acetate, b) Propionate, c) Butyrate, and d) SCFA. Data were presented as the mean±SEM (n = 3). ns-No significant differences were found in the control, and treatment groups. * and **-Significant difference (P\u0026lt;0.05) between control and aluminum chloride and D-galactose-induced rats.\u003c/p\u003e","description":"","filename":"Manuscript8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5226459/v1/7b4ba849de3210484d72dad1.jpg"},{"id":70322763,"identity":"bdb0b1f8-1065-4157-8da7-50263900e0ab","added_by":"auto","created_at":"2024-12-02 07:02:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5373890,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5226459/v1/2229fc82-1f19-48d5-ab32-851994d38793.pdf"},{"id":70322759,"identity":"4d0a083f-770f-449c-96b9-3ea35f608bf7","added_by":"auto","created_at":"2024-12-02 07:02:28","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":20921,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaldata.docx","url":"https://assets-eu.researchsquare.com/files/rs-5226459/v1/5447645b372ff1561e3f49bd.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ameliorative action of probiotics on the neurotoxicological effect of Aluminium chloride and D-galactose","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCognitive and memory diminishing as well as progressive impairment of daily living activities are the main clinical signs of Alzheimer, a chronic neurodegenerative illness with a complex pathophysiology [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Alzheimer currently has no cure or highly effective treatment due to its multifactorial nature, which results from a combination of individual factors, genetic predisposition, and environmental influences [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The marks of Alzheimer include β-amyloid plaque formation, neurofibrillary tangles, oxidative stress, neuroinflammation, and mitochondrial failure. Additionally, the pathophysiology of Alzheimer has been linked to mitochondrial dysfunction via the production of reactive oxygen species in mitochondria, amyloid plaque deposition activates microglia, which then promotes the production of pro-inflammatory cytokines in the brains, driving neuroinflammation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eVarious chemicals, such as pesticides, heavy metals, drugs, aluminium, and D-galactose, can act as neurotoxins and cause neural loss. These chemicals increase the peroxidation of lipid and protein i.e., increased malonaldehyde acrolein, neuroinflammation and apoptosis of neuron cell resulting in decreased neurons [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The main contributing element to the onset of Alzheimer is the build-up of amyloid peptide (1\u0026ndash;42) and (1\u0026ndash;40) in the brain.[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Additionally, the disruption of metal homeostasis can cause aluminum toxicity, through increasing the activity of iron, which can cause oxidative damage in Alzheimer pathogenesis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The gut microbiota dysbiosis has also been revealed to be associated with the neurological disorders such as Alzheimer's disease [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The imbalanced gut microbiota can damage the intestinal mucosal barrier, which can activate microglia (i.e., amyloids and lipopolysaccharides) and cause neuro-inflammation in the central nervous system, contributing to neurodegeneration [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The increased number of pathogenic bacteria increases the development of amyloid plaque formation in the brain [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Inhibiting the pathophysiology associated with amyloid plaque, such as oxidative stress and neuroinflammation, could be a promising approach to slowing the progression of Alzheimer. Several drugs such as Rivastigmine, Galantamine, and Donepezil (acetylcholinesterase inhibitors), Memantine (an N-methyl D-aspartate receptor antagonist), and Benzodiazepine are available in the market for the treatment of Alzheimer's [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, these drugs have several side effects, which can worsen the condition of Alzheimer's patients [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Therefore, there is a need for a protective therapy with no side effects to improve the condition of Alzheimer's.\u003c/p\u003e \u003cp\u003eProbiotics and its bioactive metabolites such as neurotransmitters, gamma-aminobutyric acid (GABA), short-chain fatty acids (SCFAs), and tryptophan has been reported to modulate peripheral and central nervous systems including Alzheimer's disease, autism, and depressive disorder [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e] in response to the immune system, the vagus nerve, the enteric nervous. Several \u003cem\u003eLactobacillus\u003c/em\u003e species have been reported to demonstrate the significant effects as neuromodulators and neurotransmitters (including monoamines, serotonin, and brain-derived neurotrophic factor) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Microbial metabolites such as SCFAs, directly alter brain neurological functioning via vagal, endocrine, humoral, and immunological pathways by entering circulation or crossing the blood-brain barrier (BBB) directly activate Treg cells, endocrine cells, and neuronal cells in order to enhance the level of regulatory cytokines that sustain brain functioning [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe detailed \u003cem\u003ein vivo\u003c/em\u003e studies to assess the anti-oxidative and anti-inflammatory effects of probiotics in the brain and gut, as well as their influence on the microbiota-gut-brain axis, are required. In previous studies, only a few researchers have done work in the area of a direct relationship between the administration of probiotics and change in alteration of neurotransmitter levels in the host. Further, the efficacy of using Probiotics in neurological disorders is not much studied. So far, indigenous probiotic cultures were not screened and evaluated earlier for anti-Alzheimer properties. This promotes the exploration of the GRAS status of probiotics over side-effects and also screens for a specific activity such as neurotransmitter and short-chain fatty acid production and antioxidant potential. To that end, we developed and tested \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701, a SCFA, GABA, and serotonin producing probiotic that could influence the gut-microbiota-brain axis by improving oxidation and inflammation in the brain, strengthening intestinal epithelial barrier integrity, and modulating microbiome composition and SCFA production in the gut.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Reagents\u003c/h2\u003e \u003cp\u003e \u003cem\u003eLimosilactobacillus fermentum\u003c/em\u003e NCDC701 was incubated with De Man, Rogosa and Sharpe agar at 37\u0026deg;C for 14 hours. The standard diets were obtained from Small Animal House, NDRI, Karnal. The catalogue numbers of chemical and ELISA kits are added to supplement material S1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Quantification of GABA production\u003c/h2\u003e \u003cp\u003eSample preparation and quantification of GABA production by \u003cem\u003eLimosilactobacillus fermentum\u003c/em\u003e NCDC701 followed by [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Preparation of Limosilactobacillus fermentum NCDC701\u003c/h2\u003e \u003cp\u003e \u003cem\u003eLimosilactobacillus fermentum\u003c/em\u003e NCDC701 was collected from the National Collection of Dairy Cultures, ICAR-NDRI, Karnal. The \u003cem\u003eLimosilactobacillus fermentum\u003c/em\u003e NCDC701 were grown at 37\u0026deg;C overnight in de Man-Rogosa-Sharpe broth (Himedia). The bacterial cells were collected by centrifugation at 8,000 \u0026times; g for 10 min, washed three times with phosphate-buffered saline and adjusted to 3\u0026times;10\u003csup\u003e\u003cem\u003e8\u003c/em\u003e\u003c/sup\u003e CFU/ml for oral administration to the rats.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Animal experimental design\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 Animal ethics declaration\u003c/h2\u003e \u003cp\u003e In the pursuit of scientific advancement and the understanding of toxicological impacts, involved following principles regarding the ethical treatment of animals such as ensured their welfare is prioritized in all aspects of our research. The #R princeiple followed such as reduction, replacement, refinement, All proposed studies involved animals that undergo thorough ethical review by an institutional animal care ICAR-NDRI, Karnal and conducted according to the guidelines of the Institutional Animal Ethics Committee in an animal house registered with CPCSEA (1705/GO/Re/SL/13/CPCSEA) to ensure adherence to ethical standards and welfare considerations. We ensured all personnel involved in the care and use of animals are adequately trained and competent in handling and administering procedures to minimize stress and harm to the animals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Euthanasia method\u003c/h2\u003e \u003cp\u003eThe euthanasia method for rat was the carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e) inhalation. The rat was placed in CO\u003csub\u003e2\u003c/sub\u003e chamber for a minute. And supply the 100% CO\u003csub\u003e2\u003c/sub\u003e with the 30\u0026ndash;70% displacement rate per minute to existing air chamber. It help to achieve balance gas mixture for rapid unconsciousness with minimal distress to the animals. The active exposure of rat was around 2\u0026ndash;3 minutes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3 Experimental design\u003c/h2\u003e \u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e experiments were conducted according to the guidelines of the Institutional Animal Ethics Committee in an animal house registered with CPCSEA (1705/GO/Re/SL/13/CPCSEA) at ICAR-NDRI, Karnal, India.\u003c/p\u003e \u003cp\u003eWistar Albino rats (48 males, 12 weeks old, weighing 155-156.5 g) were obtained from ICAR-NDRI, Karnal, and housed in polycarbonate cages with free access to food and water, and maintained at 24\u0026deg;C and 55\u0026ndash;60% humidity on a 12-hour light/dark cycle. After a week of acclimatization, the rats were randomly assigned to six groups (8 animals/ group). Group 1 was maintained on a standard diet and injected intraperitoneally with saline. Group 2, an Alzheimer's model, was injected intraperitoneally with AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal for 7 days. Group 3, a positive control, was injected with AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal (7 days) and orally administered memantine drug (21 days). Group 4 was a co-administered group injected with AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal (7 days) and \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 from the 1st to the 28th day by oral gavage. Group 5 was a prophylactic group that was administered \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 from the 1st day to the 21st day (oral gavage) and last 7 days for AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal intraperitoneal injection. Group 6 was a therapeutic group that was intraperitoneally injected with AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal for the first 7 days and administered \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 for the last 8th to 28th day (oral gavage). Grouping of experimental data and diet plan were mentioned in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eTable 1 Grouping of experimental rat and diet plan\u003c/p\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo. of rats\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDiet\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStandard diet (SD)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSD + (AlCl\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;D-gal, 7days) i.p.+ Saline by gavage\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSD + (AlCl\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;D-gal, 7 days)\u0026thinsp;+\u0026thinsp;Memantine (1.5 mg/kg), last 21 days by i.p.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eCo-administrative group\u003c/b\u003e: SD + (AlCl\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;D-gal, 7 days by i.p\u0026thinsp;+\u0026thinsp;\u003cem\u003eLimosilactobacillus fermentum\u003c/em\u003e NCDC701, 28 days) by gavage\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eProphylactic group\u003c/b\u003e: SD\u0026thinsp;+\u0026thinsp;\u003cem\u003eLimosilactobacillus fermentum\u003c/em\u003e NCDC701, first 21 days by i.p + (AlCl\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;D-gal, last 7 days) by gavage\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eTherapeutic group\u003c/b\u003e: SD + (AlCL\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;D-gal, 7 days] by i.p\u0026thinsp;+\u0026thinsp;\u003cem\u003eLimosilactobacillus fermentum\u003c/em\u003e NCDC701, last 21 days) by gavage\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Histopathological analysis\u003c/h2\u003e \u003cp\u003eThe brain and colon tissue samples were harvested and fixed in a 10% formalin solution for 12 hours. To remove any remaining moisture, the samples were soaked in xylene. The tissue samples were then cut into 4\u0026micro;m thick sections, fixed in paraffin, and stained with Congo red and haematoxylin and eosin, respectively. Detailed histopathological analysis given in supplementary material S2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Biochemical analysis\u003c/h2\u003e \u003cp\u003eELISA kits from Biolinkk were used to measure the levels of GABA and serotonin in brain homogenates, quantify β-amyloid peptide 1\u0026ndash;42 in brain and serum, assess antioxidant activity including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) in brain and colon homogenates, and detect inflammatory parameters such as IL-6, TNF, and IL-10 in brain and colon homogenates of three mice from each group. The absorbance was determined at 450nm using a microplate reader. The sample and standard preparation method given in supplemented material S3.2.6. Tight junction and inflammatory gene expression by RT-qPCR\u003c/p\u003e \u003cp\u003eRT-qPCR was used to analyse the effects of \u003cem\u003eLimosilactobacillus fermentum\u003c/em\u003e NCDC701 on gene expression of zonula occludens, occludin, and claudin-1 in colonic tissue, as well as p65, COX-2, and iNOS in both brain and colonic tissues by following the procedure of [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] given in supplemented material S4.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Analysis of microbiome\u003c/h2\u003e \u003cp\u003eThe DNA extraction conditions for faecal sample from each group of rats (Group 1\u0026ndash;6) was optimized using the MPI Bead Beater (USA) and the Quick-DNA Faecal/Soil Microbe DNA Miniprep Kit (Zymo Research, USA). The concentration and purity of the extracted DNA was assessed using the SYNERGY H1 Hybrid Reader. High-purity faecal DNA samples from each group were pooled together by following the procedure of [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] given in supplemented material S5.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Determination of SCFAs from faecal samples\u003c/h2\u003e \u003cp\u003eSCFAs were extracted from each rat faecal sample in each of the six groups using a modified method [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Detail procedure of sample preparation given in supplement material S6.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Statistical analysis\u003c/h2\u003e \u003cp\u003eThe results of each experiment were carried out in triplicate and presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. When appropriate, analysis of variance (one-way or two-way ANOVA) was performed using GraphPad Prism (version 5), followed by Bartlett\u0026rsquo;s test to assess the homogeneity of variance and determine significant differences at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Microsoft Office 2010 was used for tabulation and presentation of data.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Result","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Effects of aluminium chloride and D-galactose on histopathology\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1. Congo red staining of brain tissue\u003c/h2\u003e \u003cp\u003eHistopathological observation of brain examined the protective effect of \u003cem\u003eLimosilactobacillus fermentum\u003c/em\u003e NCDC701 on the amyloid plaque deposited in brain after the induction of AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal. The brains of the control group had a normal morphology cortical neuron, and intact cell. Amyloid plaque deposition (Red arrow), cerebral amyloid angiopathy (Yellow arrow), and neurofibrillary tangle (Black arrow) were all visible in the AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal induced group. Compared to control groups, the hippocampus and cerebral cortex of rat treated with AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal plus memantine treated displayed few amyloid plaques (Red arrow) and cerebral amyloid angiopathy (Yellow arrow) (Fig.\u0026nbsp;1). After the administration of \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 these abnormalities (neurofibrillary tangles, neuropil threads, neurotic plaques, and amyloid angiopathy) were reduced and showing normal hippocampus with intact cell (Violet arrow) and no sign of necrosis (Fig.\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2. Haematoxylin and eosin staining of colon\u003c/h2\u003e \u003cp\u003eHistopathological study demonstrated that the control group had a normal colon mucosa with intact epithelium. In the group of rats treated with AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal, an inflamed and damaged colon was observed. The colon exhibited damaged epithelial integrity (black arrow), inflammatory cell infiltration, and scattered neutrophils (red arrow). The group treated with memantine showed less damaged epithelial integrity and fewer infiltrating leukocytes. The administration of \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 resulted in an improvement in epithelial integrity. A decrease in infiltrating leukocytes and a normal colon mucosa with intact epithelium were observed in the group administered \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 (Fig.\u0026nbsp;2).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2. L. fermentum NCDC701 supplementation \u0026amp; neuromodulatory responses\u003c/h2\u003e \u003cp\u003eThe GABA production of \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 was 99.43\u0026thinsp;\u0026plusmn;\u0026thinsp;51 mM, comparable higher than another cultures. The administration of \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 was found to modulate the Alzheimer's disease-related changes by reducing the β-amyloid 1\u0026ndash;40 and 42 peptide concentration in rat tissue. The administration of \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 resulted in a decrease in β-amyloid 1\u0026ndash;40 and 42 peptide concentration in the rat brain and serum, compared to the brain and serum of the AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal-induced rats. Whereas, the positive control group, which received memantine, showed a slight decrease in β-amyloid 1\u0026ndash;40 and 42 peptide concentration in brain and serum tissue compared to the \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 treated group (Fig.\u0026nbsp;3a, b, c and d). The reduction in GABA and serotonin levels was restored by the administration of \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 in rat brain and colon tissue compared to the in brain and colon tissue of AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal induced rats There was no significant differences (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) observed in GABA and serotonin levels between the \u003cem\u003eL. fermentum\u003c/em\u003e treated groups. The memantine treated group also shown the recovery effect in restoration of GABA and serotonin levels (Fig.\u0026nbsp;3e, f, g and h).\u003c/p\u003e \u003cp\u003eThe increase in pro-inflammatory cytokines such as, IL-6 and TNF-α concentration and decreased in the level of anti-inflammatory cytokine IL-10 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), were significantly modulated by the administration of \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 in the brain and colon tissue of the co-administration group, prophylactic group and therapeutic group, compared to the brain and colon tissue of AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal-induced rats (Fig.\u0026nbsp;3i, j, k, l, m, and n).\u003c/p\u003e \u003cp\u003eThe AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal group showed a significant decrease (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in SOD, CAT, and GSH-Px, activities in the brain and colon, compared with that in the control group. The supplementation of \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 significantly improved SOD, CAT, and GSH-Px, in the co-administration group, prophylactic group and therapeutic group, compared to the brain and colon tissue of AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal-induced rat (Fig.\u0026nbsp;3o to t). The levels of SOD, CAT, and GSH-Px did not show a significant change (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) in the brain and colon of the \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 administered group. In contrast, the SOD, CAT, and GSH-Px activities of the AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal induced rats were found to be significantly lower (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than those of the control group. Furthermore, the SOD, CAT, and GSH-Px activities in the brain and colon of the rats treated with \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 were increased as compared to the AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal induced rat groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Effects of Limosilactobacillus. fermentum NCDC701 on tight junction and inflammatory response gene expression\u003c/h2\u003e \u003cp\u003eThe mRNA expression of tight junction genes, including Zoccludin-1, Claudin-1, and occludin, was significantly reduced (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the colonic tissues of the AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal group. However, supplementation with \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 increased the expressions of these genes, demonstrating a protective effect. Memantine (positive control) also improved the expression of these genes, but less than the control group (Fig.\u0026nbsp;4).\u003c/p\u003e \u003cp\u003eCompared to the control group, the AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal groups showed significantly higher expression of p65, COX-2 and iNOS in both colon and brain tissue. However, the administration of \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 and memantine (a positive control) dramatically reduced the enhanced expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) of p65, COX-2 and iNOS in both brain and colon tissue (Fig.\u0026nbsp;4).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.4 \u003cem\u003eLimosilactobacillus fermentum NCDC701 restore gut microbiota of AlCl\u003c/em\u003e\u003csub\u003e\u003cem\u003e3\u003c/em\u003e\u003c/sub\u003e \u003cem\u003eand D-gal induced rat\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe V\u003csub\u003e3\u003c/sub\u003e-V\u003csub\u003e4\u003c/sub\u003e region of the 16SrRNA gene, which has high nucleotide diversity and discriminatory power, was sequenced to assess the effect of \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 on gut microbiota composition in an AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal-induced Alzheimer rat model. More details explained in supplement material S7. There were no remarkable differences in shanon index and OTU between the control and of \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 treated groups. However, AlCl\u003csub\u003e\u003cem\u003e3\u003c/em\u003e\u003c/sub\u003e/D-gal-induced and memantine treated groups showed lower shanon index and OTU as compared to control (Fig.\u0026nbsp;5a, b). Supplement material S8 represents the Operational Taxonomic Unit (OTU) and Shannon's Diversity Index values for assessing the impact of L. fermentum NCDC701 on faecal microbial diversity.\u003c/p\u003e \u003cp\u003eThe relative abundance of taxa shared between groups is taken into consideration via a principal coordinate plot based on weighted UniFrac distance, which accounts for 39.65% of the total variance on the horizontal axis (PCoA1) and 21.09% on the vertical axis (PCoA2) Principal coordinate plot based on unweighted UniFrac distance, which assesses presence or absence, accounts for 33.64% of total variation on the horizontal axis (PCoA1) and 20.65% on the vertical axis (PCoA2) (Fig.\u0026nbsp;5c, d and ).\u003c/p\u003e \u003cp\u003eThe bacterial community of different taxonomic level was compared between the faecal sample of different groups of rats, to identify the gut microbiota changes after the administration of \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 to AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal induced Alzheimer rat group. The two major bacterial phyla were \u003cem\u003eFirmicutes\u003c/em\u003e (43%), \u003cem\u003eBacteroidetes\u003c/em\u003e (31%) in group 1, while \u003cem\u003eVerrucomicrobia\u003c/em\u003e (10.14%) and \u003cem\u003eProteobacteria\u003c/em\u003e (4.4%) in group 2 were observed, including \u003cem\u003eFirmicutes\u003c/em\u003e (38.35%), \u003cem\u003eBacteroidetes\u003c/em\u003e (28.80%), \u003cem\u003eSpirochaetes\u003c/em\u003e (13%), and \u003cem\u003eProteobacteria\u003c/em\u003e (4.7%) (Fig.\u0026nbsp;6). The \u003cem\u003eFirmicutes\u003c/em\u003e and \u003cem\u003eBacteroidetes\u003c/em\u003e (F/B) ratio were 1.435 for group 1, 1.03 for group 2, 2.57 for group 3, 1.42 for group 4, 1.4 for group 5, and 1.41 for group 6 (Fig.\u0026nbsp;6) were observed. The administration of \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 and memantine (positive control) improved the decreased F/B ratio induced by AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal. The increased (F/B) ratio was associated with the memantine administrated rat group. The relative abundance of \u003cem\u003eLactobacillaceae\u003c/em\u003e (3.37%), S24-7 (2.64%), and \u003cem\u003eRuminococcaceae\u003c/em\u003e (2.77%) families were reduced in the AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal induced rat groups, but their recovery was observed after administration of \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701. On the other hand, the levels of \u003cem\u003eErysipelotrichaceae\u003c/em\u003e (3.36%), \u003cem\u003eSpirochaetaceae\u003c/em\u003e (13.69%), \u003cem\u003eLachnospiraceae\u003c/em\u003e (4.31%), \u003cem\u003eClostridiaceae\u003c/em\u003e (6.71%), and \u003cem\u003eProphyromoadaceae\u003c/em\u003e (3.2%) families were found to be elevated in the AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal induced rat groups (Fig.\u0026nbsp;6). The abundance of genera differed across all groups at the genus level (Fig.\u0026nbsp;6). The relative abundance of \u003cem\u003eLactobacillus\u003c/em\u003e (3.37%), \u003cem\u003eRuminococcus\u003c/em\u003e (2.77%), and \u003cem\u003ePrevotella\u003c/em\u003e (13.51%), was reduced in the AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal-induced Alzheimer model compared to the control and treatment groups. However, after administration of \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 to group 4, 5, and 6, the abundance of \u003cem\u003ePrevotella\u003c/em\u003e (20.42, 14.96 and 16.56%), \u003cem\u003eLactobacillus\u003c/em\u003e (15.75, 13.78 and 14.95%), \u003cem\u003eRuminococcacus\u003c/em\u003e (4.54, 3.98 and 3.14%), and \u003cem\u003eOscillospira\u003c/em\u003e (4.25, 6.47 and 4.58%) increased. The \u003cem\u003eSpirochaetaceae\u003c/em\u003e genus was found to be highly abundant in the AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal-induced Alzheimer model.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Analysis of SCFAs after administration of Limosilactobacillus fermentum NCDC701 in the AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal-induced rat\u003c/h2\u003e \u003cp\u003eGas-liquid chromatography analysis of faecal samples found significant differences (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the concentration of SCFAs among all the groups. However, no remarkable differences were observed among the \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 treated samples. The concentration of propionate was significantly increased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in group 2 (the stressed model, i.e., Alzheimer's model) and group 3 compared to the control group. There were no remarkable differences observed in the concentration of propionate after the administration of \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 in group 4, group 5, and group 6. The total SCFA content in faecal samples of groups 1, 3, 4, 5, and 6 did not show significant differences. The gas-liquid chromatography chromatogram of all groups (Fig.\u0026nbsp;7).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe results of the present study demonstrated that GABA producing \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 treatment have anti- Alzheimer potential. The mechanism may involve the regulation of the composition of the gut microbiota, modification of the mRNA expression of tight junction and inflammatory genes, regulation of the levels of SCFAs, reduction of the levels of inflammatory and oxidative factors. Most studies have demonstrated that a weekly dose of AlCl\u003csub\u003e3\u003c/sub\u003e (50mg/kg)\u0026thinsp;+\u0026thinsp;D-gal (100mg/kg) can cause plaque formation, decrease in GABA level, abnormal cell morphology, alteration of gut microbiome. GABA is the inhibitory neurotransmitters has been demonstrated that it drastically declines in severe Alzheimer's cases, which may be the reason for the disease's emotional and behavioral symptoms [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Significant declines in serotonin levels in platelets have also been linked to aggressive behavior in Alzheimer's patients [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The injection of AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal has been reported to reduce the expression of Claudin-1 and Occludin proteins [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Studies have reported elevated levels of pro-inflammatory cytokines, such as TNF-α and IL-6 in the liver and kidney [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], as well as elevated levels of IL-6 in blood and homogenates of the liver, brain, and kidney [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], with the injection of D-galactose. Moreover, oxidative stress may also be linked to cognitive dysfunction via poor signaling and cell death, which results in neuroinflammation and plaque aggregation. The increase in oxidative stress caused by amyloid plaque development and show neurotoxic effect [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, many studies have shown that probiotics can regulate the gut microbiota and attenuate Alzheimer related symptoms. The \u003cem\u003eLactobacillus plantarum\u003c/em\u003e MTCC 1325 has been reported to reduce the neurofibrillary tangle, amyloid plaques, and raised acetylcholine levels in the hippocampus and cerebral cortex of D-Galactose-induced Alzheimer animals [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The co-administrative treatment of memantine and \u003cem\u003eL. plantarum\u003c/em\u003e showed the reduction in amyloid plaque deposition in APP/PS1 mice of hippocampus region. In contrast alone memantine not able to reduce amyloid plaque deposition as compared to co-administration treatment. [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Lactobacilli, on the other hand, appear to alter memory and plaque formation via regulating pathogenic pathways involved in Alzheimer, such as oxidative stress. Some probiotic studies have shown antioxidative capability of probiotics against oxidative stress by activating the suppressed SIRT1 gene, which codes for antioxidative system [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The activation of the SIRT1 gene has been proven to be neuroprotective. The administration of a mixture of probiotics of 10\u003csup\u003e10\u003c/sup\u003e cfm/ml \u003cem\u003eBifidobacterium infantis\u003c/em\u003e, \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e, and \u003cem\u003eLactobacillus reuteri\u003c/em\u003e has been reported to lower the level of malondialdehyde acrinols, raised the level of SOD, lower the inflammatory cytokines including TNF-α and IL-1, reduced the amyloid plaque deposition, and improved cognition [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The memantine treatment on APP/PS1 mice did not decrease the proinflammatory cytokine IL-2, IL-17 and TNF-α level as compared to the \u003cem\u003eL. plantarum\u003c/em\u003e treatment. Furthermore, the combination of memantine and \u003cem\u003eL. plantarum\u003c/em\u003e treatment can overcome with the increased proinflammatory cytokines [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Although the mechanism of lactobacilli upsides on oxidative stress has not been fully determined, there is substantial evidence that probiotics ameliorate oxidative stress by lowering cytokine production. Oxidative stress and inflammation are linked, and free radicals may cause TNF-α production by activating NF-κB [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. \u003cem\u003eLactobacilli\u003c/em\u003e have been shown in some studies to increase glutamate-cysteine ligase activity and glutathione synthesis rate; they can also block superoxide anion and hydroxyl radical production, decrease pro-oxidant, and stimulate and reinforce the immune system [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. However, this study found the \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 treated group decrease the level of proinflammatory cytokine in contrast increase in anti-inflammatory cytokine to combat the inflammation, as compared to the AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal group by reducing the mRNA expression of p65, COX-2, and iNOS, might be due to the activation of NF-κB. Moreover, the administration of \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 increased the activity of the antioxidants in both the brain and colon, enhancing the enzymatic system's capacity to withstand the harmful effects of free radicals [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eLactobacilli\u003c/em\u003e have been shown to support epithelial barrier function and tight junction protein expression in mice with colitis, such as \u003cem\u003eLactobacillus plantarum\u003c/em\u003e NCU116, which significantly increased the expression of Claudin-1, Occludin, and Zoccludin-1 [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The tight junction proteins play a crucial role in maintaining the integrity of the epithelial cell barrier by regulating its permeability through tight cell-cell junctions [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Therefore, regulating the expression and localization of tight junction genes is a potential new therapeutic target for treating such illnesses [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], and this can help maintain the function of the epithelial cell barrier. Many studies have highlighted the importance of adopting \u003cem\u003eLactobacilli\u003c/em\u003e to enhance barrier integrity.\u003c/p\u003e \u003cp\u003eThere were substantial differences in microbiota between groups in our study; for example, \u003cem\u003eLactobacillus, Oscillospora\u003c/em\u003e, and \u003cem\u003eRuminococcus\u003c/em\u003e relative abundance were higher in the treatment group as compared to the control and positive control groups. However, inflammatory and oxidative stress conditions caused by Alzheimer's disease may be associated with an increase in \u003cem\u003eTreponema, Coprococcus, Clostridium\u003c/em\u003e, and \u003cem\u003eAllobaculum\u003c/em\u003e which may lead to neuroinflammatory and oxidative stress via microglia activation [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The phylum \u003cem\u003eVerrucomicrobia\u003c/em\u003e decreased after the administration of AlCl\u003csub\u003e\u003cem\u003e3\u003c/em\u003e\u003c/sub\u003e and D-gal rats compared to the control group. The phylum \u003cem\u003eElusimicrobia\u003c/em\u003e was associated with the control and AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal induced group, but it decreased after treatment with memantine and \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701. The microbiota of the depressed rat model faeces employing the Flinders sensitive line is distinct and has relative abundances of \u003cem\u003eElusimicrobia\u003c/em\u003e and \u003cem\u003eSaccharibacteria\u003c/em\u003e that were significantly lower than those of the Flinders Resistance lines rat [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. \u003cem\u003eProteobacteria\u003c/em\u003e were found to be a potential diseased-related microbial signature, as the administration of AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal increased the level of this phylum in the gut microbiota, and it decreased following treatment with \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701, suggesting a protective effect against pathogens [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Studies found that the treatment using the memantine and lactobacilli reduced the TMA and TMAO level in plasma and liver. Increased in TMA and TMAO level indicate the alteration of gut flora. In addition, it also shown that single use of memantine have no more positive effects on TMA and TMAO levels. However, studies shown that combination treatment can modulate the gut microbiome [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e], In this study, \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 improves the gut microbiota which was disrupted by the injection of AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal at the phylum level by reducing pathogenic phyla like \u003cem\u003eProteobacteria, Tenericutes, Actinobacteria\u003c/em\u003e, \u003cem\u003eSpirochaetes\u003c/em\u003e, and TM7, and enhancing beneficial microbiota, such as \u003cem\u003eVerrucomicrobia\u003c/em\u003e. Low levels of \u003cem\u003eVerrucomicrobia\u003c/em\u003e are a sign of an unstable microbial community or gut dysbiosis [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn Alzheimer's patients, there are variations in bacterial abundance across phyla and genera, including a decline in \u003cem\u003eFirmicutes\u003c/em\u003e and an elevation in \u003cem\u003eBacteroidetes\u003c/em\u003e [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. An increase in the F/B ratio is typically associated with obesity, while a decrease is associated with inflammatory bowel disease (IBD) [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The recovery of the lowered OTU and Shannon index in AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal induced group, after the administration of \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 was consistent with a previous study that found a lower Observed species index, Shannon index, and Observed OTU index in an Alzheimer's model, suggesting decreased species abundance, in contrast to the consistent results achieved in most reports in the literature [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, \u003cem\u003eProphyromoadaceae\u003c/em\u003e family and \u003cem\u003eCoprococcus\u003c/em\u003e genus were found only in the AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal induced Alzheimer model (group 2) (3.2%) and was absent in the control and treated groups, implies that Alzheimer's disease is associated with a higher relative abundance of \u003cem\u003ePorphyromonadaceae\u003c/em\u003e of the \u003cem\u003eErysipelotrichaceae\u003c/em\u003e family, which are pathogenic and pro-inflammatory members [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In particular, \u003cem\u003eCoprococcus catus\u003c/em\u003e has been found to contribute to the psychological functioning of psychiatric disorders and was linked to the severity of depression [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The elevated relative abundance of \u003cem\u003eSpirochaetaceae\u003c/em\u003e AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal induced model, was decreased after the administration of \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701.Based on the available data, the link between spirochetes and Alzheimer's is still being evaluated [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Previous data demonstrated that senile plaques in the Alzheimer's brain were formed by \u003cem\u003eTreponema\u003c/em\u003e pallidum, a species of spirochetes. The concentration of the \u003cem\u003eTreponema\u003c/em\u003e species (genus \u003cem\u003eSpirochaetaceae\u003c/em\u003e) is higher in the AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal-induced Alzheimer model, it is likely that they play a role in plaque formation [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. The decreased abundance of \u003cem\u003eOscillospira\u003c/em\u003e, which are beneficial microbes, participating in T-cell differentiation by promoting and maintaining IL-10-producing Treg cells, in the stressed group (Group 2) was recovered with the treatment of \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. In the \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701-treated groups 4, 5, and 6, the concentration of \u003cem\u003ePrevotella\u003c/em\u003e and \u003cem\u003eLactobacillus\u003c/em\u003e species was higher compared to the other groups [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. In line with this study, oral administration of the kefir bacterium \u003cem\u003eLactobacillus. kefiranofaciens\u003c/em\u003e to mice concentration of \u003cem\u003ePrevotella, Firmicutes, Bacteroidetes, Lactobacillus\u003c/em\u003e, and the total number of bacteria were significantly higher concentrations [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. \u003cem\u003eLactobacilli\u003c/em\u003e thus influence the gut microbiota and the synthesized SCFAs content which define human health [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Breakdown of the gut epithelial barrier altered mucosal immune function, and loss of enteric neurons are pathological disorders that have been linked to a reduction in the main butyrate-producing bacteria (\u003cem\u003eFirmicutes\u003c/em\u003e) in favor of acetate and propionate producers (\u003cem\u003eBacteroidetes\u003c/em\u003e), which leads to excessive production of pro-inflammatory cytokines [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal groups were found to be higher in propionate content in this study. Similar result has been reported in Alzheimer's patients, with increased level of propionic acid compared to a healthy person [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Another study found that the levels of propionic acid (1.35%) and acetic acid (1.25%) were higher in Alzheimer's patients than in the control group [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. 16SrRNA sequencing of faecal DNA found a decreased level of \u003cem\u003eFirmicutes\u003c/em\u003e and an increased level of \u003cem\u003eBacteroidetes\u003c/em\u003e compared to the other groups. This alteration was linked with a decreased amount of butyrate, which is responsible for the pathophysiology of Alzheimer's disease, compared to the control group. SCFAs, i.e., acetate and propionate, are produced by \u003cem\u003eBacteroidetes\u003c/em\u003e, and butyrate is mainly produced by the \u003cem\u003eFirmicutes\u003c/em\u003e [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Acetic acid can cross the blood-brain barrier and modulate brain signals, reducing the permeability of the blood-brain barrier [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. The diet supplemented with \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 increased the abundance of genera such as \u003cem\u003ePrevotella\u003c/em\u003e spp, \u003cem\u003eClostridium\u003c/em\u003e, \u003cem\u003eTreponema, Oscillospira, Ruminococcus\u003c/em\u003e, and \u003cem\u003eLachnospiraceae\u003c/em\u003e that produce SCFAs and associated with the butyrate production that modulate epithelial integrity [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e] The abundant level of SCFAs content could provide a protective effect and benefited in reducing neuro-inflammation by binding to a G-coupled protein receptor that could modulate the anti-inflammatory cytokine pathway [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Previous evidence suggested that the production of SCFAs may mitigate the pathophysiology of Alzheimer. In addition, gut microbiota also modulates neurotransmitter signaling, myelination, long-term potentiation, and synaptic protein expression [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite the significant findings suggesting the anti-Alzheimer potential of \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701, it is critical to acknowledge the limitations of our study, which included only three rats in each experimental group for biochemical analysis, rather than the standard eight. In spite of financial constraints and other logistical challenges, selecting the use of a reduced sample size enabled us to complete the study within the available resources without compromising the key objectives. While it is acknowledged that a bigger sample size would improve the statistical consistency and generalizability of our findings, the observed effects in the current study serve as a platform for future research with expanded resources. Future research with standard sample size and extensive durations will be required to validate and extend the implications of our findings, ensuring a more comprehensive understanding of \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701's therapeutic potential in reducing oxidative stress-related diseases.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe results of this study indicate that \u003cem\u003eLimosilactobacillus fermentum\u003c/em\u003e NCDC701 has potential producer of GABA, SCFAs as well as an antioxidant and anti-inflammatory agent, which could be useful in the development of functional foods with anti-Alzheimer properties. Supplementation with \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 was able to mitigate the undesirable changes induced by AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal, strengthen tight junction gene expression, modulate the gut microbiota in AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal-induced rats, and increase the proportions of the \u003cem\u003eFirmicutes\u003c/em\u003e/ \u003cem\u003eBacteroidetes\u003c/em\u003e phylum while reducing harmful bacteria. These effects were linked to the protective properties of \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701, which suggests its potential use as a modulator of antioxidant enzyme system and anti-inflammatory agent, gut microbiota and short-chain fatty acid enhancer. The findings of this study indicate that \u003cem\u003eL. fermentum\u003c/em\u003e NCDC701 could be a useful therapeutic strategy for anti-Alzheimer's properties in both food and pharmaceutical applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflicts of Interest:\u003c/h2\u003e \u003cp\u003eDeclare conflicts of interest or state \u0026ldquo;The authors declare no conflict of interest.\u0026rdquo;\u003c/p\u003e \u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003e\u0026ldquo;This research received no external funding\u0026rdquo;\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eVaishali Lekchand Dasriya: Investigation, Methodology, Writing - original draft, Data curation; Manorama Kumari: Writing-review \u0026amp; editing, Methodology \u0026amp; Formal analysis; Soniya Ranveer: Methodology \u0026amp; Formal analysis, Pradip Behare: Writing-review \u0026amp; editing \u0026amp; Conceptualization; Shilpa Vij: Writing-review \u0026amp; editing, \u0026amp; Visualization;Anil Kumar Puniya: Writing - review and editing, Supervision, \u0026amp; Project administration.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eAuthors are thankful to ICAR-NDRI for providing fellowship and research facilities to VD for conducting this doctoral research.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eStogmann, E.; Moser, D.; Klug, S.; Gleiss, A.; Auff, E.; Dal-Bianco, P.; Lehrner, J. 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Journal of Chromatography B: Biomedical Sciences and Applications, 1996;681(1): 63\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJim\u0026eacute;nez-Balado, J., \u0026amp; Eich, T. S. (2021, August). GABAergic dysfunction, neural network hyperactivity and memory impairments in human aging and Alzheimer\u0026rsquo;s disease. In \u003cem\u003eSeminars in cell \u0026amp; developmental biology\u003c/em\u003e (Vol. 116, pp. 146\u0026ndash;159). Academic Press.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Toxicology](https://link.springer.com/journal/44339)","snPcode":"44339","submissionUrl":"https://submission.springernature.com/new-submission/44339/3","title":"Discover Toxicology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Limosilactobacillus fermentum NCDC701, Alzheimer, gut microbiota, neuroinflammation, oxidative stress, microbiota-gut-brain axis","lastPublishedDoi":"10.21203/rs.3.rs-5226459/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5226459/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study aimed to investigate the protective effect of \u003cem\u003eLimosilactobacillus fermentum\u003c/em\u003e NCDC701 against AlCl\u003csub\u003e3\u003c/sub\u003e and D-galactose (D-gal) -induced Alzheimer rat model. The dose of AlCl\u003csub\u003e3\u003c/sub\u003e (50mg/kg body weight) and D-gal (100mg/kg bodyweight) for 7 days induced oxidative stress such as amyloid plaque deposition, neuroinflammation, and reduction of neurotransmitters in the brain. In contrast, the supplementation of NCDC701 improved the neurotransmitter levels, antioxidants and anti-inflammatory cytokine IL-10, while decreased the amyloid peptide, pro-inflammatory cytokines, as well as LPS in colon and brain. The recovery in amyloid plaque deposition and tissue injury was also confirmed by the histopathological examinations. The supplementation of NCDC701 also improved the decreased expression levels of intestinal tight junction proteins, inhibited the up-regulation of p65, COX-2 and iNOS expression, and improved gut microbiota dysbiosis by increasing the \u003cem\u003eFirmicutes\u003c/em\u003e/\u003cem\u003eBacteroidetes\u003c/em\u003e ratio, promoted beneficial microflora (\u003cem\u003eOscillospira\u003c/em\u003e, \u003cem\u003eRuminococcus\u003c/em\u003e, \u003cem\u003eVerrucomicrobia\u003c/em\u003e, \u003cem\u003eLactobacillus\u003c/em\u003e, and \u003cem\u003ePrevotella\u003c/em\u003e), and suppressed the pathogenic microflora (\u003cem\u003eSpirochaetes\u003c/em\u003e, \u003cem\u003eTenericutes\u003c/em\u003e, \u003cem\u003eProphyromonadaceae\u003c/em\u003e, \u003cem\u003eCoprococcus, Clostridium\u003c/em\u003e, and \u003cem\u003eAllobaculum\u003c/em\u003e) by increasing the concentration of butyrate and total SCFAs compared to the AlCl\u003csub\u003e3\u003c/sub\u003e and D-gal treated model. The findings of this study provide novel insights into the effect of NCDC701 intervention on the gut\u0026ndash;microbiome\u0026ndash;brain axis and should aid future understanding of probiotics for improved host health.\u003c/p\u003e","manuscriptTitle":"Ameliorative action of probiotics on the neurotoxicological effect of Aluminium chloride and D-galactose","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-02 06:54:04","doi":"10.21203/rs.3.rs-5226459/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-02-05T05:40:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-13T17:33:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"22782069505669158647582925121895090892","date":"2024-12-12T16:15:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-10T01:41:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"218681316352842894431644439028773151074","date":"2024-12-07T05:56:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"198198634234278364184064735248707023337","date":"2024-12-06T17:19:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-25T08:09:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"30506186182012987168871794060503252612","date":"2024-11-23T02:51:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"329994178457596733814233437582021123562","date":"2024-11-21T09:37:06+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-21T09:27:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-15T14:47:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-15T14:47:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Toxicology","date":"2024-10-08T14:56:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Toxicology](https://link.springer.com/journal/44339)","snPcode":"44339","submissionUrl":"https://submission.springernature.com/new-submission/44339/3","title":"Discover Toxicology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5558189b-e5a1-45d2-93ba-7100ca85227d","owner":[],"postedDate":"December 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-03-25T08:08:35+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-02 06:54:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5226459","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5226459","identity":"rs-5226459","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}