Anti-inflammatory and Neuroprotective Effects of Acetobacter senegalensis LMG 23690T in an Escherichia coli-Induced Rat Model of Gut and Brain Inflammation

Anti-inflammatory and Neuroprotective Effects of Acetobacter senegalensis LMG 23690T in an Escherichia coli-Induced Rat Model of Gut and Brain Inflammation | 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 Anti-inflammatory and Neuroprotective Effects of Acetobacter senegalensis LMG 23690T in an Escherichia coli-Induced Rat Model of Gut and Brain Inflammation Shima Bahador, Rasoul Shafiei, Maryam Noorbakhshnia, Babak Beikzadeh, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7786634/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Lipopolysaccharides (LPSs) induced inflammation response through activation of TLR4/NF-κB pathway may results in disruption of gut barrier integrity and trigger neuroinflammation through the gut–brain axis. The present study aims to evaluate the anti-inflammatory activity of Acetobacter senegalensis LMG 23690T. Forty rats were used in this study. Rats were grouped into 4 groups: G1 (treated with physiological serum; negative control), G2 (treated with Escherichia coli O55; positive control), G3 (treated with A. senegalensis LMG 23690T; test group), G4 (treated with A. senegalensis LMG 23690T + E. coli; prevention group). Rats were gavaged with bacteria for 60 days. Behavioral tests including Morris water maze (MWM), Elevated plus maze (EPM), and Discrimination Index (DI) were performed. Functional analysis of the Liver and kidneys was examined using biochemical tests. Tissues status was studied using histopathological tests. In addition, Tumor necrosis factor (TNF-α) level was measured in the hippocampus tissue using the ELISA technique. Our results showed that A. senegalensis LMG 23690T effectively prevented the adverse effects of E. coli on spatial learning and memory, anxiety behavior, and the discriminatory ability of rats. Furthermore, A. senegalensis LMG 23690T significantly prevented liver and kidney dysfunction. Histopathological results revealed the protective activity of A. senegalensis LMG 23690T against structural damages caused by E. coli on the ileum, liver, and hippocampus tissues. Moreover, the increase in TNF-α level by E. coli was suppressed in the presence of A. senegalensis LMG 23690T. This is the first report demonstrating the anti-inflammatory and neuroprotective effects of A. senegalensis LMG 23690T, highlighting its potential as a promising candidate for next-generation probiotics targeting gut and brain inflammation. Inflammation Lipopolysaccharide (LPS) Acetobacter senegalensis LMG 23690T Gut-brain axis Rat model Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 1. Introduction Inflammation, as an important part of the immune system, plays a critical role in response to harmful stimuli, such as pathogens, damaged cells, toxic compounds, or irradiation (Chen et al. 2017 ). Despite its key role in the body’s defense system, there is a general concept that uncontrolled inflammation can be a major cause of serious conditions (Chatterjee 2016 ; Khansari, Shakiba, and Mahmoudi 2009). For instance, recent studies suggest that there is an association between inflammation and renal diseases (Andrade-Oliveira et al. 2019 ), liver fibrosis, cirrhosis (Stalnikowitz and Weissbrod 2003), and neurodegenerative diseases (Kwon and Koh 2020). An uncontrolled inflammatory process exerts its adverse effects by stimulating oxidative stress and reducing cellular antioxidant capacity (Gulec Peker and Kaltalioglu 2021; Steven et al. 2019). Lipopolysaccharide (LPS) or endotoxin is the major component of the outer surface membrane of almost all gram-negative bacteria, including Escherichia coli (Sampath 2018 ; Xi et al. 2022 ). LPS triggers acute systemic inflammation, impairing normal function in organs such as the liver, kidneys, colon, lungs, hippocampus, and cerebral cortex (Gao et al. 2021 ). The pathogenic roles of endogenous LPS are gaining much attention because they seem to play a remarkable role in the generation and progression of neuroinflammation. So that a variety of neuropsychiatric diseases, including anxiety and attention-deficit hyperactivity disorders, are related to LPS. Additionally, the role in neurodegenerative diseases, especially Alzheimer’s disease (AD), and Parkinson’s disease (PD) has been reported by several studies (Kalyan et al. 2022). Acetobacter senegalensis LMG 23690T is a gram-negative, thermo-tolerant, obligate aerobe and mesophilic bacterium that belongs to Acetic acid bacteria (AAB). This bacterium has been isolated from mango fruit and can grow and produce acetic acid at various temperatures (28–40 0 C) (Shafiei et al. 2013 ). Physiological and application of A. senegalensis LMG 23690T has been widely studied in various research (Aswini, Gopal, and Uthandi 2020; Illeghems et al. 2016 ; Shafiei et al. 2013 ). However, direct studies on the biological activities of this bacterium are limited; acetic fermentation processes involving similar bacteria have been shown to enhance the antioxidant properties of fermented products (Neffe-Skocińska et al. 2023). It shows that A. senegalensis LMG 23690T may potentially exert biological activities like anti-inflammatory properties. Therefore, we hypothesize that A. senegalensis LMG 23690T could prevent the adverse effects of LPS-induced inflammation on the blood, liver, kidneys, and brain. This study aimed to investigate the anti-inflammatory effect of A. senegalensis LMG 23690T on LPS-producing Escherichia coli O55-induced inflammation in rats as an experimental model. Integrating behavioral tests, biochemical analysis, and measurement of TNF-α levels in the hippocampus and histopathological assessment, this study highlights the potential of A. senegalensis LMG 23690T as an anti-inflammatory therapeutic agent. 2. Materials and Methods 2.1. Cultivation of A. senegalensis LMG 23690T Pre-adapted (using serial passage) A. senegalensis LMG 23690T in culture medium containing glycerol was cultivated in a 1000 ml Erlenmeyer flask containing 200 ml medium (peptone (0.5%), yeast extract (0.5%), glycerol (3%), and glucose (2%)). Incubation was conducted at 30 0 C in a shaking incubator (130 rpm). When bacterial culture reached an optical density (OD600) of 2.3, centrifugation was done at 4000 rpm for 15 min. Supernatant was discarded, and the pellet was washed twice with distilled water. 2.2. Cultivation of E. coli O55:B5 E. coli O55:B5 was provided by the Iranian Research Organization for Science and Technology. Bacterial cultivation was done in a 1000 ml Erlenmeyer flask containing 200 ml medium (peptone (0.5%), yeast extract (0.5%), glycerol (3%), and glucose (2%)). Incubation was conducted at 30 0 C in a shaking incubator (130 rpm). When bacterial culture reached an optical density (OD600) of 2.1, centrifugation was done at 4000 rpm for 15 min. Supernatant was discarded and the pellet was washed twice with distilled water. 2.3. Preparation of dead bacterial cells For this aim, bacterial cells were centrifuged at 4000 rpm for 15 min. The supernatant was removed, and bacterial pellets were incubated in a water bath (70 0 C) for 25 min. Then, bacterial pellets were cultivated on agar plates to ensure cell death. Finally, cell counting was performed using a Thoma cell counting chamber, and 10 9 CFU/ml of each dead bacteria was used to treat the rats. 2.4. Animals and in vivo study design Forty Wistar rats (weighing 200–300 g, 1-year-old) were used in this study. Four rats were housed in each cage with free access to food and water in a controlled condition under a 12-light/12-dark-hour cycle at 23°C. Animals were kept according to the guidelines for the Care and Use of Animals in Research (8th edition, National Academies Press, 2011). All the procedures were approved by the Ethics Committee of the Faculty of Biological Science and Technology, the University of Isfahan (ethical approval ID: IR.UI.REC.1403.158). Forty rats were used in this study. Rats were grouped into 4 groups: G1 (treated with physiological serum; negative control), G2 (treated with E. coli O55:B5; positive control), G3 (treated with A. senegalensis LMG 23690T; test group), G4 (treated with A. senegalensis LMG 23690T + E. coli; preventing group). Rats were gavaged with 10 9 CFU/ml of bacteria for 60 days. Rats in G4 were gavaged with A. senegalensis LMG 23690T for 30 days and subsequently gavaged with A. senegalensis LMG 23690T and E. coli on an alternating daily schedule. Finally, rats were sacrificed and biochemical and immunological studies were performed (Fig. 1 ). 2.5. Behavioral tests 2.5.1. Morris water maze test Rats ' spatial learning and memory abilities were assessed using Morris water maze (MWM) test. In this test, animals search for a hidden, fixed platform 2 cm below the water’s surface in a certain quadrant of a black pool. The pool (diameter: 150 cm) was divided into four quadrants and filled with water 25 0 C. The pool was equipped with a computerized tracking/image analyzer system. The spatial acquisition phase consisted of 4 daily training trials for 3 consecutive days. Rats were released randomly with their heads facing the pool wall from the four quadrants, allowing them to swim and locate the invisible platform for 120 s. The learning ability of rats was evaluated through measurement of escape latency and distance traveled to find the platform, and the animals then must recall the target area in the probe test (Amanzadeh Jajin et al. 2021). 2.5.2. Elevated plus maze (EPM) Anxiety behavior was evaluated using EPM test. Initially, rats were acclimated to their environment for 30 min before testing. Briefly, animals were placed in the center zone facing the open arm toward an empty wall and allowed to explore the maze for 5 minutes. Before each trial, the pool was cleaned and air dried to eliminate odor. Rats behaviors were recorded using a video tracking system embedded on above the pool. From this software, time and entries into open arms (anti–anxiety-like behavior), time and entries into closed arms (anxiety‐like behavior), and total distance traveled were quantified. The tests were recorded and monitored by an observer blinded to genotype and training status. The observer noted open‐arm freezing and falling off of open arms (Lopez, White, and Hall 2024). 2.5.3. Novel object recognition (NOR) The NOR test utilizes the natural propensity of rodents to explore new objects more than familiar ones. The test took place over two days. On the first day, each rat went through a habituation session. One at a time, the rats were placed in a square box (60 × 60 × 60 cm) with two identical objects and were given 3 minutes to explore and get used to the environment. The actual test happened the following day and was split into two parts, with a one-hour break in between. In the first part, each rat was again placed in the same box, now with two identical objects, and allowed to explore for 4 minutes. After an hour, one of those objects was swapped out for a new one. The rat was then returned to the box and given 3 minutes to explore both the familiar and the new object. Recognition memory was assessed using the discrimination index (DI), calculated as follows: DI = (time spent on the new object/total time spent exploring both objects) × 100%. An object was considered to be explored when the animal placed its nose within 2 cm of the zone where the object was located (Grayson et al. 2015 ). 2.6. Biochemical evaluations For biochemical analysis, blood samples were collected from all groups (after 60 days of treatment) into Serum Clot Activator Tubes and stored in room temperature. Tubes were centrifuged at 14000 rpm for 20 minutes in an MSC bench centrifuge (Beckman and Hirsch, Burlington, IO, USA). The prepared plasma was used in the estimation of alanine transaminase (ALT), aspartate transaminase (AST), cholesterol (TC), triglycerides (TG), urea, and creatinine content. 2.6.1. Measurement of ALT and AST activities Measurement ALT activity: ALT was measured according to Adeyemi et al. using Randox kits. Briefly, 50 µl of the sample and 500 µl of the ALT reagent were mixed in a test tube, and the initial absorbance at 340 nm was read after 1 minute. The timer was started simultaneously and further absorbance readings were taken after 1, 2, and 3 minutes. ALT activity (nm/min) = 1746 × ΔA 340 nm/min, ΔA 340 nm/min = change in absorbance per minute for the homogenate sample, 1746 = Extinction coefficient (Adeyemi et al. 2015 ). Measurement of AST activity: The same assay method described for ALT was used except that the ALT reagent was replaced with the AST reagent. AST activity (nm/min) = 1746 × ΔA 340 nm/min; ΔA 340 nm/min = change in absorbance per minute for the homogenate sample; 1746 = Extinction co-efficient (Adeyemi et al. 2015 ). 2.6.2. Measurement of serum total cholesterol (TC) and triglycerides (TG) Cholesterol and triglyceride levels were measured in the serum of rats calorimetrically by commercially available Randox kits according to the manufacturer’s instructions. 2.6.3. Measurement of urea and Creatinine levels Serum urea and creatinine estimation was carried out on the serum sample collected from both the control and the test Wistar rats using the urease kinetic method and Jaffe's reaction method, respectively, with the Roche/Hitachi Cobas C 311 auto-analyzer (Ekwempu et al. 2019 ). 2.7. Histopathological analysis Histopathological analysis of ileum, liver and hippocampus was performed according to Deori et al. Briefly, tissue samples were collected from all groups and fixed in 10% buffered formalin. After routine processing, the tissue samples were embedded in paraffin using Leica Histopathology Assembly (Leica TP1020 and Leica EG1150H). Finally, samples were sectioned at 5 µm, stained with routine hematoxylin-eosin (H-E) stain and examined using Phase contrast microscope (Leica) (Deori et al. 2016 ). 2.8. Confocal laser scanning immunofluorescence microscopy Confocal laser scanning immunofluorescence microscopy was performed for the detection of TNF-α. Initially, ileum tissue samples were washed with distilled water and different concentrations of ethanol (40%, 60%, 70%, 80%, 90%, 96%, 100%). Paraffin blocks were prepared and then samples were sectioned (thickness = 5 µm). Slides were rehydrated in xylene and ethanol. After retrieval in citrate buffer, slides were washed with TBS plus 0.03% Triton X-100. Cell blocked in 10% normal serum or with 1% BSA in TBS for 2 h at room temperature. Subsequently, cells were incubated with primary antibody. After washing with TBS plus 0.03% Triton X-100, incubation with the secondary antibody (conjugated with fluorescent dyes) was done. Slides were washed and counterstained with DAPI for 15 min. Then, slides were mounted and analyzed using a confocal microscope (Zeiss, Oberkochen, Germany). 2.9. Hippocampus tissue preparation and measurement of TNF-α According to the kit instructions, TNF-α content in the hippocampus was measured using a specific ELISA kit (KPG-TNF-α-48, Iran). Initially, 25 mg of hippocampus tissue was homogenized in 500 µl of RIPA buffer and diluted to reach an OD of 1.5. In the next step, 50 µl of standards 1, 2, 3, and 4 were added into wells 1 to 4, and well 5 was designated as the blank. Other wells were filled with 50 µl of sample and then incubated on a shaker at 200 rpm. After 50 min, wells were washed and 50 µl of detection antibody was added into all wells except well 5. After incubation under the same conditions, wells were rewashed and 50 µl of HRP-Avidin was added into all wells except well 5. After incubation under the same conditions for 30 min, the wells were washed, and 50 µl of substrate was added to all wells and incubated for 15 min. Finally, 25 µl of stop solution was added, and the optical density was measured at 450 nm using a Dynatech ELISA plate reader. 2.10. Statistical analysis Statistical analyses were performed using GraphPad Prism software. One-way ANOVA and Two-way ANOVA with suitable post hoc tests were used to compare data. P-value less than 0.05 was considered statistically significant. 3. Results 3.1. Effects of A. senegalensis LMG 23690T on behavioral parameters The effect of treatment with A. senegalensis LMG 23690T on behavioral parameters including spatial learning, anxiety behavior, and novel object recognition were assessed using MWM, EMP, and NOR tests, respectively. Our results suggested that distance traveled to reach the platform decreased from training 1 to training 3 in all groups except group 2 (treated with E. coli O55:B5) (Fig. 2 A). Furthermore, time spent in the target quadrant significantly decreased in group 2 compared to control. However, there was no considerable difference between group 3 and group 4 compared to control (Fig. 2 B). ANOVA of the times spent by rats in the open arms of elevated plus maze test showing significant differences between group 2 and all other groups (Fig. 2 C). In fact, treatment of rats with E. coli O55:B5 considerably increased the time spent on the open arms compared to the control group (P = 0.0009). Recognition memory performance was evaluated using Discrimination Index (DI) and the results revealed that treatment with E. coli O55:B5 impaired recognition memory of rats with a significant decrease in DI compared to the control condition (P = 0.001). Additionally, treatment with A. senegalensis LMG 23690T effectively reversed the the memory deficits induced by E. coli O55:B5 (Fig. 2 D). According to Fig. 3 , after 3 days training, rats treated with E. coli O55:B5 were unable to to reach the platform. However, in other groups rats normally reached the platform. In addition, in Moris test day rats treated with E. coli O55:B5 were moving around in all areas of the maze, however, in other groups, rats were more often seen in the NE area. 3.2. Effects of A. senegalensis LMG 23690T on biochemical parameters 3.2.1. Measurement of ALT and AST activities As shown in Fig. 4 , in G2 (treated with E. coli O55:B5) serum levels of AST enzyme significantly increased compared to control group (p = 0.002). However, in G3 (treated with A. senegalensis LMG 23690T) AST concentration notably decreased compared to G2 and was near to the control group. As well, AST level in G4 (preventing group) showed a greater decrease compared to G2 and was closer to its concentration observed in the control group. The analysis of AST/ALT ratio suggested that treatment with E. coli O55:B5 (G2) considerably increased this ratio compared to control (P = 0.01). Nevertheless, the presence of A. senegalensis LMG 23690T reversed this effect, leading to a restoration of normal conditions. According to these observations, A. senegalensis LMG 23690T appears to reduce the risk of liver dysfunction in both G3 and G4 groups. 3.2.2. Measurement of urea and Creatinine levels Evaluation of alteration of serum levels of urea and Creatinine revealed that treatment with E. coli O55:B5 (G2) notably increased urea and Creatinine contents compared to control condition (P < 0.0001 and P < 0.0032, respectively). On the other hand, the presence of A. senegalensis LMG 23690T reversed this effect and resulted in a significant decrease in urea and Creatinine levels in both G3 and G4 groups (Fig. 5 ). Therefore, A. senegalensis LMG 23690T effectively restored kidney dysfunction risk caused by E. coli O55:B5. 3.2.3. Measurement of serum total cholesterol (TC) and triglycerides (TG) According to Fig. 6 , comparison of TG concentration between different groups unraveled that TG significantly decreased in all groups compared to the control condition. As well, TG levels in the presence of A. senegalensis LMG 23690T (G3 and G4) were slightly lower than G2. Treatment with E. coli O55:B5 (G2) considerably increased serum level of TC compared to the control group. Nonetheless, in both G3 and G4 groups TC levels were decreased significantly (P = 0.04 and P = 0.039, respectively). 3.3. Pathological studies 3.3.1. Histopathologic changes in ileum tissue Histopathologic analysis revealed that treatment of rats with E. coli O55:B5 (G2) resulted in a significant decrease in villi length compared the control group (P < 0.0001). Also, crypt depth considerably increased in this group (P = 0.005). The villous-length-to-crypt-depth ratio (VCR) decreased significantly (P < 0.0001) in G2 compared to the control group. These observations confirmed that E. coli O55:B5 caused a villus damage. On the other hand, there was no alteration in ileum tissue in G3 and G4 groups (Fig. 7 ). Additionally, according to histopathologic images (Fig. 7 ), a superficial epithelial necrosis in villous structures was observed in samples collected from rats in G2. Villous structure was normal in other groups. 3.3.2. Histopathologic changes in hippocampus tissue Normal histopathological structure without any alteration was observed in control group. In contrast, histopathological changes in CA3 area (neuronal shrinkage) were noticed in the hippocampal sections of the G2 ( E. coli O55:B5) group. Additionally, neuronal cells were normal in samples collected from G3 and G4. Thus, A. senegalensis LMG 23690T not only did not exert any detrimental effects on hippocampal structure, but also prevented the damage induced by E. coli O55:B5 (Fig. 8 ). 3.3.3. Histopathologic changes in liver As shown in Fig. 9 , treatment of rats with E. coli O55:B5 resulted in single cell necrosis and hemorrhage. On the other hand, treatment with A. senegalensis LMG 23690T not only did not exert any detrimental effects on liver structure, but also prevented the damage induced by E. coli O55:B5. 3.4. Confocal laser scanning immunofluorescence microscopy Confocal laser scanning immunofluorescence showed that treatment of rats with E. coli O55:B5 led to a significant increase in TNF-α levels in the ileum tissue. However, in the presence of A. senegalensis LMG 23690T there was no alteration in the expression of TNF-α. Moreover, A. senegalensis LMG 23690T prevented the increased expression of TNF-α induced by E. coli O55:B5 (Fig. 10 ). 3.5. Measurement of TNF-α in hippocampus tissue According to our results, presented on Fig. 11 , a statistically significantly higher TNF-α levels were observed in the group of rats treated with E. coli O55:B5 (G2) compared to the control group. On the other hand, there was a significant decrease in TNF-α levels in the groups treated with A. senegalensis LMG 23690T (G3 and G4). Additionally, reduction of TNF-α levels in the preventing group was higher than G3. 4. Discussion Inflammation plays a vital role in the defense system. However, it may be involved in various diseases such as Alzheimer's, Parkinson's, rheumatoid arthritis, asthma, and cancer (Xie et al. 2024 ). Thus, the development of effective anti-inflammatory compounds and strategies seems to be necessary. Here, we assessed the anti-inflammatory activity of A. senegalensis LMG 23690T against the inflammatory response caused by the bacterial LPS in a rat model. A set of behavioral, biochemical, pathological, and immunological analyses was used for this aim. MWM test showed that A. senegalensis LMG 23690T could prevent a decrease in time spent in the target quadrant, which confirms its positive effects on rats' spatial learning and memory. A longer time spent in the target quadrant reflects a superior retrieval memory and vice versa (Othman, Hassan, and Has 2022). The elevated plus maze test was applied to investigate anxiety among rats, and results suggested that time spent in open arms increased in the presence of E. coli; however, treatment with A. senegalensis LMG 23690T reduced and prevented it. Reduced time spent in the open arms represents more anxious behavior (Sharma et al. 2022 ). Furthermore, the novel object recognition test revealed that A. senegalensis LMG 23690T prevented a decrease in the discrimination index (DI) caused by E. coli . In fact, a higher DI indicates a strong preference for the novel object, suggesting that the animal remembered the familiar object and can distinguish it from the novel one (Teymuori, Yegdaneh, and Rabbani 2021). Biochemical analysis through measurement of AST and ALT levels showed that A. senegalensis LMG 23690T had no adverse effects on liver function and prevented liver dysfunction caused by E. coli . Generally, AST and ALT are used as markers for liver injury and function, respectively. Elevated AST and AST/ALT ratio levels indicate increased risks of liver injury and liver dysfunction (Alghazeer et al. 2018 ). Measurement of serum levels of urea and Creatinine suggested that A. senegalensis LMG 23690T had no significant effects on urea and Creatinine levels and effectively prevented urea and Creatinine elevation by E. coli . Urea and creatinine are good indicators of a normally functioning kidney, and the serum increase indicates kidney dysfunction (Gowda et al. 2010 ; Kamal 2014 ). Histopathological studies revealed that E. coli O55:B5 caused villus damage. This observation is consistent with previous studies. For instance, Ding et al. showed that treatment with LPS caused severe damage, disorganization, decreased quantity and height, along with atrophic defects, increased mucosal collapse, and cell shedding (Ding et al. 2024 ). In a similar study, Li et al suggested that group of animals treated with LPS showed several histopathological changes including shortened ileal villi length, reduced height of colonic folds, and enhanced ileal colonic recess compared to normal group. They concluded that those observations indicated that LPS caused intestinal villi damage, recess deepening, and intestinal epithelial barrier injury among mice (Li et al. 2023 ). Our results showed that A. senegalensis LMG 23690T caused no damage to the ileum and effectively prevented histopathological damages caused by E. coli . Rehmannia glutinosa (Li et al. 2023 ), Lacticaseibacillus rhamnosus LAB3, Levilactobacillus brevis LAB20, Lactiplantibacillus plantarum LAB31 (Bhatia et al. 2022 ), and Clostridium tyrobutyricum (Xiao et al. 2021 ) are examples of bacterial strains that their protective effects against LPS-induced damages on intestine have been reported. Analysis of effects of LPS on hippocampus showed that LPS caused neuronal shrinkage in rats. Previous studies also have reported that LPS can cause damages to hippocampus tissue (Bahaidrah et al. 2022 ; Zhan et al. 2024 ). Additionally, our results suggested that treatment with A. senegalensis LMG 23690T not only didn’t cause any damage to hippocampus tissue, but also prevented histopathological damage caused by E. coli. Immunological evaluations in the current study revealed that the TNF-α level increased in rats treated with E. coli . These findings are consistent with other studies because previous studies have shown that LPS can induce the biosynthesis of this cytokine through over-expression of the TNF-α gene and enhancement of translation of TNF-α mRNA (Shen et al. 2008 ; Swantek, Christerson, and Cobb 1999). TNF-α is a cytokine that has pleiotropic effects on various cell types. It has been identified as a major regulator of inflammatory responses and is known to be involved in the pathogenesis of some inflammatory and autoimmune diseases. It is functionally known to trigger a series of various inflammatory molecules, including other cytokines and chemokines (Jang et al. 2021 ).Uncontrolled activation of TNF - α has been associated with the pathogenesis of numerous diseases, including multiple sclerosis and rheumatoid arthritis (McCarty and Chesson Jr 2011 ). A. senegalensis LMG 23690T decreased TNF-α level and prevented TNF-α elevation induced by E. coli . Finally, we suggest that A. senegalensis metabolites or cell debris (like LPS) may block Toll-Like Receptor (TLR)-mediated upregulation of TNF-α (Fig. 12 ). 5. Conclusion 1. For the first time, Bacterium A. senegalensis LMG 23690T has been shown to possess anti-inflammatory activity. 2. A. senegalensis LMG 23690T may exert its anti-inflammatory activity through prevention of overexpression of TNF-α factor. Declarations Authorship contribution statement Shima Bahador : Writing – original draft, Investigation, Visualization, Methodology. Rasoul Shafiei : Conceptualization, Supervision, Methodology, Project administration, Writing- Review & Editing. Maryam Noorbakhshnia : Methodology, Investigation. Babak Beikzadeh : Methodology, Writing - Review & Editing, Investigation, Validation. Parvin Mahzouni : Methodology, Investigation. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declaration of generative AI and AI-assisted technologies in the writing process No AI technologies have been used in the writing process. Data availability Data will be made available on request. Acknowledgment The authors gratefully appreciate the University of Isfahan for the financial support of this study. References Adeyemi OT, Osilesi O, Adebawo OO, Onajobi FD, Sunday O, Oyedemi, Afolayan AJ (2015) Alkaline Phosphatase (ALP), Aspartate Aminotransferase (AST) and Alanine Aminotransferase (ALT) Activities in Selected Tissues of Rats Fed on Processed Atlantic Horse Mackerel (Trachurus Trachurus). 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J Inflamm Res 1147–1160 Zhan F, Dong Y, Zhou L, Li X, Zheng Zhou, and Guohai Xu (2024) Minocycline Alleviates LPS-Induced Cognitive Dysfunction in Mice by Inhibiting the NLRP3/Caspase-1 Pathway. Aging 16(3):2989 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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1","display":"","copyAsset":false,"role":"figure","size":267652,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e experimental design. Rats were divided into four groups: negative control, positive control, test group and preventing group. Rats in each group were treated under different treatments and finally sacrificed for further studies\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7786634/v1/cfd085204eb8c4eb05bcce2f.png"},{"id":94704316,"identity":"1b48a168-7dfb-4bcc-8372-e0654f9465ba","added_by":"auto","created_at":"2025-10-29 21:37:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":122271,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T\u003cem\u003e \u003c/em\u003eeffects on the behavioral features of rats using MWM (A and B), EPM (C), and NOR (D) tests. Data were expressed as means ± SEM. MWM test showed that time spent in the target quadrant significantly decreased in group 2 compared to control. However, it was normal in groups 3 and 4. EPM test revealed that time spent on the open arms considerably increased in group 2 compared control. But, it was normal in groups 3 and 4. NOR test showed a significant decrease in DI index in group 2 compared to control. This condition reversed in groups 3 and 4\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7786634/v1/e023dbfa9e853f95d0e9390e.png"},{"id":94704318,"identity":"06791d2c-e93e-46a1-b787-af22de37f396","added_by":"auto","created_at":"2025-10-29 21:37:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":232062,"visible":true,"origin":"","legend":"\u003cp\u003eThe representative trajectory for each group in the probe test. Moris train (upper row) and Moris test (lower row). Rats in group 2 were unable to to reach the platform. However, in control condition and groups 3 and 4 rats normally reached the platform\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7786634/v1/6806a395c503a9d2b30e5d41.png"},{"id":94729305,"identity":"41bac93c-a278-4088-9d48-3e6a6c724664","added_by":"auto","created_at":"2025-10-30 07:04:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":77655,"visible":true,"origin":"","legend":"\u003cp\u003eAlterations of AST and AST/ALT in rats under different treatments. AST concentration and AST/ALT ratio were increased significantly in group 2 compared to control group. However, treatment with \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T\u003cem\u003e \u003c/em\u003e(groups 3 and 4) decreased them\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7786634/v1/49bb924451bee5ca877b3fa2.png"},{"id":94704336,"identity":"09291266-a3c1-42f7-804c-9a981b4220d4","added_by":"auto","created_at":"2025-10-29 21:37:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":85148,"visible":true,"origin":"","legend":"\u003cp\u003eAlterations of serum levels of urea and Creatinine in rats under different treatments. Serum levels of Urea and Creatinine were increased significantly in group 2 compared to control zgroup. However, treatment with \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T\u003cem\u003e \u003c/em\u003e(groups 3 and 4) decreased them\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7786634/v1/58ec485c2ca2a3ffaee9a18e.png"},{"id":94704334,"identity":"e21c271e-372b-4690-a1e7-dbeaf44eab05","added_by":"auto","created_at":"2025-10-29 21:37:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":62465,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of total cholesterol and triglycerides content between different groups of rats. Total cholesterol level in group 2 was significantly higher than control group. However, its level was decreased in groups 3 and 4\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7786634/v1/bf78db6b77f139848bfe5740.png"},{"id":94704324,"identity":"6499e3b1-3394-465f-928e-17643417d739","added_by":"auto","created_at":"2025-10-29 21:37:40","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":623951,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of villi length, crypt depth, and VCR index. Representative images of ileum structure were observed with 10× (G1, G3, G4) and 4× (G2) objectives. Histopathological images showed a significant decrease in villi length\u003cem\u003e villous-length-to-crypt-depth ratio (VCR) among rats in G2 compared to control condition. Also, \u003c/em\u003ea \u003cem\u003esuperficial \u003c/em\u003eepithelial\u003cem\u003e necrosis was observed \u003c/em\u003ein\u003cem\u003e villous \u003c/em\u003estructures in samples collected from rats in G2. Villous structure was normal in other groups\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7786634/v1/75c53112cb8323bada5be762.png"},{"id":94729661,"identity":"ee3223fd-2b7a-45b5-af37-53e3582a57a2","added_by":"auto","created_at":"2025-10-30 07:05:16","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1444434,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological analysis of hippocampus tissue under different treatments.Representative images of sections of hippocampus were observed with 10x objectives. Histopathological changes in CA3 area (neuronal shrinkage) were noticed in the hippocampal sections of the G2 (\u003cem\u003eE. coli\u003c/em\u003e O55:B5) group. However, such alterations were not observed in samples from control group and groups 3 and 4\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7786634/v1/b56d81045b8908f02dc1c066.png"},{"id":94704338,"identity":"67f8fc3b-274b-413d-a173-409370e56b87","added_by":"auto","created_at":"2025-10-29 21:37:40","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":724616,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological analysis of liver tissue under different treatments. (A) treated \u003cem\u003eE. coli\u003c/em\u003e O55:B5; (B) treated with \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T\u003cem\u003e.\u003c/em\u003eRepresentative images of sections of liver were observed with 10x objectives. Single cell necrosis and hemorrhage were observed among samples collected from G2 compared to control condition. However, such alterations were not observed in samples from control group and groups 3 and 4\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7786634/v1/e54522a8aa95f894b7da0d68.png"},{"id":94704340,"identity":"3416043e-170c-4ee8-8fbf-6e84cb45a433","added_by":"auto","created_at":"2025-10-29 21:37:40","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":755498,"visible":true,"origin":"","legend":"\u003cp\u003eImmunofluorescence analysis of the expression of TNF-α in the ileum tissue of rats treated under different conditions. A significant increase in TNF-α expression in ileum tissue samples collected from rats in G2 was observed. However, there was no significant alteration in TNF-α expression in samples from control group and groups 3 and 4\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-7786634/v1/500b4e3446574605e176f8e2.png"},{"id":94704345,"identity":"85900002-048b-4807-bbe2-0333927bf614","added_by":"auto","created_at":"2025-10-29 21:37:41","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":47131,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of TNF-α levels between rats treated under different conditions. A significant increase was observed in TNF-α levels among rats in G2 compared to control group. TNF-α levels among rats in groups 3 and 4 were similar to the control\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-7786634/v1/3b1edb95cd389fae2696a671.png"},{"id":94704346,"identity":"61b3d309-5dd6-469a-b2ba-5f5996d12a6c","added_by":"auto","created_at":"2025-10-29 21:37:41","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":341789,"visible":true,"origin":"","legend":"\u003cp\u003ePossible mechanism of anti-inflammation activity of \u003cem\u003eA. senegalensis.\u003c/em\u003e Induction of inflammation through TLR receptor signaling pathway in rats treated with pathogenic bacteria has proven in previous reports (Rameshrad et al. 2015; Shen et al. 2023). Therefore, it seems that \u003cem\u003eA. senegalensis\u003c/em\u003e inhibits TLR-mediated activation of immune responses by \u003cem\u003eE. coli \u003c/em\u003eLipopolysaccharide (LPS)\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-7786634/v1/acd1819cf54a87e756025a79.png"},{"id":98431356,"identity":"e3335b91-c61a-46e4-9915-8aeff06431a4","added_by":"auto","created_at":"2025-12-17 16:47:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6235667,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7786634/v1/34b756bf-65aa-4efe-92f6-8c6fba063e7f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Anti-inflammatory and Neuroprotective Effects of Acetobacter senegalensis LMG 23690T in an Escherichia coli-Induced Rat Model of Gut and Brain Inflammation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eInflammation, as an important part of the immune system, plays a critical role in response to harmful stimuli, such as pathogens, damaged cells, toxic compounds, or irradiation (Chen et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Despite its key role in the body\u0026rsquo;s defense system, there is a general concept that uncontrolled inflammation can be a major cause of serious conditions (Chatterjee \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Khansari, Shakiba, and Mahmoudi 2009). For instance, recent studies suggest that there is an association between inflammation and renal diseases (Andrade-Oliveira et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), liver fibrosis, cirrhosis (Stalnikowitz and Weissbrod 2003), and neurodegenerative diseases (Kwon and Koh 2020). An uncontrolled inflammatory process exerts its adverse effects by stimulating oxidative stress and reducing cellular antioxidant capacity (Gulec Peker and Kaltalioglu 2021; Steven et al. 2019).\u003c/p\u003e\u003cp\u003eLipopolysaccharide (LPS) or endotoxin is the major component of the outer surface membrane of almost all gram-negative bacteria, including \u003cem\u003eEscherichia coli\u003c/em\u003e (Sampath \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Xi et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). LPS triggers acute systemic inflammation, impairing normal function in organs such as the liver, kidneys, colon, lungs, hippocampus, and cerebral cortex (Gao et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe pathogenic roles of endogenous LPS are gaining much attention because they seem to play a remarkable role in the generation and progression of neuroinflammation. So that a variety of neuropsychiatric diseases, including anxiety and attention-deficit hyperactivity disorders, are related to LPS. Additionally, the role in neurodegenerative diseases, especially Alzheimer\u0026rsquo;s disease (AD), and Parkinson\u0026rsquo;s disease (PD) has been reported by several studies (Kalyan et al. 2022).\u003c/p\u003e\u003cp\u003e\u003cem\u003eAcetobacter senegalensis\u003c/em\u003e LMG 23690T is a gram-negative, thermo-tolerant, obligate aerobe and mesophilic bacterium that belongs to Acetic acid bacteria (AAB). This bacterium has been isolated from mango fruit and can grow and produce acetic acid at various temperatures (28\u0026ndash;40 \u003csup\u003e0\u003c/sup\u003eC) (Shafiei et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Physiological and application of \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T has been widely studied in various research (Aswini, Gopal, and Uthandi 2020; Illeghems et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Shafiei et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). However, direct studies on the biological activities of this bacterium are limited; acetic fermentation processes involving similar bacteria have been shown to enhance the antioxidant properties of fermented products (Neffe-Skocińska et al. 2023). It shows that \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T may potentially exert biological activities like anti-inflammatory properties.\u003c/p\u003e\u003cp\u003eTherefore, we hypothesize that \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T could prevent the adverse effects of LPS-induced inflammation on the blood, liver, kidneys, and brain. This study aimed to investigate the anti-inflammatory effect of \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T on LPS-producing \u003cem\u003eEscherichia coli\u003c/em\u003e O55-induced inflammation in rats as an experimental model. Integrating behavioral tests, biochemical analysis, and measurement of TNF-α levels in the hippocampus and histopathological assessment, this study highlights the potential of \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T as an anti-inflammatory therapeutic agent.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Cultivation of \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T\u003c/h2\u003e\u003cp\u003ePre-adapted (using serial passage) \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T in culture medium containing glycerol was cultivated in a 1000 ml Erlenmeyer flask containing 200 ml medium (peptone (0.5%), yeast extract (0.5%), glycerol (3%), and glucose (2%)). Incubation was conducted at 30 \u003csup\u003e0\u003c/sup\u003eC in a shaking incubator (130 rpm). When bacterial culture reached an optical density (OD600) of 2.3, centrifugation was done at 4000 rpm for 15 min. Supernatant was discarded, and the pellet was washed twice with distilled water.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Cultivation of \u003cem\u003eE. coli\u003c/em\u003e O55:B5\u003c/h2\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e O55:B5 was provided by the Iranian Research Organization for Science and Technology. Bacterial cultivation was done in a 1000 ml Erlenmeyer flask containing 200 ml medium (peptone (0.5%), yeast extract (0.5%), glycerol (3%), and glucose (2%)). Incubation was conducted at 30 \u003csup\u003e0\u003c/sup\u003eC in a shaking incubator (130 rpm). When bacterial culture reached an optical density (OD600) of 2.1, centrifugation was done at 4000 rpm for 15 min. Supernatant was discarded and the pellet was washed twice with distilled water.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Preparation of dead bacterial cells\u003c/h2\u003e\u003cp\u003eFor this aim, bacterial cells were centrifuged at 4000 rpm for 15 min. The supernatant was removed, and bacterial pellets were incubated in a water bath (70 \u003csup\u003e0\u003c/sup\u003eC) for 25 min. Then, bacterial pellets were cultivated on agar plates to ensure cell death. Finally, cell counting was performed using a Thoma cell counting chamber, and 10\u003csup\u003e9\u003c/sup\u003e CFU/ml of each dead bacteria was used to treat the rats.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Animals and \u003cem\u003ein vivo\u003c/em\u003e study design\u003c/h2\u003e\u003cp\u003eForty Wistar rats (weighing 200\u0026ndash;300 g, 1-year-old) were used in this study. Four rats were housed in each cage with free access to food and water in a controlled condition under a 12-light/12-dark-hour cycle at 23\u0026deg;C. Animals were kept according to the guidelines for the Care and Use of Animals in Research (8th edition, National Academies Press, 2011). All the procedures were approved by the Ethics Committee of the Faculty of Biological Science and Technology, the University of Isfahan (ethical approval ID: IR.UI.REC.1403.158).\u003c/p\u003e\u003cp\u003eForty rats were used in this study. Rats were grouped into 4 groups: G1 (treated with physiological serum; negative control), G2 (treated with \u003cem\u003eE. coli\u003c/em\u003e O55:B5; positive control), G3 (treated with \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T; test group), G4 (treated with \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T\u0026thinsp;\u003cem\u003e+\u0026thinsp;E. coli;\u003c/em\u003e preventing group). Rats were gavaged with 10\u003csup\u003e9\u003c/sup\u003e CFU/ml of bacteria for 60 days. Rats in G4 were gavaged with \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T for 30 days and subsequently gavaged with \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T and \u003cem\u003eE. coli\u003c/em\u003e on an alternating daily schedule. Finally, rats were sacrificed and biochemical and immunological studies were performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Behavioral tests\u003c/h2\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.5.1. Morris water maze test\u003c/h2\u003e\u003cp\u003eRats ' spatial learning and memory abilities were assessed using Morris water maze (MWM) test. In this test, animals search for a hidden, fixed platform 2 cm below the water\u0026rsquo;s surface in a certain quadrant of a black pool. The pool (diameter: 150 cm) was divided into four quadrants and filled with water 25 \u003csup\u003e0\u003c/sup\u003eC. The pool was equipped with a computerized tracking/image analyzer system. The spatial acquisition phase consisted of 4 daily training trials for 3 consecutive days. Rats were released randomly with their heads facing the pool wall from the four quadrants, allowing them to swim and locate the invisible platform for 120 s. The learning ability of rats was evaluated through measurement of escape latency and distance traveled to find the platform, and the animals then must recall the target area in the probe test (Amanzadeh Jajin et al. 2021).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.5.2. Elevated plus maze (EPM)\u003c/h2\u003e\u003cp\u003eAnxiety behavior was evaluated using EPM test. Initially, rats were acclimated to their environment for 30 min before testing. Briefly, animals were placed in the center zone facing the open arm toward an empty wall and allowed to explore the maze for 5 minutes. Before each trial, the pool was cleaned and air dried to eliminate odor. Rats behaviors were recorded using a video tracking system embedded on above the pool. From this software, time and entries into open arms (anti\u0026ndash;anxiety-like behavior), time and entries into closed arms (anxiety‐like behavior), and total distance traveled were quantified. The tests were recorded and monitored by an observer blinded to genotype and training status. The observer noted open‐arm freezing and falling off of open arms (Lopez, White, and Hall 2024).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.5.3. Novel object recognition (NOR)\u003c/h2\u003e\u003cp\u003eThe NOR test utilizes the natural propensity of rodents to explore new objects more than familiar ones. The test took place over two days. On the first day, each rat went through a habituation session. One at a time, the rats were placed in a square box (60 \u0026times; 60 \u0026times; 60 cm) with two identical objects and were given 3 minutes to explore and get used to the environment. The actual test happened the following day and was split into two parts, with a one-hour break in between. In the first part, each rat was again placed in the same box, now with two identical objects, and allowed to explore for 4 minutes. After an hour, one of those objects was swapped out for a new one. The rat was then returned to the box and given 3 minutes to explore both the familiar and the new object. Recognition memory was assessed using the discrimination index (DI), calculated as follows: DI = (time spent on the new object/total time spent exploring both objects) \u0026times; 100%. An object was considered to be explored when the animal placed its nose within 2 cm of the zone where the object was located (Grayson et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Biochemical evaluations\u003c/h2\u003e\u003cp\u003eFor biochemical analysis, blood samples were collected from all groups (after 60 days of treatment) into Serum Clot Activator Tubes and stored in room temperature. Tubes were centrifuged at 14000 rpm for 20 minutes in an MSC bench centrifuge (Beckman and Hirsch, Burlington, IO, USA). The prepared plasma was used in the estimation of alanine transaminase (ALT), aspartate transaminase (AST), cholesterol (TC), triglycerides (TG), urea, and creatinine content.\u003c/p\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.6.1. Measurement of ALT and AST activities\u003c/h2\u003e\u003cp\u003eMeasurement ALT activity: ALT was measured according to Adeyemi et al. using Randox kits. Briefly, 50 \u0026micro;l of the sample and 500 \u0026micro;l of the ALT reagent were mixed in a test tube, and the initial absorbance at 340 nm was read after 1 minute. The timer was started simultaneously and further absorbance readings were taken after 1, 2, and 3 minutes. ALT activity (nm/min)\u0026thinsp;=\u0026thinsp;1746\u0026thinsp;\u0026times;\u0026thinsp;ΔA 340 nm/min, ΔA 340 nm/min\u0026thinsp;=\u0026thinsp;change in absorbance per minute for the homogenate sample, 1746\u0026thinsp;=\u0026thinsp;Extinction coefficient (Adeyemi et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMeasurement of AST activity: The same assay method described for ALT was used except that the ALT reagent was replaced with the AST reagent. AST activity (nm/min)\u0026thinsp;=\u0026thinsp;1746\u0026thinsp;\u0026times;\u0026thinsp;ΔA 340 nm/min; ΔA 340 nm/min\u0026thinsp;=\u0026thinsp;change in absorbance per minute for the homogenate sample; 1746\u0026thinsp;=\u0026thinsp;Extinction co-efficient (Adeyemi et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e2.6.2. Measurement of serum total cholesterol (TC) and triglycerides (TG)\u003c/h2\u003e\u003cp\u003e Cholesterol and triglyceride levels were measured in the serum of rats calorimetrically by commercially available Randox kits according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e2.6.3. Measurement of urea and Creatinine levels\u003c/h2\u003e\u003cp\u003eSerum urea and creatinine estimation was carried out on the serum sample collected from both the control and the test Wistar rats using the urease kinetic method and Jaffe's reaction method, respectively, with the Roche/Hitachi Cobas C 311 auto-analyzer (Ekwempu et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Histopathological analysis\u003c/h2\u003e\u003cp\u003eHistopathological analysis of ileum, liver and hippocampus was performed according to Deori et al. Briefly, tissue samples were collected from all groups and fixed in 10% buffered formalin. After routine processing, the tissue samples were embedded in paraffin using Leica Histopathology Assembly (Leica TP1020 and Leica EG1150H). Finally, samples were sectioned at 5 \u0026micro;m, stained with routine hematoxylin-eosin (H-E) stain and examined using Phase contrast microscope (Leica) (Deori et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Confocal laser scanning immunofluorescence microscopy\u003c/h2\u003e\u003cp\u003eConfocal laser scanning immunofluorescence microscopy was performed for the detection of TNF-α. Initially, ileum tissue samples were washed with distilled water and different concentrations of ethanol (40%, 60%, 70%, 80%, 90%, 96%, 100%). Paraffin blocks were prepared and then samples were sectioned (thickness\u0026thinsp;=\u0026thinsp;5 \u0026micro;m). Slides were rehydrated in xylene and ethanol. After retrieval in citrate buffer, slides were washed with TBS plus 0.03% Triton X-100. Cell blocked in 10% normal serum or with 1% BSA in TBS for 2 h at room temperature. Subsequently, cells were incubated with primary antibody. After washing with TBS plus 0.03% Triton X-100, incubation with the secondary antibody (conjugated with fluorescent dyes) was done. Slides were washed and counterstained with DAPI for 15 min. Then, slides were mounted and analyzed using a confocal microscope (Zeiss, Oberkochen, Germany).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Hippocampus tissue preparation and measurement of TNF-α\u003c/h2\u003e\u003cp\u003eAccording to the kit instructions, TNF-α content in the hippocampus was measured using a specific ELISA kit (KPG-TNF-α-48, Iran). Initially, 25 mg of hippocampus tissue was homogenized in 500 \u0026micro;l of RIPA buffer and diluted to reach an OD of 1.5. In the next step, 50 \u0026micro;l of standards 1, 2, 3, and 4 were added into wells 1 to 4, and well 5 was designated as the blank. Other wells were filled with 50 \u0026micro;l of sample and then incubated on a shaker at 200 rpm. After 50 min, wells were washed and 50 \u0026micro;l of detection antibody was added into all wells except well 5. After incubation under the same conditions, wells were rewashed and 50 \u0026micro;l of HRP-Avidin was added into all wells except well 5. After incubation under the same conditions for 30 min, the wells were washed, and 50 \u0026micro;l of substrate was added to all wells and incubated for 15 min. Finally, 25 \u0026micro;l of stop solution was added, and the optical density was measured at 450 nm using a Dynatech ELISA plate reader.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Statistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using GraphPad Prism software. One-way ANOVA and Two-way ANOVA with suitable post hoc tests were used to compare data. P-value less than 0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Effects of \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T on behavioral parameters\u003c/h2\u003e\u003cp\u003eThe effect of treatment with \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T on behavioral parameters including spatial learning, anxiety behavior, and novel object recognition were assessed using MWM, EMP, and NOR tests, respectively. Our results suggested that distance traveled to reach the platform decreased from training 1 to training 3 in all groups except group 2 (treated with \u003cem\u003eE. coli\u003c/em\u003e O55:B5) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Furthermore, time spent in the target quadrant significantly decreased in group 2 compared to control. However, there was no considerable difference between group 3 and group 4 compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eANOVA of the times spent by rats in the open arms of elevated plus maze test showing significant differences between group 2 and all other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). In fact, treatment of rats with \u003cem\u003eE. coli\u003c/em\u003e O55:B5 considerably increased the time spent on the open arms compared to the control group (P\u0026thinsp;=\u0026thinsp;0.0009).\u003c/p\u003e\u003cp\u003eRecognition memory performance was evaluated using Discrimination Index (DI) and the results revealed that treatment with \u003cem\u003eE. coli\u003c/em\u003e O55:B5 impaired recognition memory of rats with a significant decrease in DI compared to the control condition (P\u0026thinsp;=\u0026thinsp;0.001). Additionally, treatment with \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T effectively reversed the the memory deficits induced by \u003cem\u003eE. coli\u003c/em\u003e O55:B5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAccording to Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, after 3 days training, rats treated with \u003cem\u003eE. coli\u003c/em\u003e O55:B5 were unable to to reach the platform. However, in other groups rats normally reached the platform. In addition, in Moris test day rats treated with \u003cem\u003eE. coli\u003c/em\u003e O55:B5 were moving around in all areas of the maze, however, in other groups, rats were more often seen in the NE area.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Effects of \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T on biochemical parameters\u003c/h2\u003e\u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\u003ch2\u003e3.2.1. Measurement of ALT and AST activities\u003c/h2\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, in G2 (treated with \u003cem\u003eE. coli\u003c/em\u003e O55:B5) serum levels of AST enzyme significantly increased compared to control group (p\u0026thinsp;=\u0026thinsp;0.002). However, in G3 (treated with \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T) AST concentration notably decreased compared to G2 and was near to the control group. As well, AST level in G4 (preventing group) showed a greater decrease compared to G2 and was closer to its concentration observed in the control group. The analysis of AST/ALT ratio suggested that treatment with \u003cem\u003eE. coli\u003c/em\u003e O55:B5 (G2) considerably increased this ratio compared to control (P\u0026thinsp;=\u0026thinsp;0.01). Nevertheless, the presence of \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T reversed this effect, leading to a restoration of normal conditions. According to these observations, \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T appears to reduce the risk of liver dysfunction in both G3 and G4 groups.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003e3.2.2. Measurement of urea and Creatinine levels\u003c/h2\u003e\u003cp\u003eEvaluation of alteration of serum levels of urea and Creatinine revealed that treatment with \u003cem\u003eE. coli\u003c/em\u003e O55:B5 (G2) notably increased urea and Creatinine contents compared to control condition (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and P\u0026thinsp;\u0026lt;\u0026thinsp;0.0032, respectively). On the other hand, the presence of \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T reversed this effect and resulted in a significant decrease in urea and Creatinine levels in both G3 and G4 groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Therefore, \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T effectively restored kidney dysfunction risk caused by \u003cem\u003eE. coli\u003c/em\u003e O55:B5.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section3\"\u003e\u003ch2\u003e3.2.3. Measurement of serum total cholesterol (TC) and triglycerides (TG)\u003c/h2\u003e\u003cp\u003eAccording to Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, comparison of TG concentration between different groups unraveled that TG significantly decreased in all groups compared to the control condition. As well, TG levels in the presence of \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T (G3 and G4) were slightly lower than G2. Treatment with \u003cem\u003eE. coli\u003c/em\u003e O55:B5 (G2) considerably increased serum level of TC compared to the control group. Nonetheless, in both G3 and G4 groups TC levels were decreased significantly (P\u0026thinsp;=\u0026thinsp;0.04 and P\u0026thinsp;=\u0026thinsp;0.039, respectively).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Pathological studies\u003c/h2\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003e3.3.1. Histopathologic changes in ileum tissue\u003c/h2\u003e\u003cp\u003eHistopathologic analysis revealed that treatment of rats with \u003cem\u003eE. coli\u003c/em\u003e O55:B5 (G2) resulted in a significant decrease in villi length compared the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Also, crypt depth considerably increased in this group (P\u0026thinsp;=\u0026thinsp;0.005). The villous-length-to-crypt-depth ratio (VCR) decreased significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in G2 compared to the control group. These observations confirmed that \u003cem\u003eE. coli\u003c/em\u003e O55:B5 caused a villus damage. On the other hand, there was no alteration in ileum tissue in G3 and G4 groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Additionally, according to histopathologic images (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), a superficial epithelial necrosis in villous structures was observed in samples collected from rats in G2. Villous structure was normal in other groups.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003e3.3.2. Histopathologic changes in hippocampus tissue\u003c/h2\u003e\u003cp\u003eNormal histopathological structure without any alteration was observed in control group. In contrast, histopathological changes in CA3 area (neuronal shrinkage) were noticed in the hippocampal sections of the G2 (\u003cem\u003eE. coli\u003c/em\u003e O55:B5) group. Additionally, neuronal cells were normal in samples collected from G3 and G4. Thus, \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T not only did not exert any detrimental effects on hippocampal structure, but also prevented the damage induced by \u003cem\u003eE. coli\u003c/em\u003e O55:B5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section3\"\u003e\u003ch2\u003e3.3.3. Histopathologic changes in liver\u003c/h2\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, treatment of rats with \u003cem\u003eE. coli\u003c/em\u003e O55:B5 resulted in single cell necrosis and hemorrhage. On the other hand, treatment with \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T not only did not exert any detrimental effects on liver structure, but also prevented the damage induced by \u003cem\u003eE. coli\u003c/em\u003e O55:B5.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Confocal laser scanning immunofluorescence microscopy\u003c/h2\u003e\u003cp\u003eConfocal laser scanning immunofluorescence showed that treatment of rats with \u003cem\u003eE. coli\u003c/em\u003e O55:B5 led to a significant increase in TNF-α levels in the ileum tissue. However, in the presence of \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T there was no alteration in the expression of TNF-α. Moreover, \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T prevented the increased expression of TNF-α induced by \u003cem\u003eE. coli\u003c/em\u003e O55:B5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec30\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Measurement of TNF-α in hippocampus tissue\u003c/h2\u003e\u003cp\u003eAccording to our results, presented on Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e, a statistically significantly higher TNF-α levels were observed in the group of rats treated with \u003cem\u003eE. coli\u003c/em\u003e O55:B5 (G2) compared to the control group. On the other hand, there was a significant decrease in TNF-α levels in the groups treated with \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T (G3 and G4). Additionally, reduction of TNF-α levels in the preventing group was higher than G3.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eInflammation plays a vital role in the defense system. However, it may be involved in various diseases such as Alzheimer's, Parkinson's, rheumatoid arthritis, asthma, and cancer (Xie et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Thus, the development of effective anti-inflammatory compounds and strategies seems to be necessary. Here, we assessed the anti-inflammatory activity of \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T against the inflammatory response caused by the bacterial LPS in a rat model. A set of behavioral, biochemical, pathological, and immunological analyses was used for this aim. MWM test showed that \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T could prevent a decrease in time spent in the target quadrant, which confirms its positive effects on rats' spatial learning and memory. A longer time spent in the target quadrant reflects a superior retrieval memory and vice versa (Othman, Hassan, and Has 2022).\u003c/p\u003e\u003cp\u003eThe elevated plus maze test was applied to investigate anxiety among rats, and results suggested that time spent in open arms increased in the presence of E. coli; however, treatment with \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T reduced and prevented it. Reduced time spent in the open arms represents more anxious behavior (Sharma et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Furthermore, the novel object recognition test revealed that \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T prevented a decrease in the discrimination index (DI) caused by \u003cem\u003eE. coli\u003c/em\u003e. In fact, a higher DI indicates a strong preference for the novel object, suggesting that the animal remembered the familiar object and can distinguish it from the novel one (Teymuori, Yegdaneh, and Rabbani 2021).\u003c/p\u003e\u003cp\u003eBiochemical analysis through measurement of AST and ALT levels showed that \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T had no adverse effects on liver function and prevented liver dysfunction caused by \u003cem\u003eE. coli\u003c/em\u003e. Generally, AST and ALT are used as markers for liver injury and function, respectively. Elevated AST and AST/ALT ratio levels indicate increased risks of liver injury and liver dysfunction (Alghazeer et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Measurement of serum levels of urea and Creatinine suggested that \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T had no significant effects on urea and Creatinine levels and effectively prevented urea and Creatinine elevation by \u003cem\u003eE. coli\u003c/em\u003e. Urea and creatinine are good indicators of a normally functioning kidney, and the serum increase indicates kidney dysfunction (Gowda et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Kamal \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHistopathological studies revealed that \u003cem\u003eE. coli\u003c/em\u003e O55:B5 caused villus damage. This observation is consistent with previous studies. For instance, Ding et al. showed that treatment with LPS caused severe damage, disorganization, decreased quantity and height, along with atrophic defects, increased mucosal collapse, and cell shedding (Ding et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In a similar study, Li et al suggested that group of animals treated with LPS showed several histopathological changes including shortened ileal villi length, reduced height of colonic folds, and enhanced ileal colonic recess compared to normal group. They concluded that those observations indicated that LPS caused intestinal villi damage, recess deepening, and intestinal epithelial barrier injury among mice (Li et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Our results showed that \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T caused no damage to the ileum and effectively prevented histopathological damages caused by \u003cem\u003eE. coli\u003c/em\u003e. \u003cem\u003eRehmannia glutinosa\u003c/em\u003e (Li et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), \u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e LAB3, \u003cem\u003eLevilactobacillus brevis\u003c/em\u003e LAB20, \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e LAB31 (Bhatia et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and \u003cem\u003eClostridium tyrobutyricum\u003c/em\u003e (Xiao et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) are examples of bacterial strains that their protective effects against LPS-induced damages on intestine have been reported. Analysis of effects of LPS on hippocampus showed that LPS caused neuronal shrinkage in rats. Previous studies also have reported that LPS can cause damages to hippocampus tissue (Bahaidrah et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhan et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Additionally, our results suggested that treatment with \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T not only didn\u0026rsquo;t cause any damage to hippocampus tissue, but also prevented histopathological damage caused by \u003cem\u003eE. coli.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eImmunological evaluations in the current study revealed that the TNF-α level increased in rats treated with \u003cem\u003eE. coli\u003c/em\u003e. These findings are consistent with other studies because previous studies have shown that LPS can induce the biosynthesis of this cytokine through over-expression of the TNF-α gene and enhancement of translation of TNF-α mRNA (Shen et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Swantek, Christerson, and Cobb 1999). TNF-α is a cytokine that has pleiotropic effects on various cell types. It has been identified as a major regulator of inflammatory responses and is known to be involved in the pathogenesis of some inflammatory and autoimmune diseases. It is functionally known to trigger a series of various inflammatory molecules, including other cytokines and chemokines (Jang et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).Uncontrolled activation of TNF\u003cem\u003e-\u003c/em\u003eα has been associated with the pathogenesis of numerous diseases, including multiple sclerosis and rheumatoid arthritis (McCarty and Chesson Jr \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T decreased TNF-α level and prevented TNF-α elevation induced by \u003cem\u003eE. coli\u003c/em\u003e. Finally, we suggest that \u003cem\u003eA. senegalensis\u003c/em\u003e metabolites or cell debris (like LPS) may block Toll-Like Receptor (TLR)-mediated upregulation of TNF-α (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003e1. For the first time, Bacterium \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T has been shown to possess anti-inflammatory activity.\u003c/p\u003e\n\u003cp\u003e2. \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T may exert its anti-inflammatory activity through prevention of overexpression of TNF-\u0026alpha; factor.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthorship\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003econtribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShima Bahador\u003cstrong\u003e:\u003c/strong\u003e Writing \u0026ndash; original draft, Investigation, Visualization, Methodology. Rasoul Shafiei\u003cstrong\u003e:\u003c/strong\u003e Conceptualization, Supervision, Methodology, Project administration, Writing- Review \u0026amp; Editing. Maryam Noorbakhshnia\u003cstrong\u003e:\u003c/strong\u003e Methodology, Investigation. Babak Beikzadeh\u003cstrong\u003e:\u003c/strong\u003e Methodology, Writing - Review \u0026amp; Editing, Investigation, Validation. Parvin Mahzouni\u003cstrong\u003e:\u003c/strong\u003e Methodology, Investigation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of generative AI and AI-assisted technologies in the writing process\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo AI technologies have been used in the writing process.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully appreciate the University of Isfahan for the financial support of this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdeyemi OT, Osilesi O, Adebawo OO, Onajobi FD, Sunday O, Oyedemi, Afolayan AJ (2015) Alkaline Phosphatase (ALP), Aspartate Aminotransferase (AST) and Alanine Aminotransferase (ALT) Activities in Selected Tissues of Rats Fed on Processed Atlantic Horse Mackerel (Trachurus Trachurus). 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Aging 16(3):2989\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Inflammation, Lipopolysaccharide (LPS), Acetobacter senegalensis LMG 23690T, Gut-brain axis, Rat model","lastPublishedDoi":"10.21203/rs.3.rs-7786634/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7786634/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLipopolysaccharides (LPSs) induced inflammation response through activation of TLR4/NF-κB pathway may results in disruption of gut barrier integrity and trigger neuroinflammation through the gut\u0026ndash;brain axis. The present study aims to evaluate the anti-inflammatory activity of \u003cem\u003eAcetobacter senegalensis\u003c/em\u003e LMG 23690T. Forty rats were used in this study. Rats were grouped into 4 groups: G1 (treated with physiological serum; negative control), G2 (treated with \u003cem\u003eEscherichia coli\u003c/em\u003e O55; positive control), G3 (treated with \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T; test group), G4 (treated with \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T\u0026thinsp;\u003cem\u003e+\u0026thinsp;E. coli;\u003c/em\u003e prevention group). Rats were gavaged with bacteria for 60 days. Behavioral tests including Morris water maze (MWM), Elevated plus maze (EPM), and Discrimination Index (DI) were performed. Functional analysis of the Liver and kidneys was examined using biochemical tests. Tissues status was studied using histopathological tests. In addition, Tumor necrosis factor (TNF-α) level was measured in the hippocampus tissue using the ELISA technique. Our results showed that \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T effectively prevented the adverse effects of \u003cem\u003eE. coli\u003c/em\u003e on spatial learning and memory, anxiety behavior, and the discriminatory ability of rats. Furthermore, \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T significantly prevented liver and kidney dysfunction. Histopathological results revealed the protective activity of \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T against structural damages caused by \u003cem\u003eE. coli\u003c/em\u003e on the ileum, liver, and hippocampus tissues. Moreover, the increase in TNF-α level by \u003cem\u003eE. coli\u003c/em\u003e was suppressed in the presence of \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T. This is the first report demonstrating the anti-inflammatory and neuroprotective effects of \u003cem\u003eA. senegalensis\u003c/em\u003e LMG 23690T, highlighting its potential as a promising candidate for next-generation probiotics targeting gut and brain inflammation.\u003c/p\u003e","manuscriptTitle":"Anti-inflammatory and Neuroprotective Effects of Acetobacter senegalensis LMG 23690T in an Escherichia coli-Induced Rat Model of Gut and Brain Inflammation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-29 21:37:35","doi":"10.21203/rs.3.rs-7786634/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3564c79b-5371-437f-867e-9a2469ef43e0","owner":[],"postedDate":"October 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-14T05:08:25+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-29 21:37:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7786634","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7786634","identity":"rs-7786634","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}