Effect of mild moxibustion on intestinal microbiota and NLRP6 inflammasome signaling in rats with post-inflammatory irritable bowel syndrome

Abstract

Background: About one-third of refractory irritable bowel syndrome (IBS) cases are caused by gastrointestinal (GI) infection/inflammation, known as post-infectious/post-inflammatory IBS (PI-IBS). Although it is known that intestinal microbiota and host NOD-like receptor family pyrin domain containing 6 (NLRP6) inflammsome signaling are closely related to PI-IBS and moxibustion has a therapeutic effect on PI-IBS, whether moxibustion regulates the intestinal flora and host NLRP6 events in PI-IBS remains unclear.

Aim: To examine the regulatory effect of moxibustion on intestinal microbiota and host NLRP6 inflammatory signaling in PI-IBS.

Methods: Sprague-Dawley rats were divided into a normal control group, a model control group, a mild moxibustion group, and a sham mild moxibustion group. PI-IBS rats in the mild moxibustion group were treated with moxibusiton at bilateral Tianshu (ST 25) and Zusanli (ST36) for 7 consecutive days for 10 min each time. The sham group rats were given the same treatment as the mild moxibustion group except the moxa stick was not ignited. Abdominal withdrawal reflex (AWR) score was measured to assess the visceral sensitivity, and colon histopathology and ultrastructure, colonic myeloperoxidase (MPO) activity, and serum C-reactive protein (CRP) level were measured to evaluate low-grade colonic inflammation in rats. The relative abundance of selected intestinal bacteria in rat feces was detected by 16S rDNA PCR and the NLRP6 inflammsome signaling in the colon was detected by immunofluorescence, qRT-PCR, and Western blot.

Results: The AWR score was significantly decreased and the low-grade intestinal inflammation reflected by serum CRP and colonic MPO levels was inhibited in the mild moxibustion group compared with the sham group. Mild moxibustion remarkably increased the relative DNA abundances of Lactobacillus, Bifidobacterium, and Faecalibacterium prausnitzii but decreased that of Escherichia coli in the gut of PI-IBS rats. Additionally, mild moxibustion induced mRNA and protein expression of intestine lectin 1 but inhibited the expression of IL-1β, IL-18, and resistance-like molecule β by promoting the NLRP6 and reducing the mRNA and protein expression of apoptosis-associated speck-like protein containing CARD (ASC) and cysteinyl-aspartate-specific proteinase 1 (Caspase-1). The relative DNA abundances of Lactobacillus, Bifidobacteria, Faecalibacterium prausnitzii, and Escherichia coli in each group were correlated with the mRNA and protein expression of NLRP6, ASC, and Caspase-1 in the colon.

Conclusion: These findings indicated that mild moxibustion can relieve low-grade GI inflammation and alleviate visceral hypersensitivity in PI-IBS by regulating intestinal microbes and controlling NLRP6 inflammasome signaling.

Keywords: Intestinal inflammation; Intestinal microbes; Moxibustion; NLRP6 inflammasome; Post-inflammation irritable bowel syndrome; Traditional Chinese medicine; Visceral hypersensitivity.

Figure 1

Diagram of moxibustion in rats. Rats were immobilized on a fixing frame. A: A moxa stick of 5 mm in diameter was ignited and placed 2-3 cm above the bilateral Zusanli (ST26) and Tianshu (ST25) for 10 min per day with 7 d of continuous treatment in the mild moxibustion group; B: The sham moxibustion group was given the same treatment as the mild moxibustion group except that moxibustion was treated with an unlit moxa stick.

Figure 2

A rat post-infectious/post-inflammatory irritable bowel syndrome model established by 2,4,6-trinitrobenzenesulfonic acid induction. A: Gross structure; B: Pathology of the normal control group and model control group on days 7, 14, 21, and 28; C: Abdominal withdrawal reflex scores of the normal and model control groups under different pressure stimulations on day 28 after model generation. ^bP < 0.01 vs normal control group. AWR: Abdominal withdrawal reflex.

Figure 3

Effects of mild moxibustion on abdominal withdrawal reflex scores and low-grade inflammation of the colon in each group. A: Abdominal withdrawal reflex score; B: Observed gross structure; C: Histological score; D: Observed histopathology; E: Pathological score. ^bP < 0.01 vs normal control group; ^dP < 0.01 vs model control group. AWR: Abdominal withdrawal reflex.

Figure 4

Effects of mild moxibustion on colonic ultrastructure of rats with post-infectious/post-inflammatory irritable bowel syndrome.

Figure 5

Effects of mild moxibustion on serum C-reactive protein levels and colonic myeloperoxidase activities in post-infectious/post-inflammatory irritable bowel syndrome rats in each group. A: Serum C-reactive protein levels; B: Myeloperoxidase activities. ^bP < 0.01 vs normal control group; ^dP < 0.01 vs model control group; ^fP < 0.01 vs sham moxibustion group. CRP: C-reactive protein; MPO: Myeloperoxidase.

Figure 6

Effects of mild moxibustion on the relative DNA abundances of targeted bacteria in feces of post-infectious/post-inflammatory irritable bowel syndrome rats in each group. A: Relative DNA abundance of Lactobacillus; B: Relative DNA abundance of Bifidobacterium; C: Relative DNA abundance of Faecalibacterium prausnitzii; D: Relative DNA abundance of Escherichia coli. ^bP < 0.01 vs normal control group; ^cP < 0.05 and ^dP < 0.01 vs model control group; ^fP < 0.01 vs sham moxibustion group.

Figure 7

Effects of mild moxibustion on the mRNA and protein expression of NLRP6, ASC, and Caspase-1 in the colon of post-infectious/post-inflammatory irritable bowel syndrome rats in each group. A: The mRNA expression levels of NLRP6, ASC, and Caspase-1; B: The protein expression of NLRP6, ASC, and Caspase-1. ^aP < 0.05 and ^bP < 0.01 vs normal control group; ^dP < 0.01 vs model control group; ^eP < 0.05 and ^fP < 0.01 vs sham moxibustion group.

Figure 8

Effects of mild moxibustion on the protein expression of NLRP6, ASC, and Caspase-1 in the colon of post-infectious/post-inflammatory irritable bowel syndrome rats. NLRP6, ASC, and Caspase-1 are localized in the cytoplasm of mucosal cells in the mucosal layer of rats in each group.

Figure 9

Effects of mild moxibustion on the mRNA and protein expression of IL-1β and IL-18 in the colon of post-infectious/post-inflammatory irritable bowel syndrome rats in each group. A: The mRNA expression levels of IL-1β and IL-18; B: Protein expression levels of IL-1β and IL-18. ^aP < 0.05 and ^bP < 0.01 vs normal control group; ^dP < 0.01 vs model control group; ^fP < 0.01 vs sham moxibustion group.

Figure 10

Effects of mild moxibustion on the mRNA and protein expression of ITLN1 and RELMβ in the colon of post-infectious/post-inflammatory irritable bowel syndrome rats in each group. A: The mRNA expression levels of ITLN1 and RELMβ; B: Protein expression levels of ITLN1 and RELMβ. ^aP < 0.05 and ^bP < 0.01 vs normal control group; ^dP < 0.01 vs model control group; ^eP < 0.05 and ^fP < 0.01 vs sham moxibustion group.