Effect of Electroacupuncture in Mice with Dextran Sulfate Sodium-Induced Colitis and the Influence of Gut Microbiota

Abstract

Background: The relationship between inflammatory bowel disease and gut microbiota is inextricable. Electroacupuncture (EA) can alleviate acute experimental colitis, but the performance of intestinal microorganisms and the mechanism are still not fully understood. We investigated the relationship between the EA and gut microbes and clarified the role of tight junction and adiponectin in the anti-inflammatory effect of EA.

Methods: Male C57BL/6 mice were randomized into three groups: normal control, dextran sulfate sodium- (DSS-) induced ulcerative colitis (DSS), and DSS with EA ST36 (DSS + EA). Mice body weight, DAI score, colon length, and histological score were evaluated for colitis severity. Colonic inflammation and tight junctions were demonstrated by the immunohistochemical (IHC) method. Systemic responses were confirmed by plasma cytokines and adiponectin with multiplex immunoassays. Gut microbiome profiling was conducted by 16S rRNA gene sequencing.

Results: EA had benefit in relieving both macroscopic and microscopic colonic inflammation. It can reduce disease activity, maintain colon length, and ameliorate histological inflammatory reaction. In IHC stain, EA decreased CD11b, F4/80, TLR4, and MyD88 and preserved claudin-1 and ZO-1 expression. Compared with the control group, the DSS group showed elevated levels of CRP, IFN-γ, TNF-α, and IL-6, but decreased adiponectin. These changes were reversed by EA, accompanied by modulation of the overall structure of gut microbiota.

Conclusion: Our findings suggest that EA exerts its therapeutic effect by TLR4 signaling via the MyD88-dependent pathway. EA could increase adiponectin, maintain mucosal tight junctions, and modulate gut microbiota.

Figure 1

Electroacupuncture ameliorates dextran sulfate sodium- (DSS-) induced colitis in C57BL/6 mice. (a) Experimental protocol of 3.5% DSS colitis and electroacupuncture (EA) ST36 treatment course. (b) Mice body weights change from baseline after 3.5% DSS induction of colitis. (c) Intestine photograph of colorectum length in each group. (d) Representative H&E-stained colorectum sections (200x magnification) in mice with acute colitis. (e) Average daily chow diet consumption in each group. Statistics of (f) colon length, (g) histological score, and (h) disease activity index (DAI) in each group. ^∗Results of three-group comparison (represented by bold line segments with endpoints). ^#,^✠, ^✟Results of the DSS group versus DSS + EA group, DSS group versus control group, and DSS + EA group versus control group, respectively (represented as thin lines without endpoints). ^∗#✠†P < 0.05; ^{∗∗##✠✠††}P < 0.01; ns: no significance. Data were presented as mean ± SEM of six mice in each group.

Figure 2

Immunohistochemical (IHC) staining of colon sections (200x magnification). Upper part: expression of (a) CD11b, (b) F4/80, (c) TLR4, (d) MyD88, (e) claudin-1, and (f) ZO-1 antibody in colon tissue of different groups. The arrowheads in the DSS group indicate the positive yellow and brown staining for these proteins, which mean that macrophages infiltrated bowel inflammation (a, b) through TLR4 signaling via MyD88-dependent pathway (c, d). The arrows in DSS + EA group indicate the positive brown staining for these proteins, which mean tight junction preservation (e, f) when compared to the DSS group. Scale bar: 50 μm. TLR4: Toll-like receptor 4. EA: electroacupuncture ST36. Lower part: quantification of area percentage of IHC staining by true color image analysis with the application of adjusted thresholds. ^∗Results of three-group comparison (represented by bold line segments with endpoints). ^#, ^✠, ^✟Results of the DSS group versus DSS + EA group, DSS group versus control group, and DSS + EA group versus control group, respectively (represented as thin lines without endpoints). ^∗#✠†P < 0.05; ^∗∗P < 0.01; ns: no significance. Data were presented as mean ± SEM of repeated adjusted thresholds in each group.

Figure 3

Overall structural modulation of gut microbiota after EA ST36 treatment. (a) Rarefaction analysis on Chao1 curves for nine samples, a measure of species richness. (b) Rarefaction analysis on Shannon curves for nine samples, a measure of species diversity. (c) The rank abundance of samples. (d) Multiple-sample PCA. (e) Venn diagram of OTUs in the three groups. (f) Multiple-sample similarity tree. (A) Samples of the control group, (B) samples of the DSS group, (C) samples of the DSS + EA group (a–f, n = 3). EA: electroacupuncture ST36.

Figure 4

Gut microbial community structure in mice after EA treatment. (a) Microbial community bar plot by phylum. (b) Microbial community bar plot by class. (c) Microbial community bar plot by order. (d) Microbial community bar plot by family. (e) Microbial community bar plot by genus. (f) Microbial community bar plot by species. From left to right, control, DSS, and DSS + EA groups, respectively. N = 3 in each group. EA: electroacupuncture ST36.

Figure 5

Electroacupuncture decreases plasma inflammatory marker/cytokines and increases adiponectin in dextran sulfate sodium- (DSS-) induced colitis in C57BL/6 mice. (a) C-reactive protein (CRP), as an inflammatory biomarker, was elevated in the DSS group and declined in DSS + EA group. Similar trends were observed in other proinflammatory cytokines, including (b) IFN-γ, (c) TNF-α, and (d) IL-6. (e) Adiponectin was decreased in DSS group and increased in DSS + EA group. ^∗Results of three-group comparison (represented by bold line segments with endpoints). ^#, ^✠, ^✟Results of the DSS group versus DSS + EA group, DSS group versus control group, and DSS + EA group versus control group, respectively (represented as thin lines without endpoints). ^∗#✠†P < 0.05; ^{∗∗##✠✠}P < 0.01; ns: no significance. Data were presented as mean ± SEM of six mice in each group.