Abdominal ultrasound stimulation alleviates DSS-induced colitis and behavioral disorders in mice by mediating the microbiota-gut-brain axis balance

Abstract

Inflammatory bowel disease (IBD) has the potential to induce neuroinflammation, which may increase the risk of developing neurodegenerative disorders. Ultrasound stimulation to the abdomen is a potential treatment for dextran sulfate sodium (DSS)-induced acute colitis. The present study aimed to investigate whether abdominal low-intensity pulsed ultrasound (LIPUS) can alleviate DSS-induced neuroinflammation through the microbiota-gut-brain axis. Male mice were fed DSS to induce ulcerative colitis. LIPUS stimulation was then applied to the abdomen at intensities of 0.5 and 1.0 ​W/cm². Mouse biological samples were analyzed, and behavior was evaluated. [¹⁸F]FEPPA PET/CT imaging was employed to track and quantify inflammation in the abdomen and brain. Changes in the gut microbiota composition were analyzed using 16S rRNA sequencing. Abdominal LIPUS significantly inhibited the DSS-induced inflammatory response, repaired destroyed crypts, and partially preserved the epithelial barrier. [¹⁸F]FEPPA accumulation in the colitis-induced neuroinflammation in the abdomen and specific brain regions significantly decreased after LIPUS treatment. LIPUS maintained intestinal integrity by increasing zonula occludens and occludin levels, reduced lipopolysaccharide-binding protein and lipopolysaccharide levels in the serum, and improved behavioral dysfunctions. Moreover, LIPUS, at an intensity of 0.5 ​W/cm², reshaped the gut microbiota in colitis-induced mice by increasing the relative abundance of the Firmicutes and decreasing the relative abundance of the Bacteroidota. Our findings demonstrated that abdominal LIPUS stimulation has the potential to be a novel therapeutic strategy to improve colitis-induced behavioral disorders through microbiota-gut-brain axis signaling.

Keywords: Abdominal ultrasound; IBD; Intestinal inflammation; Microbiota; Neuroinflammation.

Fig. 1

LIPUS mitigated colon damage in DSS-induced acute colitis in mice. (A) Changes in body weight in different groups. (B) The disease activity index (DAI) score for each group. (C) Representative images of the colon. (D) Measurement of colon length. LIPUS reduced the increase in the spleen weight/body weight ratio in DSS-induced acute colitis. (F) Representative hematoxylin and eosin–stained images of the colon. (G) Histological score of each group. In (A) and (B), ∗, ^#, and ^† denote a significant difference between the DSS group and the Sham group, DSS ​+ ​LIPUS 0.5 group, and DSS ​+ ​LIPUS 1.0 group, respectively. In (C), (D), and (E), ∗ and ^# denote significant differences from the Sham group and the DSS group, respectively (∗^,#, p ​< ​0.05; ^##,††, p ​< ​0.01; ∗∗∗^,###, p ​< ​0.001; n ​= ​6–8).

Fig. 2

LIPUS inhibited inflammatory cytokine expression in DSS-induced acute colitis in mice. Quantification of the mRNA levels of (A) TNF-α, (B) IL-1β, and (C) IL-6 in colon tissue using qRT-PCR. Quantification of the mRNA levels of (D) TNF-α, (E) IL-1β, and (F) IL-6 in the cortex using qRT-PCR. Measurement of the mRNA levels of (G) TNF-α, (H) IL-1β, and (I) IL-6 in the hippocampus using qRT-PCR.∗, ^#, and ^† denote significant difference from the Sham group, DSS group, and DSS ​+ ​LIPUS 0.5 group, respectively (^#, p ​< ​0.05; ^##, p ​< ​0.01; ∗∗∗^,###, p ​< ​0.001; n ​= ​6).

Fig. 3

PET/CT imaging evaluated the effects of abdominal LIPUS stimulation on the level of inflammation in the abdomen and brain of mice. (A) Mice were given DSS for 7 days to induce an IBD disease model. Abdominal LIPUS stimulation was performed from day 4 to day 7. A PET/CT scan was performed on day 7 to evaluate the inflammation levels after LIPUS treatment (DSS: dextran sulfate sodium; PET: positron emission tomography; CT: computed tomography). (B) Representative PET/CT images of [¹⁸F]FEPPA were obtained for the Sham, DSS, and DSS ​+ ​LIPUS groups on day 7. PET/CT images of the levels of inflammation in the abdomen and brain were obtained simultaneously. The white-dashed area shows the contour of the abdomen for quantitative analysis. (C) Results of quantitative analysis of the averaged standard uptake value (SUV) of [¹⁸F]FEPPA in the abdomen showed that abdominal LIPUS stimulation improved DSS-induced abdominal inflammation in the DSS ​+ ​LIPUS group compared to the DSS group. The SUV of each group is color-coded within the specified range. These images show the levels of inflammation after LIPUS treatment. (D) PET/CT images of the brain were obtained 30 ​min after intravenous injection of [¹⁸F]FEPPA. The different colored lines indicate the contours of the respective brain sections used for quantitative analysis. Quantitative analysis of [¹⁸F]FEPPA uptake in (E) the whole brain, (F) cortex, (G) striatum, (H) midbrain, (I) hippocampus, (J) thalamus, (K) hypothalamus, (L) amygdala, (M) cerebellum, and (N) brain stem. [¹⁸F]FEPPA uptake was lower in all brain regions after abdominal LIPUS stimulation compared to the uptake in the DSS group. ∗ and ^# denote significant differences from the Sham group and DSS group, respectively (∗,^#, p ​< ​0.05; ∗∗^,##, p ​< ​0.01; ^###, p ​< ​0.001; n ​= ​6).

Fig. 4

LIPUS inhibited the neuroinflammatory response and alleviated tight junction protein degradation in the colon of mice with DSS-induced acute colitis. Serum levels of (A) lipopolysaccharide-binding protein (LBP), (B) lipopolysaccharide (LPS), and (C) interleukin-6 (IL-6) were measured. The relative levels of (D) zonula occludens (ZO-1), (E) occludin, and (F) inducible nitric oxide synthase (iNOS) in colon tissue were assessed. ∗, ^#, and ^† denote significant differences from the Sham group, DSS group, and DSS ​+ ​LIPUS 0.5 group, respectively (∗,^#, p ​< ​0.05; ^##,††, p ​< ​0.01; ∗∗∗^,###, p ​< ​0.001; n ​= ​6).

Fig. 5

LIPUS alleviated DSS-induced behavioral disorders in mice. (A) Representative movement paths in the open field test (OFT). (B) Total distance traveled in the entire OFT area. (C) Distance traveled in the central OFT area. (D) Dwell time results. (E) The novel object recognition task (NORT). (F) Exploration ratios of the four groups. DSS-treated mice exhibited significant object memory impairment compared to Sham mice, while LIPUS treatments ameliorated these deficits. (G) The number of object encounters in the four groups. (H) The Y-maze test. (I) Preference ratios of the four groups. DSS-treated mice had a reduced preference ratio compared to Sham mice; the preference ratio was significantly improved by LIPUS treatment. (J) The total number of arm visits in the four groups. ∗, ^#, and ^† denote significant differences from the Sham group, DSS group, and DSS ​+ ​LIPUS 0.5 group, respectively (^#, p ​< ​0.05; ∗∗∗^{, ###}, p ​< ​0.001; n ​= ​8–10).

Fig. 6

Effects of LIPUS treatment on the gut microbiota structure in mice with DSS-induced colitis. (A) A Venn diagram was used to assess the shared and group-specific operational taxonomic units of the four groups. (B) Principal coordinate analysis (PCoA) based on the Bray–Curtis distance of the four groups. Each symbol represents an individual mouse. Relative abundance bar plots show bacterial composition at the (C) phylum, (D) genus, and (E) species level of each group. (F) The ratio of Firmicutes to Bacteroidota of the four groups at the phylum level. LIPUS significantly increased the F/B ratio at an intensity of 0.5 ​W/cm². (G) The phylum Firmicutes. (H) The phylum Bacteroidota. (I) The genus Bacteroides. (J) The genus Anaerobacterium. (K) The species Bacteroides caccae. ∗, ^#, and ^† denote significant differences from the Sham group, DSS group, and DSS ​+ ​LIPUS 0.5 group, respectively (∗,^#, p ​< ​0.05; ∗∗^,##,††, p ​< ​0.01; ∗∗∗, p ​< ​0.001; ∗∗∗∗^{,####,††††}, p ​< ​0.0001; n ​= ​6).