Gamma -aminobutyric acid ameliorates neurological impairments in type 1 diabetes mellitus mice by regulating the “gut flora-LPS-TLR4-NF-ΚB” signalling Axis

Abstract

This study examined the potential impact of gamma-aminobutyric acid (GABA) supplementation on the progression of type 1 diabetes mellitus (T1DM) through alterations in gut flora and its associated effects on neurological functions. A T1DM mouse model was created using streptozotocin. The study employed flow cytometry to analyze colonic Th17/Treg cells, 16 S rRNA sequencing to analyze microbiota, and western blot to evaluate colonic proteins. Neurological impairments were assessed through various tests. GABA intervention improved blood glucose levels, body weight, and oral glucose tolerance test (OGTT) results in T1DM mice. It also reduced serum LPS, IL-6, and TNF-α levels. GABA mitigated changes in the expressions of Th17 and Treg cells in T1DM mice. GABA-treated mice had more intestinal flora than T1DM mice. TLR4, MyD88, and NF-κB levels decreased with GABA, while Occludin and ZO-1 expressions increased. GABA improved neurological assessments, reduced neuronal damage and apoptosis, and lowered hippocampal LPS, IL-6, and TNF-α levels in T1DM mice. These findings indicated that GABA can manage T1DM by ameliorating hyperglycemia, reducing inflammation, regulating intestinal microbiota, modulating colonic protein expression, and alleviating neurological impairment.

Introduction

Type 1 diabetes mellitus (T1DM) is an autoimmune disease in which the immune system attacks and destroys pancreatic β-cells, resulting in insulin deficiency and impaired glucose metabolism [1]. The global incidence of T1DM is currently increasing every year, and it is most common in children or adolescents. Over time, children with T1DM may experience a decline in cognitive function, such as deficits in memory, attention, problem solving, and other neurological functions, which in turn may lead to further deterioration of subsequent T1DM glycaemic control and additional T1DM complications, placing a heavy burden on the patient’s family and society [2, 3]. Recent research has shown that these deficits are likely caused by direct hyperglycemic damage to neuronal tissue and indirect effects, such as microvascular changes and chronic inflammation [4]. In addition, recent studies have begun to delineate the pathways by which metabolic control influences neurological health [5], suggesting that better glycemic control may ameliorate some of these adverse effects. Glucose-lowering agents, such as insulin or metformin, are used to control blood glucose levels in diabetic patients. However, adverse effects, such as hypoglycaemia, increased body mass, and allergies, can affect treatment outcomes. Therefore, clinical research focuses on finding new drugs or drugs with fewer side effects.

In recent years, numerous studies have confirmed that the development of T1DM is closely related to inflammation, particularly the intestinal flora [6–9]. Clinically, pediatric patients with T1DM have increased intestinal permeability and structural dysregulation of intestinal flora, including decreased diversity and altered commensal-to-pathogen ratios [2, 10]. Animal studies have shown that disrupting the gut microbiota of T1DM rats leads to cognitive decline [11]. In addition, a study reported that the use of a specific drug modulated cognitive decline in T1DM mice by altering their gut flora [12]. Therefore, targeting the regulation of gut flora and ameliorating the inflammatory response is a promising therapeutic strategy to combat neurological impairment in T1DM.

Gamma-aminobutyric acid (GABA) is widely distributed in mammals and is an important inhibitory neurotransmitter in the central nervous system. GABA has been reported to prevent neurological disorders and has antihypertensive, antioxidant, anti-inflammatory, antimicrobial, gut protective, and therapeutic effects against diabetes mellitus [13–19]. In addition, GABA is an extracellular signalling molecule that is synthesized and released by pancreatic β-cells to regulate pancreatic islet cell secretion and function [20]. GABA supplementation has been reported to influence disease progression by modulating intestinal immunity and changes in intestinal flora [21, 22]. It is still unknown whether GABA regulates changes in neurological function in T1DM by modulating gut flora. Khalili et al. [23] showed that cucurbitacine could attenuate LPS-induced cognitive impairment in rats by inhibiting the inflammatory response of hippocampal neurons to TLR4/NF-κB. Endogenous and microbiota‑derived GABA can influence intestinal epithelial homeostasis, attenuate LPS translocation, and thereby suppress TLR4‑driven inflammatory signaling [24]. This opens the door to the possibility that GABA could protect against T1DM-induced neurological damage by regulating the “gut flora-LPS-TLR4-NF-κB” axis. Therefore, elucidating the mechanism of action of GABA at the animal level is important for preventing and treating neurological dysfunction in patients with T1DM.

Lipopolysaccharide (LPS), also known as endotoxin, is an integral component of the cell wall of Gram-negative bacteria and the most potent inducer of inflammation. Studies have shown that an increased abundance of Gram-negative bacteria, such as Vibrio spp. and Enterobacteriaceae species, in the intestinal tract of diabetic patients can lead to significant LPS secretion of LPS from Gram-negative bacterial cell walls, resulting in “metabolic endotoxaemia” [25]. In addition, chronic high levels of LPS can induce a chronic low-grade inflammatory response that may contribute to the development of diabetes mellitus and its complications. LPS activates immune cells by interacting with Toll-like receptors (TLRs), thereby increasing the levels of inflammatory factors such as tumour necrosis factor (TNF-α), interleukin (IL)-1, IL-6. These inflammatory factors can directly cause damage to hippocampal neurons and induce cognitive impairment [26].

In conclusion, we hypothesise that GABA protects against T1DM neuropathy by regulating the “gut flora-LPS-TLR4-NF-κB” axis. We intend to investigate the regulatory effects of GABA on gut flora and its metabolite pathways from the perspective of the “gut flora-brain-gut axis”, and the protective effects of GABA on neurological impairment in T1DM mice. On this basis, we will further elucidate the specific communication pathways between gut flora and brain through the mechanisms of neuroimmunity, endocrinology and bacterial metabolites, and conduct more in-depth studies on the mechanisms of antidiabetic and neuroprotective effects of GABA.

Materials and methods

Establishment of the T1DM mouse model

Eighteen male SPF-grade NOD mice, aged 4 weeks and weighing 15–20 g, were obtained from the College of Veterinary Medicine at Yangzhou University (Institute of Comparative Medicine) in Yangzhou, China. The mice were acclimatized and fed for 1 week, given food and water ad libitum, kept at 23℃~25℃ temperature, 40%~70% relative humidity, and kept in alternating light and dark for 12 h. Twelve mice were taken for modelling, the mice were injected intraperitoneally with freshly prepared streptozotocin (200 mg/kg) once a day for 3 consecutive days to construct T1DM mouse model, after the injection, the mice were free to drink water and eat food freely, and the modelling mice were observed urinate. After 5 days, blood was collected from the tail vein, and the mice were tested for glucose with a glucometer, and the mice were considered to be T1DM modelling mice with stable glucose of > 11.1mmol/L on fasting blood glucose (FBG) for 2 consecutive days. For accurate FBG measurements, mice were fasted for 12 h prior to blood sampling. All animal experiments were approved by the Animal Care and Use Committee of the First Affiliated Hospital of Jiamusi University (approval number 202406).

Grouping and intervention methods

T1DM mice were randomly divided into two groups (T1DM and GABA; n = 6 each). Starting one day after the model was established, the GABA group was gavaged with 2.3 mg/ml/day of GABA for two consecutive weeks, as described in a previous study [27]. The T1DM and control groups (n = 6) were administered 5 mL/kg of double-distilled water daily via gavage for two consecutive weeks. The day after the treatment period ended, fresh fecal samples were collected from all mice and stored at -80 °C for subsequent 16 S ribosomal RNA gene sequencing. At the conclusion of the experiment, euthanasia was performed using sodium pentobarbital (200 mg/kg). The weight of the mice was measured during the experiment. Blood was then collected from the orbital sinus of all mice, and tissue from the hippocampus and colon was rapidly collected and stored at -80 °C for subsequent experiments. A portion of the colon tissue was used for flow cytometry and the rest was stored at -80 °C for further analysis.

Shuttle box test for assessing neurological impairment

A shuttle box test was conducted to evaluate neurological impairment using a standard shuttle box apparatus (Model XYZ, Manufacturer) with two compartments separated by a grid floor that delivers mild electrical foot shocks. Mice were acclimated to the shuttle box for 5 min without shocks on the day before testing. On the test day, each mouse was placed in one compartment. A mild electrical foot shock (0.5 mA) was administered through the grid floor if the mouse did not cross to the other compartment within 10 s. The active evasion time, defined as the time taken to escape from the shock by moving to the other compartment, was recorded for each of the 6 trials, and the average evasion time was calculated. The active evasion number, representing the number of successful escapes (crossing within 10 s) out of the 6 trials, was also recorded. These indices were compared among the control, T1DM, and GABA-treated groups to assess the impact of GABA on neurological function.

Oral glucose tolerance test (OGTT) experiment

Fourteen days following GABA intervention, the mice were administered 150 µl of 20% glucose solution (1 g/kg; Sigma-Aldrich, MO, USA) via oral gavage. Blood glucose values were determined at 0, 30, 60, 90, and 120 min after glucose loading using glucometer (Sinocare, Changsha, China).

ELISA

The hippocampal tissues were extracted using the mechanical crushing method and centrifuged at 300 ×g for 5 min at 21 °C. The supernatant was then discarded and the pellet resuspended in phosphate-buffered saline (PBS; Gibco, Thermo Fisher, MA, USA) to prepare a single-cell suspension. To further lyse the tissue cells, the homogenate was subjected to ultrasonication and then centrifuged at 5000×g for 5 min. The supernatant was collected and stored at -20 °C. Both the tissue samples and serum collected from the orbital sinus were used for subsequent ELISA analyses. According to the manufacturer’s instructions, the concentrations of LPS, IL-6, and TNF-α were measured using the LPS ELISA kit (Cusabio, TX, USA), the IL-6 ELISA kit (Elabscience, TX, USA), and the TNF-α ELISA kit (Elabscience, TX, USA), respectively. Absorbance values were detected using a microplate reader (Thermo Fisher).

Flow cytometry

The colon tissues of the mice were extracted using the mechanical crushing method and then centrifuged at 300 ×g for 5 min at 21 °C. The supernatant was then discarded, and the pellet was resuspended in PBS to create a single-cell suspension. Next, antibodies were added. The antibodies used were as follows: For the detection of Th17 cells, RORγT-PE (12-6988-82, Invitrogen, Thermo Fisher) and CD4-FITC (11-0041-82, Invitrogen) were used; for the detection of Treg cells, CD4-PerCP (46-0041-82, Invitrogen), Foxp3-PE (12-5773-82, Invitrogen), and CD25-FITC (11-0251-82, Invitrogen) were used. The cells were incubated on ice in the dark for 30 min and then analyzed for changes in the proportion of Th17 or Treg cells using a flow cytometer (FACScalibur, BD Biosciences, CA, USA).

16 S ribosomal RNA gene sequencing

First, total microbial DNA was extracted using a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). The quality of the DNA extraction was then assessed using agarose gel electrophoresis with a 1% agarose gel (Bio-Rad, CA, USA). PCR amplification of the target fragments was then performed using Platinum PCR SuperMix (Invitrogen). The amplified products were purified using AMPure XP magnetic beads (Beckman Coulter, CA, USA) and quantified for fluorescence using the Qubit dsDNA HS Assay Kit (Thermo Fisher). Sequencing libraries were prepared using Illumina’s TruSeq Nano DNA LT Library Prep Kit (CA, USA), and the libraries were quality checked on an Agilent Bioanalyzer. High-throughput sequencing was then performed on an Illumina NovaSeq 6000 system (Illumina).

Western blot

Proteins were extracted by grinding colon tissue with a mixture containing PMSF, cell lysate and protease inhibitor (Solarbio, Beijing, China). Protein quantification was performed using BCA kit (Beyotime, Shanghai, China), protein separation by SDS-PAGE electrophoresis, constant flow membrane (Millipore, MA, USA) at 320 mA for 2 h, 5% skimmed milk powder (BD Biosciences) for 1 h, primary antibodies TLR4 (1:1000, GTX13556, Gene Tex, TX, USA), MyD88 (1:1000, ab219413, Abcam, Cambridge, UK), NF-κB (1:1000, ab32536, Abcam), Occludin (1:1000, ab216327, Abcam), ZO-1 (1:1000, ab307799, Abcam), β-actin (1:5000, ab8227, Abcam) were incubated at 4 ℃ overnight, and then the secondary antibody Goat Anti-Rabbit IgG H&L (HRP) (1:5000, ab6721, Abcam) was incubated for 2 h at room temperature. The chemiluminescence was performed by ECL (Tanon, Shanghai, China), which was operated according to the instruction of the kit, and the pictures were taken under the Tanon 5200 chemiluminescence imager to obtain the pictures, which were analyzed by Image J software (National Institutes of Health [NIH], MD, USA).

HE staining

4% paraformaldehyde (Boster, Beijing, China)-fixed mouse hippocampal tissues were made into paraffin sections after dehydration in a gradient of alcohol (Beyotime), transparency in xylene (Beyotime), and embedding in paraffin. The sections were stained with hematoxylin (Beyotime), differentiated by hydrochloric acid in alcohol, stained by eosin (Beyotime), dehydrated by gradient alcohol and made transparent by xylene, sealed by neutral resin (Solarbio), and observed and photographed under a light microscope (Olympus, Tokyo, Japan).

TUNEL staining

Hippocampal tissues were incubated in a proteinase K solution (Invitrogen) for 30 min at 37 °C. The sections were then incubated in a TUNEL reaction mixture (Beyotime) at 37 °C for 1 h, protected from light, and sections were incubated with DAB substrate solution (Beyotime) for 5 min, and apoptosis of mouse brain tissues in each group was observed under the microscope (Olympus).

Statistics

The data obtained from the experiment were expressed as mean ± standard deviation (Mean ± SEM), and all data were processed and statistically analysed using GraphPad Prism 6. Comparisons between sample means of multiple groups were performed using the ANOVA test, with P < 0.05 indicating a statistically significant difference. Principal Coordinates Analysis (PcoA) was conducted using QIIME 2 (Quantitative Insights Into Microbial Ecology 2) software to evaluate the differences in gut microbial communities between the groups.

Result

Successful establishment of the T1DM mouse model

To confirm the successful establishment of the T1DM mouse model, we monitored the general condition, blood glucose levels, and body weight of the mice. The model mice were injected intraperitoneally with freshly prepared streptozotocin (200 mg/kg) once a day for three consecutive days (Fig. 1A). The control group mice were in good mental state, with neat and lustrous fur. Compared with the control group, the mice in the T1DM and GABA groups were in poorer mental state, slow in activity, showed obvious polydipsia, polyphagia and polyuria, and had rough and dull fur, higher FBG (Fig. 1B). Additionally, GABA treatment alleviated the significant reduction in body weight observed in T1DM mice (Fig. 1C). This confirms that the T1DM model was successfully established in the mice.

Fig. 1.

Establishment of T1DM mouse model. (A) Model diagram for establishing a mouse T1DM model. (B) FBG values of mice in each group. (C) Changes in body weight of mice in each group (*P < 0.05; ** P < 0.01; ***<0.001)

3.2 Results of glucose metabolism, inflammatory microenvironment, and peripheral immune system in groups of mice

We then examined the effects of GABA treatment on these factors in T1DM mice. Compared to the control group, OGTT levels in the T1DM group were significantly increased. After GABA treatment, OGTT levels decreased (Fig. 2A). ELISA experiments revealed that serum levels of LPS, IL-6, and TNF-α were significantly increased in the T1DM group than in the control group. After GABA treatment, these cytokine levels decreased significantly (Fig. 2B-D). The expression level of Th17 cells in single-cell suspensions of colonic tissues is shown in Fig. 2E-F, in T1DM mice, the proportion of Th17 cells increased, while the proportion of Treg cells decreased. After GABA treatment, the balance of Th17/Treg cells was significantly restored. These results indicate that GABA treatment improved glucose metabolism and modulated the inflammatory microenvironment and peripheral immune system in T1DM mice.

Fig. 2.

Results of Glucose Metabolism, Inflammatory Microenvironment, and Peripheral Immune System in Groups of Mice. (A) OGTT assay to assess glucose tolerance in groups of mice. (B) Changes in serum levels of LPS, TNF-α and IL-6 in mice in each group by ELISA. (C) Flow assay for TH17 cells in the colonic tissues of mice in each group. (D) Flow assay for Treg cells in the colonic tissues of mice in each group (*P < 0.05; ** P < 0.01; ***<0.001)

Changes in the intestinal microbiota in various groups of mice

To explore the impact of GABA on gut microbiota, we performed 16 S rRNA sequencing. Through this method, we obtained the species composition of the intestinal flora of each group of mice. First, we obtained the specific composition of each microbial community at the category level (Fig. 3A). Then, we analyzed each sample at the genus level (Fig. 3B) and analyzed the abundance of each sample (Fig. 3C). These results showed that the abundance of intestinal flora increased significantly in the GABA group compared to the T1DM group. Finally, PcoA analyses were used to respond to community differences between samples (Fig. 3D), and PcoA analyses were supplemented by a significance analysis of differences between groups (Fig. 3E). To compare species composition differences between samples, we performed VENE plot analysis of the mouse gut flora in each group, followed by further analysis species composition heatmaps and screening for differential markers using LDA Effect Size (LEfSe) (Fig. 3F-H). Finally, we performed KEGG enrichment analysis (Fig. 3I). These findings suggest that GABA treatment significantly alters the composition and diversity of intestinal microbiota in T1DM mice.

Fig. 3.

Species richness and composition of intestinal flora. (A) Statistics of the number of microbial taxonomic units at each level (Phylum, Class, Order, Family, Genus, Species) in each group of mice. (B) Distribution of intestinal flora at the genus level in various groups of mice. (C) Abundance plots of the flora for each sample. (D) Two-dimensional ordination plot of samples analyzed by PcoA. (E) Significance analysis of intergroup distances for PcoA (permanova P = 0.003). (F) VENE plots for each sample group. (G) The taxonomic branching diagram illustrates the taxonomic hierarchical relationships of the major taxonomic units from phylum to genus (from inner circle to outer circle) in the sample community. Node size corresponds to the mean relative abundance of that taxonomic unit; hollow nodes represent taxonomic units that do not differ significantly between groups, whereas nodes of other colors indicate that these taxonomic units reflect significant between-group differences and are more abundant in the subgroup sample represented by that color. Letters identify the names of taxonomic units with significant intergroup differences. (H) Presentation diagram of classification units for intergroup differences based on classification hierarchy tree (*P < 0.05; ** P < 0.01; ***<0.001)

TLR4, MyD88, NF-κB, occludin and ZO-1 expression levels in mouse colon tissues

To understand the molecular mechanisms underlying GABA’s effects, we examined the protein expression levels of TLR4, MyD88, NF-κB, Occludin, and ZO-1 in colonic tissues. Western blot results showed that the levels of TLR4, MyD88, and NF-κB proteins increased and the levels of Occludin and ZO-1 proteins decreased in the colonic tissues of T1DM mice compared to the control group. After GABA treatment, the levels of these proteins were significantly restored (Fig. 4). These results indicate that GABA modulates the expression of key proteins involved in inflammation and intestinal barrier integrity in T1DM mice.

Fig. 4.

Western blot assay for protein levels in colonic tissues of various groups of mice. (A) TLR4, MyD88 and NF-κB protein expression level of mice in each group. (B) Data analysis was conducted on the protein levels of TLR4, MyD88, and NF-κB. (C) Occludin and ZO-1 protein expression level of mice in each group. (D) Data analysis was conducted on the protein levels of Occludin and ZO-1 (*P < 0.05; ** P < 0.01; ***<0.001)

Assessment of neurological impairment in various groups of mice

To evaluate the effects of GABA on neurological function, various neurological tests were conducted. Compared to the control group, the active evasion time index of the T1DM group was significantly higher, while the active evasion number index was significantly lower. Compared to the T1DM group, the active evasion time index of the GABA group was significantly lower, while the active evasion number index was significantly higher (Fig. 5A-B). The results of HE staining of the brain tissue of mice in each group are shown in Fig. 5C. As seen under the light microscope: In control group, the number of cells in CA1 area of hippocampus was high, the arrangement was regular, and the distribution of cells was even. Compared with control group, in T1DM group, the nuclei of neurons were solidified and densely stained, the number of cells in CA1 area of hippocampus was decreased, and the size of many cells could be seen to decrease, and the arrangement of cells was irregular, and the distribution of cells was not even, and compared with T1DM group, the number of neurons in GABA group increased to some extent, and the arrangement of cells in CA1 area of hippocampus improved.Under the light microscope, it can be seen that: The number of apoptotic cells in the hippocampus of the brain tissue of mice in the control group was low; the number of apoptotic cells in the hippocampus of the brain tissue of mice in the T1DM group was significantly increased compared with that in the control group; the number of apoptotic cells in the hippocampus of the brain tissue of mice in the GABA group was relatively reduced compared with that in the T1DM group (Fig. 5D).

Fig. 5.

Effects of GABA treatment on neurologic impairment in mice. (A) Indicator of active escape time in mice. (B) Indicator of the number of active evasions in mice. (C) HE staining of mouse hippocampal tissues in various groups. (D) TUNEL staining of mouse hippocampal tissues in various groups (*P < 0.05; ** P < 0.01; ***<0.001)

ELISA to assess inflammation in the hippocampus of various groups of mice

Lastly, we evaluated hippocampal inflammation in T1DM mice and the effect of GABA treatment using ELISA. The results of ELISA experiments showed that the contents of LPS, IL-6, and TNF-α in the hippocampal tissues of mice in the T1DM group were significantly increased when compared with those in the control group; the contents of LPS, IL-6, and TNF-α in the hippocampal tissues of mice in the GABA group were significantly decreased when compared with those in the T1DM group (Fig. 6A-C). This indicates that GABA reduces hippocampal inflammation in T1DM mice.

Fig. 6.

Effects of GABA treatment on inflammatory factors in mice. (A-C) Determination of the concentration of inflammatory factors (A) LPS, (B) TNF-α, (C) IL-6 in mouse hippocampal tissue by ELISA assay

Discussion

In recent years, numerous studies have highlighted the diverse pharmacological properties of GABA, rendering it a potential therapeutic agent for the prevention and treatment of various diseases [17]. There are currently no research reports addressing whether GABA ameliorates neurofunctional impairment in T1DM through the modulation of gut flora. Our research, approached from the perspective of the “gut-brain axis,” represents the first identification of GABA’s capacity to ameliorate neurofunctional impairment in T1DM mice through the modulation of the “gut flora–LPS–TLR4–NF‑κB” signalling axis. Initially, GABA treatment significantly improved glucose tolerance in T1DM mice, indicating its potential therapeutic role in T1DM. Subsequently, GABA treatment induced alterations in the composition and abundance of the gut microbiota, affected the immune microenvironment as well as the inflammatory response in T1DM mice, suggesting that GABA exerts its effects by influencing the gut microbiota and subsequently impacting the inflammatory response. Finally, GABA ameliorated the histopathological damage in the hippocampus and improved inflammation in the hippocampus of T1DM mice, suggesting that GABA significantly ameliorated neurological impairment in T1DM mice.

In recent years, there has been increasing evidence that intestinal flora plays a crucial role in promoting human health, and the dysbiosis of the intestinal microbiota is implicated in the pathogenic mechanisms of various diseases [28–31]. Related study shows differences in bacterial composition between diabetic and non-diabetic rats [32]. In addition, a study demonstrated that the bacterial profiles of the gut flora of patients with T1DM and T2DM exhibited significant differences by 16s RNA sequencing [33]. Recent studies have shown an association between reduced microbiome diversity and T1DM [34]. The above evidence suggests that the development of T1DM is closely associated with disturbances in the gut flora. In order to further elucidate the impact of GABA on the gut microbiota of T1DM mice, we conducted high-throughput sequencing of 16sRNA genes to analyze the gut microbiota in each group of mice. Our findings demonstrate significant differences in the composition and microbial diversity of the gut microbiota between the T1DM group and the GABA group. Lactobacillus is a beneficial bacterial genus that plays a crucial role in maintaining intestinal health and balancing the gut microbiota. Several studies using mouse models have reported the beneficial effects of different Lactobacillus spp. in delaying the onset of T1DM or reducing its complications [35]. It is worth noting that our research has revealed a significant reduction in Lactobacillus in T1DM mice. However, when subjected to GABA treatment, there is an observed increase in the abundance of Lactobacillus in the gut microbiota of these mice. This finding hints at a potential constructive impact of GABA in promoting the proliferation and presence of Lactobacillus, which may play a role in reinstating gut health in T1DM.

LPS, a constituent of the cell wall of Gram-negative bacteria, is the most potent inducer of inflammation. Extensive research has confirmed that the dysbiosis of the gut microbiota in diabetic patients results in a significant increase in LPS levels [36, 37], and that long-term high levels of LPS can induce a chronic low-level inflammatory response in the body, which contributes to the development of diabetes mellitus and its complications. LPS activates immune cells by interacting with Toll-like receptors [38], modulation of the TLR4/MYD88/NF-κB signalling pathway improves LPS-induced neuroinflammation and cognitive function in mice [39]. In the present study, we found that GABA treatment significantly reduced the protein expression levels of TLR4, MYD88, and NF-κB in the colonic tissues of T1DM mice, suggesting that GABA may ameliorate neurological injury by modulating the intestinal flora to influence inflammation. In addition, this study found that GABA treatment significantly increased the protein levels of Occludin and ZO-1 in the colonic tissues of T1DM mice, suggesting that GABA helps maintain or repair the integrity of the intestinal barrier and reduces the penetration of harmful substances, thereby reducing chronic inflammation and immune system activation.

The TLR4-NF-κB signalling pathway can alter the proportion of immune cells and the release of inflammatory factors. Increasing the expression of inflammatory factors, such as TNF-α, IL-6, and LPS, which can directly damage hippocampal neurons and induce cognitive impairment. Our results showed that after treatment with GABA, the level of LPS in the GABA group was significantly lower than that in the T1DM group, and the level of inflammatory factors was reduced at the same time, and the neurological damage was reduced in the mice, which further suggests that GABA inhibits neurological damage in T1DM mice through the modulation of the “intestinal flora-LPS-TLR4-NF-κB” signalling axis.

This study has limitations. First, we used a relatively small sample size of mice, which may limit the generalizability of the results. Second, our focus was primarily on male mice, and it remains unclear whether similar results would be observed in female mice or across different age groups. Third, the duration of GABA treatment was short, which may be insufficient to fully understand the long-term effects of GABA on neurological impairment and gut microbiota modulation in T1DM. Four, Our study did not include direct correlation analyses between central nervous system biomarkers (e.g., hippocampal TLR4/MyD88/NF‑κB expression) and gut microbiota diversity or metabolite profiles. Finally, other potential pathways by which GABA might exert its effects, such as its direct neuroprotective effects, were not explored. These limitations suggest future research directions and objectives.

Conclusion

In conclusion, continuous GABA treatment in T1DM mice improved neurological function by regulating the gut flora–LPS–TLR4–NF‑κB axis. The results showed that GABA treatment is associated with improvements in neurological function that correlate with modulation of the gut-LPS–TLR4–NF‑κB axis. Specifically, GABA increased the diversity of intestinal flora, decreased the secretion of LPS, which further led to the reduction of the expression of “TLR4-NF-κB” axis proteins, and ultimately led to the attenuation of neurological impairments in mice. These findings provide valuable insights into the ability of GABA to alleviate T1DM-induced neurological impairments and underline the potential therapeutic role by affecting the intestinal flora.

Author contributions

Jiao Wang– research concept and design; Lihai Zhang, Xianhe Wang– collection and/or assembly of data; Jing Dong, Jiaxin Li– data analysis and interpretation; Jiao Wang, Lihai Zhang– writing the article; Xianhe Wang, Jing Dong, Jiaxin Li– critical revision of the article. All authors read and approved the final version of the manuscript.

Funding

The study was supported by National Specialty Construction Project of Excellent Research Team in the First Hospital Affiliated to Jiamusi University (GJ202301) and Heilongjiang Provincial Higher Educational Institutions’ Basic Research Operating Expenses Scientific Research Programs (2022-KYYWF-0631).

Data availability

All data generated or analyzed in this study are included in the present manuscript.

Declarations

Ethics approval and consent to participate

All animal experiments were approved by the Animal Care and Use Committee of the First Affiliated Hospital of Jiamusi University (approval number 202406).

Competing interests

The authors declare no competing interests.