Changes of intestinal microbiome and its relationship with painful diabetic neuropathy in rats

Abstract

Objective: To analyze the gut bacterial microbiome in rats with painful diabetic neuropathy (PDN) compared to normal rats.

Methods: Type 2 diabetes was induced in rats via a high-fat and high-sugar diet combined with a low dose of streptozotocin. Glucose metabolism and insulin sensitivity were evaluated using intraperitoneal glucose tolerance tests and insulin tolerance tests. The progression of peripheral neuropathy was assessed using the mechanical withdrawal threshold and thermal withdrawal latency. Histopathological analysis of rat colon tissues was performed using hematoxylin-eosin staining to observe morphological changes. The expression levels of pro-inflammatory cytokines TNF-α and IL-1β in spinal cord tissues were measured using enzyme-linked immunosorbent assay (ELISA). Fecal samples were then collected for metagenomic sequencing and analysis.

Result: Behavioral tests revealed reduced mechanical withdrawal threshold and thermal withdrawal latency in PDN rats. Histological analysis showed significant colonic mucosal damage and inflammatory cell infiltration, suggesting impaired intestinal barrier function. Elevated TNF-α and IL-1β levels in spinal cord tissues further highlight peripheral inflammation's role in PDN. Sequencing analysis revealed significant differences in gut microbiota composition between PDN and control rats, with altered Bacillota/Bacteroidota ratios and increased Lactobacillus abundance. Functional annotation analysis, based on the KEGG, EggNOG, and CAZy databases, indicated significant enrichment of metabolic pathways related to carbohydrate and amino acid metabolism, energy metabolism, and cell structure biogenesis in PDN rats. Cluster analysis identified higher functional clustering in Metabolism and Genetic Information Processing pathways in PDN rats.

Conclusion: This study demonstrates that PDN leads to altered gut microbiota composition, disrupted metabolic pathways, and increased inflammation, contributing to the pathological progression of diabetic neuropathy. This study provides new insights into the interplay between gut microbiota and diabetic neuropathy, offering potential avenues for therapeutic interventions targeting microbiome and metabolism.

Keywords: Collinsella; Fecal metagenomic sequencing; Gut microbiota; Lactobacillus; Neuropathic pain; Painful diabetic neuropathy.

Fig. 1

Changes in body weight, blood glucose, intraperitoneal glucose tolerance test (IPGTT), and insulin tolerance test (ITT) results in rats. A The trend of body weight in Group C (n = 5) and Group PDN (n = 5) over 15 weeks (measurements taken weekly); B The trend of blood glucose levels in Group C (n = 5) and Group PDN (n = 5) over 15 weeks (measurements taken weekly); C Statistics of 24-h food intake after STZ injection (days 1, 4, 7 post-injection, n = 5 per group); D Statistics of 24-h water intake after STZ injection (days 1, 4, 7 post-injection, n = 5 per group); E IPGTT results at the 15 th week after STZ injection (n = 5 per group).; F ITT results at the 15 th week after STZ injection (n = 5 per group). Data are presented as mean ± SDs. Compared with the group C, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 2

Evaluation of peripheral neuropathy in rats. A The trend of MWT in Group C (n = 5) and Group PDN (n = 5) over 15 weeks (measurements taken weekly from week 1 to week 15); B The trend of TWL in Group C (n = 10) and Group PDN (n = 5) over 15 weeks (measurements taken weekly from week 1 to week 15). Data are presented as mean ± SDs. Compared with the group PDN, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 3

Pathological Changes and Detection of Inflammatory Factors. A The HE staining of colonic tissue (x200). Representative images from Group C and Group PDN are shown, Scale bar: 100um; B The content of pro-inflammatory cytokines TNF-α and IL-1β in spinal cord tissue. Values were presented as mean ± SDs. Compared to the group PDN, * P < 0.05, **** P < 0.0001

Fig. 4

Analysis of intestinal flora diversity in group C and group PDN. A-E Bacterial alpha diversity analysis. F-G Bacterial beta diversity analysis. Group C, n = 5; Group PDN, n = 5)

Fig. 5

Relative abundance of species statistical figure (group). A Clustering of intestinal flora OTUs in each group of rats Venn diagram. B Statistical analysis of the Bacillota/Bacteroidota ratio in the group C and the group PDN. C Statistical analysis of bacteria at the genus level that may be associated with the pathogenesis of PDN in the group C and the group PDN. D Histogram of relative abundance of species at the phylum level. E Histogram of relative abundance of species at the genus level

Fig. 6

Analysis of species differences across groups. A Histogram of the distribution of LDA values for differing species. B Evolutionary branching diagrams for divergent species, circles radiating from inside to outside represent taxonomic levels from phylum to species

Fig. 7

Histogram of relative abundance of functional annotations on level 1.The results of KEGG, eggNOG, and CAZy are shown in order. A: Histogram of relative abundance of functional annotations in KEGG; B: Histogram of relative abundance of functional annotations in eggNOG; C: Histogram of relative abundance of functional annotations in CAZy. The vertical axis indicates the relative proportion of annotations to a functional class; the horizontal axis indicates the sample name; the functional class corresponding to each colour block is shown in the legend on the right

Fig. 8

Functional abundance clustering heat map. A Functional abundance clustering heat map in KEGG (level 1), B is for level 2, C is for level 3; D Functional abundance clustering heat map in eggnog (level 1), E is for level 2; F: Functional abundance clustering heat map in CAZy. Sample information is shown horizontally; functional annotation information is shown vertically; the clustering tree on the left side of the graph is the functional clustering tree; the values corresponding to the heat map in the middle are the Z-values obtained from normalising the relative abundance of the functions in each row