The Animal Ethics Committee of the Animal Experiment Centre at Dalian Medical University (Approval No. AEE24088) granted approval for this research. C57BL/6 mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (Number of quality certification: 210726241102709171) and subsequently reared in the SPF-grade breeding facility of the SPF Animal Experiment Centre at Dalian Medical University. The mother mice were allowed to nurse their offspring, access water and consume food freely. A 12-h light/dark cycle was established to simulate the diurnal pattern, with the ambient temperature maintained at 23 °C ± 1 °C and the relative humidity sustained at 45% ± 5%.

Seven-day-old (P7) male mouse pups (weight: 3.2–5.5 g) were randomly divided into four groups (n = 6 in each group): Sham group, Sepsis group, Glycine group, and Placebo group. Another group of healthy P15 mice (sham2 group, n = 6) was included in the study as a baseline reference for the analysis of the gut microbiota. There was no significant difference in the initial body weight among the groups (P > 0.05). According to the previous method, an LPS-induced sepsis model was established (Chen et al., 2022; Mao et al., 2023). Mice were intraperitoneally injected with lipopolysaccharide (LPS, 10 mg/kg) once, while the sham operation group was given the same amount of normal saline (NS). Subsequently, for seven consecutive days, the glycine group was treated qd with a glycine solution (0.125 mg/g) via oral gavage, whereas the placebo group received an equivalent volume of NS (Rom et al., 2020) (Figure 1A). Following administration, the pups were returned to their mother for nursing. Their survival status and body weight gains were monitored daily (Figures 1B,C).

Mice were deeply anesthetized by inhaling isoflurane (3%–5%) in 100% oxygen, followed by cardiac perfusion with 4% paraformaldehyde (PFA) or normal saline solution. After hematoxylin-eosin (H&E) staining and immunohistochemistry (IHC), the tissue sections were preserved in 4% PFA for histological and molecular studies. Samples were stored at −80 °C for Enzyme-Linked Immunosorbent Assay (ELISA), Transcriptomics analysis and Western blotting. Intestinal fecal samples were aseptically collected, quickly transferred into sterile EP tubes, snap-frozen in liquid nitrogen, and preserved at −80 °C within 1 h of collection for further analyses.

In this study, fecal samples were collected at P8 and P15. P8 was selected to capture early alterations in the gut microbiota during the acute phase of sepsis. Glycine was administered daily from P8 to P14, and P15 was chosen to assess the cumulative effects of treatment after its completion. By P15, the gut microbiota typically reaches higher biomass and a more stable community structure, which yields higher quality and more reproducible 16S rRNA sequencing data.

After fixation in 4% PFA, brain and ileal tissues were embedded in paraffin. Sections (4 μm thick) were cut and stained for histological analysis. Pathological changes in ileal tissue sections were examined under light microscopy. Histopathological evaluation was performed using a standardized histological scoring system. Five distinct parameters were assessed: (1) severity of epithelial injury; (2) depth of ulceration; (3) degree of submucosal edema; (4) extent and depth of lymphomonocytic infiltration; (5) intensity and pattern of neutrophilic and eosinophilic infiltration (Tang et al., 2019). Ileal tissue sections were immunostained with primary antibodies against occludin (1:1000, Abcam, United Kingdom), C5aR1 (1:1000, Abcam, United Kingdom). Brain sections were stained with antibodies against MBP (1:2000, Abcam, United Kingdom), Iba-1 (1:1000, Abcam, United Kingdom), and C5aR1 (1:1000, Abcam, United Kingdom). For each mouse, three white matter regions were randomly selected, and the percentage of immunopositive area was quantified using Fiji (version 2.3.0; https://fiji.sc/).

Due to animal mortality, fecal samples from five mice per group were used to ensure equal sample sizes across all groups. Total fecal microbial DNA was obtained through the Fecal Genome DNA Extraction Kit (AU46111-96, BioTeke, China) according to the manufacturer’s instruction manual. The DNA was quantified by Qubit (Invitrogen, United States). Total DNA was amplified by PCR using the universal primer 341F/805R (341F: 5′-CCTACGGGNGGCWGCAG-3’; 805R: 5′-GACTACHVGGGTATCTAATCC-3′). The PCR amplification conditions were pre-denaturation at 98 °C for 30 s, denaturation at 98 °C for 10 s, annealing at 54 °C for 30 s, extension at 72 °C for 45 s and 32 cycles. The final extension was at 72 °C for 10 min. The PCR product was purified using AMPure XP Beads (Beckman Coulter Genomics, Danvers, MA, United States) and quantified using Qubit (Invitrogen, United States). Qualified PCR products were evaluated using an Agilent 2100 Bioanalyzer (Agilent, United States) and Illumina library quantitative kits (Kapa Biosciences, Woburn, MA, United States), which were further pooled together and sequenced on an Illumina NovaSeq 6000 (PE250).

Sequencing primer were removed from de-multiplexed raw sequences using cutadapt (v1.9). Paired-end reads were merged using FLASH (v1.2.8). The low-quality reads (quality scores < 20), short reads (<100 bp), and reads containing more than 5% “N” records were trimmed by using the sliding-window algorithm method in fqtrim (v0.94). Quality filtering was performed to obtain high-quality clean tags according to fqtrim. Chimeric sequences were filtered using Vsearch software (v2.3.4). DADA2 was applied for denoising and generating amplicon sequence variants (ASVs). The sequence alignment of species annotation was performed by QIIME2 plugin feature-classifier, and the alignment database was SILVA and NT-16S. Alpha and beta diversities were calculated using QIIME2. Relative abundance was used in bacteria taxonomy. The wilcox test was used to identify the differentially abundant genus, and significances were declared at P < 0.05. LDA effect size (LEfSe, LDA≥3.0, P value < 0.05) was performed using nsegata-lefse. Other diagrams were implemented using the R package (v3.4.4).

The concentrations of IL-1β, IL-6, IL-10, and TGF-β in ileal and brain tissues were quantified using ELISA kits according to the manufacturer’s instructions.

RNA was extracted from brain tissues of LPS-treated mice (n = 6 biological replicates per group), and transcriptome sequencing was performed by Shanghai Biotree Biotechnology Co., Ltd. (http://www.biotree.com.cn/). Following mRNA enrichment, RNA concentration was measured by Nanodrop (OD260/280 and OD260/230 ratios), RNA integrity was assessed using the Agilent 5400, and double-stranded cDNA fragments of 250–300 bp were size-selected with AMPure XP beads. After PCR amplification and purification, strand-specific libraries were constructed and sequenced on an Illumina Novaseq platform with PE150 strategy, generating 150 bp paired-end reads. Raw data were filtered to obtain clean data for subsequent bioinformatics analysis, and data quality was evaluated by FastQC (v0.23.4). Reads were mapped to gene exons for genome annotation using Hisat2 (v2.1), and genes with fewer than 15 reads per sample were excluded from further analysis. Differentially expressed transcripts (DETs) were identified using the DESeq2 package (v1.38.3) in R software. Functional enrichment analysis was performed with the clusterProfiler package (v4.6.2). Genes and gene sets with false discovery rate (FDR)-adjusted p-values < 0.05 were considered statistically significant, determined by multivariate analysis of variance (MANOVA). FDR correction was conducted using the Benjamini–Hochberg method after edgeR and MANOVA tests.

Western blot analysis was performed according to established protocols (Scandura et al., 2022). Tissues stored at −80 °C were ground in liquid nitrogen and homogenized in RIPA lysis buffer supplemented with protease inhibitors. The homogenates were centrifuged at 12,000×g for 15 min at 4 °C, and the supernatants were collected. Total protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific) following the manufacturer’s instructions. Equal amounts of protein were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (Green et al., 2019) and transferred onto polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% skim milk in TBST for 1 h at room temperature, followed by overnight incubation at 4 °C with primary antibodies against MBP (1:2000, Abcam, United Kingdom), Iba-1 (1:500, Abcam, United Kingdom), occludin (1:500, Abcam, United Kingdom), and C5aR1 (1:500, Abcam, United Kingdom). After three washes with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG secondary antibody (1:10000, Proteintech, China) for 1 h at room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL) detection and quantified with ImageJ software (v2.3.0, NIH, United States). Expression levels of target proteins were normalized to GAPDH as an internal loading control.

All data are presented as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism (version 9.5.1, United States). The t-test or one-way analysis of variance (ANOVA) was used for comparison between groups. P < 0.05 was considered statistically significant.

H&E staining revealed that brains from the sham group exhibited well-preserved white matter architecture, characterized by tightly packed, regularly arranged myelinated fibers and intact oligodendroglial nuclei. In contrast, the sepsis group displayed severe white matter pathology, including disorganized fiber tracts, axonal swelling, vacuolar degeneration, oligodendroglial pyknosis, and loss of structural integrity. Glycine treatment significantly attenuated these degenerative changes in septic mice, preserving white matter organization and markedly reducing edema in axons and surrounding tissue (Figure 1D).

IHC results revealed that in a mouse model of sepsis, the expression level of MBP in the cerebral white matter region was significantly decreased, with its positive staining intensity notably weakened, which suggests myelin sheath loss or damage. After glycine treatment, the expression level of MBP was restored (p < 0.05), indicating that glycine may have a certain repair effect on the myelin sheath (Figures 1E,G,I,K).

The IHC analysis of Iba-1 showed significant variations among the groups. The paraventricular white matter in the sepsis group had a considerably higher rate of Iba-1-positive areas than the placebo group. The glycine treatment significantly decreased microglial activations, as indicated by a marked reduction in the Iba-1-positive area rate (p < 0.05). The findings indicate that glycine may have neuroprotective effects in septic mice by modulating microglial activation in white matter regions (Figures 1F,H,J,L).

Compared to the sham group, the levels of IL-6, IL-1β, IL-10 and TGF-β in the sepsis group and the placebo group were also significantly elevated (p < 0.01). No significant difference was observed between the sepsis group and the placebo group (p > 0.05). After glycine treatment, the levels of IL-6 and IL-1β in the brain tissue were significantly reduced compared to the placebo group (p < 0.01). Compared to the placebo group, TGF-β levels were also significantly reduced (p < 0.05), while IL-10 levels decreased but remained higher than those in the sham group (p < 0.05). These findings suggest that glycine regulates inflammatory factors in brain tissue. Glycine treatment enables brain tissue to maintain a certain level of anti-inflammatory compensatory capacity, thereby preserving inflammatory homeostasis (Figures 1M–P).

We administered an orally gavaged glycine solution to septic mice for 7 days consecutively to investigate its effects on gut microbiota, followed by 16S rDNA sequencing of fecal samples (Figure 2). Figure 2A illustrates the Venn diagram depicting ASV distribution. The abundance and diversity indices of the gut microbiota significantly decreased in the sepsis group. To comprehensively assess the α-diversity of the gut microbiota in mice, we evaluated both the Chao1 and Shannon indices. Notably, following glycine intervention, septic mice exhibited improved microbial abundance and diversity compared to their pre-intervention state (Figures 2B,C). PCoA analysis of β-diversity revealed significant differences in the structure and community composition of gut microbiota across the groups (Figure 2D). Figures 2E–H depict the distribution of the five predominant microbial taxa at both the phylum and genus levels. Stacked bar charts at the phylum and genus levels illustrate notable differences in microbial composition across the groups (Figures 2G,H). Furthermore, these data are represented as heatmaps to facilitate a more detailed analysis (Figures 2I,J). At the phylum level, Bacteroidota predominated in the sham1 group; however, Bacteroidota decreased while Proteobacteria increased in both the sepsis and placebo groups. The glycine treatment group of the sham2 group demonstrated a sustained develop in Bacteroidota and a decrease in Proteobacteria compared to the placebo group. At the genus level, Rodentibacter and Clostridium exhibited an increase in the sepsis group relative to the sham1 group; conversely, Bacteroides, Parabacteroides, Lactobacillus, and Eisenbergiella showed a growing pattern in the glycine group compared to both the sham2 and placebo groups, whereas Escherichia-Shigella and Rodentibacter displayed a decline. These modifications signify a notable dysbiosis of intestinal microbiota in septic mice, with glycine able to restore beneficial microbiota and suppress harmful microbiota. LEfSe analysis revealed species exhibiting significant abundance differences between glycine-treated and untreated groups. After glycine treatment, in comparison to the untreated group, the abundances of Bacteroidales, Parabacteroides, and Lactobacillus were significantly increased. Conversely, in the untreated group, the levels of Firmicutes, Enterobacteriaceae, and Escherichia - Shigella were notably elevated (Figure 2K). The findings indicate that glycine can effectively improve gut microbiota dysbiosis in septic mice.

We conducted a detailed analysis of the 30 most abundant species to identify potential biomarkers. In the sepsis group, there was a significant increase in Alistipes, Eisenbergiella, and Colidextribacter. The administration of glycine resulted in a significant elevation of Bacteroides levels (Figure 2L). KEGG pathway analysis revealed that the MAPK signaling pathway, glycine/serine/threonine metabolism, and cysteine/methionine metabolism were significantly upregulated in the sepsis group compared to the sham group (Figure 2M).

Preliminary observations showed that septic mice began to display clinical signs of distress, including lethargy and tremors, within 12 h after LPS administration. Hematochezia occurs within 24–48 h after LPS modeling. Histopathological analysis indicates that septic mice exhibit severe intestinal tissue damage, characterized by mucosal bleeding, infiltration of inflammatory cells, and loss of epithelial cells. Although glycine treatment significantly reduced pathological changes, mucosal loss remained evident in the LPS-induced ileum. The sepsis group exhibited extensive mucosal erosion within the inflammatory lesions, along with dense infiltration of inflammatory cells. Glycine significantly enhanced the histological injury score induced by LPS (p < 0.05) (Figures 3A,D).

In the ileal tissue of LPS-induced septic mice, the expression of occludin protein in the intestinal mucosa was markedly diminished compared to the sham group, indicating impairment of the intestinal mucosal barrier. The positive localization of occludin protein transferred from the cell membrane to the cytoplasm, indicating potential malfunctions of the occludin protein. The expression of the positive area in the glycine group markedly increased, suggesting that glycine treatment effectively restored the impaired intestinal mucosa (p < 0.05) (Figures 3B,C,E,F).

After LPS induction, the concentrations of IL-6, IL-1β, IL-10 and TGF-β in the ileal tissues of the sepsis group and the placebo group were significantly increased compared to the sham group (p < 0.01), with no difference observed between the sepsis group and the placebo group (p > 0.05). 7 days after glycine treatment, the levels of IL-6 and IL-1β in the ileal tissue were significantly reduced compared to the placebo group (p < 0.01), indicating a marked attenuation of the pro-inflammatory response. Comparing to the placebo group, the levels of TGF-β and IL-10 decreased (p < 0.05), with both cytokines showing a synchronous trend. These findings suggest that glycine attenuates both hyperactive pro-inflammatory signaling and exaggerated anti-inflammatory compensation in the ileum, facilitating a balanced resolution of inflammation (Figures 3G–J).

Transcriptome analysis of brain tissues from WMI mice and healthy controls identified differentially expressed genes (DEGs). Principal component analysis (PCA, Figure 4A) shows a separation between the two groups in the transcriptome space, suggesting that they have different global gene expression profiles. The hierarchical clustering heatmap (Figure 4B) displays differentially expressed genes (DEGs). The red and blue clusters represent high and low expression in the sepsis group and the placebo group respectively, demonstrating the substantial transcriptional changes associated with WMI. The volcano map (Figure 4C) shows all genes, with red dots indicating considerable upregulation, confirming the major DEGs contributing to individual segregation. The top ten differentially expressed genes in this study include Gm29879, Lcn2, Cxcl10, Zbp1, Lbhd2, C5aR1, Cxcl10, Lbhd2, Cxcl9, and Fpr1, all of which might play a role in the pathophysiology of sepsis-induced WMI.

GO and KEGG annotations allow for further investigation into the complex biological behaviors of genes. GO annotation describes the molecular functions (Molecular Function, MF), cellular components (Cellular Component, CC), and involved biological processes (Biological Process, BP) of genes (Figure 4D). KEGG annotation reveals that genes are primarily involved in the immune system, immune diseases, infectious diseases, signal transduction, signal molecules and interactions, as well as transport and catabolism (Figure 4E). GO Enrichment reflects that DEGs were enriched in biological processes (BP) such as innate immune response and defense response to bacterium (Figure 4F), reflecting robust immune activation in septic brain injury. Molecular function (MF) and cellular component (CC) enrichments supported dysregulated immune signaling (Figures 4F,G).

Figure 4H shows the KEGG gene set enrichment analysis (GSEA). Genes in the gene set are mainly concentrated near the peak of the enrichment score (ES) curve, indicating that these genes exhibit strong enrichment in gene regions highly relevant to the pathway. This suggests that the concentrated gene set has a positive correlation with the KEGG pathway, corresponding to an upregulated pathway.

According to the results of KEGG pathway enrichment, the Complement and coagulation cascades pathway was significantly enriched (Figure 4G, p < 0.05), a central mediator of septic inflammation and coagulopathy. Pathway mapping (Figure 4I) positioned C5aR1 at a key node, where its upregulation likely amplifies complement activation, aggravating inflammatory cascades and brain injury.

The expression of C5aR1 in intestinal mucosal epithelial cells of mice in the sepsis group was significantly upregulated (Figures 5A,C,E,G). The positive signals were mainly located in the cytoplasm and cell membrane of intestinal villus epithelial cells. The total positive area was significantly larger than that in the sham group. Glycine intervention significantly decreased C5aR1 immunoreactivity, resulting in a smaller positively stained region compared to the sepsis group (P < 0.05; Figures 5A,E). Western blot analysis revealed that the C5aR1 protein expression in ileum tissue increased significantly in the sepsis group and placebo group (P < 0.01), while in mice with glycine treatment, it decreased significantly (P < 0.05; Figures 5C,G).

Mice in the sepsis group had significantly higher levels of C5aR1 in their brain tissues, according to IHC analysis (Figures 5B,D,F,H). Both the cytoplasm and the cell membrane showed clear localization of the positive signals, which were mainly found in the neuroglial cells in the brain’s white matter region. After intervention with glycine, the positive expression of C5aR1 in the neuroglial cells of the brain white matter region was significantly weakened, the signal intensity in the cytoplasm and cell membrane decreased, and the positively stained area was significantly smaller than that in the sepsis group (P < 0.05; Figures 5B,F). These findings were corroborated by western blot analysis, which showed a significant increase in C5aR1 protein levels in the brain tissue of the sepsis group compared to the sham group (P < 0.01), and a significant reduction following glycine administration (P < 0.05; Figures 5D,H).