Background and Aims:
Gut microbiota affect the development of visceral hypersensitivity and gut dysmotility in irritable bowel syndrome (IBS). Recent studies found the mechanoreceptor Piezo2 affects gastrointestinal functions and may also affects visceral sensitivity. Whether the gut microbiota participates in the development of visceral hypersensitivity and gut dysmotility by regulating Piezo2 deserves further investigation.
Methods:
Pseudo-germ-free (pGF) mice was constructed by administering 4 antibiotics to 8-week-old male C57BL/6 mice. Then pGF mice randomly received fecal microbiota transplantation (FMT) from healthy volunteers or diarrhea-predominant IBS patients. Visceral hypersensitivity was detected by colorectal distension-electromyography (CRD-EMG). Whole-intestinal transit time, colonic transit time, number of fecal pellets, fecal water content were measured. Piezo2 expression in colon and dorsal root ganglion (DRG) neurons was detected by western blot. Piezo2 shRNA was used to established colon or DRG Piezo2 knock down mice. Gut microbiota was detected by 16s rRNA. Akkermansia muciniphila (AKK) and Fusobacterium varium (Fuso) at 10⁹ CFU/ml were gavaged to the pGF mice every other day for 2 weeks.
Results:
Compared with FMT-control group, FMT-IBS group had increased visceral sensitivity, shortened colonic transit time, increased number of fecal pellets, and increased fecal water content. But whole-intestinal transit time was not significantly different between the two groups (Fig. 1A-C). Piezo2 expression was elevated in the distal colon and DRG of FMT-IBS group (Fig. 1D). Compared with the FMT-IBS group injected with control shRNA, the FMT-IBS group injected with Piezo2 shRNA into either the colon or DRG could alleviate visceral hypersensitivity, prolong colonic transit time, and reduce the number of fecal pellets. However, there was no significant difference in whole-intestinal transit time and fecal water content (Fig. 1E-J). Fecal 16s rRNA detection found that Akkermansia and Fusobacterium are enriched in FMT-IBS group (Fig. 2A-E). After administration of AKK, the pGF mice had decreased visceral sensitivity, prolonged colonic transit time, and reduced fecal pellets number (Fig 2F). But after Fuso is administrated, the pGF mice had increased visceral sensitivity, shortened colonic transit time, increased fecal pellets number and increased fecal water content (Fig. 2G). Knocking down Piezo2 in the colon or DRG can significantly alleviate the visceral hypersensitivity, shortened colonic transit time, and increased number of fecal pellets caused by Fuso gavaged (Fig. 2H-I).
Conclusions:
Piezo2 is involved in the regulation of colon motility and visceral sensitivity;
Piezo2 receptor may be regulated by gut microbiota;
Fusobacterium varium may affect colon dysmotility and visceral hypersensitivity through affecting Piezo2.

<b>Figure 1 Piezo2 receptor is involved in the development of IBS-like behavior induced by FMT of IBS patients. </b><br /> A) CRD-EMG results, B) Colon transit time, number of fecal pellets output and water content of fecal pellets, C) Whole-intestinal transit time after FMT in pGF mice. D) Piezo2 expression in distal colon and DRG neurons innervating the colon of pGF mice after FMT. E) CRD-EMG results, F) Colon transit time and number of fecal pellets output, G) Whole-intestinal transit time and water content of fecal pellets after FMT in colon Piezo2 knock down pGF mice. H) The CRD-EMG results, I) The colon transit time and the number of fecal pellets output, J) The whole-intestinal transit time and the water content of fecal pellets after FMT in DRG Piezo2 knock down pGF mice. Data are presented as the mean ± SEM. A) n = 11; B-C) n = 23; D) n = 9 – 10; E-G) n = 8; H-J) n = 7 – 10.

Figure 1 Piezo2 receptor is involved in the development of IBS-like behavior induced by FMT of IBS patients.
A) CRD-EMG results, B) Colon transit time, number of fecal pellets output and water content of fecal pellets, C) Whole-intestinal transit time after FMT in pGF mice. D) Piezo2 expression in distal colon and DRG neurons innervating the colon of pGF mice after FMT. E) CRD-EMG results, F) Colon transit time and number of fecal pellets output, G) Whole-intestinal transit time and water content of fecal pellets after FMT in colon Piezo2 knock down pGF mice. H) The CRD-EMG results, I) The colon transit time and the number of fecal pellets output, J) The whole-intestinal transit time and the water content of fecal pellets after FMT in DRG Piezo2 knock down pGF mice. Data are presented as the mean ± SEM. A) n = 11; B-C) n = 23; D) n = 9 – 10; E-G) n = 8; H-J) n = 7 – 10.

<b>Figure 2 Piezo2 receptor is involved in the development of IBS-like behavior induced by <i>Fusobacterium varium</i> gavaged</b><br /> A) The α - diversity. B) The β – diversity. C) PCA. D) The gut microbiota composition in genus level. E) Differential expression of gut microbiota at genues level. F) CRD-EMG results, number of fecal pellets output, colon transit time and whole-intestinal transit time after AKK gavaged in pGF mice. G) CRD-EMG results, number of fecal pellets output, colon transit time and whole-intestinal transit time after Fuso in pGF mice. H) CRD-EMG results, number of fecal pellets output, colon transit time and whole-intestinal transit time after Fuso in colon Piezo2 knock down pGF mice. I) CRD-EMG results, number of fecal pellets output, colon transit time and whole-intestinal transit time after Fuso in DRG Piezo2 knock down pGF mice. Data are presented as the mean ± SEM. A - E) n = 10, F) n = 10 – 11, G) n = 10, H) n = 8, I) n = 10.

Figure 2 Piezo2 receptor is involved in the development of IBS-like behavior induced by Fusobacterium varium gavaged
A) The α - diversity. B) The β – diversity. C) PCA. D) The gut microbiota composition in genus level. E) Differential expression of gut microbiota at genues level. F) CRD-EMG results, number of fecal pellets output, colon transit time and whole-intestinal transit time after AKK gavaged in pGF mice. G) CRD-EMG results, number of fecal pellets output, colon transit time and whole-intestinal transit time after Fuso in pGF mice. H) CRD-EMG results, number of fecal pellets output, colon transit time and whole-intestinal transit time after Fuso in colon Piezo2 knock down pGF mice. I) CRD-EMG results, number of fecal pellets output, colon transit time and whole-intestinal transit time after Fuso in DRG Piezo2 knock down pGF mice. Data are presented as the mean ± SEM. A - E) n = 10, F) n = 10 – 11, G) n = 10, H) n = 8, I) n = 10.

Si-Qi Lu

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