Journal: Food Science and Human Wellness, Vol. 12, No. 5, pp. 1526-1537

Akkermansia muciniphila, one of the most promising next-generation probiotics, was reported to exhibit beneficial modulatory effects on the gut barrier. However, the strain-specific and underlying regulatory mechanisms of this species on gut barrier function were not well studied. Therefore, this study evaluated the protective effect of A. muciniphila strains on the intestinal barrier and investigated the mode of action and material basis of this modulatory effect. We first confirmed the strain-specific effects of A. muciniphila on intestinal barrier regulation and found that this phenomenon may be explained by the different abilities of strains to affect tight junction protein expression in enterocytes. Comparative genomic analysis proved that the ability of A. muciniphila to regulate the intestinal barrier was exerted in part by the functional genes (such as COG0438, COG0463, and COG2244) related to the synthesis of cellular surface proteins. The role of these surface proteins in intestinal barrier regulation was further verified by strain-comparative experiments in animal and cell models and surface protein removal trials. This study confirmed the different effects of A. muciniphila strains on gut barrier modulation and provided molecular and genetic targets for the screening of A. muciniphila strains with superior protection against gut barrier dysfunction.
 

Studies have shown that cannabinoid CB2 receptors are involved in wound repair, however, its physiological roles in fibrogenesis remain to be elucidated. In the present study, the capacity of cannabinoid CB2 receptors in the regulation of skin fibrogenesis during skin wound healing was investigated. To assess the function of cannabinoid CB2 receptors, skin excisional BALB/c mice were treated with either the cannabinoid CB2 receptor selective agonist, GP1a, or antagonist, AM630. Skin fibrosis was assessed by histological analysis and profibrotic cytokines were determined by immunohistochemistry, immunofluorescence staining, reverse transcription‑quantitative polymerase chain reaction and immunoblotting in these animals. GP1a decreased collagen deposition, reduced the levels of transforming growth factor (TGF)‑β1, TGF‑β receptor I (TβRI) and phosphorylated small mothers against decapentaplegic homolog 3 (P‑Smad3), but elevated the expression of its inhibitor, Smad7. By contrast, AM630 increased collagen deposition and the expression levels of TGF‑β1, TβRI and P‑Smad3. These results indicated that cannabinoid CB2 receptors modulate fibrogenesis and the TGF‑β/Smad profibrotic signaling pathway during skin wound repair in the mouse.

Cell Invasion and Migration Assays

Boyden chamber (CORNING, USA) was coated with 30 μl rat tail tendon collagen type I (PERFEMIKER, #C20-200110), incubated for 30 min at 37°C.  cells were seeded into chambers and cultured in DMEM/F12 with 2% FBS, and the lower chamber medium contains 10% FBS. After 20 hr, cells were fixed with 4% paraformaldehyde, and the nonmigrated cells were removed with cotton swabs. The migrated cells on the bottom were stained with crystal violet and counted. Invasion assays were repeated six times over multiple days.

For the wound-healing assay, horizontal lines were drawn on the back of 12-well plates at a spacing of 0.5~1 cm using a marker pen, with at least 5 lines pass through each well plate. When cells were grown to 70% confluence in 12 well tissue culture dish, a single scratch was made perpendicular to the horizontal lines, using a 10 μl pipette tip. Cells were then rinsed with PBS and incubated in serum-free medium with different concentrations of ITE (0.1 nM, 1 nM, 10 nM, 100 nM, 1000 nM, and 100000 nM) or 0.02% DMSO (SIGMA, #D5879) for 20 hr. Images were captured at 0 hr and 20 hr at the same position of cross points of horizontal lines and scratch. Using ImageJ, the wound in each image was identified using the magic wand tool, and the area measured by the ROI function. Wound-healing assays were carried out in three batches of the experiment. In a batch, each treatment was replicated in three wells, quantified by measuring two or three fields per well.