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

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.