Electroacupuncture at Tianshu (ST25) and Zusanli (ST36) alleviates stress-induced irritable bowel syndrome in mice by modulating gut microbiota and corticotropin-releasing factor

Abstract

Objective: To investigate whether electroacupuncture (EA) at bilateral Tianshu (ST25) and Zusanli (ST36) acupoints could alleviate stress-induced irritable bowel syndrome (IBS) and evaluate its effect on gut microbiota and corticotropin-releasing factor (CRF).

Methods: Thirty C57BL/6 mice were randomly divided into the normal, water avoidance stress (WAS), and WAS+EA groups (10 mice per group). An experimental model of IBS was established by exposing the animals to WAS. The mice were treated with EA at the bilateral Tianshu (ST25) and Zusanli (ST36) acupoints. The abdominal withdrawal reflex test was conducted to evaluate visceral sensitivity in IBS. Gut microbiota was analyzed using 16S rRNA sequencing and analysis. The expression of CRF was determined using immune-ofluorescence and quantitative real-time polymerase chain reaction.

Results: EA alleviated visceral hypersensitivity in a mouse model of WAS-induced IBS. It modulated the dysbiosis of gut microbiota induced by WAS. Moreover, it suppressed the WAS-induced overexpression of CRF in colon tissues.

Conclusion: The findings of this study suggest that EA alleviated WAS-induced IBS mechanisms possibly involving the modulation of the dysbiosis of gut microbiota and suppression of CRF expression.

Keywords: corticotropin-releasing hormone; electroacupuncture; gastrointestinal microbiome; irritable bowel syndrome.

Figure 1. α-diversity of the gut microbiota

A: Chao1 index in normal, WAS and WAS + EA groups; B: Ace index in normal, WAS and WAS + EA groups; C: Simpson index in normal, WAS and WAS + EA groups; D: Shannon index in normal, WAS and WAS + EA groups. Normal group: without treatment; WAS group: subjected to WAS for 1 h daily on 10 consecutive days; WAS + EA group: subjected to WAS for 1 h daily on 10 consecutive days and EA at bilateral Tianshu (ST25) and Zusanli (ST36) for 7 consecutive days. WAS: water avoidance stress; EA: electroacupuncture. ^aP < 0.01 versus normal group; ^bP < 0.01 versus WAS group.

Figure 2. Structure of the gut microbiota

A, B: PCoA plots of the unweighted and weighted UniFrac distances in normal, WAS and WAS + EA groups; C: NMDS ordination in normal, WAS and WAS + EA groups. Stress < 0.2 shows that NMDS can accurately reflect the difference between samples; D-E: the relative abundances of the microbial communities for each group at the phylum and genus levels. Normal group: without treatment; WAS group: subjected to WAS for 1 h daily on 10 consecutive days; WAS+EA group: subjected to WAS for 1 h daily on 10 consecutive days and EA at bilateral Tianshu (ST25) and Zusanli (ST36) for 7 consecutive days. WAS: water avoidance stress; EA: electroacupuncture; PCoA: principal co-ordinate analysis; NMDS: non-metric multidimensional scaling.

Figure 3. Composition of the gut microbiota

A-B: MetaStat analysis at the genus and order level in normal, WAS and WAS+EA groups; C: histogram of LEfSe in normal, WAS and WAS+EA groups; D: cladogram of LEfSe in normal, WAS and WAS+EA groups. LDA score > 3.0. The red, green, or blue shading in the cladogram depicts bacterial taxa that were significantly higher in either the normal, WAS or WAS+EA groups, as indicated. p: phylum; c: class; o: order; f: family; g: genus; s: species. Normal group: without treatment; WAS group: subjected to WAS for 1 h daily on 10 consecutive days; WAS+EA group: subjected to WAS for 1 h daily on 10 consecutive days and EA at bilateral Tianshu (ST25) and Zusanli (ST36) for 7 consecutive days. WAS: water avoidance stress; EA: electroacupuncture; LDA: linear discriminant analysis.

Figure 4. Effect of EA on the expression of CRF in colon tissues (magnification, × 200)

A: relative mRNA expression of CRF in normal, WAS and WAS + EA groups; B: relative fluorescence intensity of CRF in normal, WAS and WAS + EA groups; C1-C3: confocal images of CRF, DAPI and merge (CRF + DAPI) in normal group; C4-C6: confocal images of CRF, DAPI and merge (CRF + DAPI) in WAS group; C7-C9: confocal images of CRF, DAPI and merge (CRF + DAPI) in WAS + EA group. Micrographs represent labeling of CRF (green) in mice colon tissues. Nuclear staining was conducted with DAPI (blue). Data were mean ± standard deviation (n = 3/ group). Normal group: without treatment; WAS group: subjected to WAS for 1 h daily on 10 consecutive days; WAS + EA group: subjected to WAS for 1 h daily on 10 consecutive days and EA at bilateral Tianshu (ST25) and Zusanli (ST36) for 7 consecutive days. EA: electroacupuncture; WAS: water avoidance stress; CRF: corticotropin-releasing factor; DAPI: 4',6-diamidino-2-phenylindole. ^aP < 0.001, ^cP < 0.01 versus normal group; ^bP < 0.001, ^dP < 0.05 versus WAS group.