*Contributed equally
Evidence has demonstrated that the gut microbiota, which consists of probiotics and pathogenic microorganisms, is involved in the initiation of ulcerative colitis (UC) via the dysregulation of intestinal microflora and normal immune interactions, which ultimately leads to intestinal mucosal dysfunction. Irisin is released from muscle cells and displays anti-inflammatory effects; however, the mechanisms underlying irisin-mediated anti-inflammatory effects in UC have not been previously reported. In the present study, mice were divided into the following four groups: i) Control; ii) irisin; iii) dextran sulfate sodium (DSS) salt; and iv) DSS + irisin. Subsequently, the effects of irisin were investigated by observing alterations in intestinal microbes. Irisin significantly reduced the degree of inflammation in UC by reversing alterations to the macroscopic score, histological score, number of CD64+ cells and inflammatory cytokine alterations (P<0.05). Analysis of the microbial diversity in the stools of mice with active UC indicated that the five bacteria that displayed the greatest alterations in relative abundance were
Ulcerative colitis (UC), a chronic non-specific inflammatory bowel disease, typically starts in the rectum and spreads to the proximal colon in a continuous manner, involving the area surrounding the appendix (
First identified in 2012, irisin is a myogenic factor (
A previous study revealed that plasma irisin levels in sedentary mice with UC fed a high-fat diet were decreased compared with those in mice fed a standard diet (
Dextran sodium sulphate (DSS) salt (colitis grade; lot no. Q5756) was obtained from MP Biomedicals, LLC. Recombinant irisin (cat. no. 067-29A) was obtained from Phoenix Pharmaceuticals, Inc. The CD64 antibody (cat. no. B166615) was obtained from BioLegend, Inc.
Mice were purchased from the Henan Experimental Animal Center [license no. SCXK (Yu) 2015-0004]. A total of 40 C57/BL mice (age, 6 weeks; average weight, 37.767±4.487 g) were randomly divided into the following four groups (n=10 per group; five males and five females): i) Control; ii) irisin; iii) UC; and iv) UC + irisin. The mice in each group were kept in different cages and administered the corresponding treatments. The control and irisin groups had access to normal autoclaved water. The UC and UC + irisin groups had access to 4% DSS (dissolved in autoclaved water) via drinking water. From day 1, the irisin and the UC + irisin groups received an intraperitoneal injection of irisin (0.0075 µg/g; dissolved in PBS) once a day, the UC group and the control group received an intraperitoneal injection of 0.2 ml of PBS, as described by Asadi
Mice were weighed daily and the spleen weight of each mouse was compared at the end of the experiment. The mice were evaluated using macroscopic and histological scoring systems. The histological score was assessed by hematoxylin and eosin staining of colon sections. Colon tissues were fixed in 4% paraformaldehyde overnight at room temperature and embedded in paraffin. The paraffin embedded colon tissues were cut into 5 µm thick slices. From each colon sample 5 sections were evaluated and this was repeated in 10 mice per group. The slices were stained at room temperature for 5 min with hematoxylin and 1 min with eosin. Stained sections were observed under a light microscope with a magnification of x200 in four randomly selected fields of view to evaluate inflammatory cell infiltration and mucosal and epithelial destruction. Tissues were histologically scored by two experienced pathologists independently in a blinded manner, from 0 (no change) to 6 (extensive cell infiltration and tissue damage), according to the Siegmund method (
Colon tissues were fixed in 4% paraformaldehyde at room temperature overnight, paraffin-embedded, sectioned into 5 µm slices, and incubated for 2 h at 60˚C. The sections were dewaxed with xylene, hydrated with a descending alcohol series and blocked using 5% BSA (cat. no. 9048-46-8; Sigma-Aldrich; Merck KGaA) at room temperature for 1 h. Antigen retrieval was performed by microwaving the sections. Subsequently, the sections were incubated with a PE-labelled CD64 primary antibody (cat. no. 139303, BioLegend, Inc.; 0.2 mg/ml) at room temperature for 1.5 h, followed by nuclear staining with DAPI for 5 min at room temperature. The sections were sealed with glycerol and observed using a fluorescence microscope with a magnification of x200. CD64 expression was quantified using ImageJ software (version 1.51; National Institutes of Health) according to the following formula: Fluorescence integrated density=region of interest area x mean fluorescence intensity.
Prior to sacrifice on day 10, whole blood (0.5 ml) was collected from the submandibular vein of each mouse. Blood samples were centrifuged at 1,509.3 x g for 10 min at 4˚C to separate the plasma. Subsequently, plasma levels of IL-12 and IL-23 were measured using Hermes Criterion Biotechnology IL-12 mouse ELISA kits and Hermes Criterion Biotechnology IL-23 mouse ELISA kits respectively (cat. nos. I085 and I114; Elixir Canada Medicine Company Ltd.) according to the manufacturer's protocol.
On day 10, fecal samples were collected from each mouse. Fecal bacterial DNA was extracted using the TIANamp Stool DNA kit (cat. no. DP328-02; Tiangen Biotech Co., Ltd.) according to the manufacturer's instructions.
MetaVx™ library preparation and Illumina MiSeq sequencing were conducted at Genewiz, Inc. DNA quantity of the fecal bacterial DNA was determined using a Qubit 2.0 fluorometer (Invitrogen; Thermo Fisher Scientific, Inc.) and a library was prepared using the MetaVx™ Library Preparation kit (Genewiz, Inc.). V3 and V4 are two hypervariable regions of the prokaryotic 16S rDNA (
Statistical analyses were performed using SPSS software (version 19.0; IBM Corp.). Data are presented as the mean ± SD. Values were obtained from 10 animals per group. For normally distributed data, comparisons among multiple groups were analysed by one-way ANOVA followed by Tukey's post hoc test. For non-normally distributed data, comparisons among multiple groups were analysed by the Kruskal-Wallis test followed by the Dunn-Bonferroni post hoc test. Correlations between specific bacteria and the histological score or IL-12/23 levels were determined by Pearson correlation analysis. P<0.05 was considered to indicate a statistically significant difference.
In the UC group, body weight was significantly decreased from day 4-7 compared with the control group (P<0.01;
Compared with the control group, the number of CD64+ cells in colonic tissues in the irisin group was not significantly altered; however, the UC group displayed a significantly increased number of CD64+ cells compared with the control group (P<0.01;
The levels of IL-12 and IL-23 in the plasma of normal mice were 893.15±222.76 and 685.67±137.55 pg/ml, respectively. There was no significant difference in IL-12 and IL-23 levels between the control and irisin (911.63±129.57 and 698.69±163.15 pg/ml, respectively) groups. However, the levels of IL-12 (1,147.37±233.92; P<0.05) and IL-23 (1,137.27±156.49 pg/ml; P<0.01) in the UC group were significantly increased compared with the control group. Irisin significantly reduced UC-mediated upregulation of IL-12 (911.04±122.78 pg/ml; P<0.05). In addition, irisin decreased UC-mediated upregulation of IL-23 levels, but the decrease was not significant (
A total of 6,064,158 reads and 1,516,039,500 base pairs were analysed in the four groups. Quality filtering on joined sequences was performed, and sequences that did not fulfil the criteria were discarded. Finally, a total of 2,409,318 reads with an average length of 450.78 base pairs were obtained. The multivariate analysis of variance of the PCoA matrix scores indicated that primary coordinate 1 and primary coordinate 2 accounted for 22.94 and 9.84% of the total structural changes, respectively (
The gut microbiota community structure histograms indicated the microbial species present and their relative abundance (
In all samples, 20 main families were identified, including
Sequencing data identified 31 genera of microbial flora. The abundance of
Furthermore, 31 main species were identified in all four groups. For example, the relative abundance of
At the phylum level,
A positive association between the histopathological score and IL-12/23 levels was identified (
The composition of the gut flora was significantly different among the four groups (P=0.001; R=0.515;
UC is a chronic non-specific inflammatory bowel disease with a high recurrence rate, for which the current therapies are largely ineffective (
Intestinal microorganisms consist of commensal bacteria, probiotics and pathogenic bacteria (
In the present study, the symptoms of the UC model mice were consistent with the clinical symptoms of patients with UC, and the macroscopic and histological scores of UC model mice were increased compared with the control mice. The relative abundance of
Intestinal microbiota belong to a diverse anaerobic species and extraction may be challenging (
A number of studies have indicated that the occurrence of UC is not only related to intestinal microbiota alterations but is also related to alterations in the microbiota of the intestinal mucosal (
Due to the differences in UC course, stages, treatments and analytical methods in each patient, as well as the complex structure and function of the intestinal flora, at present, there is no consensus regarding alterations in intestinal aerobic bacteria and common anaerobic bacteria in patients with UC. As a result, the specific roles of bacteria in the progression of the disease have not yet been fully elucidated, and alterations in the gut flora and their relationship with pathological changes in the intestinal mucosa remain unclear. To date, the majority of studies have primarily focused on the relationship between a certain type of intestinal flora and UC; however, a comprehensive comparison between common intestinal flora and UC has not been performed. In the present study, a relatively thorough analysis of the intestinal microbiota in mice in each of the four groups was conducted.
To the best of our knowledge, a consensus regarding the anti-inflammatory mechanism underlying irisin has not been reached. Studies have indicated that irisin inhibits the inflammatory response and reduces pathological alterations during the progression of acute lung injury in a concentration-dependent manner (
IL-12 is a heterodimer molecule composed of p40 and p35 subunits connected by disulfide bonds. Under physiological conditions, IL-12 is produced by mature dendritic cells and activated monocyte macrophages and provides important signals that promote the differentiation and proliferation of T helper (Th)1 cells, thereby participating in the inflammatory response (
During the present study, three mice died. The first death occurred in the UC group on day 5 of the experiment, and on day 6, two further deaths were recorded (one in the UC group and one in the UC + Irisin group). According to previous study (
The present study demonstrated the anti-inflammatory effects of irisin, as well as the accompanied alterations to the intestinal microbiome. In addition, a possible preliminary relationship between the anti-inflammatory effect of irisin and alterations to the intestinal flora was identified. Future studies using specific order or gene knockout mice should be conducted to support the results of the present study. A limitation of the present study is that the relationship between alterations to the intestinal microbiota and inflammation is controversial. For example, colonic infusion of donor human intestinal flora can reverse UC in selected patients, which suggests that differences in the gut microbiota may be the cause of milder inflammation (
In conclusion, the present study suggested that irisin ameliorated gut inflammation potentially by altering the gut microbiota in UC model mice. Therefore, irisin may serve as a novel therapeutic agent for UC.
Not applicable.
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.
LXH and XTC performed the histopathological examination and immunofluorescence experiment, and contributed equally to this paper. XHL designed the research study. RLY conducted IL-12 and IL-18 measurement, and the correlation analysis. RLY and LXH confirmed the authenticity of the raw data. JNY and HCW helped to interpret and analyze the data. ZFD and YXL conducted high throughput sequencing experiments. XTC and XHL revised the work critically for important intellectual content. All authors read and approved the final manuscript.
The animal experiments were approved by the Medical Ethics Committee of Henan University (approval no. HUSOM2020-103).
Not applicable.
The authors declare that they have no competing interests.
Alterations to body weight, spleen weight, macroscopic score and histological score. (A) Body weight. (B) Spleen weight. (C) Macroscopic score. (D) Histological score. (E) Hematoxylin and eosin staining. x200, black arrows indicate congestion, edema and inflammatory cell infiltration in the colon tissue. **P<0.01 vs. control; #P<0.05 vs. UC. UC, ulcerative colitis.
Abundance of CD64+ cells in the colonic tissues of mice in the four groups. (A) Representative images of CD64 expression in the colonic tissues of the mice x200. (B) Quantification of CD64 expression. **P<0.01 vs. control; #P<0.05 vs. UC. UC, ulcerative colitis.
IL-12 and IL-23 levels in the plasma of mice in in the four groups. The level of (A) IL-12 and (B) IL-23 in the plasma. *P<0.05 and **P<0.01 vs. control; #P<0.05 vs. UC. IL, interleukin; UC, ulcerative colitis.
Comparison of β and α diversity index in the four groups. (A) A principal coordinate analysis was performed using the β diversity metrics of the weighted UniFrac distance in the four groups. There were apparent differences in the composition of the microbial population between the control, UC and UC + irisin groups. (B) Analysis of the unweighted pair group method with arithmetic mean clustering in the four groups. ‘0.02’ represented the scale of the difference between samples. (C) The dilution curve of each sample. (D) Shannon-Wiener, one of the α diversity indexes, could directly reflect the heterogeneity of a community based on the number of species present and their relative abundance. (E) Chao 1, another α diversity index, reflects the richness of the community in the sample. The green dot in the UC group represented an outlier. UC, ulcerative colitis; con, control; OUT, operational taxonomic unit; PC, primary coordinate.
Gut microbial community structure in the four groups. Microbial community structures by (A) phylum, (B) family, (C) genus and (D) species. Con, control; UC, ulcerative colitis.
Heatmaps of the gut microbiota in the four groups. A heatmap of the gut microbiota in the four groups at the (A) phylum and (B) class levels. The different shades of blue represented the abundance of different bacteria, the dark shade was high, and the light shade was low. Con, control; UC, ulcerative colitis.
Association between the histopathological score and IL-12/23 levels. IL, interleukin; UC, ulcerative colitis; RDA, redundancy analysis.
Differences in the microflora composition among the four groups. There was a significant difference among the four groups of samples. The circle in the control group represented an outlier. The R value was 0.515, and the P-value was 0.001, which indicates that the difference among the four groups of samples was significantly greater than those within the group. UC, ulcerative colitis.
Microbiota that serve vital roles in each group. The UC + irisin group had no dominant bacteria. Con, control; UC, ulcerative colitis; LDA, linear discriminant analysis.