Role of GDNF, GFRα1 and GFAP in a Bifidobacterium-Intervention Induced Mouse Model of Intestinal Neuronal Dysplasia

Abstract

Objective: To investigate the effects of glial cell-derived neurotrophic factor (GDNF), GDNF family receptor alpha 1 (GFRα1), and glial fibrillary acidic protein (GFAP) on colonic motility in a mouse model of intestinal neuronal dysplasia by intervention with Bifidobacterium and to explore the influence of Bifidobacterium on enteric glial cells (EGCs).

Methods: Western blotting and qRT-PCR were employed to detect the expression of GFRα1 and GFAP in colonic tissues of mice with or without Tlx2 mutations, and ELISA was used to detect the expression of GDNF in serum. IHC was used to detect the appearance of the ganglion cells. Subsequently, Tlx2 homozygous mutant (Tlx2^-/-) mice were treated with Bifidobacterium. Colonic motility was measured before and after intervention by measuring the glass bead expelling time. The variations in abdominal circumference and GDNF, GFRα1, and GFAP expression were measured. In addition, 16SrRNA gene sequencing was performed to detect the abundance of the intestinal microbiota.

Results: The mRNA and protein expression of GFRα1 and GFAP was decreased in the colonic tissues of Tlx2^-/- mice and GDNF expression was decreased in serum compared with Tlx2^+/- and WT mice. After confirming the colonization of Bifidobacterium by 16S rRNA gene sequencing, the expelling time and abdominal distension were ameliorated, and the expression of GFAP, GDNF, and GFRα1 was increased.

Conclusions: The expression of GDNF, GFRα1, and GFAP is associated with colonic motility. The altered expression of EGC-related factors suggested that Bifidobacterium may be involved in the EGC activation process. The amelioration of IND symptoms after intervention with Bifidobacterium prompted the elicitation of adjuvant therapy.

Keywords: Bifidobacterium; GDNF; gut microbiota; homozygous mutant mice of Tlx2; intestinal neuronal dysplasia (IND).

Figure 1

Example genotyping and decreased expressions of GDNF, GFRα1 and GFAP in Tlx2^−/− mice. (A) Genotyping; the wild-type allele is 568 bp, and the mutant allele is 395 bp. (B,C) Western blot indicated that the protein expressions of GFAP and GFRα1 in colonic tissues of Tlx2^−/− mice were decreased (n = 7 for each group). (D–F) The mRNA expressions of GDNF, GFRα and GFAP were decreased in colon tissues of Tlx2^−/− mice (n = 9 for each group). (G) The expression level of GDNF in serum of Tlx2^−/− mice was lower than that in Tlx2^+/− mice and WT mice (n = 8 for each group). (H) IOD of positive staining indicated that GFAP was significantly downregulated in the colonic tissues of Tlx2^−/− mice (n = 6 for each group). (I) Representative western blot analysis and detail bands of other mice were attached in Supplementary Figure 1. (J) GFAP was mainly expressed in EGCs of the colonic myenteric plexus (ns P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; GAPDH, glyceraldehyde-3-phosphate; Scale bar: 100 μm).

Figure 2

Measurement of the colonic motility and abdominal circumference of mice in groups BB, NM and NS. (A) Hierarchical clustering heatmap of the top 30 bacterial species of each group. (B) The relative abundance of Bifidobacterium among groups BB, NM and NS. (C,F) T After Bifidobacterium intervention, the expelling time in BB group was shortened compared to groups NM and NS. (D,F,G) The abdominal circumference after Bifidobacterium intervention among the three groups. (E,F) Differences in abdominal circumference before and after Bifidobacterium intervention in group BB was smaller than the other groups. (ns P > 0.05; *P < 0.05; **P < 0.01).

Figure 3

Expression of GDNF, GFRα1 and GFAP was increased in group BB. (A–C) The protein expression of GFAP and GFRα in the colonic tissues of group BB was higher than those in the other two groups (n = 8 for each group), detail bands of other mice can be found at Supplementary Figure 1. (D–F) The mRNA expressions of GDNF, GFRα and GFAP in colon tissues of group BB were increased (n = 7 for each group). (G) The serum levels of GDNF in group BB increased compared to those in groups NM and NS after intervention (n = 7 for each group). (H) The IOD of positive staining in group BB was increased compared to that of the NM and NS groups (n = 6 for each group). (I) Representative results of IHC staining of GFAP in different groups under 20 × microscope (ns P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; GAPDH, glyceraldehyde-3-phosphate; Scale bar: 100 μm).