Background and aims: Patients with slow transit constipation (STC) have abnormal intestinal microbiota and metabolites, in which short-chain fatty acids (SCFAs) are of great importance. Colonic ICCs injury by abnormal microbiota may play a role in pathogenesis of STC. It’s suggested that intestinal muscularis macrophages (MMφ) converting to M1 type could change gastrointestinal motility regulated by unbalanced intestinal microbiota. MMφ is close to ICCs in space. Our aim was to investigate the effect of fecal metabolites from STC and SCFAs supplementation on colonic ICC, MMφ activation and related inflammatory factors as well as intestinal motility.
Methods: Fecal supernatant (FS) from healthy and STC subjects were collected and SCFA levels were measured by GC-MS. We detected the effects of FS (STC: n=3 and HC: n=3) and SCFAs on gastrointestinal transit, c-Kit expression of colons, numbers of ICCs and MMφ and M1 related inflammatory factors (IL-6 and TNF-α) in vivo. In vitro, the levels of TNF-α and IL-6 in bone marrow derived Mφ (BMDM) were measured after the intervention of STC-FS and SCFAs for 24h. HC-FS, STC-FS and different BMDM conditioned mediums (BMDM-CM) were used to stimulate the colonic ICCs of mice and then c-Kit mRNA and protein expression were detected.
Results: The fecal SCFA levels from STC patients were lower especially in acetic and propionic acid (Fig 1A). Mice receiving STC-FS intracolonic injection had fewer feces and delayed gastrointestinal transit. After supplementing SCFAs, the above performances were improved (Fig 1B-E). STC-FS reduced c-Kit expression and ICC number while SCFAs could partially protect ICC in mice’s colon (Fig 2B). However, STC-FS had little influence on ICC when macrophages were absent. ICC and MMφ are in close contact in muscular laye(Fig 2A). STC-FS promoted the expression and secretion of TNF-α and IL-6 while SCFAs can inhibit the IL-6 mRNA transcription and secretion by macrophages (Fig 1F). STC-BMDM-CM and exogenous IL-6 reduced c-Kit expression whereas SCFAs+STC- BMDM-CM and IL-6 neutralizing antibody could up-regulate the c-Kit mRNA and protein (Fig 2C-D).
Conclusions: STC-FS which is low in SCFAs damaged ICCs and gastrointestinal transit via upregulating IL-6 released by MMφ, instead of impairing ICCs directly. Supplementing SCFAs could protect ICCs and restore intestinal motility by inhibiting IL-6 secreted by MMφ.

<b>Figure 1.</b> <b>Changes and effect of short-chain fatty acids (SCFAs) in STC</b>

Figure 1. Changes and effect of short-chain fatty acids (SCFAs) in STC

Figure 2. STC-FS decreased c-Kit expression via inhibiting IL-6 by macrophages.

Weichen Liu

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