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. 2024 Jun 4;21(1):147.
doi: 10.1186/s12974-024-03116-5.

Prevotella copri transplantation promotes neurorehabilitation in a mouse model of traumatic brain injury

Nina Gu  1 , Jin Yan  1 , Wei Tang  1 , Zhaosi Zhang  1 , Lin Wang  1   2 , Zhao Li  1   3 , Yingwen Wang  1 , Yajun Zhu  1 , Shuang Tang  1   4 , Jianjun Zhong  1 , Chongjie Cheng  5 , Xiaochuan Sun  6 , Zhijian Huang  7
Affiliations

Prevotella copri transplantation promotes neurorehabilitation in a mouse model of traumatic brain injury

Nina Gu et al. J Neuroinflammation. .

Abstract

Background: The gut microbiota plays a critical role in regulating brain function through the microbiome-gut-brain axis (MGBA). Dysbiosis of the gut microbiota is associated with neurological impairment in Traumatic brain injury (TBI) patients. Our previous study found that TBI results in a decrease in the abundance of Prevotella copri (P. copri). P. copri has been shown to have antioxidant effects in various diseases. Meanwhile, guanosine (GUO) is a metabolite of intestinal microbiota that can alleviate oxidative stress after TBI by activating the PI3K/Akt pathway. In this study, we investigated the effect of P. copri transplantation on TBI and its relationship with GUO-PI3K/Akt pathway.

Methods: In this study, a controlled cortical impact (CCI) model was used to induce TBI in adult male C57BL/6J mice. Subsequently, P. copri was transplanted by intragastric gavage for 7 consecutive days. To investigate the effect of the GUO-PI3K/Akt pathway in P. copri transplantation therapy, guanosine (GUO) was administered 2 h after TBI for 7 consecutive days, and PI3K inhibitor (LY294002) was administered 30 min before TBI. Various techniques were used to assess the effects of these interventions, including quantitative PCR, neurological behavior tests, metabolite analysis, ELISA, Western blot analysis, immunofluorescence, Evans blue assays, transmission electron microscopy, FITC-dextran permeability assay, gastrointestinal transit assessment, and 16 S rDNA sequencing.

Results: P. copri abundance was significantly reduced after TBI. P. copri transplantation alleviated motor and cognitive deficits tested by the NSS, Morris's water maze and open field test. P. copri transplantation attenuated oxidative stress and blood-brain barrier damage and reduced neuronal apoptosis after TBI. In addition, P. copri transplantation resulted in the reshaping of the intestinal flora, improved gastrointestinal motility and intestinal permeability. Metabolomics and ELISA analysis revealed a significant increase in GUO levels in feces, serum and injured brain after P. copri transplantation. Furthermore, the expression of p-PI3K and p-Akt was found to be increased after P. copri transplantation and GUO treatment. Notably, PI3K inhibitor LY294002 treatment attenuated the observed improvements.

Conclusions: We demonstrate for the first time that P. copri transplantation can improve GI functions and alter gut microbiota dysbiosis after TBI. Additionally, P. copri transplantation can ameliorate neurological deficits, possibly via the GUO-PI3K/Akt signaling pathway after TBI.

Keywords: Prevotella copri; GUO-PI3K/Akt pathway; Guanosine; Gut microbiota; Neurorehabilitation; Traumatic brain injury.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Schematic diagram of the different experimental protocols and setups used in this study. P. copri, Prevotella copri; CCI, controlled cortical impact; TBI, traumatic brain injury; WB, western blot; IF, immunofluorescence; GUO, guanosine; BBB, blood-brain barrier; TME, transmission electron microscopy
Fig. 2
Fig. 2
P. copri treatment improved neurological function in TBI mice. A. Time course of P. copri abundance after TBI by qPCR. B-C. Quantitative analysis of short-term neurological function by NSS scores (B) and wire grip scores (C). *, p < 0.05 vs. sham; #, p < 0.05 vs. TBI. n = 8 per group. D. Representative images of the movement path of mice in the open field test 14 days after TBI. E-I. Quantitative analysis of central time (E), periphery time (F), total distance(G), time immobile (H) and mean speed (I) travelled by mice in the open field test. *, p < 0.05 vs. Sham; #, p < 0.05 vs. TBI, n = 8 per group. J. Representative images of the swim path of mice in the Morris water maze. K-N. Quantitative analysis of the latency in the learning test (K), the time in the target quadrant (L), the latency (M) and the average swimming speed (N) in the probe trial. *, p < 0.05 vs. Sham; #, p < 0.05 vs. TBI, n = 8 per group. Two-way repeated-measures ANOVA with Tukey’s post hoc multiple-comparisons test was used to analyze continuously measured data. One-way analysis of variance (ANOVA) was used to compare means of different groups followed by a Tukey post hoc multiple-comparisons test
Fig. 3
Fig. 3
P. copri treatment improved body weight, food intake, intestinal permeability and intestinal motility of TBI mice. A-B. Changes of body weight (A) and food intake (B) over time. *, p < 0.05 vs. sham; #, p < 0.05 vs. TBI. n = 8 per group. C-E. Effect of P. copri on gastrointestinal motility. Representative images after administration of barium (C), the filling of the intestinal tract was measured by radiological methods (D), the size of the intestinal tract was determined using ImageJ (E). *, p < 0.05 vs. Sham; #, p < 0.05 vs. TBI. n = 4 per group. F. Intestinal permeability by measuring FITC intensity in serum after oral gavage of FITC-dextran. *, p < 0.05 vs. sham; #, p < 0.05 vs. TBI. n = 8 per group. G-I. Effect of P. copri treatment on tight junction proteins expression. Representative images of immunofluorescence staining for Occludin (green) and ZO-1 (red), nuclei were stained with DAPI (blue) (G). Scale bar = 100 μm. Quantitative analysis of relative fluorescence intensity of Occludin (H) and ZO-1 (I) in different groups. *, p < 0.05 vs. Sham; #, p < 0.05 vs. TBI, n = 4 per group. Two-way repeated-measures ANOVA with Tukey’s post hoc multiple-comparisons test was used to analyze continuously measured data. One-way ANOVA was used to compare means of different groups followed by a Tukey post hoc multiple-comparisons test
Fig. 4
Fig. 4
P. copri treatment reshaped the gut microbiota of TBI mice. A-C. Bar plot analysis of gut microbiota relative abundance of bacterial phylum (A), family (B) and genus (C) in the sham, TBI, TBI + P. copri groups. Different colours represent different phyla, family and genus. D-F. Significant differences in the abundance of phyla (D) in each group, and significant differences in the families (E) and genus (F) with high abundance (top 15) (n = 6 per group). Data are presented as mean ± SD. *, p < 0:05. **, p < 0:01. ***, p < 0:001. The Kruskal‒Wallis H-test with false discovery rate (FDR), multiple comparisons correction and Tukey’s post hoc comparisons test were calculated at the phylum, family, and genus levels among each group
Fig. 5
Fig. 5
P. copri treatment caused the changes in serum metabolomics in TBI mice. A. Hierarchical clustering analysis of serum metabolite biomarkers. The abscissa is the clustering of samples and the ordinate is the clustering of differential metabolites. The coloured blocks in different positions represent the content of the metabolites in the corresponding parts. Red means that the substance is highly expressed in the group where it is located, while blue means that the sense is poorly represented in the group where it is located. B. Venn diagram of differential metabolites in serum. C-D. Matchstick analysis of serum metabolite biomarkers. TBI + P. copri group vs. sham group (C), TBI + P. copri group vs. TBI group (D). Red nodes represent significantly upregulated metabolites (top 25), blue nodes represent significantly downregulated metabolites (top 25) (VIP > 1 and P < 0.05)
Fig. 6
Fig. 6
P. copri treatment increased the presence of GUO in the feces, serum and brain tissue of TBI mice. A-C. The changes of GUO level in feces (A), serum (B) and brain (C) after TBI were detected by ELISA. *, p < 0.05 vs. sham; #, p < 0.05 vs. day 1. n = 6 per group. D-F. Changes in GUO level in feces (D), serum (E) and brain (F) after
Fig. 7
Fig. 7
P. copri treatment attenuated oxidative stress after TBI. A-E. The changes of oxidative stress markers in the brain lesion area after TBI were detected by spectrophotometer. A. SOD activity. B. CAT activity. C. ROS level. D. GSSG level. E. GSH/GSSG ratio. *, p < 0.05 vs. sham; #, p < 0.05 vs. TBI. n = 6 per group. One-way ANOVA was used to compare the means of different groups followed by a Tukey post hoc multiple-comparisons test
Fig. 8
Fig. 8
P. copri treatment attenuated BBB disruption after TBI. A. Representative horizontal and coronal images of brain slices after EB injection. B. Quantitative analysis of EB leakage intensity. *p, < 0.05 vs. sham; #, p < 0.05 vs. TBI, n = 6 per group. C. Red fluorescence of EB was observed by fluorescence microscopy in different groups. Cell nuclei were stained with DAPI (blue). Scale bar = 100 μm. n = 4 per group. D. Brain sample with a schematic illustration showing the area (indicated by the black box) that was used for the immunofluorescence analysis. E. Representative images of double immunofluorescence staining for Occludin and CD31, nuclei were stained with DAPI (blue). Scale bar = 100 μm. F. Quantitative analysis of relative Occludin fluorescence intensity in different groups. *, p < 0.05 vs. sham; #, p < 0.05 vs. TBI, n = 4 per group. G. Representative Western blot bands of ZO-1, Occludin and β-actin at the lesion sites after TBI. H-I. Quantitative analysis of relative ZO-1 (H), Occludin (I) density. *, p < 0.05 vs. sham; #, p < 0.05 vs. TBI, n = 6 per group. J. Transmission electron microscopy showed the ultrastructure of tight junctions. Scale bar = 500 nm. EC: Endothelial cells. TJ: Tight junction. K. Quantitative data show a decreased number of organized tight junctions. *, p < 0.05 vs. sham; #, p < 0.05 vs. TBI, n = 4 per group. One-way ANOVA was used to compare the means of different groups followed by a Tukey post hoc multiple-comparisons test
Fig. 9
Fig. 9
P. copri treatment attenuated neuronal apoptotic death and expression of apoptosis-related proteins after TBI. A. Representative images of dead cells (TUNEL, red) and neurons (NeuN, green) surrounding the lesion sites. Scale bar = 100 μm. B. Quantitative analysis of TUNEL-positive neurons surrounding lesion sites after TBI. *, p < 0.05 vs. sham; #, p < 0.05 vs. TBI, n = 4 per group. C. Representative Western blot bands of Bcl-2, Bax and β-actin at the lesion sites after TBI. D-E. Quantitative analysis of Bcl-2 (D) and Bax (E) density at the lesion sites after TBI. *, p < 0.05 vs. sham; #, p < 0.05 vs. TBI, n = 6 per group. One-way ANOVA was used to compare the means of different groups followed by a Tukey post hoc multiple-comparisons test
Fig. 10
Fig. 10
The GUO-PI3K/Akt axis was involved in the neuroprotective effects of P. copri. A. Representative Western blot bands of total PI3K, p-PI3K, total Akt, p-Akt and β-Actin at the lesion sites after TBI. B-C. Quantitative analysis of p-PI3K (B) and p-Akt (C) density at the lesion sites after TBI. *, p < 0.05 vs. sham; #, p < 0.05 vs. TBI + P. copri or TBI + GUO, n = 6 per group. One-way ANOVA was used to compare the means of different groups followed by a Tukey post hoc multiple-comparisons test
Fig. 11
Fig. 11
LY294002 inhibited the neuroprotective effects of P. copri. A-B. Quantitative analysis of short-term neurological function using NSS scores (A) and wire grip scores (B). *, p < 0.05 vs. sham; #, p < 0.05 vs. TBI + P. copri or TBI + GUO. n = 8 per group. C. Representative images of the movement path of mice in the open field test 14 days after TBI. D-H. Quantitative analysis of central time (D), periphery time (E), total distance (F), time immobile (G) and mean speed (H) travelled by mice in the open field test. *, p < 0.05 vs. sham; #, p < 0.05 vs. TBI + P. copri or TBI + GUO, n = 8 per group. I. Representative images of the swim path of mice in the Morris water maze. J-M. Quantitative analysis of latency in the learning test (J), the time in the target quadrant (K), the latency (L) and the average swimming speed (M) in the probe trial. *, p < 0.05 vs. sham; #, p < 0.05 vs. TBI + P. copri or TBI + GUO, n = 8 per group. Two-way repeated-measures ANOVA with Tukey’s post hoc multiple-comparisons test was used to analyze continuously measured data. One-way ANOVA was used to compare means of different groups followed by a Tukey post hoc multiple-comparisons test
Fig. 12
Fig. 12
The GUO-PI3K/Akt axis was involved in the effects of P. copri on oxidative stress, blood-brain barrier and neuronal apoptosis in TBI mice. A-B. Oxidative stress after TBI. SOD activity (A) and ROS level (B). *, p < 0.05 vs. Sham; #, p < 0.05 vs. TBI + P. copri, n = 6 per group. C. Representative images of double immunofluorescence staining for Occludin and CD31, nuclei were stained with DAPI (blue). Scale bar = 100 μm. D. Quantitative analysis of relative Occludin fluorescence intensity in different groups. *, p < 0.05 vs. sham; #, p < 0.05 vs. TBI + P. copri, n = 4 per group. E. Representative Western blot bands of ZO-1, Occludin, Bcl-2, Bax and β-Actin at the lesion sites after TBI. F-I. Quantitative analysis of the relative density of ZO-1 (F), Occludin (G), Bcl-2 (H) and Bax (I). *, p < 0.05 vs. sham; #, p < 0.05 vs. TBI + P. copri, n = 6 per group. One-way ANOVA was used to compare means of different groups followed by a Tukey post hoc multiple-comparisons test
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