He staining

After 1, 2, and 4 weeks, the rats were euthanized using an intraperitoneal overdose of anesthetic (Pentobarbital sodium salt, Nacalai tesque, Inc., Kyoto, Japan). Skin tissue, including wounds, was collected from rats for histological evaluation. The excised tissues were fixed with 20% paraformaldehyde-containing phosphate buffer for 48 h. The fixed tissues were then embedded in a paraffin wax block. The specimens were sectioned into 6–8 µm thicknesses from the block. The sections were then stained using hematoxylin and eosin staining (HE staining: Muto Pure Chemicals, Co., Ltd., Tokyo, Japan) and Masson’s trichrome staining (MT staining: Muto Pure Chemicals, Co., Ltd., Tokyo, Japan). Cells and collagen generated during wound healing were observed on the stained sections with an optical microscope (BX53, OLYMPUS, Tokyo, Japan).

The procedures of myoblastic cell cultivation were following Chang et al. (2012). Briefly, myoblastic C2C12 cells (5×10⁵) were seeded in each of a 6-well plate for 24 hours. The media were changed to DMEM with 2% horse serum (HS, Gibco, USA) and at the same time treated with arecoline (Tokyo Chemical Industries, Tokyo, Japan) and NAC for 7 days. The cells were then fixed with 4% paraformaldehyde (J.T. Baker, Radnor Township, Pennsylvania, USA) for 30 min and stained with hematoxylin and eosin (H&E staining, Muto Pure Chemicals, Tokyo, Japan). The myoblast differentiation was morphologically determined by measuring the total number of multinucleated myotubes and the average number of nuclei in each myotube. The cells were imaged by a phase contrast microscope Nikon Diaphot TMD (Nikon, Nikon Corporation, Tokyo, Japan) at 100x magnification.